UNIVERSIDAD DE GRANADA - hera.ugr.eshera.ugr.es/tesisugr/18583477.pdf · A todos mis compañeros y...

UNIVERSIDAD DE GRANADA FACULTAD DE FARMACIA

Departamento de Bioquímica y Biología Molecular II

TESIS DOCTORAL MECANISMO DE ACCIÓN DEL GLUCOMACROPÉPTIDO

COMO AGENTE ANTIINFLAMATORIO INTESTINAL

Memoria presentada por la Licenciada María del Pilar Requena Méndez para optar al grado de Doctor por la Universidad de Granada.

Fdo. María del Pilar Requena Méndez

VºBº de los directores:

Fdo.Olga Martínez Augustin Fdo. María Dolores Suárez Ortega

Fdo. Fermín Sánchez de Medina López-Huertas

Granada, 2009.

Editor: Editorial de la Universidad de GranadaAutor: María del Pilar Requena MéndezD.L.: GR 2673-2010ISBN: 978-84-693-2000-6

Campus de Cartuja 18071 Granada

Tfno.34 958 243 838 Fax.34 958 248 960

Facultad de Farmacia Departamento de Bioquímica y Biología Molecular II

Dña. Olga Martínez Augustin, Profesora Titular del Departamento de Bioquímica y Biología Molecular II de la Universidad de Granada CERTIFICA: Que el trabajo titulado “ MECANISMO DE ACCIÓN DEL GLUCOMACROPÉPTIDO COMO AGENTE ANTIINFLAMATORIO INTESTINAL” ha sido realizado por María del Pilar Requena Méndez bajo su dirección, y que reúne todos los requisitos para ser defendido y optar al grado de doctor. Y para que conste donde proceda, se firma este certificado en Granada a 12 de Noviembre de 2009. Fdo. Dra. Olga Martínez Augustin


Tfno.34 958 243 838 Fax.34 958 248 960


D. Fermín Sánchez de Medina López-Huertas, Profesor Titular del Departamento de Famacología de la Universidad de Granada CERTIFICA: Que el trabajo titulado “ MECANISMO DE ACCIÓN DEL GLUCOMACROPÉPTIDO COMO AGENTE ANTIINFLAMATORIO INTESTINAL” ha sido realizado por María del Pilar Requena Méndez bajo su dirección, y que reúne todos los requisitos para ser defendido y optar al grado de doctor. Y para que conste donde proceda, se firma este certificado en Granada, a 12 de Noviembre de 2009. Fdo. Dr. Fermín Sánchez de Medina López-Huertas


Tfno.34 958 243 838 Fax.34 958 248 960


Dña. María Dolores Suárez Ortega, Profesora Catedrática del Departamento de Bioquímica y Biología Molecular II de la Universidad de Granada CERTIFICA: Que el trabajo titulado “ MECANISMO DE ACCIÓN DEL GLUCOMACROPÉPTIDO COMO AGENTE ANTIINFLAMATORIO INTESTINAL” ha sido realizado por María del Pilar Requena Méndez bajo su dirección, y que reúne todos los requisitos para ser defendido y optar al grado de doctor. Y para que conste donde proceda, se firma este certificado en Granada a 12 de Noviembre de 2009. Fdo. Dra. María Dolores Suárez Ortega


Tfno.34 958 243 838 Fax.34 958 248 960


D. Alberto Vargas Morales, Profesor Catedrático y Director del Departamento de Bioquímica y Biología Molecular II de la Universidad de Granada CERTIFICA: Que el trabajo titulado “MECANISMO DE ACCIÓN DEL GLUCOMACROPÉPTIDO COMO AGENTE ANTIINFLAMATORIO INTESTINAL” ha sido realizado por María de Pilar Requena Méndez, Licenciada en Farmacia, en los laboratorios de este departamento. Y, a efectos legales, se firma la siguiente constancia en Granada, a 12 de noviembre de 2009. Fdo. Dr. Alberto Vargas Morales

AGRADECIMIENTOS Esta Tesis Doctoral recoge los frutos del trabajo de estos años de investigación y supone, al menos en parte, el final de una etapa. En cierto modo, el fin de este proyecto asusta, porque implica que toca tomar decisiones y que hay que enfrentarse a un futuro muy incierto. Pero al mismo tiempo, me siento afortunada por haber tenido la oportunidad de trabajar en los que más me gusta, la investigación. Ahora bien, si miro atrás, tengo que admitir que, en realidad, lo que más me llena de alegría es saber que durante este tiempo nunca estuve sola. Por eso, porque esta Tesis tiene realmente muchísimos “coautores”, que han colaborado directa o indirectamente en la elaboración de este trabajo, quiero expresar mi más profundo agradecimiento a las siguientes personas. A la Dra. Olga Martínez Augustin, al Dr. Fermín Sánchez de Medina López-Huertas y a la Dra. María Dolores Suárez Ortega, mis directores de tesis, por la confianza depositada en mí durante estos años. Me habéis dado lo mejor de cada uno y por eso he aprendido mucho junto a vosotros. Y nunca podré olvidar el sprint final que os habéis metido para que pudiera pedir la beca. Gracias, Olga, por haberme enseñado a investigar, en todos los planos y con todo lo que eso conlleva, y por haber cuidado mi formación. Gracias, Fermín, por todos los sabios consejos, ideas y diseños experimentales, así como por tu disponibilidad en cada momento. Y Gracias, Mª Lola, por la cálida acogida en tu grupo de investigación en aquellos comienzos tempranos y por todas tus enseñanzas. Al Ministerio de Educación, por financiar mis años de doctorado y mis estancias en Boston mediante una beca FPU. Al departamento de Bioquímica y Biología Molecular II, por permitirme realizar mi trabajo aquí y avalarlo en las convocatorias de becas. A los profesores, por vuestro apoyo y por enseñarme en mis primeros pasos en la Docencia. Y especialmente, gracias a Mª Carmen y Jose Manuel, por vuestra ayuda con el papeleo y las prácticas, respectivamente. Al departamento de Farmacología, por dejarme usar sus instalaciones. A todos sus miembros, pero de manera especial al “Pollo grupo”: Rocío, Isa, Cristi, Raquel, Mercedes y Borja, por la acogida en vuestro laboratorio y las risas. A Mercedes, por enseñarme a trabajar con ratitas y por tu ayuda en tantas ocasiones. Y a Rocío, por las técnicas que me has enseñado y por todos estos años. Al departamento de Bioquímica y Biología Molecular III e Inmunología, por dejarme usar sus instalaciones. Especialmente, a la Dra. Ana Abadía, por tus enseñanzas en citometría, y a los becarios, por la acogida dentro y fuera del laboratorio. A todos mis compañeros y amigos del departamento, a los que aún andan por aquí y a los que trabajan ya en otro sitio, porque en mayor o menor medida, de todos he aprendido. Gracias a Laura, Natalia, Dámaso, Paty, Mer, Fer, Alí, Victor, Kike, Mª José, Olga, Raúl, Anabel, Sergio, María, y a los que me dejo en el tintero, que espero disculpen mi despite. A Laura y Natalia, por vuestra amistad, y porque sois en parte culpables de que haya llegado hasta aquí. Os echaré de menos el gran día. A Dámaso, por toda tu ayuda técnica (¿cuántas veces has salvado mi ordenador?), por tu apoyo y

por las risas durante las comidas. A Kike, por hacer del laboratorio un lugar de “buen rollo”. Y a los nuevos, Paty, Mer y Fer, por “llenar” de nuevo este lab y por vuestra ayuda científica. A mis amigos, de manera especial a cada uno de ellos. Gracias por los ratos de desconexión, los viajes, las tapas, las juergas, el hombro amigo, la paciencia con mis agobios, las llamadas oportunas... A las niñas: Isa, Raquel y Bea. Por tantos años de amistad, por escuchar incansables. ¡Que sigamos echando más cervezas “online”! A mis amigos de Sanfran, con quien comparto tantos proyectos de vida, especialmente a Paco, Patri Ga y Patri Ro, por cuidar nuestra relación en la lejanía. A la gente de la erasmus, por aquel año increible y la amistad que aún perdura. A los amigos de la Facultad. Y especialmente, a Ester, por haber compartido este proceso conmigo. Gracias por tu ayuda en algún experimento, pero sobretodo, gracias por estar siempre ahí y entenderme como nadie. A la Comunidad de San Francisco, por ser referencia en mi proceso personal. A mi familia. A mis abuelos Román y Concha, por preocuparse y alegrarse por mí, y por mimarme tanto. Y a mis abuelos Pepe y Ascención por confiar en mis posibilidades, aunque nunca ganaré el Nobel (ni nada que se le parezca, jeje). A mis hermanas, Patri y Ana, porque pese a la distancia siempre os he sentido cerca, siempre me habéis cuidado y os habéis alegrado por mí. He aprendido mucho de vosotras y aunque no lo diga muy a menudo, os echo de menos en Granada. A Ingo y Ricky, mis cuñados, porque siempre habéis estado pendientes de mí. A Pablo y Lucía, mis sobrinos, por alegrar los ratos de trabajo al estar retratados en el fondo de escritorio de mi ordenador. ¡Ojalá os tuviera más cerquita! A mis padres, por la educación que he recibido y de la que me siento muy orgullosa aunque no siempre consiga ponerla en práctica. Por vuestro apoyo en cada proyecto en el que me embarco, sufriéndolo más que yo misma, ¿verdad, mamá? Sois ejemplo de conciliación de las ambiciones profesionales y personales, y por eso, siempre me habéis animado a persiguir mi sueño sin dejar por el camino a los amigos, la familia o la pareja. Cuando no queda nadie, siempre, siempre, estáis ahí. Y eso es algo que nuncá podré pagaros. A Baldo, por tu apoyo incondicional durante esta Tesis, por tu paciencia, por llenar de risas mi vida, por enseñarme a relativizar aunque yo nunca aprenda, por darme alas, y por elegirme para nuestro proyecto más ambicioso. ¡Juntos somos más que dos! Al Padre, por todo lo que me regalas.

ACKNOWLEDGEMENTS With these words, I would like to express my acknowledgement to the people who contributed to make me feel like at home during my two stays in Boston. To Dr. William Allan Walker, for accepting and guiding me during those periods at the Mucosal Immunology Laboratory in Harvard Medical School. Those stays have been a great professional and personal experience. To Dr. Nanda Nanthakumar, from Mucosal Immunology Laboratory, for trusting me as a researcher and being always so kind. Thank you for the miRNA project, with which I have learned so much. To Dr. Charles R. Vanderburg, from Harvard NeuroDiscovery Center, for leading me into the amazing world of miRNAs. To Suzzette and Maureen, for their help with the paperwork. Specially, thanks to Maureen for receiving me at her place in Christmas Day. I will never forget it. To Sam, Alix and Krinsgton, the postdocs who “suffered” all my questions and kindly took care of me. To the labmates, particularly to Linda, for the lunchtime together and the great trip to Chicago. To the friends in Boston, who supported me in the sad and happy moments. To Marie-Anne and the Spanish group, for all the parties and good talks. To Tamara, a good friend with who you can talk about science, philosophy, parties and dances.

“La inspiración existe,

pero tiene que encontrarte trabajando”

(Pablo Picasso)

ÍNDICE ABREVIATURAS.............................................................................................................i CAPÍTULO 1: Resumen. New insights into the immunological effects of food bioactive peptides in animal models of intestinal inflammation. .....................................................1 In press. Proc Nutr Soc 2009. CAPÍTULO 2: Antecedentes bibliográficos y objetivos................................................15

1. El sistema inmune gastrointestinal......................................................................17 2. La enfermedad inflamatoria intestinal.................................................................33 3. El glucomacropéptido bovino..............................................................................47 Objetivos....................................................................................................................69

CAPÍTULO 3: Bovine glycomacropeptide has intestinal antiinflammatory effects in dextrane sulfate sodium rat colitis...................................................................................71 Submitted for publication. CAPÍTULO 4: Bovine glycomacropeptide ameliorates experimental rat ileitis by mechanisms involving downregulation of interleukin 17...............................................85 British Journal of Pharmacology (2008) 154, 825–832. CAPÍTULO 5: Bovine glycomacropeptide induces cytokine production in human monocytes through the stimulation of the MAPK and the NF-kB signal transduction pathways........................................................................................................................101 British Journal of Pharmacology (2009), 157, 1232–1240. CAPÍTULO 6: The intestinal antiinflammatory agent glycomacropeptide has immunomodulatory actions on rat splenocytes.............................................................117 Submitted for publication. CAPÍTULO 7: Effect of BGMP on intestinal epithelial cells as a part of the immune system............................................................................................................................133 CAPÍTULO 8: Pathways mediating the anti-inflammatory effect of bovine glycomacropeptide in hapten-induced colitis in the rat involve the upregulation of IL-10 expression......................................................................................................................139 Submitted for publication. CAPÍTULO 9: Discusión..............................................................................................159 CONCLUSIONES.........................................................................................................177

ABREVIATURAS

5-ASA AINE APC BGMP CAM CAS CD ConA COX DAI DC DSS ERK GM-CSF IBD IFNγ IKK IL iNOS IκB JNK LDH LPS LT MAPK MHC MPO NFAT NFκB NK NO PPARγ STAT TCR TGFβ TLR TNBS TNF Treg UC

ácido 5-aminosalicílico 5-aminosalicylic acid antiinflamatorio no esteroideo célula presentadora de antígenos antigen presenting cell glucomacropéptido bovino bovine glycomacropeptide molécula de adhesión celular cellular adhesion molecule casoplatelina enfermedad de Crohn Crohn’s disease Concanavalina A cicloxigenasa índice de actividad de la enfermedad disease activity index célula dendrítica dendritic cell dextrán sultafo sódico dextran sulfate sodium kinasa de proteínas activada por señales extracelulares extracellular signal-regulated kinase factor estimulante de colonias de granulocitos y macrófagos granulocyte-macrophages colony stimulating factor enfermedad inflamatoria intestinal inflammatory bowel disease interferón gamma kinasa de IκB IκB kinase interleuquina sintetasa inducible de óxido nítrico inducible nitric oxid synthetase inhibidor de κB kinasa c-Jun N-terminal c-Jun N-terminal kinase lactato deshidrogenasa lipopolisacárido linfotoxina kinasa de proteínas activada por mitógenos mitogen-activated protein kinase complejo mayor de histocompatibilidad mayor histocompatibility complex mieloperoxidasa factor nuclear de linfocitos activados nuclear factor of activated T- lymphocytes factor nuclear κB nuclear factor κB célula asesina natural natural killer óxido nítrico nitric oxid receptor activado por el proliferador de peroxisomas-gamma peroxisome proliferator-activated receptor gamma transductor de señal y activador de la transcripción signals transducer and activator of transcription receptor de los linfocitos T T cell receptor factor de crecimiento transformante-beta transforming growth factor-beta receptor tipo Toll Toll-like receptor ácido 2,4,6-trinitrobenceno sulfónico 2,4,6-trinitrobenzene sulfonic acid factor de necorosis tumoral tumor necrosis factor linfocitos T reguladores colitis ulcerosa ulcerative colitis

i

RESUMEN

New insights into the immunological effects of food bioactive peptides in animal models of intestinal inflammation.

Fermín Sánchez de Medina, Abdelali Daddaoua, Pilar Requena, Fermín Capitán-Cañadas, Antonio Zarzuelo, María D. Suárez, Olga Martínez-Augustin.In press. Proc Nutr Soc 2009.

CAPÍTULO 1

Abstract: Bioactive peptides have proven to be active in several conditions, including inflammatory bowel disease. This is a chronic and relapsing condition of unknown etiology that comprises chiefly ulcerative colitis and Crohn’s disease. Although there are treatments for inflammatory bowel disease, they have frequent side effects and they are not always effective; therefore there is a need for new therapies that could alleviate this condition. Two bioactive peptides present in milk (transforming growth factor-beta and casein macropeptide, also named glycomacropeptide) have been shown to have intestinal anti-inflammatory activities. In fact, transforming growth factor-beta is currently added to formulas intended for patients with IBD, and several studies indicate that these formulas could induce clinical remission. In this article, the evidences that guarantee the anti-inflammatory effect of transforming growth factor-beta and bovine glycomacropeptide, as well as their mechanisms of action, are reviewed. A special attention will be paid to evidences described in animal models of inflammatory bowel disease. Food bioactive peptides There is a series of specific food peptides, referred to as bioactive peptides that, besides their nutritional value, modulate biological processes. Bioactive peptides can be present in food as such or can be the result of in vivo or in vitro protein digestion [1]. To obtain the latter, both proteolytic enzymes from animal or bacterial origin can be used. Many of the bioactive peptide enriched functional foods currently marketed are obtained by bacterial fermentation. The low cost of these products and the positive image associated to fermented drinks and foods make these products very attractive [1,2]. Any source of food proteins can produce bioactive peptides. Thus, they have been isolated from sardine, corn, soy, egg, gelatine, etc. [3]. Nevertheless, the main source of food bioactive peptides is milk [1]. Several studies have demonstrated that food bioactive peptides can reach the intestine, after resisting digestion, go through the intestinal barrier, and hence exert effects both at the intestinal and systemic levels [2]. Accordingly, intestinal [1,4,5], cardiovascular [6,7] and immunological [4] effects have been described [1,3,8]. In addition, a group of peptides can also act as biocarriers for calcium and other minerals increasing their biodisponibility [8,9]. The activities of milk peptides and products containing them have been recently reviewed elsewere [1,2]. Inflammatory bowel disease Inflammatory bowel disease (IBD) is a chronic and relapsing condition that severely affects the quality of life of patients. The term IBD refers to a group of diseases such as microscopic colitis, collagenous colitis, pseudomembranous colitis, ulcerative colitis (UC) and Chrohn’s disease (CD). UC and CD are the most relevant conditions, both in incidence and prevalence [10]. They share histological and clinical similarities, nevertheless there are clear differences between them; while in UC only the colonic mucosa is damaged, in CD any part of the gastrointestinal tract from the mouth to the anus can be affected (although mostly the ileum and colon), and damage concerns not only the intestinal mucosa but the whole intestinal wall [10]. On the other hand, differences in the pathophysiology of these diseases have been observed that indicate a preponderance of T helper (Th) 1 response in CD patients, with an increased production of interleukin (IL)-12, interferon (IFN)-γ and IL-18, while a Th2 response seems to be operating in UC patients (see below). The etiology of IBD is unknown but genetic [11], environmental and life style factors (dietary habits, smoking, stress) have been associated with this condition [10,12-14]. Genetic disorders have been linked to intestinal microflora and intestinal permeability alterations in IBD patients, and currently the most accepted theory indicates that in humans intestinal inflammation can result from an inappropriate immune response by the host (driven by genetic and environmental factors) to the intestinal microflora.

Resumen

3

IBD can be currently treated with drugs, among which immunosuppressors, like azathioprine and corticoids, and aminosalicylates, together with anti-TNFα antibodies, are the treatment of choice in acute episodes and/or for delaying relapses in the disease [10]. Unfortunately, most of these treatments can lead to severe side effects [15]. Therefore, the search for new treatments that could alleviate or treat the disease is guaranteed. Pathophysiology of IBD Every type of intestinal cell is involved in the pathophysiology of IBD. Epithelial cells constitute the first line of defense. Studies in which the NFkB response in the intestinal epithelium of mice was specifically ablated showed that colonic inflammation and pancolitis developed after 3 weeks of age [16]. The authors of this study indicated that the inhibition of the NFkB response favours the disruption of the intestinal barrier integrity, resulting in bacterial translocation and intestinal inflammation [16]. In accordance with this indication, low levels of defensins in intestinal epithelial cells have been observed in IBD patients [17]. Macrophages and neutrophils are the first cells attracted to the focus of inflammation. There macrophages produce mainly TNF, IL-1β, IL-6 and IL-12. These cytokines attract neutrophils, T and B lymphocytes and regulate the inflammatory response. At least four different types of CD4+ Th lymphocytes (Th1, Th2, Th17 and Treg) are involved in the immune response in IBD. These are the result of naive Th cell (Th0) differentiation that ultimately depends on the cytokines produced in the inflammatory site (Figure 1). Differentiated T helper cells in turn produce cytokines that regulate the immune response activating or directing B lymphocytes, macrophages, neutrophils and cytotoxic cells. Tregs are CD4+ Foxp3+ regulatory T cells that play an essential role in intestinal homeostasis. Tregs are characterized by the expression of Foxp3, which is considered to confer their regulatory activity [18]. The expression of Foxp3 in peripheral Th0 is dependent on transforming growth factor (TGF)-β. Tregs appear in the intestine after the oral administration of antigens and produce IL-10 and TGF-β1, that in general terms downregulate inflammation. Several proinflammatory cytokines inhibit Treg induction including IL-6, IL-21, IL-23 and IL-27. Among them IL-23 is the key for the inhibition of Treg during inflammation [19]. IBD patients appear to have lower numbers of Tregs both in blood and colon, nevertheless it is important bear in mind that Tregs isolated from these patients are functionally active in vitro [19]. Th17 cells are a newly described subpopulation of T helper cells, involved in the pathogenesis of inflammatory bowel disease. These proinflammatory lymphocytes are characterized by the expression of the master transcription factor ROR-γt as well as IL-17A, IL17F, IL-21, IL-22 and IL-26 [21]. Th17 cells differentiate under the influence of IL-1 β, IL-6, IL-21 and IL-23 and TGF-β. The latter has been shown to be essential for their differentiation [21]. Th17 response appears to be involved in the pathophysiology of IBD, particularly in Crohn’s disease, in which Th1 cell response is also implicated [22-24]. Th1 cells express STAT-4 and produce IL-2, IL-12, IL-18 and IFNγ. While IFNγ, IL-12 and IL-18 drive Th1 differentiation, IL-10, TGF-β and IL-4 inhibit it. IL-2 acting in an autocrine fashion induces Th1 proliferation. The Th1 response implicates mainly the activation of macrophages increasing the production of IFNγ and in turn the production of IL-12 by macrophages and dendritic cells. Th2 cells express GATA-3 and produce IL-4, IL-5, IL-6 and IL-13. Th2 cells proliferate in response to IL-4 and IFNγ inhibits them. The activation of the Th2 response implicates B cells, increasing antibody production. As indicated above, Th1 response is predominant in CD patients while in UC colitis Th2 response seems to dominate [10].

Capítulo 1

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Figure 1. Molecular requirements for Th-cell differentiation. Adapted from Dennick and Tangye [20]. Animal models of IBD In general, animals do not suffer spontaneous chronic intestinal inflammation. Therefore, several animal models that mimic different aspects of the disease have been developed, including gene knockout (KO), transgenic, chemical, and adoptive transfer models [25]. Chemical models use different substances that disrupt the intestinal barrier or induce an immune reaction to induce colonic inflammation [26]. Among these the models of murine colitis induced by the administration of trinitrobenzene sulfonic acid (TNBS) or the dextran sodium sulphate (DSS) are the most widely used [27,28]. The first is a simple and convenient model based on one single rectal administration of TNBS dissolved in ethanol. TNBS is a hapten that elicits an immune response when bound with high molecular weight tissue proteins, while ethanol contributes to the disruption of the intestinal barrier. The result is a severe and prolonged degenerative inflammation of large parts of the colon that shares several clinical and molecular characteristics with CD. In particular, the inflammation produced by the administration of TNBS-ethanol involves all the layers of the intestinal mucosa and produces long lasting damage with cell infiltration and ulcers [29]. Furthermore, the administration of TNBS-ethanol to mice, as observed in human Crohn’s disease, produces Th1 driven inflammation characterized at the initial stage by the infiltration of macrophages and neutrophils releasing high levels of proinflammatory cytokines such as TNF, IL-1β or IL-6, followed by T cell infiltration, mainly of the CD4+ phenotype, that produce IL-2 and IFN-γ [27,29]. It is interesting to notice that, when administered directly into the ileum of rats, TNBS also induces ileitis [30]. The administration to mice or rats of DSS in the drinking water gives rise to an inflammation

Resumen

5

characterized by bloody diarrhoea, ulcerations and granulocyte infiltration. DSS is thought to be directly toxic to epithelial cells, disrupting the intestinal mucosal barrier. Both the TNBS and DSS model can be reproduced successfully in the absence of adaptive immunity, although this does not mean that the latter does not contribute to pathology in normal conditions [27]. The involvement of Th17 cells in these models is unclear or controversial at this time. Besides their similarities to multiple aspects of human IBD, the DSS and the TNBS murine models have several outstanding characteristics: the onset and duration of inflammation is immediate and controllable and there are no artificial genetic deletions or manipulations that are not found in human IBD [31]. The most used knock out model of IBD is the IL10-/- mice. These animals develop a spontaneous cecal inflammation and colitis at 2-4 month of age that features many characteristics observed in human IBD. The inflammation in this model is Th1 driven, similar therefore to CD inflammation in humans. Transgenic models of IBD are well represented by the HLA-B27 model of rat colitis. In this model HLA-27 and human microglobulin 2 mu transgenic rats develop chronic colitis that, among other characteristics, is accompanied by some extra intestinal complications resembling those seen in IBD patients, like spondyloarthropathy with peripheral and axial joint, dermatologic complications and male genital inflammation [32]. Finally, in the transference model CD4+ CD45RBhigh T cells (or equivalent) from healthy wild type mice are transferred to mice lacking functional B and T cells [33]. After 5-8 weeks pancolitis and intestinal inflammation is observed in recipient mice with features that are similar to those of human CD. This model presents the advantages that both early symptoms of inflammation and the perpetuation of the disease can be studied [33]. Bioactive peptides and inflammatory bowel disease Until now, the evidence indicates that at least two milk bioactive peptides could be useful in the treatment of IBD: TGF-β and bovine glycomacropeptide (BGMP), also denominated bovine casein macropeptide. TGF-β is a growth factor present in high concentrations in the milk of several species, including human milk, and it is also produced in small amounts in the intestine of newborn infants. There are at least 3 isoforms of TGF-β (TGF-β1, TGF-β2 and TGF-β3). These isoforms have high homology among them (70% TGF-β1 vs TGF-β2 and 74% TGF-β3 vs TGF-β2), and the amino acid sequence is highly conserved in different species (<94%) [34]. It has been described that TGF-β1 and TGF-β2 are generally equivalent in their functionality both in vivo and in vitro [35]. Human milk contains mainly TGF-β1, TGF-β2, being the last the major isoform (95%) [34,36]. Recent studies indicate that TGF-β is expressed as a pre-pro-factor and, after intracellular hydrolysis, the profactor is included with other proteins in a latent complex that is secreted. The latent form in milk is finally activated by gastric acid [37]. TGF-β has pleiotropic functions including the regulation of the immune function as well as functions related to cellular growth and differentiation. TGF-β1 transferred from the mother via placenta or milk is so essential that mouse in which this gene is disrupted survive until weanling, and then succumb to a wasting syndrome accompanied by a multifocal, mixed inflammatory cell response and tissue necrosis, leading to organ failure and death [38]. It has been widely demonstrated in rodents that the oral administration of TGF-β induces oral tolerance and inhibits allergic reactions [35]. When TGF-β is orally administered to TGF-β null mice it prevents their death and can be localized in internal organs such as lung, indicating that it can be active not only at the intestinal level [38,39]. The fast absorption in newborns together with the detection of higher TGF-β levels in the plasma of human healthy volunteers reinforce this hypothesis [35,40].

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Alterations in TGF-β signalling can play an important role in IBD. In fact, in a study carried out in mice in which a dominant negative mutant form of the TGF-β type II receptor was overexpressed specifically in the intestine, the animals spontaneously developed diarrhoea and under specific pathogen free (SPF) conditions were more susceptible to the induction of colonic inflammation by DSS [41]. In these mice treatment with DSS produces an increased expression of major histocompatibility complex class II, an exacerbated generation of autoantibodies against intestinal goblet cells, and an augmented activity of matrix metalloproteinase in intestinal epithelial cells, compared with wild-type littermates [41]. On the other hand, there is strong evidence that indicates the importance of TGF-β in the development of IBD. The induction of TGF-β brought about by the oral administration of haptenated colonic proteins (to induce oral toleration) protects mice from the induction of colitis by the administration of TNBS. This preventive effect is also obtained after the systemic administration of TGF-β. Conversely, the administration of anti-TGF-β antibodies reverses the protective effect of oral haptenated colonic proteins [42]. These studies indicate that TGF-β is a major regulator of intestinal mucosal homeostasis, acting in fact by regulating suppressive cells in an autocrine fashion, preventing the expression of IFNγ and therefore inhibiting the activation of T (mainly Th1) and B cells, and protecting the epithelial monolayer from the permeability enhancing effects of TNF [40]. Several authors have shown that the oral administration of TGF-β has anti-inflammatory effects at the intestinal level in animal models of colitis. Recently, a study has been published in which animals were fed 500 µl of cow’s milk containing TGF-β (3 µg/L) daily for 2 wk, before the induction of colitis by the administration of DSS. The authors observed that the animals that received TGF-β lost less weight and had a decreased degree of intestinal inflammation [35]. Furthermore, in 2005 Schiffrin et al. carried out a study to determine the effect of a casein based formula containing TGF-β (1 ng per mg of protein) to treat inflammation in HLA-B27 transgenic rats [15]. These authors described that the administration of TGF-β reduced white blood cells and the acute phase reactants fibrinogen and orosomucoid. In addition, colonic weight and wall thickness as well as the mRNA for IFNγ were reduced. Finally, there was an increase in MUC2 production in the cecum of the animals that received the TGF-β containing formula and a normalization of the muscle proteolytic activity. A formula with the same protein composition has also been proven to be anti-inflammatory in pediatric patients with Crohn’s disease, in which a reduction in the expression of IFNγ production was also observed [43]. In this study, seven children with Crohn’s disease received a TGF-β2 enriched formula for 8 weeks. In spite of the small number of patients, the results were quite clear since all the children showed a significant improvement of the disease activity, with C-reactive protein returning to normal, an increase in serum albumin and a substantial weight gain. Furthermore, ileal biopsies of these children showed reduced mucosal inflammation in six of seven children, with complete healing in two [43]. However, the lack of a control group limits the usefulness of this study. This was followed by other study with 29 children with Crohn’s disease in which a 79% remission was achieved, with complete healing in 10 cases, after 8 weeks administration of the TGF-β enriched formula [44]. In this study, Fell et al. described a fall in ileal IFNγ, indicating a decrease in Th1 response and a strong fall in ileal and colonic IL-1β and in colonic IL-8 mRNA [44]. This and several other studies have indicated that formulas enriched in TGF-β could induce clinical remission associated to mucosal healing in IBD patients [43-46]. These formulas are casein based, lactose free formulas that are enriched in TGF-β and are currently in the market. Although several studies have shown that the formulas are less effective that corticosteroids, they have proven to be more active in pediatric than in adults [45]. As a result, TGF-β enriched formulas are used in pediatric patients in which the administration of coricosteroids could have deleterious effects on linear growth [45].

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On the other hand, BGMP is a 64 amino acid peptide derived from the digestion of milk k-casein. This peptide is produced physiologically in vivo, as a result of k-casein hydrolysis, and in vitro. Actually, BGMP is one of the components that elutes with the whey that results after chymosin digestion during the cheese-making process. This peptide can be purified and is found in the market with a high purity degree. BGMP is heavily glycosylated and many of its actions depend on these glycosylations [47]. A wide variety of actions and applications have been described for BGMP. Because it does not contain phenylalanine in its sequence, BGMP has been proposed to be useful in the making of products for individuals with phenylketonuria [48-50]. On the other hand, this peptide has anticariogenic properties and has been proposed to be used in tooth pastes [47]. Its mechanism of action is related to the inhibition of bacterial growth, the prevention of tooth demineralisation and the promotion of enamel remineralisation [47]. In addition, BGMP may combat infection since it has been reported to bind to cholera and Escherichia coli enterotoxins, to inhibit bacterial and viral adhesion, to promote bifidobacteria growth and to modulate the immune system response (2, 6, 7). Because of its antimicrobial, mineral absorption [51] and possible prebiotic effects, BGMP is added to infant formulas [52]. It is important to indicate that, although as indicated above there are some studies showing that this peptide promotes the growth of bifidobacteria in vitro [53,54], so far there is not definitive evidence of the prebiotic effect of BGMP, since the studies carried out with Rhesus monkeys and human infants were hampered by a high initial level of bifidobacteria [51,55]. BGMP has been shown to affect both innate and adaptive immunity. Thus BGMP increases the proliferation of concanavalin A stimulated rat splenocytes (unpublished results), and induces the expression and production of iNOS, COX-2, IL-10 and FoxP3 at the concentration of 1 g/L. However, there is also evidence that BGMP inhibits mouse splenocyte proliferation induced by LPS and PHA [56]. While an inhibition in the production of IFNγ has been observed when splenocytes were co-stimulated with ConA (unpublished results). In macrophages BGMP seems to have also a stimulatory effect. For instance, BGMP increased the proliferation and phagocytic activity in a human macrophage cell line [57]. Our research group has shown, both in THP-1 cells (a monocyte/macrophage cell line) and in human peripheral blood macrophages, that BGMP stimulates the production of several cytokines (TNF, IL-1β and IL-8) [58]. In these experiments the mechanism of action of this peptide was further studied, showing that the whole peptide is needed, since it was active when protease inhibitors were added to the culture medium [58], and no effect was observed when it was hydrolysed (unpublished results). The effect of BGMP was prevented by the addition of MAPK or NF-kB inhibitors and the addition of BGMP induced the phosphorylation of IκB-α. Therefore, an involvement of these signal transduction pathways in the stimulatory effect of BGMP on cytokine production by macrophages was hypothesized [58]. In addition, studies by Monnai and Otani indicated that GMP increases the expression of an IL-1 receptor antagonist-like component in mouse spleen cells, involving probably monocytes [59]. In general, these results suggest that BGMP modulates the immune/inflammatory response by the activation of macrophages, favouring the differentiation of Treg cells, and hampering the activation of Th1 cells. As stated above, an exacerbated immune response and an imbalance in the intestinal flora have been blamed, among other factors, for the etiology of IBD. The fact that BGMP exerts immunomodulatory, antimicrobial and possibly prebiotic activities makes it a very good candidate to modulate intestinal inflammation. In fact, our research group has described that the oral

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administration of BGMP results in substantial anti-inflammatory effects in experimental colitis and ileitis [30,60]. In a first study we used the model of colitis induced by the administration of TNBS to rats. Rats were administered BGMP daily starting either 2 d before (pre-treatment) or 3 h after (post-treatment) colitis induction [60]. The effect of BGMP (500 mg/kg·day) was compared to that of sulfasalazine, an established drug used in the treatment of IBD. We found that pre-treatment with glycomacropeptide had a dose-dependent anti-inflammatory effect, characterized by lower body weight loss, decreased anorexia (57%), colonic damage (65%), and weight to length ratio (32%), as well as a reduction in colonic alkaline phosphatase activity (42%) and IL-1β, trefoil factor 3, and iNOS mRNA levels (P<0.05) [60]. The magnitude of the anti-inflammatory effect was generally comparable to that of sulfasalazine [60]. To further ascertain the mechanism involved in the anti-inflammatory effect of BGMP and to find new beneficial applications for this peptide, we studied its effect in a model of ileitis induced by the injection of TNBS in the ileum of rats [30]. Since CD affects any part of the intestine, ileitis frequently affects CD patients. Furthermore, ileitis can be also de result of ileal infections [61,62] and is a frequent complication of the ileal pouch-anal anastomosis interventions practiced to treat ulcerative colitis [63]. While lymphocyte Th1 and Th2 responses are predominant in colonic CD and UC patients, respectively, it seems that at least in Crohn’s ileitis both types of T cells are involved in the inflammatory response. Our results indicated that BGMP pre-treatment (500 mg/kg·day) results in a marked reduction of inflammatory injury, as assessed by lower extension of necrosis and diminished damage score, myeloperoxidase, alkaline phosphatase, iNOS, IL-1β and IL-17. Again, the effects of BGMP were similar to those observed in the same experiment for 5-aminosalicylic acid (200 mg/kg·day, 5-ASA). 5-ASA is the active part of sulfasalazine, which is release from the prodrug by colonic bacteria. From the above mentioned studies with BGMP we can conclude that BGMP has an anti-inflammatory effect in animal models of ileitis and colitis induced by TNBS, and that this effect is comparable to that of drugs frequently used to treat IBD. We have showed that BGMP decreases the expression of IL-1β, IL-1ra, TNF, and IL-17 in ileitis/colitis and therefore we can concluded that the mechanism of action of GMP probably involves Th17 cells, macrophages and T cells, probably excluding Th1 cells [30,60]. In a third study, we obtained mRNA from the colon of control and TNBS colitic rats administered vehicle or BGMP (500 mg/kg·day) as above and carried out microarrays, allowing the simultaneous measurement of over 30000 genes, and real time PCR for 96 selected genes (unpublished results). The use of powerful genomic and postgenomic techniques gave us further insight into the mechanism of action of this peptide. The processing of our results from microarrays and real time PCR with the Ingenuity Pathway Analysis software indicated that the Il1b (IL-1β) and Il6 (IL-6) genes, both of which are downregulated by BGMP, are key points in its mechanism of action. We observed also a decrease in IL-17, in neutrophil and macrophage infiltration, and in tissue remodeling genes, as well as an increase in Il10 gene and genes related to lymphocyte infiltration. IL-6 is a key factor in the development of inflammation and specifically in the uncontrolled inflammatory process in IBD [64-66]. In fact, increased blood and mucosal levels of this cytokine, related to the severity of the disease, have been described in UC and CD patients [64]. Accordingly, antibodies against IL-6 or its soluble receptor have been shown to be useful in the treatment of IBD in humans [65]. There is growing evidence indicating that this cytokine, produced mainly by macrophages and CD4+ T cells, is one of the main ones in the chronic phase of colitis [64]. IL-6 has three main roles in inflammation: (1) the recruitment of neutrophils to the inflammation site and

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activation to produce matrix metalloproteinases, contributing to severe tissue damage. (2) The regulation of CD4+ leukocyte apoptosis. IL-6, together with IL-12 and TNF, is an important antiapoptotic cytokine involved in the pathogenesis of CD, whose inflammatory response, like that of the TNBS model of colitis, is driven by Th1 (and possibly Th17) cells. And, (3) the induction of Th17 cell differentiation [64-66]. From our results we could hypothesize that downregulation of Il6 gene expression could: (1) specifically decrease Th17 cell populations, (2) reduce neutrophil infiltration at the site of inflammation, (3) lower the expression of tissue remodelation genes, probably as a consequence of the decrease neutrophil infiltration, and (4) probably allow the expression of Il10, which could also contribute to the resolution of colitis. In this study control rats were fed with BGMP and Il6 gene expression was also downregulated in these rats, reinforcing the idea that IL-6 downregulation could be key point in the anti-inflammatory effect of BGMP. It is known that in acute inflammation leukocyte recruitment to the site of inflammation is characterized by an initial infiltration of neutrophils, which are later replaced by a more sustained influx of mononuclear cells, initially macrophages and then lymphocytes (15). Therefore, the presence of increased amounts of lymphocytes together with a decrease in neutrophils and macrophages may be interpreted as indicative of an advanced stage of recovery from inflammation. Our results show just that, i.e. a decrease in the expression of genes related to macrophage and granulocyte activation, movement, recruitment and/or accumulation with BGMP treatment, together with an upregulation in both B and T cell related genes, indicating that the inflammatory state of BGMP treated animals was closer to resolution (unpublished results). These results are further sustained by the finding that Il1b (mainly produced by macrophages) [58], and Il6 (a neutrophil recruitment cytokine) (18) are key genes in the effect of BGMP as indicated by the analysis of microarray data using the Ingenuity Pathway analysis software. Because of the nature of our studies we cannot ascertain whether BGMP prevents the inflammatory damage induced by TNBS administration or accelerates the recovery from injury. Nevertheless, when BGMP was administered to TNBS rats before (pre-treatment) or after (post-treatment) the TNBS challenge, we found that the pre-treatment was more effective than the post-treatment, indicating that the preventive action possibly predominates. As alluded to before, studies carried out with splenocytes have shown that the addition of BGMP to culture medium increases the production of macrophage produced cytokines (IL-1β, TNF and IL-8) in a fashion that seems to be dependent on the activation of MAKP and NF-kB [58]. These results are apparently at odds with the anti-inflammatory effect of BGMP. However, the very complexity of IBD pathology makes it possible that monocyte activation may be involved in the anti-inflammatory activity of BGMP. Thus several studies suggest that defective response of the mucosa to harmful stimuli may actually worsen the outcome, at least in some cases [16,67]. We have already described the study of Nenci et al. [16] in which reduced epithelial activation of the NF-kB pathway, results in spontaneous severe colonic inflammation. Similarly, the absence of monocytes and dendritic cells aggravates rather than ameliorates experimental colitis [67]. Because it is well established that intestinal inflammation is strongly dependent on the presence of non-pathogenic bacteria, it may be concluded that the microbiota constitutes the challenge that must be met by the intestinal mucosa in just adequate terms: not too soft, not too hard [68]. Thus, defects in immune function may impair a prompt resolution of intestinal injury, triggering a more robust reaction to a normally trivial challenge. If so, monocyte stimulation would result in a more efficient and prompt response to luminal antigens as they gain access to the subepithelial milieu. In this regard, it is tempting to speculate that BGMP may exert monocyte/ macrophage-stimulating actions.

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Other biopeptides with anti-inflammatory potential As indicated above, a number of milk derived peptides with antimicrobial and immunomodulatory effects have been described. These properties could be very useful in the amelioration of intestinal inflammation. The properties of these peptides have been discussed in several reviews [1,8]. Recent studies have shown in the HLA-B27 model of intestinal inflammation that calcium supplemented diets ameliorate several important aspects of colitis, reducing IL-1β expression and histological scores and increasing the expression of extracellular matrix remodelling genes [69]. Since many caseinphosphopeptides have been shown to enhance Ca2+ absorption in vivo [8,9], these could be good anti-inflammatory candidates. On the other hand, several peptides and fermented milk products have been reported to have antioxidant effects. Antioxidants including glutathione and flavonoids [70-73] have proven to be effective in the treatment of IBD in several animal models, indicating that perhaps antioxidant peptides derived from milk could be also beneficial. Conclusions Research is demonstrating that milk derived peptides offer a wide variety of actions and some of them are good candidates to attenuate intestinal inflammation. Among these peptides TGF-β and BGMP have proven to be useful in animal models of intestinal inflammation and TGF-β has successfully been used in the treatment of Crohn’s disease patients. While TGF-β is a factor involved naturally in intestinal inflammation, its anti-inflammatory mechanism of action, that seems to be related at least in part to the inhibition of the Th1 response, needs to be better studied. The mechanism of action of BGMP remains to be fully described. Nevertheless, evidence indicates that it could have preventive effects, activating macrophages and, in inflammatory conditions, it could directly or indirectly downregulate IL-6 and upregulate IL-10. No clinical studies have been carried out with BGMP although it is known to be a safe food ingredient and is currently being added to infant formulas. References 1. Hayes M, Stanton C, Fitzgerald GF, Ross RP: Putting microbes to work: dairy fermentation, cell factories and bioactive peptides. Part II: bioactive peptide functions. Biotechnol J 2007;2:435-449. 2. Hayes M, Ross RP, Fitzgerald GF, Stanton C: Putting microbes to work: dairy fermentation, cell factories and bioactive peptides. Part I: overview. Biotechnol J 2007;2:426-434. 3. Korhonen H, Pihlanto A: Food-derived bioactive peptides--opportunities for designing future foods. Curr Pharm Des 2003;9:1297-1308. 4. Politis I, Chronopoulou R: Milk peptides and immune response in the neonate. Adv Exp Med Biol 2008;606:253-269. 5. Baldi A, Ioannis P, Chiara P, Eleonora F, Roubini C, Vittorio D: Biological effects of milk proteins and their peptides with emphasis on those related to the gastrointestinal ecosystem. J Dairy Res 2005;72 Spec No:66-72. 6. Erdmann K, Cheung BW, Schroder H: The possible roles of food-derived bioactive peptides in reducing the risk of cardiovascular disease. J Nutr Biochem 2008;19:643-654. 7. Saito T: Antihypertensive peptides derived from bovine casein and whey proteins. Adv Exp Med Biol 2008;606:295-317. 8. Martinez Augustin O, Martinez de Victoria Munoz E: Proteins and peptides in enteral nutrition. Nutr Hosp 2006;21 Suppl 2:1-13, 11-14. 9. Shimizu M: Food-derived peptides and intestinal functions. Biofactors 2004;21:43-47. 10. Bernstein CN, Fried M, Krabshuis JH, Cohen H, Eliakim R, Fedail S, Gearry R, Goh KL, Hamid S, Khan AG, Lemair AW, Ouyang Q, Rey JF, Sood A, Steinwurz F, Thomsen OO, Thomson A, Watermeyer G: World Gastroenterology Organisation Practice Guidelines for the Diagnosis and Management of IBD in 2010. Inflamm Bowel Dis 2009. 11. Achkar JP, Fiocchi C: Gene-gene interactions in inflammatory bowel disease: biological and clinical implications. Am J Gastroenterol 2009;104:1734-1736. 12. Gassull MA, Mane J, Pedrosa E, Cabre E: Macronutrients and bioactive molecules: is there a specific role in the management of inflammatory bowel disease? JPEN J Parenter Enteral Nutr 2005;29:S179-182; discussion S182-173, S184-178.

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13. Regueiro M, Kip KE, Cheung O, Hegazi RA, Plevy S: Cigarette smoking and age at diagnosis of inflammatory bowel disease. Inflamm Bowel Dis 2005;11:42-47. 14. Levenstein S, Prantera C, Varvo V, Scribano ML, Andreoli A, Luzi C, Arca M, Berto E, Milite G, Marcheggiano A: Stress and exacerbation in ulcerative colitis: a prospective study of patients enrolled in remission. Am J Gastroenterol 2000;95:1213-1220. 15. Schiffrin EJ, El Yousfi M, Faure M, Combaret L, Donnet A, Blum S, Obled C, Breuille D: Milk casein-based diet containing TGF-beta controls the inflammatory reaction in the HLA-B27 transgenic rat model. JPEN J Parenter Enteral Nutr 2005;29:S141-148; discussion S149-150, S184-148. 16. Nenci A, Becker C, Wullaert A, Gareus R, van Loo G, Danese S, Huth M, Nikolaev A, Neufert C, Madison B, Gumucio D, Neurath MF, Pasparakis M: Epithelial NEMO links innate immunity to chronic intestinal inflammation. Nature 2007;446:557-561. 17. Ramasundara M, Leach ST, Lemberg DA, Day AS: Defensins and inflammation: the role of defensins in inflammatory bowel disease. J Gastroenterol Hepatol 2009;24:202-208. 18. Gupta S, Shang W, Sun Z: Mechanisms regulating the development and function of natural regulatory T cells. Arch Immunol Ther Exp (Warsz) 2008;56:85-102. 19. Boden EK, Snapper SB: Regulatory T cells in inflammatory bowel disease. Curr Opin Gastroenterol 2008;24:733-741. 20. Deenick EK, Tangye SG: Autoimmunity: IL-21: a new player in Th17-cell differentiation. Immunol Cell Biol 2007;85:503-505. 21. Korn T, Bettelli E, Oukka M, Kuchroo VK: IL-17 and Th17 Cells. Annu Rev Immunol 2009;27:485-517. 22. Brand S: Crohn's disease: Th1, Th17 or both? The change of a paradigm: new immunological and genetic insights implicate Th17 cells in the pathogenesis of Crohn's disease. Gut 2009;58:1152-1167. 23. Sakuraba A, Sato T, Kamada N, Kitazume M, Sugita A, Hibi T: Th1/Th17 Immune Response is Induced by Mesenteric Lymph Node Dendritic Cells in Crohn's Disease. Gastroenterology 2009. 24. Leppkes M, Becker C, Ivanov, II, Hirth S, Wirtz S, Neufert C, Pouly S, Murphy AJ, Valenzuela DM, Yancopoulos GD, Becher B, Littman DR, Neurath MF: RORgamma-expressing Th17 cells induce murine chronic intestinal inflammation via redundant effects of IL-17A and IL-17F. Gastroenterology 2009;136:257-267. 25. Jurjus AR, Khoury NN, Reimund JM: Animal models of inflammatory bowel disease. J Pharmacol Toxicol Methods 2004;50:81-92. 26. Izcue A, Coombes JL, Powrie F: Regulatory lymphocytes and intestinal inflammation. Annu Rev Immunol 2009;27:313-338. 27. Wirtz S, Neufert C, Weigmann B, Neurath MF: Chemically induced mouse models of intestinal inflammation. Nat Protoc 2007;2:541-546. 28. Sanchez de Medina F, Martinez-Augustin O, Gonzalez R, Ballester I, Nieto A, Galvez J, Zarzuelo A: Induction of alkaline phosphatase in the inflamed intestine: a novel pharmacological target for inflammatory bowel disease. Biochem Pharmacol 2004;68:2317-2326. 29. Martinez-Augustin O, Merlos M, Zarzuelo A, Suarez MD, de Medina FS: Disturbances in metabolic, transport and structural genes in experimental colonic inflammation in the rat: a longitudinal genomic analysis. BMC Genomics 2008;9:490. 30. Requena P, Daddaoua A, Martinez-Plata E, Gonzalez M, Zarzuelo A, Suarez MD, Sanchez de Medina F, Martinez-Augustin O: Bovine glycomacropeptide ameliorates experimental rat ileitis by mechanisms involving downregulation of interleukin 17. Br J Pharmacol 2008;154:825-832. 31. Alex P, Zachos NC, Nguyen T, Gonzales L, Chen TE, Conklin LS, Centola M, Li X: Distinct cytokine patterns identified from multiplex profiles of murine DSS and TNBS-induced colitis. Inflamm Bowel Dis 2009;15:341-352. 32. Sartor RB: Colitis in HLA-B27/beta 2 microglobulin transgenic rats. Int Rev Immunol 2000;19:39-50. 33. Ostanin DV, Bao J, Koboziev I, Gray L, Robinson-Jackson SA, Kosloski-Davidson M, Price VH, Grisham MB: T cell transfer model of chronic colitis: concepts, considerations, and tricks of the trade. Am J Physiol Gastrointest Liver Physiol 2009;296:G135-146. 34. Massague J: The transforming growth factor-beta family. Annu Rev Cell Biol 1990;6:597-641. 35. Ozawa T, Miyata M, Nishimura M, Ando T, Ouyang Y, Ohba T, Shimokawa N, Ohnuma Y, Katoh R, Ogawa H, Nakao A: Transforming growth factor-beta activity in commercially available pasteurized cow milk provides protection against inflammation in mice. J Nutr 2009;139:69-75. 36. Cheifetz S, Hernandez H, Laiho M, ten Dijke P, Iwata KK, Massague J: Distinct transforming growth factor-beta (TGF-beta) receptor subsets as determinants of cellular responsiveness to three TGF-beta isoforms. J Biol Chem 1990;265:20533-20538. 37. Nakamura Y, Miyata M, Ando T, Shimokawa N, Ohnuma Y, Katoh R, Ogawa H, Okumura K, Nakao A: The latent form of transforming growth factor-beta administered orally is activated by gastric acid in mice. J Nutr 2009;139:1463-1468. 38. Letterio JJ, Geiser AG, Kulkarni AB, Roche NS, Sporn MB, Roberts AB: Maternal rescue of transforming growth factor-beta 1 null mice. Science 1994;264:1936-1938. 39. Letterio JJ: Murine models define the role of TGF-beta as a master regulator of immune cell function. Cytokine Growth Factor Rev 2000;11:81-87.

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40. Donnet-Hughes A, Duc N, Serrant P, Vidal K, Schiffrin EJ: Bioactive molecules in milk and their role in health and disease: the role of transforming growth factor-beta. Immunol Cell Biol 2000;78:74-79. 41. Hahm KB, Im YH, Parks TW, Park SH, Markowitz S, Jung HY, Green J, Kim SJ: Loss of transforming growth factor beta signalling in the intestine contributes to tissue injury in inflammatory bowel disease. Gut 2001;49:190-198. 42. Neurath MF, Fuss I, Kelsall BL, Presky DH, Waegell W, Strober W: Experimental granulomatous colitis in mice is abrogated by induction of TGF-beta-mediated oral tolerance. J Exp Med 1996;183:2605-2616. 43. Beattie RM, Schiffrin EJ, Donnet-Hughes A, Huggett AC, Domizio P, MacDonald TT, Walker-Smith JA: Polymeric nutrition as the primary therapy in children with small bowel Crohn's disease. Aliment Pharmacol Ther 1994;8:609-615. 44. Fell JM, Paintin M, Arnaud-Battandier F, Beattie RM, Hollis A, Kitching P, Donnet-Hughes A, MacDonald TT, Walker-Smith JA: Mucosal healing and a fall in mucosal pro-inflammatory cytokine mRNA induced by a specific oral polymeric diet in paediatric Crohn's disease. Aliment Pharmacol Ther 2000;14:281-289. 45. Fell JM: Control of systemic and local inflammation with transforming growth factor beta containing formulas. JPEN J Parenter Enteral Nutr 2005;29:S126-128; discussion S129-133, S184-128. 46. Afzal NA, Van Der Zaag-Loonen HJ, Arnaud-Battandier F, Davies S, Murch S, Derkx B, Heuschkel R, Fell JM: Improvement in quality of life of children with acute Crohn's disease does not parallel mucosal healing after treatment with exclusive enteral nutrition. Aliment Pharmacol Ther 2004;20:167-172. 47. Aimutis WR: Bioactive properties of milk proteins with particular focus on anticariogenesis. J Nutr 2004;134:989S-995S. 48. Laclair CE, Ney DM, MacLeod EL, Etzel MR: Purification and use of glycomacropeptide for nutritional management of phenylketonuria. J Food Sci 2009;74:E199-206. 49. van Calcar SC, MacLeod EL, Gleason ST, Etzel MR, Clayton MK, Wolff JA, Ney DM: Improved nutritional management of phenylketonuria by using a diet containing glycomacropeptide compared with amino acids. Am J Clin Nutr 2009;89:1068-1077. 50. Ney DM, Hull AK, van Calcar SC, Liu X, Etzel MR: Dietary glycomacropeptide supports growth and reduces the concentrations of phenylalanine in plasma and brain in a murine model of phenylketonuria. J Nutr 2008;138:316-322. 51. Bruck WM, Kelleher SL, Gibson GR, Nielsen KE, Chatterton DE, Lonnerdal B: rRNA probes used to quantify the effects of glycomacropeptide and alpha-lactalbumin supplementation on the predominant groups of intestinal bacteria of infant rhesus monkeys challenged with enteropathogenic Escherichia coli. J Pediatr Gastroenterol Nutr 2003;37:273-280. 52. Sandstrom O, Lonnerdal B, Graverholt G, Hernell O: Effects of alpha-lactalbumin-enriched formula containing different concentrations of glycomacropeptide on infant nutrition. Am J Clin Nutr 2008;87:921-928. 53. Idota T KH, Nakajima I. : Growth-promoting effects of N-acetylneuraminic acid-containing substances on Bifidobacteria. Biosci Biotech Biochem 1994;58:1720-1722. 54. Yakabe T KH, Idota T. Japanese patent.: Growth stimulation agent for bifidus and lactobacillus. 1994:07-267866. 55. Bruck WM, Redgrave M, Tuohy KM, Lonnerdal B, Graverholt G, Hernell O, Gibson GR: Effects of bovine alpha-lactalbumin and casein glycomacropeptide-enriched infant formulae on faecal microbiota in healthy term infants. J Pediatr Gastroenterol Nutr 2006;43:673-679. 56. Otani H MM, Kawasaki Y, Kawakami H, Tanimoto M. : Inhibition of mitogen-induced proliferative responses of lymphocytes by bovine kappa-caseinoglycopeptides having different carbohydrate chains. J Dairy Res 1995;62:349–357. 57. Li EW, Mine Y: Immunoenhancing effects of bovine glycomacropeptide and its derivatives on the proliferative response and phagocytic activities of human macrophagelike cells, U937. J Agric Food Chem 2004;52:2704-2708. 58. Requena P, Daddaoua A, Guadix E, Zarzuelo A, Suarez MD, de Medina FS, Martinez-Augustin O: Bovine glycomacropeptide induces cytokine production in human monocytes through the stimulation of the MAPK and the NF-kappaB signal transduction pathways. Br J Pharmacol 2009. 59. Monnai M OH: Effect of bovine k-caseinoglycopeptide on secretion of interleukin-1 family cytokines by P388D1 cells, a line derived from mouse monocyte/macrophage. . Milchwissenschaft. 1997;52:192–196. 60. Daddaoua A, Puerta V, Zarzuelo A, Suarez MD, Sanchez de Medina F, Martinez-Augustin O: Bovine glycomacropeptide is anti-inflammatory in rats with hapten-induced colitis. J Nutr 2005;135:1164-1170. 61. Sands BE: From symptom to diagnosis: clinical distinctions among various forms of intestinal inflammation. Gastroenterology 2004;126:1518-1532. 62. Navarro-Llavat M, Domenech E, Masnou H, Ojanguren I, Manosa M, Lorenzo-Zuniga V, Boix J, Gassull MA: Collagenous duodeno-ileo-colitis with transient IgG deficiency preceded by Yersinia enterocolitica intestinal infection: case report and review of literature. Gastroenterol Hepatol 2007;30:219-221. 63. Alexander F: Complications of ileal pouch anal anastomosis. Semin Pediatr Surg 2007;16:200-204. 64. Mudter J, Neurath MF: Il-6 signaling in inflammatory bowel disease: pathophysiological role and clinical relevance. Inflamm Bowel Dis 2007;13:1016-1023. 65. Mitsuyama K, Sata M, Rose-John S: Interleukin-6 trans-signaling in inflammatory bowel disease. Cytokine Growth Factor Rev 2006;17:451-461. 66. Carey R, Jurickova I, Ballard E, Bonkowski E, Han X, Xu H, Denson LA: Activation of an IL-6:STAT3-dependent transcriptome in pediatric-onset inflammatory bowel disease. Inflamm Bowel Dis 2008;14:446-457.

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67. Qualls JE, Kaplan AM, van Rooijen N, Cohen DA: Suppression of experimental colitis by intestinal mononuclear phagocytes. J Leukoc Biol 2006;80:802-815. 68. Seksik P, Sokol H, Lepage P, Vasquez N, Manichanh C, Mangin I, Pochart P, Dore J, Marteau P: Review article: the role of bacteria in onset and perpetuation of inflammatory bowel disease. Aliment Pharmacol Ther 2006;24 Suppl 3:11-18. 69. Schepens MA, Schonewille AJ, Vink C, van Schothorst EM, Kramer E, Hendriks T, Brummer RJ, Keijer J, van der Meer R, Bovee-Oudenhoven IM: Supplemental calcium attenuates the colitis-related increase in diarrhea, intestinal permeability, and extracellular matrix breakdown in HLA-B27 transgenic rats. J Nutr 2009;139:1525-1533. 70. Mane J, Loren V, Pedrosa E, Ojanguren I, Xaus J, Cabre E, Domenech E, Gassull MA: Lactobacillus fermentum CECT 5716 prevents and reverts intestinal damage on TNBS-induced colitis in mice. Inflamm Bowel Dis 2009;15:1155-1163. 71. Sanchez de Medina F, Galvez J, Gonzalez M, Zarzuelo A, Barrett KE: Effects of quercetin on epithelial chloride secretion. Life Sci 1997;61:2049-2055. 72. Sanchez de Medina F, Galvez J, Romero JA, Zarzuelo A: Effect of quercitrin on acute and chronic experimental colitis in the rat. J Pharmacol Exp Ther 1996;278:771-779. 73. Sanchez de Medina F, Vera B, Galvez J, Zarzuelo A: Effect of quercitrin on the early stages of hapten induced colonic inflammation in the rat. Life Sci 2002;70:3097-3108.

Capítulo 1

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Antecedentes bibliográficos y objetivos.

CAPÍTULO 2

1. EL SISTEMA INMUNE GASTROINTESTINAL

1.1. La mucosa intestinal.

La mucosa intestinal es la capa más externa de las cuatro del tracto gastrointestinal (GI), y representa la mayor área corporal en contacto con el entorno externo. En esta zona, no sólo la digestión y la absorción tienen lugar, sino que funciona como una barrera contra agentes nocivos e infecciones (5). En su superficie apical, la mucosa intestinal está cubierta por moco y glicocálix: un gel compuesto por mucinas, rodeado por un esqueleto de proteínas y cadenas de oligosacáridos (4). Debajo del glicocálix, la capa más externa de la mucosa está constituida por una monocapa de células (epitelio) que incluye células columnares (enterocitos), células caliciformes, células pluripotentes de las criptas, células de Paneth, células enteroendocrinas y los linfocitos intraepiteliales (IEL) (5). Debajo del epitelio se encuentra el tejido conjuntivo y de apoyo llamado lamina propria (LP), que tiene también una función inmunológica, debido a la presencia de diferentes células inmunes. Finalmente, la LP está rodeada por una capa de músculo liso denominada muscularis mucosae.

1.1.1 Células epiteliales - Enterocitos: Estas células son las mayoritarias en el epitelio de la mucosa

intestinal y su función más importante es la absorción de alimentos. Su membrana apical posee numerosos microvilli cubiertos con moco. En sus ápices, los enterocitos están conectados a las células vecinas por tres tipos de complejos de unión: zonula ocludens, zonula adherens y mácula adherens. Además, las membranas laterales interactúan por medio de moléculas de adhesión celulares (CAM) y otras estructuras (13). Todas estas conexiones confieren al epitelio la propiedad de barrera. Por otro lado, los enterocitos mantienen una polaridad característica, especialmente en la membrana.

- Células caliciformes: son las productoras del moco, que es liberado en gránulos

en respuesta a diversos estímulos, como la acetilcolina. Sus microvilli son irregulares. El moco está formado principalmente por mucinas, pero contiene también albúmina, immunogloglobulinas (principalmente IgA secretora, S-IgA), α1-antitripsina, lisozima, lactoferrina, factor de crecimiento epidérmico (EGF) y factores trefoil (TFFs).

- Células pluripotenciales de las criptas: están ubicadas en la porción media de las

criptas y producen células que se diferencian en enterocitos, células caliciformes, células Paneth y células enteroendocrinas. Se cree que la tasa de migración depende de factores extraluminales como el factor de crecimiento epitelial (EGF), el factor de crecimiento transformante (TGF), citoquinas, factores neurales y vasculares; y factores luminales como la nutrición, la motilidad y la microflora.

- Células de Paneth: comúnmente están ubicadas en las criptas del intestino

delgado, pero pueden aparecer en el estómago e intestino grueso en algunas enfermedades. Tienen una forma piramidal muy característica. Son también células secretoras: contienen gránulos con lisozima, factor de necrosis tumoral (TNF) (14), criptidinas (15) e inmunoglobulinas (16).

Antecedentes bibliográficos

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- Células enteroendocrinas: liberan hormonas en los capilares de la LP en respuesta a cambios en el entorno externo.

- Linfocitos intraepiteliales (IELs): tienen características especiales que los

distinguen de los linfocitos normales. Serán descritos más adelante.

1.1.2 Funciones de las células epiteliales. La principal función del tracto gastrointestinal es la digestión y absorción de nutrientes, que tiene lugar principalmente en los enterocitos. En cualquier caso, la mucosa intestinal tiene otras funciones importantes.

Por un lado el epitelio hace de barrera de entrada a patógenos. Así, en el lumen intestinal, el jugo biliar y pancreático, el moco y el glicocálix, la motilidad intestinal y la flora bacteriana interactúan para limitar la colonización por enteropatógenos (5). Se ha demostrado además que los IEL pueden matar células infectadas por virus y bacterias en animales de experimentación (19), pero no existe información disponible acerca de esa función en seres humanos. Por último, los complejos de unión de los enterocitos forman una barrera física a la entrada de los agentes patógenos.

Por otro lado, el epitelio tiene también función de presentación de antígenos e

inmunológica. A fin de trabajar como células presentadoras de antígenos (APC), las células epiteliales deberían expresar MHC-II y tener capacidad fagocítica. Efectivamente los enterocitos humanos expresan en su membrana basolateral MHC-I, así como MHC-II (20, 21). El HLA-DR (molécula del tipo MHC-II) presenta máxima expresión en íleon, mientras que su expresión es escasa en colon (21). Los IEL CD8+, que secretan IFNγ (22), podrían estimular a los enterocitos para expresar MHC II (23) y protegerse contra los virus.

Finalmente, las células epiteliales de colon producen una variedad de

interleuquinas (IL) como IL1, IL8, IL10, TNF, y TFG-β, que funcionan como señales a la LP cuando se produce una perturbación en la barrera epitelial (24).

1.2 Tejido linfoide asociado al intestino (GALT) El sistema inmunológico intestinal es el más extenso del cuerpo y tiene mecanismos únicos de defensa. Está permanentemente expuesto a estímulos externos y antígenos de microorganismos y alimentos y, como resultado, la mucosa intestinal se ha adaptado a distinguir entre señales bacterianas patógenas y no patógenas. Esta elevada “presión” antigénica hace necesaria la presencia de una gran cantidad de células inmunes, que se organizan formando el GALT (1). La existencia de células inmunes en estado activo a lo largo del tracto gastrointestinal ha hecho que algunos científicos y autores hablen de un estado de “inflamación controlada o fisiológica”. Ya que la mayoría de los componentes celulares y mecanismos implicados en esta inflamación fisiológica actúan también en condiciones patológicas, como en la enfermedad inflamatoria intestinal (IBD) (2-3), una mejor comprensión de estos mecanismos naturales proporcionará un mejor conocimiento de los mecanismos que intervienen en el estado patológico.

Capítulo 2

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El GALT en los seres humanos está formado por las placas de Peyer (PP), los folículos aislados del intestino delgado y colon, y el apéndice. Además, se incluyen como parte del GALT los linfocitos aislados de la LP y los IEL. Aunque algunos autores consideran que los ganglios linfáticos mesentéricos forman parte del GALT, en realidad muestran características a caballo entre los sistemas inmunes centrales y de mucosas. Pero por razones prácticas, se tratarán en este apartado.

1.2.1. Placas de Peyer Las PP son áreas organizadas de tejido inmune situadas a lo largo de la mucosa intestinal, que no están delimitadas por una cápsula fibrosa (a diferencia de los ganglios linfáticos). El epitelio en las PP (denominado epitelio asociado a folículo, FAE) es especial ya que no muestra las criptas o vellosidades típicas. El FAE contiene unas células epiteliales características llamadas células M. Las células M representan el 10% de células epiteliales en FAE y desempeñan un papel importante como transportadoras de antígenos, captando partículas del lumen y transportándolas a los macrófagos y linfocitos que están contenidos en los “bolsillos” de sus membranas basolaterales (25). Además, la membrana de estas células tiene sitios de unión para S-IgA (26) y HLA-DR (27), lo que sugiere, al menos en íleon, una función no sólo de transporte sino de presentación de antígenos. Desde el punto de vista anatómico, las PP tienen forma de cúpula, con el área justo debajo de la FAE enriquecida en células B productoras de IgA, linfocitos Th1, macrófagos y células dendríticas (DC) (28) (Figura 1).

Figura 1. Esquema de una placa de Peyer. DC: célula dendrítica; M:macrófago; T:linfocito T; B:linfocito B. Adaptado de Kato T, Owen RL. Structure and function of intestinal mucosal epithelium. In Ogra PL, Mestecky J, Lamm ME, Strober W, Bienenstock J, McGhee JR. Mucosal Immunology, 2nd edn. San Diego: Academic Press, 1999.


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1.2.2 Folículos linfoides aislados (ILF) Existen otras agregaciones celulares denominadas folículos linfoides aislados (ILF) que son macroscópicamente invisibles (a diferencia de las PP) y están distribuidos a lo largo de todo el intestino, siendo más numerosos en el íleon distal y colon. Los ILF se asemejan al compartimento de linfocitos B de las PP, pero les falta las zonas de células T (28). 1.2.3 Apéndice Aunque no se conoce bien el papel del apéndice en la inmunidad de la mucosa intestinal, algunos estudios han sugerido que la apendicectomía protege contra la aparición de la enfermedad de Crohn (CD) y colitis ulcerosa (UC), y disminuye la gravedad en los casos de UC (29, 30). 1.2.4 Linfocitos de la lamina propria y linfocitos intraepiteliales. La LP posee células inmunes, a saber: linfocitos B activados, macrófagos, DC, eosinófilos, mastocitos y linfocitos T. La mayoría de los linfocitos T son CD4+ y tienen un fenotipo activado. Como se mencionó anteriormente, el intestino también contiene linfocitos activados en el epitelio denominados IEL, que son en su mayoría CD8+. 1.2.5. Ganglios linfáticos mesentéricos Los ganglios linfáticos son tejidos linfoides secundarios, donde se inician y desarrollan las respuestas a los antígenos. El epitelio intestinal contiene numerosos capilares linfáticos que absorben y drenan el líquido de los espacios intercelulares. El líquido linfático fluye a través de dicho capilares y se introduce en los ganglios linfáticos a través del conducto aferente. Dentro de los ganglios, los antígenos epiteliales recogidos son mostrados a los linfocitos T o B vírgenes, pudiéndose iniciar una respuesta inmune innata.

1.3. Sistema inmune innato Las células más importantes del sistema inmune innato GI son las siguientes.

- Los macrófagos o fagocitos mononucleares son importantes en la primera

respuesta tras la invasión microbiana debido a su papel como células fagocíticas que destruyen microorganismos, pero también son importantes para la inmunidad adaptativa debido a su capacidad para presentar antígenos. En condiciones normales, los precursores de los macrófagos (monocitos) están circulando en la sangre (o en tejidos inmunes especializados). Cuando sus receptores reconocen ciertos estímulos, los monocitos abandonan la circulación siguiendo agentes quimiotácticos, se diferencian a macrófagos y activan sus mecanismos de defensa. Los monocitos humanos se dividen en dos grupos: monocitos CD14hiCD16- que actúan como centinelas, y monocitos CD14+16+, con actividad pro-inflammatoria (32).

- Los neutrófilos, también llamados fagocitos polimorfonucleares, son las

células inmunológicas más abundantes en sangre periférica, pero

Capítulo 2

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- Las células natural-killer (NK) son un subconjunto de linfocitos que

espontáneamente lisan células infectadas por virus y células tumorales. No son muy frecuentes en la mucosa, así que su contribución a la inmunidad innata GI es incierta.

- Los eosinófilos son los leucocitos más abundantes en la mucosa tras los

linfocitos T y B y los macrófagos, seguidos por los mastocitos y, rara vez, basófilos. Se sabe poco sobre el papel de estas células en el mantenimiento de la inflamación gastrointestinal controlada pero probablemente participen en las defensas contra la alergia y los parásitos (3).

- Las células dendríticas (DC) desempeñan papeles importantes en las

respuestas innatas a las infecciones, y en la vinculación entre las respuestas inmunes innatas y adaptativas. Poseen proyecciones en la membrana hacia la luz intestinal así como actividad fagocítica, y están ampliamente distribuidas por la mucosa intestinal. Expresan receptores de reconocimiento de patrones (PPRs) y secretan citoquinas. Las DC reconocen antígenos microbianos y los presentan a los linfocitos.

- Las células epiteliales desempeñan una función inmunológica activa y, por

tanto, deben considerarse parte del sistema inmune innato GI aunque clásicamente no hayan sido clasificadas como células inmunológicas.

Todas estas células pueden actuar de manera directa o a través de mediadores solubles como los que se describen a continuación. Algunas citoquinas involucradas en la respuesta inmune innata del intestino son el TNF, (también llamado TNFα para distinguirlo del TNFβ), la interleuquina (IL)1α /β, el interferón (IFN)α / β / γ, la IL12, la IL8, la IL6, la IL5, la IL17, la IL23, la IL10 y el TGF-β. Su implicación se describirá más adelante. Otros mediadores producidos durante la inflamación son las cuatro familias de eicosanoides (prostaglandinas, prostaciclinas, leucotrienos y tromboxanos), que son responsables en parte de los signos clásicos de la inflamación (calor, tumor, rubor y dolor). El intestino produce también metabolitos reactivos tóxicos, como el O2

-, que contribuyen a la defensa contra patógenos potenciales. Además, el intestino produce moléculas antioxidantes como el urato y el glutation, que mantienen el equilibrio entre agentes anti-oxidantes y pro-oxidantes. El NO, producido por la sintetasa de NO, es otro mediador importante en la inflamación. En la mucosa sana existe sintetasa de NO endotelial (eNOS), que es constitutiva, mientras que la inducible (iNOS) está ausente y sólo aparece en procesos inflamatorios. Por lo tanto el NO parece ser beneficioso para el tejido en condiciones fisiológicas y nocivo durante la inflamación, y esto puede explicarse por el efecto opuesto que pueden tener diferentes concentraciones de NO en un tejido (33).


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Algunos de los componentes del complemento también son detectados en el intestino delgado de personas sanas (34).

1.4. Sistema inmune adaptativo El objetivo de la inmunidad adaptativa es establecer una respuesta inmune de carácter específico. Los responsables últimos de esta función son dos tipos de células: los linfocitos B y los linfocitos T. A menudo, un sólo antígeno puede estimular tanto la producción de anticuerpos como una respuesta celular. Sin embargo, un tipo de inmunidad (humoral o celular) suele predominar sobre la otra, y esto dependerá del tipo de antígenos y el tejido donde el contacto tiene lugar, dado que la distribución de células B y T no es igual en cada órgano linfoide. Éstas células también juegan un papel importante en el intestino en condiciones fisiológicas debido a su participación en la tolerancia oral.

1.4.1 Inmunidad humoral La inmunoglobulina A secretora (S-IgA) es la principal inmunoglobulina de la mucosa. Es muy estable, quizá porque el componente secretor protege el sitio susceptible de proteolisis. Entre sus funciones se encuentran inhibir la adhesión bacteriana (35) (un primer paso crítico para la colonización bacteriana), inducir aglutinación bacteriana y atrapar bacterias junto al moco. Una propiedad interesante de la S-IgA, a diferencia de otras Ig, es su escasa o nula capacidad para activar el complemento por cualquiera de las dos vías. Pero en condiciones patológicas, se produce un cambio en el tipo de Ig, de tal forma que la IgA da paso a la IgG en la mucosa intestinal. 1.4.2 Inmunidad celular Los linfocitos T del intestino pueden encontrarse en sitios inductivos como las PP y los folículos linfoides, donde las células CD45RA+ vírgenes son estimuladas por antígenos, y en sitios efectores como la localización intraepitelial (IEL) o la LP (LPL), donde residen las células de memoria CD45RO+. Ambos tipos de linfocitos, IEL y LPL, son células T activas, pero su fenotipo y propiedades son bastante diferentes (3).

- Los linfocitos T de las PP y los ILF son vírgenes, inmaduros (a excepción de los linfocitos situados en el FAE de las PP). La relación CD4:CD8 es de 3’5:1. Derivan de timo y emigran a los sitios de inducción a través de interacciones entre la integrina y la L-selectina de las células T, y las moléculas de adhesión celular (CAM) de los capilares. La mayoría de los linfocitos T dentro de las PP tienen un TCRαβ. En cualquier momento, esas células pueden encontrar sus antígenos específicos, migrar al FAE y permaner en los “bolsillos” de las células M como células de memoria (CD45RO+).

- Los IELs son principalmente células T CD8+, α/β-TCR y oligoclonales, lo

que disminuye la frecuencia de respuestas inmunitarias específicas, protegiendo a las células epiteliales vecinas (36). La proporción de receptores-γδ de las células T (γδ-TCR) es más alta que en la sangre

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periférica (17), sugiriendo un papel especial en intestino. Su función no está clara porque muestran una baja tasa de proliferación tras exposición a mitógenos, pero responden vigorosamente frente a los glóbulos rojos (37). Debido a su fenotipo CD8+, se piensa que los IEL son células citotóxicas. También tienen actividad quimiotáctica en respuesta a IL8 y RANTES, y pueden movilizarse a los sitios de inflamación (38).

- Los LPL son principalmente (60-70%) células T CD4+, oligoclonales y con

fenotipo altamente activado en comparación con sus homólogos en sangre periférica, ya que proliferan más rápidamente en condiciones basales. Además responden a mitógenos, antígenos bacterianos y LPS (39), y producen citoquinas del tipo Th1 y Th2 (40).

1.5 Microflora Un enorme número de microorganismos, principalmente bacterias, colonizan y forman complejas comunidades, o microfloras, en diversos sitios dentro del cuerpo humano. Un total de 1014 células bacterianas se estima que cohabitan en el seno del huésped humano. La mayor y más compleja microbiota es la intestinal, que comprende 1012 células por gramo de heces, como media en el individuo humano (41). Aunque la respuesta dominante del GALT a los microorganismos es la tolerancia, el sistema inmune GI también puede iniciar una respuesta local o sistémica a determinados antígenos de bacterias patógenas. El mecanismo por el cual el GALT distingue las señales de los microorganismos comensales de los patógenos, elaborando tolerancia o una respuesta inmune respectivamente no es bien conocido, y se examinará con detalle más abajo. Se calcula que hay entre 500 y 1000 especies bacterianas diferentes en el intestino, y su ubicación anatómica varía. Aunque no se han identificado muchas de esas especies, su relevancia y efecto en el hospedador, en su fisiología y patología, ha sido bien documentado. En condiciones fisiológicas, las bacterias y el hospedador conviven en perfecta simbiosis. El hospedador proporciona “hospedaje” a las bacterias: un lugar donde habitar y el alimento. Y las bacterias ejercen muchos beneficios sobre el hospedador, los cuales se deben en gran parte (pero no únicamente) a la producción de ácidos grasos de cadena corta (AGCC), que se originan tras la digestión de algunos glúcidos no digeribles por el hospedador. Los AGCC que se producen más abundantemente en el lumen son el acetato, el propionato y el butirato. Y sus principales funciones son: ser fuente de energía para los colonocitos, aumentar el volumen de las heces y disminuir el pH del lumen intestinal (lo que en teoría favorece el crecimiento de bacterias beneficiosas) (84). Para que la flora bacteriana ejerza algunos de sus efectos beneficiosos, o para que los patógenos microbianos inicien la infección, es necesaria a veces una interacción directa con el hospedador mediante receptores. La mayoría de los organismos de la microbiota son reconocidos por algunas células del individuo humano mediante receptores especiales que reconocen patrones moleculares. Los receptores tipo Toll (TLR) desempeñan un papel esencial en esta señalización.


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1.5.1. Receptores tipo Toll (TLR)

Las respuestas inmunes innatas son iniciadas por receptores de reconocimiento de patrones (PRR), que reconocen componentes de los microorganismos y conducen a una respuesta anti-patógeno. Los TLR detectan diversos microorganismos, como bacterias, hongos, virus y protozoos, y desempeñan un papel importante en la inmunidad (76). Hasta la fecha han sido identificados 11 TLRs humanos y 13 de ratón (75). Pueden presentarse en la superficie celular (TLR1, TLR2, TLR4, TLR5 y TLR6) o bien intracelularmente (TLR3, TLR7, TLR8, y TLR9). Los TLRs comparten dos características estructurales: un dominio de unión de ligando, con varias regiones ricas en leucina (Leu), y un dominio responsable de la transducción de señales Toll/IL1 receptor (TIR). Tras el reconocimiento del ligando, la señalización es iniciada por una homo/heterodimerización del receptor. Después al menos 5 proteínas adaptadoras diferentes continúan la señalización: MyD88, Mal/TIRAP, TRIF/TICAM-1, TRAM/Tirp/TICAM-2, y SARM (77). Todos los TLRs, excepto el TLR3, señalan a través de MyD88, mientras que el TLR4 puede usar rutas de señalización tanto dependientes como independientes de MyD88. El reclutamiento de MyD88 activa algunas moléculas como TRAF6 y TAK1, lo que conduce a la activación final de NF-κB o factores de regulación de IFN (77). Otros factores de transcripción como AP-1, Elk-1, CREB y STAT también pueden ser activados tras el reconocimiento de patrones moleculares. Todas estas señales finalmente sirven para regular la expresión de varios citoquinas y/o marcadores inflamatorios, que representan la primera respuesta a un estímulo microbiano. Los ligandos TLR más frecuentes se muestran en la siguiente tabla: TLR Ligando TLR1 Triacil-lipopéptidos bacterianos (forma un heterodímero con TLR2)

(79) TLR2 Lipoproteínas/lipopéptidos, peptidoglicanos y ácido lipoteicoico

(76) TLR3 ARN viral de doble hebra (dsRNA) (82) TLR4 LPS de bacterias gram negativas (80) TLR5 Flagelina del flagelo bacteriano (81) TLR6 Diacil-lipopéptidos bacterianos (forma un heterodímero con TLR2)

(79) TLR7 Y TLR8 Análogos de ARN de única hebra (ssRNA) (83, 84) TLR9 Motivos CpG de ADN no metilados (85)

La familia TLR está expresada en una amplia gama de células, incluidas las implicadas en la respuesta inmune innata (DC, monocitos/macrófagos e IECs), así como en las células del sistema adaptativo, como los linfocitos T.

1.6. Citoquinas Las citoquinas son pequeñas proteínas producidas por las células inmunológicas que facilitan la comunicación entre células, estimulan la proliferación de células efectoras y median las manifestaciones de la inflamación, tanto locales como sistémicas, actuando de manera autocrina, paracrina y endocrina (54, 55). Sus acciones son

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pleiotrópicas (una citoquina puede ejercer varios efectos) y redundantes (varias citoquinas pueden tener el mismo efecto), y se ejercen mediante unión a receptores. Las citoquinas generalmente regulan la producción de otras citoquinas o de ellas mismas, y su secreción es breve y autolimitante. La respuesta celular a esta secreción normalmente consiste en cambios en la expresión de genes en las células diana, modificando su fenotipo. A continuación se describen las citoquinas cuya implicación en IBD ha sido más estudiada.

- El TNF es el principal mediador de la respuesta inflamatoria aguda a infecciones y

es responsable de muchas de las complicaciones sistémicas durante infecciones graves. La principal fuente celular son los fagocitos mononucleares, aunque también la producen los linfocitos T activados por antígeno, las células NK, y los mastocitos. El estímulo más potente para su producción es LPS (y otros productos microbianos), ya que estas células poseen TLR4. El IFNγ potencia la secreción de TNF inducida por LPS en macrófagos. La función principal del TNF es reclutar neutrófilos y monocitos a los sitios de infección y activar estas células para eliminar los microorganismos. En cantidades adecuadas, el TNF resulta esencial para contener las infecciones; pero cuando su nivel es muy alto en infecciones severas o en disfunciones del sistema inmune, ejerce una plétora de reacciones perjudiciales (52). Los efectos secundarios incluyen inducción de fiebre, aumento de proteínas de fase aguda, caquexia, disminución de la presión sanguínea (choque), trombosis y trastornos metabólicos. Una importante complicación de la sepsis por G- es un síndrome denominado shock séptico, que es debido a altas concentraciones de TNF (y otras citoquinas) en el suero sanguíneo.

- La IL1 trabaja junto al TNF en la inmunidad innata y la inflamación. Su principal fuente celular son los macrófagos, aunque otras células como los neutrófilos, las células epiteliales y las células endoteliales también la producen. El estímulo más potente para su secreción son el LPS y el TNF. La IL1 existe en dos formas: IL1α e IL1β, que tienen menos de un 30% de homología, pero se unen al mismo receptor y tienen las mismas funciones biológicas. IL1 tiene el mismo efecto biológico que el TNF, y aunque resulta también tóxica en grandes cantidades a causa de los efectos sistémicos, no induce apoptosis en células y no causa el típico shock séptico (51).

- La IL6 forma parte de la inmunidad innata y adaptativa. Es sintetizada por

diferentes células, tales como los fagocitos mononucleares, células endoteliales vasculares, fibroblastos, enterocitos y algunas células T activadas. La IL6 se produce en respuesta a infecciones microbianas, TNF e IL1. Entre sus acciones están: aumento de las proteínas de fase aguda y diferenciación de neutrófilos desde la médula ósea (inmunidad innata); así como la estimulación del crecimiento de células plasmáticas (51).

- La IL12 es la principal mediadora de la respuesta innata temprana a los

microorganismos intracelulares y además es un elemento clave inductor de la inmunidad celular. Las principales fuentes celulares son las DC activadas y los macrófagos. La IL12 se produce tras la activación de varios TLR. También las células Th activadas pueden inducir su producción. La IL12 fue originalmente identificada como un activador de células NK y CD8+, pero su acción más


25

importante es estimular la síntesis de IFNγ y promover la diferenciación de células Th0 al subtipo Th1 (51).

- El IFNγ es el principal activador de los macrófagos y cumple funciones críticas en

la inmunidad innata y adaptativa contra microorganismos intracelulares. Es la citoquina característica de la subpoblación Th1, aunque las células NK y las CD8+ la producen también. Las células T sintetizan IFNγ en respuesta al reconocimiento antigénico, y su producción se incrementa con la IL12 y la IL18 (51). El efecto proinflamatorio directo del IFNγ consiste en la activación de los macrófagos para destruir microorganismos fagocitados. Pero también promueve la activación de monocitos mediante mecanismos indirectos. Así, el IFNγ promueve la diferenciación de células T CD4+ vírgenes a Th1, inhibe la subpoblación Th2 y estimula la expresión de moléculas MHC de clase I y II y de coestimuladores en las APC (51).

- La IL5 es un activadora de eosinófilos. Los eosinófilos activados son capaces de

matar helmintos y estimular la proliferación de células B así como la producción de IgA. La IL5 es producida por el subtipo de células Th2 y por mastocitos activados (51).

- La IL2 es un factor de crecimiento, supervivencia y diferenciación de linfocitos T,

y desempeña un papel importante en la regulación de las respuestas linfocitarias a través de sus acciones sobre las células T reguladoras (Treg). Sus funciones son autocrinas y paracrinas. La principal fuente celular son los linfocitos T CD4+ (51). Y aunque su papel es fundamentalmente proinflamatorio, la IL2 es necesaria también para la actividad inmunosupresora de las células Treg (51, 56).

- La familia de citoquinas IL-17, descubierta recientemente, incluye seis miembros:

IL17A, B, C, D, IL17E (o IL25) e IL17F. La IL17 (IL17A) es el miembro original de esta familia. Diferentes tipos celulares, incluidas las células T, NK, γδT y neutrófilos producen IL17A e IL17F (58). En linfocitos T CD4+, la IL17A se expresa específicamente en un subconjunto de células llamado Th17. La IL17 estimula a las células endoteliales y macrófagos para producir IL1, TNF, y diversas quimioquinas que promueven el reclutamiento de neutrófilos (51).

- La IL23 comparte la subunidad p40 con la citoquina IL12 y como ella, también

induce la producción de IFNγ en linfocitos T humanos. Sin embargo, en contraste con la IL12, que es importante para la diferenciación de células T vírgenes, la IL23 actúa principalmente en linfocitos T memoria para inducir su proliferación (78). En cualquier caso, la función más importante de la IL23 es ser inductora de la citoquina proinflamatoria IL17 (79). La IL23 se produce en monocitos activados, APC activadas, linfocitos T y B, y células endoteliales (78, 80).

- La IL10 es un inhibidor de macrófagos y DC activadas. Se produce

principalmente en macrófagos y Treg. La IL10 inhibe la expresión de IL12, coestimuladores y moléculas MHC de clase II en macrófagos y DC activadas.

- La principal función del TGFβ en el sistema inmune es inhibir la proliferación y la

activación de los linfocitos y otros leucocitos, aunque también puede ejercer efectos proinflamatorios. El TGFβ es una familia de citoquinas compuesta por TGFβ1, TGFβ2 y TGFβ3, si bien las células del sistema inmune producen

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principalmente TGFβ1. Las fuentes celulares de TGFβ1 son los linfocitos T activados, los macrófagos activados por LPS y algunas células Treg. El TGFβ modula específicamente la diferenciación en distintos subconjuntos de células T, dependiendo de la combinación con otras citoquinas. Y controla la reparación tisular después de un proceso inflamatorio (51, 57).

- La IL1ra es un inhibidor natural de la IL1 que reprime su señalización mediante

competición con el receptor. Es producida por los fagocitos mononucleares. 1.7. Señalización celular en las respuestas inmunes.

Existen muchas rutas de señalización dentro del sistema inmune que controlan las respuestas inmunológicas y los procesos de tolerancia. Revisarlas todas no es el objetivo de este capítulo; sin embargo, algunos factores de transcripción desempeñan un papel fundamental en la inflamación, y concretamente, en la IBD, por lo que su mención resulta esencial para un mejor entendimiento de la enfermedad.

1.7.1 NFκB. El factor nuclear-kappa B (NF-κB) es un regulador central de las respuestas inmunes e inflamatorias. Entre otros estímulos, la activación celular mediante IL1, TNF y LPS conduce a la activación de NF-κB. La familia NF-κB tiene cinco miembros en mamíferos: c-Rel, RelA (p65), RelB, NF-κB1 (p50/p105) y NF-κB2 (p52/p100), que forman homodímeros o heterodímeros. Cuando NF-κB se encuentra en estado inhibido está situado en el citoplasma, unido a una proteína inhibitoria: IκB. La acción de estímulos específicos (IL1, TNF y LPS entre otros) conduce a la activación de una enzima clave, IκB-kinasa (IKK), que está compuesta por la subunidad reguladora IKKγ (NEMO) y las dos subunidades efectoras, IKKα y IKKβ. La activación de IKK produce a su vez la activación de NF-κB mediante dos rutas. La ruta canónica de activación de IKK, que generalmente tiene al heterodímero p50/RelA como diana, implica a IKKβ y resulta en la fosforilación de IκB, ubiquitinación, degradación proteasómica del inhibidor y posterior disociación de IκB de NF-κB (81). Al separarse IκB, queda expuesta la secuencia de translocación al núcleo y por tanto NF-κB se dirige al núcleo (Figura 2), donde regula la expresión génica. En la señalización alternativa, se activa IKKα y ésta causa la fosforilación y procesamiento de p100, lo que conduce a la formación de dímeros p52/RelB (Figura 2). Tras la activación, los dímeros de NF-κB se translocan al núcleo, donde se unen a los promotores de los genes diana y median la activación o, en algunos casos, represión transcripcional de numerosos (>200) genes involucrados en respuestas inmunes innatas y adaptativas (70). Los genes clásicamente regulados por NF-κB, entre los que se encuentran IL-1, TNF, IL-6 e IL-12 p40, sugerían un papel proinflamatorio de este factor de transcripción. Pero algunos estudios más recientes han mostrado que NF-κB promueve también respuestas protectoras frente a la inflamación, lo cual depende del tipo celular y de la condición fisiopatológica concreta (70). La actuación concreta de NF-κB en IBD será discutida más adelante.


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p50

Ruta canónica

CITOPLASMA Ub no proteasómica

RelA

TNFR1

NEMO

IKKβ IKKα

PNEMO

IKKβ IKKα

P

IkB

RelA

IkB

p50

P Ub

PROTEASOMA

RelA p50

RelA p50

RelB

IkB

RelB

IkB

p100

Ub

P

P

gen

Ruta alternativa

IL-1R/TLR4

RelBp52

RelBp52

gen

MEMBRANA PLASMÁTICA

p100

NÚCLEO

Figura 2 Esquema de la ruta de señalización NF-κB. Se muestran las rutas canónica y alternativa. IKK:IκB-kinasa; P: grupo fosfato; IκB: inhibidor de κB; Ub:ubiquitina. Adaptado de Scheidereit C. IκB kinase complexes: gateways to NF-jB activation and transcription. Oncogene (2006) 25, 6685–670. 1.7.2 MAP kinasas (MAPK). Un grupo de kinasas importante en las rutas de señalización inmunológicas son las kinasas de proteínas activadas por mitógenos (MAPK). Existen 4 MAPK: kinasa

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c-Jun N-terminal activated kinase (JNK), p38-kinase (p38), extracellular signal regulated kinase (Erk 1/2) y big MAP-kinase (BMK1). Las MAPK se activan por la acción de estímulos moleculares como las citoquinas, los factores de crecimiento y las hormonas; pero también se activan en respuesta a estímulos físicos patológicos como la isquemia, los rayos ultravioleta (UV), los daños neuronales o el shock osmótico. Aunque cada MAPK ejerce efectos característicos, y cada ruta de señalización regula la expresión de genes específicos, se ha demostrado que existe redundancia entre las distintas vías de señalización. Se sabe además que entre ellas mismas regulan su expresión y/o activación; por ejemplo, una potente activación de JNK induce la inhibición de ERK (82).

Las MAPK se encuentran en realidad en el centro de rutas de señalización más amplias. A nivel celular, por encima de las MAPK encontramos otras kinasas que se fosforilan y, por tanto, se activan unas a las otras en serie. Estas cascadas de kinasas se activan en respuesta a los estímulos específicos comentados anteriormente y convergen en la fosforilación/activación de las MAPK centrales: JNK, Erk 1/2, p38 y BMK1. Por debajo de las MAPK, encontramos los factores de transcripción regulados por estas enzimas y que son los responsables últimos de la regulación génica ejercida mediante estas rutas (Figura 3). Las rutas MAPK pueden resumirse así, a nivel muy esquemático:

ESTÍMULO

Figura 3. Esquema de las rutas de señalización de las MAPK. 1.7.3 NFAT

El factor nuclear de las células T activadas (NFAT) juega un papel central en la transcripción de genes inducibles durante la respuesta inmune. NFAT es una familia de factores de transcripción compuesto por cinco proteínas: NFAT1 (NFATp, NFATc2), NFAT2 (NFATc, NFATc1), NFAT3 (NFATc4), NFAT4 (NFATx, NFATc3), y NFAT5 (TonEBP) (75). Pese a su nombre, NFAT no sólo se expresa en las células T sino en otras células inmunológicas. En condiciones normales, NFAT se encuentra en el citoplasma en forma inactiva, unido a un grupo fosfato. Se activa por la calcineurina-fosfatasa dependiente de calcio-calmodulina, la cual desfosforila NFAT permitiendo la traslocación al núcleo y la subsiguiente regulación génica (76).

MAPKKKEstrés, UV, citoquinas, hormonas, etc.

MAPKK MAPK kinasa kinasa MAPK kinasa

EFECTO BIOLÓGICO MAPK Erk 1/2, JNK, p38 y

BMK1.

F.TRANCRIPCIÓN Crecimiento, diferencia- ción, inflamación, etc.

Myc, Elk-1, ATF-2, c-FOS, p53, c-Jun, etc.


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1.7.4 Rutas de señalización en la diferenciación de linfocitos T Los linfocitos T suelen clasificarse en función de la ausencia/presencia de los marcadores de superficie CD4 y CD8. Las células CD4+ “ayudan” a los linfocitos B y T en la elaboración de la respuesta inmune, razón por la cual se llaman T helper (Th). Las células T CD4+ vírgenes (Th0) son células inexpertas que todavía deben ser estimuladas por su antígeno específico. Cuando lo hacen en presencia de determiandas citoquinas, cambian su fenotipo y se diferencian en distintos subconjuntos. El subconjunto de células Th1 produce una gran cantidad de IFNγ y dirige la respuesta ante patógenos intracelulares, a través de la activación de macrófagos dependiente de IFNγ. El factor de transcripción T-bet, perteneciente a la familia de factores T-box, parece ser esencial para la diferenciación a Th1, y lo hace en parte al inducir la expresión de IFNγ. T-bet no sólo induce la diferenciación Th0→Th1, sino que cuando es introducido en linfocitos Th2, éstos cambian su fenotipo hacia Th1 (83). Otro factor de transcripción importante en la señalización Th1 es STAT4, que también estimula la producción de IFNγ. Si a nivel transcripcional T-bet y STAT4 son importantes para la diferenciación Th1, la IL12 es la citoquina que mejor dirige dicha diferenciación. Secuencialmente, la IL12 activa la vía de señalización de STAT4, resultando en el aumento de expresión de IFNγ y T-bet (59). A su vez, Tbet estimula la expresión del receptor de la IL12 (IL12R), haciendo así a las células Th1 más sensibles a la acción de esta citoquina polarizante (61). En cualquier caso, algunos modelos animales han demostrado que sólo Tbet (que se induce también al estimular el TCR), y no la IL12, es esencial para la diferenciación Th1 (60) (Figura 4). En condiciones fisiológicas, las células Th1 son detectadas en la LP, en un número tal vez mayor que en otros tejidos (198). Las células Th2 expresan IL4, IL10, IL5 e IL13 y son importantes para la eliminación de helmintos y gusanos, ya que activan a los linfocitos B. Los factores de transcripción GATA-3 y STAT6 son fundamentales para la diferenciación Th2. La IL4 es la citoquina que más conduce a la diferenciación Th2 y lo hace a través de la activación de STAT6, que a su vez aumenta la expresión de GATA-3 (62). GATA-3 aumenta aún más la expresión de IL4, creando así un ciclo de retroalimantación positiva que promueve la diferenciación Th2 (Figura 4). La IL4 también inhibe la diferenciación de las células Treg y, junto a TGFβ, promueve la generación de un conjunto de linfocitos T IL9+IL10+Foxp3- (63, 64). Algunos autores defienden que estos linfocitos pueden ser clasificados como un nuevo subconjunto de linfocitos T CD4+ efectores, Th9, pero aún no se conocen marcadores específicos de este subconjunto (198). Al contrario que las células Th1, las Th2 no se encuentran (o lo hacen en número muy pequeño) en el intestino sano. Las células Th17 producen IL17A, IL17F, TNF, IL21 e IL22 y se consideran responsables de la defensa contra patógenos extracelulares ya que sus productos reclutan y activan neutrófilos. El desarrollo de la subpoblación Th17 puede dividirse en tres pasos: diferenciación (impulsado por el TGFβ y la IL6), amplificación (desencadenado por la IL21) y estabilización (mantenido por la IL23) (60). El TGFβ dirige la señalización Smad, y la IL6 y la IL21 activan el factor de transcripción STAT3. A su vez STAT3 promueve la expresión de dos factores de transcripción esenciales para las Th17: RORγ y RORα (65). Aunque originalmente se pensó que

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la IL23 era esencial para la diferenciación de las Th17, no es en realidad necesaria para esta función pero sí para el mantenimiento de este subconjunto. Los datos recientes sugieren que la subpoblación Th17 no corresponde a una única clase de células, sino que dependiendo de las citoquinas en el ambiente cercano, se observan diferentes linfocitos Th17 con diferentes funciones (60) (Figura 4). Al igual que los linfocitos Th1, los Th17 son detectables en la LP de individuos sanos. Las células Treg tienen propiedades reguladoras que les permiten controlar (reprimir) la respuesta de las células Th, y son esenciales para mantener la homeostasis inmunológica. Definidas, al menos en ratones, por la expresión del factor de transcripción FoxP3 (66), estas células expresan otros marcadores como CD25, factor de necrosis tumoral inducido por glucocorticoides (GITR) y proteína asociada a linfocitos citotóxicos (CTLA)-4 (60). La IL2 y el TGFβ desencadenan la expresión de FoxP3 mediante mecanismos dependientes de STAT5 y Smad3 (68). Algunos estudios han vinculado el desarrollo de los subconjuntos celulares Treg y Th17, ya que TGFβ puede conducir a la co-expresión de RORγ y FoxP3 en linfocitos vírgenes y en función de otras señales, como la IL6, las células pueden llegar a ser Th17 o Tregs (69). Algunos estudios sugieren un papel fundamental de los linfocitos T productores de IL-10 en la inhibición intestinal de los linfocitos Th en condiciones fisiológicas, es decir, un papel fundamental en la tolerancia oral (198).

T-betSTAT-4

IFNγ, TNF

Th1

Figura 4.Esquema de la diferenciación de linfocitos CD4+ en respuesta a los estímulos de citoquinas, y factores de transcripción implicados.

FoxP3STAT5

STAT3RORγT

GATA-3STAT-6

Th2

Th17

IL4

IL17, IL23

IL10 TGFβ

Th0 activado

Macrófago

IL6

IL12

IL10

IL4

TGFβ IL21

TNF

Treg


31

1.8 Tolerancia oral La tolerancia inmunológica se define como la falta de respuesta a un antígeno provocada por la exposición previa a dicho antígeno. La tolerancia a antígenos propios, llamada autotolerancia, es una propiedad fundamental del sistema inmune. Tiene lugar en timo y médula ósea, por lo que se denomina igualmente tolerancia central. Pero también puede inducirse tolerancia a antígenos extraños (44). Así, la administración oral de un antígeno proteico conduce a menudo a una inhibición de las respuestas sistémicas humorales y celulares. Este fenómeno, llamado tolerancia oral, es muy importante para evitar las respuestas inmunes contra alimentos y microflora bacteriana. En la tolerancia oral participan los mecanismos de tolerancia periférica, que difieren un poco de los centrales.

1.8.1. Linfocitos T y tolerancia La tolerancia periférica es el mecanismo por el cual los linfocitos T maduros que reconocen antígenos en tejidos periféricos se vuelven incapaces de responder a estos antígenos (44), y se debe a procesos de anergia, supresión o eliminación de las células T. a) La anergia es el proceso por el cual las células T CD4+ se vuelven incapaces de responder a un antígeno determinado debido a la falta de coestimulación (49). Puede ser el resultado de alteraciones bioquímicas y/o genéticas. De hecho, las células anérgicas muestran un bloqueo en la señalización a través del TCR, y aunque el mecanismo exacto se desconoce, se ha visto una disminución en la expresión (o aumento de la degradación) del TCR. La presencia de moléculas inhibidoras como CTLA-4 también ha sido descrita (44). Por otra parte, las DC que están en un estado de reposo expresan pocos (o ningún) coestimuladores. Cuando estas células presentan un antígeno a linfocitos CD4+ pueden contribuir a mantener el estado de anergia (44). b) La tolerancia periférica puede producirse tras la supresión de células Th mediada por células Treg. Aunque se desconoce el mecanismo exacto, se ha propuesto la existencia de linfocitos Treg memoria en el intestino que reconocen antígenos específicos de los alimentos. Cuando se activan las Treg, inhiben el sistema inmune mediante la secreción de IL-10, que inhibe macrófagos y células dendríticas, y/o TGFβ, que inhibe linfocitos y macrófagos. Algunos estudios sugieren contacto directo entre las Treg y las células diana (44). c) La eliminación de células Th mediante apoptosis ocurre tras una exposición recurrente a un antígeno en ausencia de marcadores inflamatorios (44). El mecanismo sugerido es la activación de proteínas proapoptóticas como Bim, dando lugar a la apoptosis por la ruta mitocondrial. Por otra parte, la estimulación repetida de las células T resulta en la coexpresión de receptores de muerte y sus ligandos (Fas y FasL) (44). La tolerancia periférica en los linfocitos CD8+ es menos conocida, pero parecen estar implicados los tres mecanismos anteriormente mencionados. 1.8.2. Linfocitos B y tolerancia

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Los linfocitos B maduros que reconocen antígenos en tejidos periféricos en ausencia de linfocitos Th pueden volverse funcionalmente insensibles o morir por apoptosis (44). Además, las células B que encuentran autoantígenos tienen menos probabilidades de migrar a folículos linfoides ya que tienen una expresión disminuida del receptor CXCR5. Algunas moléculas inhibidoras que se unen al receptor Fc parecen estar igualmente implicadas en la tolerancia de los linfocitos B.

2. LA ENFERMEDAD INFLAMATORIA INTESTINAL

2.1 Aspectos generales La enfermedad inflamatoria intestinal (IBD) consiste en una inflamación crónica, idiopática y recurrente del tracto gastrointestinal, que deteriora significativamente la calidad de vida de los pacientes. Su incidencia ha aumentado considerablemente en el siglo XX, y los pacientes que la padecen tienen un riesgo mayor de desarrollar cáncer gastrointestinal (261). La IBD se refiere en realidad a varias condiciones estrechamente relacionadas, siendo las más importantes la colitis ulcerosa (UC) y la enfermedad de Crohn (CD). Estas dos formas de IBD tienen muchas similitudes, pero también existen características clínicas específicas de cada (101,241). La inflamación en la UC comienza generalmente en el ano y se extiende de manera continua, pero nunca más allá del colon. La inflamación es superficial (sólo afecta a la mucosa), con infiltración de linfocitos y granulocitos, pérdida de células caliciformes y frecuentes ulceraciones y abscesos en las criptas. Los síntomas más comunes son la diarrea (a menudo sangrienta), urgencia en defecación, cólicos abdominales, y tenesmo. Si la inflamación afecta a un tramo más o menos largo del colon, la UC puede venir acompañada de síntomas sistémicos como anorexia, fatiga, fiebre baja y pérdida de peso (240,241). La CD afecta a cualquier parte del tracto gastrointestinal, pero más comúnmente al íleon terminal, ciego, colon y zona perianal. Se caracteriza por la presencia de segmentos de intestino sano entre las regiones afectadas (a diferencia de UC, en la que la inflamación se extiende de manera continua). La inflamación es de tipo transmural (afecta a todas las capas de la pared intestinal), con una densa infiltración de linfocitos y macrófagos, así como ulceración productora de fisuras y fibrosis submucosa. La diarrea es frecuente, aunque menos graves que en UC. Otros síntomas son dolor, estenosis y obstrucción intestinal, formación de abscesos, y fistulización de la piel y de los órganos internos (101,241). Pese a las intensas actividades de investigación, la etiología de la IBD sigue sin conocerse en su totalidad. En cualquier caso, se cree que implica una interacción de factores genéticos, bacterianos, ambientales e inmunológicos. De hecho, se piensa que la IBD se produce tras una respuesta exacerbada del sistema inmune a los antígenos luminales (239). El aumento de células B en la mucosa de pacientes con IBD sugiere que la enfermedad está mediada en parte por anticuerpos, pero la principal anomalía fisiopatológica es la presencia de un número exagerado de linfocitos T (208). Debido en parte al desconocimiento de su causa, no existe cura para la IBD, si bien algunos tratamientos mejoran la enfermedad, produciendo remisión y prevención de las recaídas. Sin embargo, estos fármacos tienen muchos efectos adversos, tanto


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locales como sistémicos, por lo que se hace necesaria la búsqueda de nuevos tratamientos. 2.2. Epidemiología y factores de riesgo La incidencia de IBD ha aumentado considerablemente en el sigo XX, sobre todo a partir de la Segunda Guerra Mundial, siendo estimada actualmente en 6 por 100.000 para CD y 15-20 por 100.000 para UC, aunque se han descrito mayores tasas de incidencia para ambas enfermedades (85,86). La incidencia específica por edades ha variado con el tiempo y según los países, pero parece ser que el aumento más pronunciado se ha dado en el rango de edad de 20-40 años (87). La suposición de que hay otro pico de edad en los 60 años es controvertida, ya que algunos autores defienden que este aumento es consecuencia de un diagnóstico tardío (88,89). Durante muchos años se sugirió la existencia de un gradiente norte-sur en la prevalencia de la IBD, pero estudios más exhaustivos han descartado esta hipótesis. En cambio, ahora hay fuertes indicios de la existencia de un gradiente oriente-occidente (90,91). La etnia judía fue considerada durante mucho tiempo como un grupo de riesgo, pero algunos autores coinciden en que esos estudios no fueron muy estrictos desde el punto de vista estadístico. Otras publicaciones mostraron que las minorías con bajos ingresos eran más propensas a padecer IBD; sin embargo, estas observaciones fueron dependientes del momento del estudio (87). Otra conclusión de los estudios de observación de UC y CD fue la asociación entre estatus socioeconómico alto y un aumento del riesgo (8, 9). Pero, de nuevo, los datos recientes no han logrado encontrar esa observación (10, 11). Con respecto a los factores de riesgo, la dieta ha sido uno de los más estudiados por razones obvias. La leche o sus derivados primero, (96) y alimentos occidentales (como la margarina (97), maíz (98), “fast food” (99) y bebidas de cola (100) después, fueron propuestos como alimentos de riesgo, pero aún no se han descubierto los mecanismos subyacentes. En cualquier caso, los síntomas de IBD podrían influir en los hábitos alimentarios de los pacientes, por lo que resulta difícil comprobar si la dieta es la causa o la consecuencia de la enfermedad (87). Fumar aumenta el riesgo de sufrir CD (104); no obstante los ex fumadores y las personas que nunca han fumado tienen el mismo riesgo (105), sugiriendo que fumar no es un factor iniciante sino potenciante. Curiosamente, el tabaco ha sido establecido como un factor de protección contra UC. Esta protección se produce en ambos sexos, en todos los grupos de edad y en todos los grados de enfermedad (87,103). Especial atención requiere la conclusión de que la apendicectomía protege contra la UC. Hay dos interpretaciones para esta función protectora: 1) la eliminación del apéndice (especialmente antes de la edad de 20) disminuye el riesgo de la UC y 2) los pacientes que sufren de apendicitis son menos propensos a sufrir de UC (87).

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2.3 Etiología de la IBD.

La etiología de la IBD no es del todo conocida, pero en general se considera que consiste en una respuesta inmune aberrante contra las bacterias del lumen del tracto intestinal en un grupo de personas con una predisposición genética para desarrollar inflamación crónica de la mucosa intestinal (205). Algunos factores frecuentemente asociados con la IBD incluyen el hábito de fumar, el uso de AINEs y problemas psicológicos, pero sin duda el microbiano es el factor más estudiado hasta ahora.

2.3.1 Bacterias intestinales, TLRs e IBD

Hoy día parece claro que las bacterias de la flora intestinal son esenciales para el desarrollo de la IBD. Así, en los modelos animales de inflamación inducida, dicha inflamación no se produce en ambientes libres de gérmenes (GF) o libres de patógenos específicos (SPF). Otros datos que apoyan esta teoría son los estudios que han mostrado la mejoría que produce el uso de antibióticos en humanos (206,210) y prebióticos/probióticos en animales.

Ya que la mayoría de individuos cohabitan en homeostasis con la microflora del

intestino, el comprender por qué y cómo algunas personas muestran una respuesta anormal sigue siendo central en la investigación en IBD (208). Algunos autores han propuesto que esta respuesta exagerada es consecuencia de una alteración en dichas bacterias, ya sea por la presencia de algún patógeno específico o porque varía la composición de la flora bacteriana (208,209). Pero estas teorías no han podido ser demostradas y más bien parece que la respuesta aberrante se produce ante bacterias comunes de la microflora que no estimulan respuesta inmune alguna en individuos sanos.

Además, los receptores TLR, importantes en la interacción bacterias-huésped parecen estar implicados en la IBD. En principio, dado que la estimulación de los TLR conlleva casi siempre la activación de NF-κB, que clásicamente se ha considerado un factor de transcripción proinflamatorio (aunque hoy día existen datos que contradicen esa afirmación), cabría esperar que el efecto de los TLR en la IBD fuera promotor. De hecho, el uso de antagonistas de los TLR ha resultado eficaz en la colitis en ratones (161). Y la expresión de TLR4 en IEC está aumentada en pacientes de CD y UC (280). Pero existen datos que muestran que los TLR ejercen también un papel protector en la IBD. Dos estudios demostraron que un polimorfismo del TLR4 denominado TLR4 Asp299Gly es factor de riesgo en UC y CD (174,194). Curiosamente un estudio in vitro con células epiteliales respiratorias mostró que esta variante del TLR4 responde con menos potencia al LPS (279), lo que podría sugerir un factor protector del TLR4 en IBD. Pero otro estudio elaborado en células mononucleares de sangre periférica (PBMC) procedentes de personas con una variante u otra, mostró que no había diferencia en la producción de citoquinas. Así pues, pese a que hay evidencias de que el TLR4 Asp299Gly es factor de riesgo en IBD, la implicación biológica no está clara. Otros estudios han vinculado polimorfismos de otros TLR con IBD (174,175), subrayando la idea de que un funcionamiento aberrante del sistema inmune innato podría estar implicado en IBD. Esta hipótesis será discutida más adelante.


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Por otro lado, se ha descubierto que la activación de linfocitos T en respuesta a los TLR puede ser indirecta, mediante células dendríticas, o directa, ya que los linfocitos T CD4+ expresan TLR también (176). Esto es importante en el contexto de IBD porque los frecuentes daños en la barrera epitelial durante las crisis podrían resultar en la exposición anormal de las células T con TLR a los antígenos bacterianos. En muchos casos, la unión de ligando a TLR aumenta la supervivencia y acelera la inducción de respuestas adaptativas efectoras, lo que agravaría la IBD (177). Sin embargo, la estimulación de los TLR puede regular también la función de los linfocitos Treg (178), por lo que el efecto global no está claro. En resumen, los TLR tienen doble función en IBD: son necesarios para el mantenimiento de la tolerancia y equilibrio bacteriano en la interfaz intestinal, pero también pueden amplificar respuestas inadecuadas y contribuir a la perpetuación de inflamación crónica. Como resultado, los investigadores se han centrado en los TLR como posibles objetivos terapéuticos para IBD, y algunos agonistas y antagonistas para estos receptores han demostrado ser eficaces en IBD (examinado en ref. 77).

2.3.2 Genética Tal y como se mencionaba anteriormente, hay fuertes indicios de que la IBD está, en gran parte, determinada genéticamente. Algunas elementos que avalan esta suposición incluyen las diferencias étnicas en la prevalencia de la enfermedad, la agregación familiar, la mayor concordancia en la enfermedad entre gemelos monocigóticos (37%) que dicigóticos (7%) (186), las asociaciones entre marcadores genéticos y diversas manifestaciones de IBD, y la vinculación entre regiones cromosómicas específicas e IBD en ciertas familias (179). Así, un historial familiar positivo se encuentra en el 5-20% de los pacientes con IBD, y un primer grado familiar aumenta en 10-15 veces el riesgo de desarrollar IBD, algo más en CD que en UC (181-185). El aumento del riesgo no se encuentra en cónyuges o hijos adoptivos, sugiriendo una explicación genética más que ambiental para esta agregación familiar (181). Además de todo lo anterior, se ha demostrado que existe un grupo de genes que confieren susceptibilidad a IBD (187), pero la manera en que interactúan con los factores ambientales y el sistema inmune para desarrollar la enfermedad inflamatoria es aún desconocida. Algunos de esos genes y su implicación en IBD se mencionan a continuación: - NOD2: localizado en el cromosoma 16q, NOD2 es seguramente el gen de

susceptibilidad a IBD mejor definido (188,189). Dos mutaciones que codifican aminoácidos diferentes (R702W y G908R) y una alteración del marco de lectura (Leu1007fs) han sido fuertemente asociados con la CD en la población caucasiana, ya que un 25-45% de los pacientes tienen estas variedades en comparación con un 15-20% en los controles (185). Este gen codifica la proteína NOD2, que es un receptor de reconocimiento de patrones (PPR) que se une al muramildipéptido (MDP) y que se expresa en muchas células, tales como los monocitos, las células dendríticas, las células de Paneth y los enterocitos (185,190).

Las mutaciones de NOD2 se han asociado con una reducción de la expresión de defensinas y activación de NF-κB (189,191). También los monocitos y DCs de

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pacientes con mutaciones en NOD2 tienen respuestas menores tras la estimulación de los TLR. Estas observaciones van en contraposición del aumento de actividad NF-κB observado en la mucosa de pacientes con IBD (192), pero concuerdan con un estudio en el que la represión condicional de NF-κB en células epiteliales produjo inflamación colónica grave espontánea (193). El controvertido papel del sistema inmune innato en IBD se seguirá discutiendo más adelante.

- TLR4: como se explicó antes, se ha descrito una asociación entre un polimorfismo del TLR4 (D299G) e IBD (1174,194).

- Región IBD5: la región IBD5, en el cromosoma 5q31, ha sido asociada a susceptibilidad a la IBD (195) y confirmada en varios estudios. Esta región contiene genes que codifican varias citoquinas, pero la identificación de los genes individuales no ha sido posible por el momento. Sin embargo, algunas mutaciones en dos transportadores de cationes orgánicos (OCTN) situados en el locus IBD5 se han asociado con una mayor susceptibilidad a la CD (196).

- MDR1: el gen de multiresistencia a fármacos/drogas (MDR1), en el cromosoma 7q21, ha sido vinculado a la UC tras un meta-análisis que confirmó que la mutación SNP C3435T en MDR1 está asociada a un mayor riesgo de contraer la enfermedad (202). Otro SNP en MDR1 (G2677T) ha sido asociado a la IBD, pero los datos son contradictorios. MDR1 codifica la P-glicoproteína-1, un transportador ATP-dependiente que bombea toxinas anfipáticas (201).

- IL23R: un informe de un estudio amplio de asociación genómica (GWAS) mostró en 2006 (203) 3 polimorfismos de un sólo nucleótido (SNP) que estaban asociados con la CD. Uno de ellos, el rs11209026, localizado en el cromosoma 1p31 está situado en el gen del receptor de IL23 (IL23R), y está asociado con un menor riesgo de desarrollar IBD. Los otros dos SNP son variantes en el gen NOD2 descritos anteriormente. La asociación del SNP con CD rs11209026 fue confirmada posteriormente, y otros 8 SNPs asociados con CD fueron descubiertos dentro del gen IL23R (185). Algunos modelos en ratón han confirmado el importante papel del eje IL23-Th17 en la patogénesis de IBD, si bien esta vinculación no está clara en la actualidad.

- Genes de autofagia: otro GWAS sobre la CD mostró una SNP en el gen ATG16L1, situado en el cromosoma 2q37.1. Este gen codifica la proteína ATG16L1, que participa en la autofagocitosis. Esta mutación se ha asociado con un mayor riesgo de padecer CD en varios estudios, incluso en ausencia de mutaciones en NOD2 (204).

- Otros genes como NOD1, HLA o DLG5 presentan alguna relación con IBD, pero los datos son todavía muy contradictorios (185).

2.4 Signos y síntomas en IBD Aunque la CD y la UC son enfermedades diferentes, tienen muchas similitudes. Ambas suelen alternar recaídas y remisiones. Los síntomas de la UC tienden a ser más uniformes y las recaídas tienen características clínicas similares entre sí, mientras que los síntomas de la CD varían mucho entre pacientes y a lo largo del tiempo. Los pacientes con CD no suelen tener diarrea sanguinolenta, sino más bien dolor abdominal y síntomas abdominales inespecíficos (261). Las principales características clínicas de la IBD son dolor abdominal, diarrea y desnutrición.


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2.4.1 Dolor abdominal El 50%-70% de los pacientes con IBD en estado activo padecen dolor abdominal, y hasta una sexta parte de los pacientes crónicos son tratados con opioides. El dolor es normalmente un síntoma de alarma de la inflamación, y a veces es el único signo de la actividad de la enfermedad (149). Sin embargo, el dolor no está relacionado sólo con las recaídas ya que el 20% de los pacientes en remisión siguen experimentando importantes molestias abdominales (150), lo que implica una componente emocional y psicológica en la percepción del dolor en la IBD (151). En ambos casos, el dolor disminuye la calidad de vida de los pacientes y su tratamiento no es fácil. Los AINEs tradicionales y los inhibidores específicos de la COX-2 parecen agravar la enfermedad (152,153); y los opioides presentan el considerable problema del abuso, más los aumentos de morbilidad y mortalidad que se han asociado al uso crónico de estos fármacos en la IBD (149). 2.4.2 Diarrea La diarrea se define como un aumento del peso de las heces diarias por encima de 200 g. Es el síntoma principal y más común de IBD. La diarrea está relacionada con la gravedad de la recaída y puede servir como un marcador de la actividad inflamatoria (147). Se considera que la causa más frecuente de diarrea en la IBD es la pérdida de superficie de absorción de agua, lo que sucede cuando la mucosa intestinal sufre un daño o bien después de una resección. Además, la malabsorción provoca exceso de nutrientes en el lumen, que producen un gradiente osmótico que empeora la diarrea. Y el tránsito rápido inhibe más aún la absorción de agua.

Otro mecanismo importante implica mediadores inmunes que afectan a las IEC y a las células de la LP, y que resultan en una alteración del transporte de sodio y cloruro (147). En principio, era de esperar que se produjera una mayor secreción iónica durante el transcurso de la enfermedad, especialmente en la UC. Sin embargo, el examen de muestras intestinales ha mostrado que en realidad la secreción iónica está inhibida, tanto en modelos animales como en pacientes humanos (111). El mecanismo propuesto para esta disminución en la secreción está relacionado con NO (112, 113) y cAMP (114). Una inhibición de los procesos secretores durante la inflamación crónica puede ser tal vez una respuesta del organismo para evitar la excesiva pérdida de líquido debido a la diarrea (111). La absorción de Na+ está también disminuida en la IBD. Estudios en animales han mostrado que esta inhibición se debe a la alteración en la expresión de los transportadores involucrados en dicho transporte: NHE3, EnaC y Na+/K+ ATPasa. Además, la regulación nerviosa gastrointestinal podría estar implicada igualmente en los cambios observados en la absorción de Na+ (111). La diarrea en los pacientes con UC se caracteriza por heces con sangre. La urgencia, el tenesmo y una sensación de evacuación incompleta son comunes. Cuando la inflamación está muy extendida, se produce diarrea sanguinolenta, con pus y materia fecal. Los coágulos no son frecuentes (147).

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Los patrones de diarrea en la CD varían dependiendo de la parte anatómica afectada. Cuando se trata del colon y recto los síntomas son similares a la UC. Cuando es el intestino delgado el afectado, las heces son mayores en volumen y no están asociadas con tenesmo, urgencia ni sangre. 2.4.3 Malnutrición. La malnutrición es muy común en pacientes con IBD, especialmente en la CD (158). Aunque la mejora en la medicación y en la cirugía ha reducido considerablemente los casos de desnutrición, todavía hoy sigue siendo un problema importante en la IBD, particularmente en el área pediátrica, y contribuye enormemente a la mala calidad de vida de los pacientes (147). La desnutrición en los pacientes de IBD tiene un origen multifactorial. Por una lado, es común que se produzca una ingesta calórica insuficiente. Una ingesta baja de alimentos puede deberse al temor al dolor o diarrea postprandiales, pero la causa más importante de anorexia es la propia inflamación. De hecho, la IL1 y el TNF inhiben el apetito cuando son administrados a nivel central o periférico (242). La otra causa importante de malnutrición en la IBD es la malabsorción, que afecta principalmente a la absorción de grasas y puede producir un déficit de vitaminas A, D y E.

2.4.4 Otros síntomas. - Lesiones anorrectales: la presencia de lesiones anorrectales, incluyendo las

hemorroides, marcas perianales en la piel, fisuras anales y abscesos perianales, es mucho más común en los pacientes con CD que en la UC (240).

- Megacolon tóxico: se refiere a la dilatación del colon en casos graves de UC,

generalmente pancolitis fulminante, y normalmente requiere cirugía. Aunque la incidencia de este síntoma se desconoce, se ha descrito que entre el 15 y el 25% de pacientes que sufren megacolon tóxico fallecen (240).

- Perforación: está asociada con megacolon tóxico; sin embargo puede ocurrir en

pacientes con UC en ausencia de dilatación tóxica. El sitio más común de perforación es el colon sigmoide (240).

- Cáncer: los pacientes con UC y CD ileocolítica poseen un elevado riesgo de

desarrollar cáncer de colon, mientras que los pacientes con CD y enteritis tienen un elevado riesgo de desarrollar cáncer de intestino delgado (261). La exploración colonoscópica debería realizarse por primera vez de 8 a 10 años después de la aparición de la enfermedad.

La IBD puede cursar además con síntomas extraintestinales. La respuesta sistémica a la inflamación comienza con un cambio en la concentración de proteínas de fase aguda. Esto no es específico de la IBD, sino que ocurre en otras situaciones clínicas caracterizadas por lesiones tisulares, como trauma, isquemia, quemaduras, enfermedades autoinmunes, infecciones y tumores (131). Algunos de los síntomas extraintestinales son:


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- Fiebre: un alto porcentaje de pacientes con IBD presentan fiebre, usualmente baja (137). Algunas de las citoquinas elevadas en la enfermedad (IL1, TNF, linfotoxina-α (LTα), IFNα e IL6) pueden producir fiebre por interacción directa con el hipotálamo.

- Anemia: entre un tercio y la mitad de los pacientes de IBD están anémicos

(138-140). La anemia puede contribuir a la fatiga y la mala calidad de vida en este grupo de pacientes, y algunos estudios han demostrado que la corrección de la anemia mejora la sensación de bienestar, el humor y las habilidades sociales (141). El origen de la anemia en pacientes de IBD es multifactorial: la ulceración intestinal con pérdidas de sangre, la mala absorción de hierro y vitamina B12, y algunos fármacos pueden contribuir a su instauración (131).

- Trastornos de coagulación: el porcentaje de población con IBD que sufre un

episodio trombótico es casi 10-20 veces superior al de la población sana (142,143). Aunque la hipercoagulación en IBD puede ser de origen hereditario, es cierto que los cambios en factores hemostáticos producidos en las recaídas podrían provocar la manifestación de dichos trastornos hereditarios (131).

- Amiloidosis: es una consecuencia rara pero peligrosa de IBD que causa

nefrotoxicidad letal en 1 de cada 4 pacientes que la padecen (146).

2.5 La respuesta inflamatoria mucosa en la IBD. Como decíamos anteriormente, se considera que en condiciones fisiológicas el intestino se encuentra en un estado de “inflamación controlada”, dada la gran carga antigénica del lumen intestinal. Pero existen fenómenos patológicos que cursan con procesos inflamatorios intestinales graves, como es el caso de la IBD. El objetivo de este apartado es describir el tipo de respuestas inmunológicas que se dan en la CD y en la UC.

2.5.1 Sistema inmune innato e IBD. No está claro el papel que desempeñan las células inmunes innatas en la IBD; por un lado la hiperreactividad es importante para el mantenimiento de la inflamación, pero por otro, una capacidad insuficiente para eliminar bacterias está relacionada con el establecimiento de la enfermedad (215). Muchas de las citoquinas típicas del sistema innato se encuentran elevadas tanto en la CD como en la UC, en modelos animales y en pacientes, lo que sugiere un papel importante de las células de este sistema en ambas patologías. Así, los ratones que sobrexpresan TNF desarrollan espontáneamente una enfermedad parecida a la CD, mientras que la colitis inducida no se produce en animales TNF-/- (136). Además, la expresión de IL12 está aumentada tanto en la UC como en la CD, y se correlaciona con el índice de actividad (221). Por otro lado, la IL1 está notablemente aumentada en LPMC, pero no en IEC, de pacientes de IBD (225). Y un estudio demostró que el equilibrio entre la IL18 y su inhibidor natural, la proteína de unión a IL18 (IL18BP), puede contribuir a la patogénesis de IBD (223). La IL6 se encuentra elevada en las zonas de inflamación intestinal (226), así como el receptor para IL6 soluble (sIL6R) (227). Y finalmente la IL10 parece desempeñar un papel

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importante en la IBD ya que los ratones IL10-/- desarrollan espontáneamente colitis (229). Sin embargo, los pacientes de IBD presentan niveles elevados de mRNA y proteína de IL10 en la mucosa intestinal. Con la excepción de la IL10 y el TGFβ, las citoquinas del sistema inmune innato producen de una manera u otra inflamación. Esto hizo pensar durante muchos años que el sistema inmune innato tenía un efecto promotor de la IBD. Pero algunos datos recientes han señalado en cambio que tiene una función protectora, al menos en la CD. Hace algunos años, un ensayo clínico destacó la falta de neutrófilos y la baja producción de IL8 e IL1β en pacientes con CD (216), así como disfunciones en la actividad neutrofílica (215). Otro estudio demostró que el sargramostim, un factor estimulante de colonias de granulocitos-macrófagos (GM-CSF) produce mejoría en la CD (217). Por lo tanto respuestas innatas débiles están asociadas a la aparición de la CD, y estimular dichas respuestas parece beneficioso. Puede resultar lógico, ya que un sistema innato imperfecto es incapaz de eliminar rápidamente las bacterias que han sobrepasado la barrera epitelial, permitiendo la activación del sistema inmune adaptativo, el cual es el responsable último de la inflamación crónica (214,215). Algunos otros datos apoyan esta teoría. Como se mencionó anteriormente, mutaciones en NOD2 asociadas a CD muestran una capacidad disminuida para activar NFκB en respuesta a MDP (214). Además las mutaciones NOD2 están asociadas con una capacidad disminuida de las mucosas para producir α-defensinas. Y la represión condicional de NEMO y, por tanto, de NF-κB en células epiteliales produce inflamación colónica de manera espontánea (193). Finalmente, el epitelio intestinal (como parte del sistema inmune innato) también se encuentra alterado en la IBD ya que la permeabilidad del epitelio parece ser mayor (281), por lo que los antígenos luminales podrían entrar en contacto más fácilmente con el sistema inmune y activar más las respuestas inmunológicas. Además, las IEC pueden potenciar o modular los procesos inflamatorios. Así, durante el transcurso de la IBD, las IEC preferentemente estimulan células T CD4+ en lugar de CD8+, lo que puede contribuir a la respuesta inflamatoria (117,87). Además, las células epiteliales producen una gran variedad de citoquinas, de acción local o sistémica, a saber: IL1α, GM-SCF, G-SCF, IL6 e IL8 (87). 2.5.2. Sistema inmune adaptativo Aunque la alteración de la barrera epitelial y del sistema inmune innato son esenciales para el inicio de la IBD, los linfocitos son necesarios para el mantenimiento de la enfermedad y el establecimiento de la inflamación crónica (282).

2.5.2.1. Respuestas de linfocitos T en la CD: linfocitos Th1 y Th17.

Durante muchos años se creyó que la CD estaba mediada por células Th1, debido a los altos niveles de TNF, IFNγ, IL18 e IL12 encontrados en pacientes con CD, así como los efectos beneficiosos de los anticuerpos anti-TNF y anti-IL12p40. Además algunos modelos animales apoyaron esta hipótesis. Así, el tratamiento con anticuerpos anti-IFNγ previno casi completamente la inducción de colitis en el modelo KO de IL10-/- y en el modelo de transferencia


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de linfoctios CD45RBhi (284,285). Aunque no se debe restar importancia a la implicación de los linfocitos Th1 en la patogénesis de la CD, el subconjunto de células Th17 ha surgido como un importante mediador de la inflamación en CD. En primer lugar, la IL17 y la IL23 están incrementadas en muestras de pacientes de CD (235,236). Además, ciertos estudios en animales han sugerido que la mejoría producida por la terapia anti-IL12 podría deberse a la inhibición de IL23, ya que ambas comparten la subunidad p40. Y la delección del gen que codifica la IL-23p19 inhibe la colitis producida en el modelo KO de IL10-

/-, mientras que la delección de IL-12p35 no, sugiriendo que la IL23 y no la IL12 es importante en este modelo (283).

2.5.2.2. Respuestas de linfocitos T en la UC: linfocitos Th2 y NKT. Hace algunos años se pensaba que las células Th2 estaban implicadas en la patogénesis de la UC, pero no se encontraron datos que confirmaran esta hipótesis. De hecho, se vio que el número de linfocitos T productores de IL-4 es menor en la UC, aunque se observó un aumento de la IL5 (286). Más tarde se vio que la IL13 también estaba aumentada en pacientes de UC y que las células responsables de esta producción aumentada de IL13 eran linfocitos NKT (237). Aunque se sigue asociando la UC con los linfocitos Th2, o al menos con las citoquinas Th2, existe mucho desconocimiento sobre los mecanismos subyacentes de la inflamación en la UC, en parte, debido a la ausencia de modelos animales apropiados para la UC. En cualquier caso, se ha descrito que los genes inducidos por el virus Epstein-Barr (EBI3) y la IL27 parecen estar involucrados (238). Esto es coherente con el aumento de los niveles de EBI3 en la mucosa de pacientes con UC con respecto a CD o controles sanos. 2.5.2.3 Linfocitos Treg en la IBD En la IBD se ha observado un número reducido de cálulas Treg en sangre; sin embargo, el número de células CD25brightFoxP3+ en LP, ganglios linfáticos mesentéricos y mucosa de pacientes con IBD está aumentado (177,244). No está claro si estos hallazgos representan una redistribución de estos linfocitos al sitio de la inflamación. Por otro lado, el análisis funcional in vitro de linfocitos Treg de sangre o mucosa procedentes de pacientes con IBD ha revelado que, pese a los cambios en el número de células, la actividad inmunosupresora se mantiene normal (245), al contrario de lo que ocurre en la artritis reumatoide.

2.6 Modelos Animales Existe un único modelo animal de inflamación intestinal crónica espontánea, que se produce en los monos cottou-top tamarin en condiciones de cautividad. Pero este modelo no puede usarse en investigación al tratarse de una especie en peligro de extinción. Además, la frecuencia de colitis es relativamente alta en los ratones C3H/HeJBir y SAMP1/Yit bajo ciertas condiciones (247,248). Aparte de esos tres modelos de colitis espontánea, se han conseguido múltiples modelos animales de IBD por manipulación química o genética. Estos modelos reproducen muy bien los síntomas característicos de la enfermedad por lo que son adecuados para el estudio de los

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mecanismos subyacentes a la inflamación y la enfermedad, así como la investigación de nuevos tratamientos. Dividiremos en 4 grupos los modelos animales de IBD:

- Modelos “knockout” (KO), en los que la deficiencia de un mediador, generalmente implicado en la regulación de linfocitos T, causa una respuesta inflamatoria intestinal. Los modelos KO más típicos de IBD son IL2-/-, IL10-/-, ratones deficientes en la cadena α del TCR y ratones deficientes en TGFβ1 (247,248).

- Ratones y ratas transgénicos, en los que la mutación o sobreexpresión de

elementos potenciadores del sistema inmune provoca la aparición de una condición similar a la IBD. Los modelos más estudiados son los transgénicos para STAT 4, IL7, HLA B27 y TNF (llamando TNFΔARE) (247,248).

- Modelos de transferencia, en los que la transferencia de subpoblaciones Th1

específicas a ratones inmunodeficientes (SCID) produce colitis. El modelo mejor estudiado es el de transferencia de linfocitos CD4+CD45bright (247,248).

- Modelos inducidos químicamente, que usan un agente químico, estrés u otras

lesiones para causar la enfermedad. Dentro de este grupo hay varios modelos, siendo los más usados las colitis inducidas por la administración de ácido acético, iodoacetamida, indometacina, oxazolona, peptidoglicano-polisacárido (PG-PS), dextrán sulfato sódico (DSS) y ácido trinitrobenceno sulfónico (TNBS). Por ser los más usados y por la importancia en elaboración de esta tesis doctoral, nos centraremos de manera específica en los modelos del DSS y del TNBS.

2.6 1. Colitis inducida por el DSS La adición de DSS al agua potable en proporción del 3 al 10% induce colitis en ratas y ratones (253), con diarrea sanguinolenta, pérdida de peso, acortamiento del colon, ulceración mucosa e infiltración de neutrófilos. El primer signo de la colitis es la depleción progresiva de criptas intestinales, sugiriendo un primer efecto directo tóxico del DSS sobre las células epiteliales. Después se producen erosiones y se forman abscesos en las criptas del colon ascendente. En el modelo del DSS se considera que existen dos fases: una fase aguda, en los primeros 5-7 días, y una fase crónica, producida por la administración de DSS durante un tiempo mayor o bien en ciclos. La colitis aguda se caracteriza por una respuesta inmune innata que se autolimita cuando se retira el DSS. En la fase aguda aumenta la producción de citoquinas producidas por los macrófagos: IL1β, IL6, TNF y GM-CSF. Además, la colitis aguda inducida por el DSS se produce también en ratones SCID, indicando que las células T y B no son necesarias (254). El DSS puede inducir también colitis crónica cuando es administrado en ciclos. En un estudio publicado por Okayasu et al., los ratones recibieron el DSS en 5 ciclos que consistían cada uno en 7 días de ingesta de agua con DSS al 5% seguidos de 10 días con agua normal. Los animales que desarrollaron colitis crónica mostraron signos de erosión, zonas prominentes de regeneración de la mucosa


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colónica incluyendo displasia, acortamiento del intestino grueso y formación de folículos linfoides (253). Se considera que esta fase está mediada por los linfocitos que han sido activados por las citoquinas proinflamatorias producidas durante la fase aguda. Así, parece que la respuesta crónica está mediada por linfocitos Th1 y Th2, con aumento de IFNγ e IL4 (288). 2.6.7. Colitis inducida por TNBS En este modelo, la colitis ocurre en ratas y ratones por la administración del TNBS mediante un enema, previa alteración de la barrera epitelial con etanol (255). Este modelo es un híbrido ya que mezcla agentes químicos (etanol) y agentes haptenos (TNBS). El TNBS establece una necrosis aguda en la pared del colon provocada por estrés oxidativo, lo cual parece esencial para la instalación de la inflamación. A ésta, le sigue una inflamación crónica submucosa asociada a dirarrea, anorexia y pérdida de peso, por lo que este modelo posee muchas características similares a la CD. En ratones, las reacciones están mediadas por linfocitos B y T. De hecho, tras la administración del TNBS la mucosa de animales se encuentra infiltrada por células T CD4+ (256) y células B productoras de IgG e IgA (257). El infiltrado de linfocitos CD4+ es claramente del subtipo Th1, ya que producen grandes cantidades de IFNγ e IL2 pero no IL4 (256). La interacción entre células APC productoras de IL12 y células Th1 parece esencial para el desarrollo y la progresión de la colitis inducida por TNBS, como se demuestra por varios experimentos en los que el bloqueo de la IL12 impide el establecimiento de la colitis (256, 258-260). La administración directa de TNBS en el íleon de ratas produce ileitis (277). En ratas, la respuesta parece estar mediada principalmente por macrófagos y neutrófilos, con un aumento claro de IL1, IL6 y TNF, y por linfocitos Th1 y Th17, ya que se produce un aumento de IFNγ e IL17, pero no IL-4 (289).

2.7 Tratamientos actuales en IBD. Los objetivos más importantes del tratamiento farmacológico en ambas variedades de IBD son la rápida inducción y mantenimiento de la remisión, idealmente sin recurrir a esteroides, así como la prevención de las complicaciones de la enfermedad en sí y su tratamiento. Como regla general, el tratamiento es elegido sobre la base de la magnitud y la gravedad de la enfermedad, su capacidad de respuesta a los tratamientos anteriores, y la situación del paciente (261). El tratamiento farmacológico actual para la IBD consta de fármacos antiinflamatorios e inmunosupresores y agentes biológicos.

2.7.1 Fármacos antiinflamatorios - Aminosalicilatos

La sulfasalazina y su metabolito activo, el ácido 5-aminosalicílico (5-ASA o mesalamina) son los principales fármacos utilizados para la inducción y mantenimiento de la remisión en UC y CD de intensidad ligera a moderada (262,263). El 5-ASA se usa en los casos de inflamación del intestino delgado pero

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no en la inflamación colónica, ya que cuando es administrado por vía oral, al ser absorbido por el epitelio intestinal, no llega al colon. La sulfasalazina es una sulfonamida modificada que casi no es absorbida por el epitelio intestinal, por lo que se usa en los casos de UC o CD colónica. Una vez en colon, las bacterias hidrolizan la sulfasalazina, dando lugar al 5-ASA (la molécula activa) y sulfapiridina, que actúa únicamente como molécula transportadora. Dado que la sulfapiridina se considera la responsable de la mayoría de los efectos secundarios de la sulfasalazina, se han desarrollado nuevos aminosalicilatos desprovistos de la misma, como la balsalazina, olsalazina o la propia mesalamina. El mejor perfil de efectos secundarios, ha convertido a la nueva generación de aminosalicilatos en los fármacos más utilizados en la IBD ya que, efectivamente, el 80% de pacientes que sufren intolerancia o alergia a la sulfasalazina toleran otros aminosalicilatos (266). El mecanismo de acción del 5-ASA no se conoce completamente, aunque se ha propuesto que la inhibición de las rutas del ácido araquidónico a través de la inhibición de las enzimas 5-lipooxigenasa y ciclooxigenasa, y la inhibición del factor de transcripción NF-κB, son importantes para la acción antiinflamatoria del 5-ASA. Más recientemente se ha postulado que el 5-ASA ejerce una activación de PPARγ, aunque un estudio genómico realizado en nuestro laboratorio ha indicado que, al menos en ratas, este mecanismo parece no ser importante para el efecto del 5-ASA (290). - Glucocorticoides Los corticoides son parte de la terapia en UC y CD moderadas a graves. Sin embargo, son ineficaces como terapias de mantenimiento, lo que resulta enormemente llamativo (262). Su uso sistémico está asociado a varios efectos secundarios importantes, y su uso presenta además el problema de la dependencia, mecanismo por el cual en determinados individuos que han usado estos fármacos, su retirada provoca el agravamiento de la enfermedad, por lo que “necesitan” usar estos fármacos. Los corticosteroides mejoran la inflamación por varios mecanismos, incluyendo la disminución de la transcripción de múltiples genes pro-inflamatorios (citoquinas, quimioquinas, enzimas inflamatorias y moléculas de adhesión) y el aumento de la expresión de genes antiinflamatorios. También inhiben el reclutamiento de leucocitos en el sitio de la inflamación e interfieren con las acciones de los fibroblastos y las células endoteliales (266). La prednisona es el glucocorticoide oral más utilizado en IBD, mientras que la prednisolona, metilprednisolona e hidrocortisona son los más usados vía parenteral por su eficacia terapéutica (266). La budesonida es un corticoide relativamente nuevo con baja biodisponibilidad sistémica debido a la alta metabolización de primer paso en el hígado. Estas propiedades favorecen el uso de la budesonida vía tópica (mucosa) ya que posee elevada potencia con menos actividad sistémica (262). Además, la budesonida ha sido eficaz para el mantenimiento de la remisión en pacientes con CD por periodos de 3 a 6 meses (aunque no por periodos más largos) (267). 2.7.2 Otros fármacos inmunosupresores


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La tiopurina 6-mercaptopurina (6-MP) y su profármaco azatioprina (AZA) son eficaces en la IBD (268). Son útiles en pacientes de IBD resistentes a glucocorticoides y en la CD perianal. Los mecanismos pueden estar relacionados con la citotoxicidad directa, inhibición de la proliferación e inhibición de la producción de citoquinas (262,266). La eficacia del 6-MP y AZA se ve menoscabada por los efectos secundarios, que provocan el abandono del tratamiento en el 15-30% de pacientes (266). El metotrexato, un inhibidor de la dihidrofolato reductasa y otras enzimas folatodependientes, tiene propiedades antiinflamatorias e inmunosupresoras relacionadas con la disminución de la producción de IL1 y eicosanoides, y el bloqueo de la quimiotaxis de neutrófilos (269). Su efecto es más rápido que el de las tiopurinas y es utilizado en casos de resistencia a 6-MP y AZA en la CD. Su uso está limitado por los efectos secundarios, especialmente hepatotoxicidad. La ciclosporina es otro agente inmunosupresor importante en la IBD que actúa selectivamente sobre las células T inhibiendo respuestas mediadas por la calcineurina, como la transcripción de IL2, IL4, IFN y TNF (270). Ha sido utilizada como terapia de rescate en pacientes hospitalizados por UC en estado grave, resistentes a corticoides y candidatos a cirugía; pero no está indicada en pacientes con CD, excepto las condiciones perianales. También posee graves efectos secundarios asociados, como nefrotoxicidad (266). 2.7.3 Terapia biológica La denominada “terapia biológica” hace referencia a los fármacos obtenidos mediante ingeniería genética, generalmente anticuerpos u otras proteínas. Este grupo incluye bloqueadores de citoquinas como TNF, IL12, IL6 e IFNγ, y los anticuerpos anti-moléculas de adhesión, que interfieren en el reclutamiento leucocitario. Como se mencionó anteriormente, la citoquina TNF es un mediador crucial en la CD. Infliximab es un anticuerpo monoclonal anti-TNF quimérico (75% humano, 25% ratón) de alta afinidad, que produce mejoría en la IBD. Se une al TNF soluble y lo neutraliza, pero también provoca la lisis de las células que expresan TNF en la membrana e induce apoptosis de las células T activadas (272). Actualmente no está claro cuál de estos dos mecanismos es el más importante, pero en cualquier caso produce un efecto inmunosupresor claro. Actúa muy rápidamente, produciendo inhibición de la gran mayoría de parámetros inflamatorios tras 24 horas y una buena mejoría clínica después de 7-14 días. Los efectos secundarios son más frecuentes en los tratamientos crónicos e incluyen mayor susceptibilidad a infecciones, y en algunos casos, hipersensibilidad. Se han comercializado otros bloqueadores de TNF, tales como adalimumab y CDP571. CDP571 contiene un 95% de fracción humana por lo que es menos inmunogénico que el infliximab. Adalimumab no es más eficaz que infliximab pero puede utilizarse como alternativa a ella en pacientes que desarrollan anticuerpos anti-infliximab (262,266). Un reciente estudio meta-análisis ha demostrado la eficacia de la terapia anti-TNF en la inducción y mantenimiento de la remisión en CD (273).

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Otras citoquinas proinflamatorias son blancos potenciales en la terapia IBD. MRA es un anticuerpo humanizado contra sIL6R (274), que ha sido ensayado en la CD con éxito, así como los anticuerpos anti-IFNγ y anti-IL12 (275,276).

3. EL GLUCOMACROPÉPTIDO BOVINO

3.1. Péptidos bioactivos. La degradación enzimática de los alimentos en el intestino libera pequeños péptidos de las proteínas. Además de su valor meramente nutricional, se ha contrastado que estos péptidos tienen efectos biológicos y/o farmacológicos, por lo que se les suele denominar péptidos bioactivos. Estos péptidos pueden estar presentes en los alimentos como tal o bien pueden producirse in vivo o in vitro tras la digestión de proteínas. Su presencia en determinados alimentos puede hacer que éstos se consideren alimentos funcionales y, por tanto, tengan un gran valor añadido desde el punto de vista nutricional, terapéutico e industrial (327). En principio, cualquier fuente de proteínas puede producir péptidos bioactivos. Sin embargo, la fuente principal es la leche (293). Las proteínas de la leche suelen clasificarse en tres fracciones o compartimentos:

- Caseínas: Son proteínas que precipitan formando micelas en un medio ácido (pH 4 ó 5). Las caseínas son de 5 tipos: αS1-caseína, αS2-caseína, β-caseína, κ-caseína y γ-caseína, aunque la proporción de esta última en la leche de vaca es muy escasa. Las caseínas se agrupan en forma de polímeros denominados micelas, que se componen a la vez de varias submicelas. La submicela de caseína está compuesta por los 4 tipos de caseínas más abundantes, en proporciones variables. Al estructurarse la micela, las α y β-caseínas, de carácter lipófilo, se sitúan en el centro, formando un núcleo hidrofóbico, mientras que la κ-caseína, hidrofílica, se sitúa en la superficie (Figura 5A) (328).

- Proteínas del suero: son solubles en la fracción líquida (suero) que queda tras

precipitar las caseínas. Se pueden distinguir: α-lactoalbúmina, β-lactoglobulina, albúmina sérica, proteasas-peptonas, lactoferrina e inmunoglobulinas (328).

- Proteínas de la membrana de los glóbulos de grasa, que forman parte de la

membrana de los glóbulos de grasa en la crema de la leche (1).

Todas las fracciones de la leche contienen péptidos bioactivos. Y aunque los componentes biológicamente activos de la leche suponen menos del 0.08% del total de sólidos, excluyendo la α-lactoalbúmina, algunas de las fracciones pueden llegar a tener tal valor añadido que hacen rentable su obtención. Es importante destacar que hay evidencias de que los péptidos bioactivos pueden atravesar el epitelio intestinal y llegar a tejidos periféricos por medio de la circulación, por lo que pueden ejercer efectos locales a nivel del tracto GI, así como efectos sistémicos. Así, se han demostrado diversas acciones en al ámbito de los sistemas digestivo, inmune y cardiovascular (327). A continuación se describen algunas de ellas.

- Péptidos con efectos sobre el sistema digestivo. Péptidos opioides.


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Existen péptidos derivados del gluten y de las α y β-caseínas que demuestran actividad opiácea y se denominan exorfinas. Éstos se unen a los receptores de la luz intestinal y actúan como moduladores exógenos de la motilidad intestinal, de la permeabilidad epitelial y de la liberación de hormonas intestinales. Entre ellos se encuentran los péptidos llamados casomorfinas, derivados de las α y β-caseína (327).

- Péptidos con efectos sobre el sistema cardiovascular.

Los principales efectos descritos sobre la actividad cardiovascular son de actividad antihipertensiva y antitrombótica. Los péptidos que poseen actividad antihipertensiva lo hacen por inhibición de la enzima convertidora de angiotensina. Se han descrito tres péptidos de la αS1-caseína y dos de la β-caseína que muestran esta actividad. El efecto antitrombótico de otra serie de péptidos procedentes fundamentalmente de la κ-caseína parece venir dado por la similitud estructural de éstos con la cadena γ del fibrinógeno, de forma que entran en competición con los receptores plaquetarios, inhibiendo así la agregación plaquetaria (327).

- Péptidos con efectos inmunomoduladores y antimicrobianos.

Los hidrolizados de αS1-caseína modulan las funciones inmunes de una manera muy diferente dependiendo de los péptidos liberados. Así, las digestiones producidas por la pancreatina y tripsina inhiben las respuestas proliferativas en linfocitos de roedores y conejos, mientras que la digestión con pepsina y quimotripsina parece no tener efecto (298). Y los hidrolizados de pepsina/tripsina inhiben la proliferación de PMBC (299), mientras que el tratamiento con tripsina solamente produce un hexapéptido con propiedades inmunoestimulantes (292). Como con la αS1-caseína, los hidrolizados de la β-caseína producidos con pancreatina y tripsina tienen efecto inhibitorio sobre las respuestas proliferativas en linfocitos de ratón y conejo, mientras que los de pepsina y quimotripsina no tienen efecto (298). También el hexapéptido de la β-caseína PGPIPN y el tripéptido LLY han demostrado promover la formación de anticuerpos y acelerar la fagocitosis en células inmunes (299). Las actividades biológicas de los hidrolizados de la αS1-caseína y β-caseína péptidos están revisadas de manera más extensa en la ref #292.

Dos péptidos bioactivos de la leche de vaca merecen especial atención por sus propiedades inmunomoduladoras: el TGF-β y el glucomacropéptido bovino (BGMP). El BGMP, objeto de estudio de esta tesis doctoral, será descrito con mayor extensión más adelante.

EL TFG-β es un factor de crecimiento presente a alta concentración en la leche de varias especies animales, incluida la humana, y también se produce en pequeñas cantidades en el intestino de los recién nacidos. Existen al menos 3 isoformas del TFG-β ((TGF-β1, TGF-β2 y TGF-β3). Algunos estudios recientes han indicado que el TFG-β se expresa como un pre-pro-factor que, tras la hidrólisis celular, es secretado en la leche como un profactor junto a otras proteínas. Esta forma latente que se encuentra en la leche es finalmente activada y transformada en TFG-β por el ácido gástrico (329).

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El TGF-β que la madre transfiere a la progenie a través de la placenta y la leche materna es muy importante para el desarrollo de los recién nacidos, ya que los ratones que presentan una alteración en el gen del TGF-β sobreviven hasta el destete, después de lo cual desarrollan un síndrome acompañado de inflamación, necrosis y fallo multiorgánico que causa la muerte de los animales (330). La administración oral de TGF-β a ratones TGF-β-/- previene su muerte, observándose el péptido en tejidos internos como el pulmón, lo cual indica que puede ser activo no sólo a nivel GI (330, 331).

Algunos autores han mostrado además que la administración oral de TGF-β tiene efecto antiinflamatorio en modelos animales de colitis (332, 333). Y un estudio realizado en pacientes pediátricos de CD mostró que la administración de una fórmula enriquecida en TGF-β produjo una reducción en la expresión de IFNγ (334), y aunque el número de pacientes en el estudio, siete, fue muy bajo, los resultados fueron muy claros ya que todos los niños mostraron una mejoría del índice de actividad de la enfermedad. Más tarde se realizó un estudio similar con 29 pacientes, en los que se observó remisión de la enfermedad en el 79% de los casos (335). Estos estudios han demostrado que las fórmulas enriquecidas en TGF-β pueden inducir la remisión clínica y la curación de la mucosa en pacientes, siendo más activas en pacientes pediátricos que en adultos, por lo que aun siendo menos eficaces que los corticoides, se usan en pacientes pediátricos en los que el uso de corticoides está contraindicado por su efecto en el crecimiento (336).

3.2. El BGMP El BGMP es un péptido de 64 aminoácidos producido en la digestión de la k-caseína de la leche de vaca. Cuando la k-caseína es sometida a la acción de la quimosina, surgen dos péptidos: un péptido de mayor tamaño que contiene los aminoácidos 1-105 y que se llama para-k-caseína, y un glicofosfopéptido (BGMP) menor que contiene los aminoácidos 106-169 (Figura 5B). El BGMP es relativamente pequeño, con un peso molecular de 8000 Dalton; sin embargo, debido a las glicosilaciones, su tamaño real puede variar entre 25000 y 30000 Dalton. La secuencia de aminoácidos del BGMP (con sus dos variantes) ha sido definida (315, 316) (Figura 5B). El BGMP está altamente glucosilado y la composición del componente glucídico también se ha estudiado (300). Se han encontrado cinco grupos sacáridos diferentes (300), siendo el más destacado el ácido N-acetilneuramínico, comúnmente conocido como ácido siálico:

1) Monosacárido GalNAc-O-R 2) Disacárido Gal β1→3 GalNAc-O-R 3) Trisacárido NeuAc α2→ Gal β1→3 Gal-Nac-O-R 4) Trisacárido Gal β1→3 (NeuAc α2→6) Gal-Nac-O-R 5) Tetrasacárido NeuAc α2→ Gal β1→3 (NeuAc α2→6) Gal-Nac-O-R

Donde Gal = galactosa, GalNAc = N-acetylgalactosamine y NeuAc = ácido siálico.


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Centro Hidrofóbico: α-caseínas y β−caseína

B)

A)

Agregados de fosfato y cálcio

K-caseína

Submicela Micela

BGMP

Para-k-caseína

1 10 20 30 H-MAIPPKKNQDKTEIPTINTIASGEPTSTPT(T)EA

Variante B (I )

40 50 60 VESTVATLE(D)SPEVIESPPEINTVQVTSTAV-OH

Variante B (A)

Figura 5. A) Esquema de la micelas de la leche. B) Estructura primaria del BGMP, con ambas variantes. El número pequeño sobre las letras hace referencia a la numeración secuencial de los residuos aminoácidos. Las estructuras glucídicas son importantes, ya que de ellas dependen muchas de las actividades biológicas del BGMP; de hecho, el efecto del BGMP puede variar en función de los polisacáridos a los que se encuentre unido. Además es importante señalar que dicho componente glucídico varía entre los GMP bovino y humano. Por otra parte, la composición y la posición del glúcido dentro del BGMP pueden variar en cada molécula, dando lugar a distintas fracciones del BGMP. En contraste con esta variabilidad, el grupo fosfato se encuentra siempre en la serina 44 (316). La leche contiene BGMP tal cual en baja proporción; en cambio, se produce una cantidad mayor en el tracto gastrointestinal humano después de la ingestión de leche, como resultado de la hidrólisis de la k-caseína (301, 302). Además, en la industria el BGMP se obtiene como subproducto en la fabricación de queso, al ser soluble en el suero (300). Este péptido puede ser purificado y se encuentra en el mercado con una alta pureza. Como nutriente, el BGMP se considera muy seguro. Así, un estudio reciente ha demostrado que carece de propiedades inmunogénicas al no inducir respuestas específicas en linfocitos T de ratones, aun administrándose polimerizado, al contrario que la κ-caseína (306). Además, actualmente ya se añade a algunas fórmulas infantiles

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y, debido a su bajo contenido en aminoácidos aromáticos incluida la fenilalanina, es muy útil en la elaboración de dietas para las personas con fenilcetonuria (303-305). El BGMP posee muchas acciones biológicas. Actualmente es añadido a las pastas dentífricas debido a sus propiedades anticariogénicas: posee actividad antibacteriana, reduce la caries dental y la desmineralización de los dientes, promoviendo la mineralización del esmalte (307). Además el BGMP inhibe la hemaglutinación provocada por las bacterias cariogénicas S. mutans, S. sanguis y A. viscosa (318), por lo que se cree que su efecto protector frente a la caries dental se debe a que produce un cambio en la composición microbiana de la placa dental. El BGMP también podría combatir infecciones, ya que se ha visto que se une a las enterotoxinas del cólera y E.coli, inhibe la adherencia de bacterias y virus, promueve el crecimiento de bifidobacterias y modula las respuestas inmunitarias (300, 308-309). La actividad del BGMP de bloqueo de la toxina del cólera depende de la cadena glucídica del péptido, ya que el tratamiento con sialidasa revierte el efecto, aunque la cadena peptídica también participa en menor medida en la actividad inhibidora (309). Otro estudio mostró que el BGMP capta las enterotoxinas LT-I y LT-II de E. coli in vitro, y que este efecto inhibitorio se produce también in vivo, puesto que la administración de 1 mg de BGMP al día protege al 100% de los ratones contra la diarrea producida por la toxina del cólera y LT-II (317). En otro estudio se observó que el BGMP inhibe la hemaglutinación producida por cuatro cepas del Influenzavirus humano (319).

Por otra parte, aunque hay algunos estudios que demuestran que este péptido promueve el crecimiento de bifidobacterias in vitro, hasta ahora no hay evidencia definitiva del efecto prebiótico del BGMP. Se han hecho estudios in vivo con monos Rhesus y bebés humanos, pero el alto nivel inicial de bifidobacterias hace que los resultados obtenidos no pudieran ser evaluados correctamente (293, 310, 311).

Además de todas estas actividades biológicas, el BGMP desempeña acciones inmunomoduladoras muy importante. 3.3 Efectos inmunomoduladores del BGMP. Los efectos inmunomoduladores del BGMP han sido estudiados tanto a nivel del sistema inmune innato como del adaptativo. Antes de nuestro propio estudio, sólo hubo dos que se centraron en el efecto del BGMP sobre los macrófagos. El primero de ellos demostró que el BGMP aumenta la secreción de IL1ra sin que ello afecte a la IL1β, en una línea celular monocítica de ratón (312). En el segundo, el BGMP se estableció como un potente reforzador de la actividad de los macrófagos humanos. A concentraciones tan bajas como 10 μg/mL, aumentó considerablemente la proliferación y capacidad fagocítica de células U937, una línea celular humana de macrófagos. La digestión con pepsina aumentó significativamente estos efectos, mientras que el hidrolizado con tripsina no tuvo efecto significativo, lo que sugiere que algunos pequeños péptidos podrían ser responsables de las acciones de BGMP. Además, las fracciones con diferentes cadenas glucídicas mostraron diferentes capacidades inmunomoduladoras, siendo la fracción rica en ácido siálico la más activa (313).


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Con respecto a las respuestas inmunes adaptativas, el BGMP ha demostrado inhibir la proliferación de esplenocitos estimulada por LPS, siendo la para-κ-caseína inactiva. Este efecto inmunosupresor se inhibe completamente tras el tratamiento con sialidasa (321), y se reduce tras la digestión con quimotripsina, pero aumenta con el tratamiento con tripsina y pronasa, sugiriendo que la cadena peptídica también es importante para este efecto. El BGMP también inhibió la proliferación de esplenocitos cuando éstos fueron estimulados con fitohemaglutinina (PHA) (322) pero, curiosamente, no cuando el mitógeno empleado fue concanavalina A (ConA) (322, 323). Varios mecanismos han sido propuestos para el efecto inhibitorio del BGMP sobre las respuestas inmunes adaptativas. Por un lado, la mayor producción de IL1ra impediría que la IL1 ejerciera su efecto proinflamatorio, es decir, linfoproliferativo. Además, se ha visto que el BGMP se une a la membrana de células T CD4+ y reprime la expresión de CD25 (IL2R), sin ningún efecto sobre las células T CD8+ (324). Por otra parte, el BGMP aumenta la producción de IgA en esplenocitos estimulados por LPS, así como el número de linfocitos B IgA+ (325). Y se demostró que el BGMP acelera la proliferación de linfocitos B pero no linfocitos T en humanos (326). References 1. Brandtzaeg P, Farstad IN, Haraldsen G. Regional specialization in the mucosal immune system: primed cells do not always home along the same track. Immunol Today. 1999 Jun;20(6):267-77. 2. Ogra PL, Mestecky J, Lamm ME, Strober W, Bienenstock J, McGhee JR. Mucosal Immunology, 2nd edn. San Diego: Academic Press, 1999. 3. Fiocchi C. The normal intestinal mucosa: a state of “controlled inflammation”. In Targan SR, Shanahan F, Karp LC. Inflammatory Bowel Disease: From Bench to Bedside, 2nd edn. Springer, 2005. 4. Chang SK, Dohrman AF, Basbaum CB, Ho SB, Tsuda T, Toribara NW, Gum JR, Kim YS. Localization of mucin (MUC2 and MUC3) messenger RNA and peptide expression in human normal intestine and colon cancer. Gastroenterology. 1994 Jul;107(1):28-36. 5. Kato T, Owen RL. Structure and function of intestinal mucosal epithelium. In Ogra PL, Mestecky J, Lamm ME, Strober W, Bienenstock J, McGhee JR. Mucosal Immunology, 2nd edn. San Diego: Academic Press, 1999. 6. Targan SR, Shanahan F, Karp LC. Inflammatory Bowel Disease: From Bench to Bedside, 2nd edn. Springer, 2005. 7. Rhodes JM. Mucus and inflammatory bowel disease. In Allan RN, Keighley MRB, Alexander-Williams J, Hawkins CF. Inflammatory Bowel Diseases, 2nd edn. London, Churchill Livingstone, 1990. 8. Magnusson KE, Stjernström I. Mucosal barrier mechanisms. Interplay between secretory IgA (SIgA), IgG and mucins on the surface properties and association of salmonellae with intestine and granulocytes. Immunology. 1982 Feb;45(2):239-48. 9. Chadee K, Petri WA Jr, Innes DJ, Ravdin JI. Rat and human colonic mucins bind to and inhibit adherence lectin of Entamoeba histolytica. J Clin Invest. 1987 Nov;80(5):1245-54. 10. Mantle M, Basaraba L, Peacock SC, Gall DG. Binding of Yersinia enterocolitica to rabbit intestinal brush border membranes, mucus, and mucin. Infect Immun. 1989 Nov;57(11):3292-9. 11. Sajjan SU, Forstner JF. Role of the putative "link" glycopeptide of intestinal mucin in binding of piliated Escherichia coli serotype O157:H7 strain CL-49. Infect Immun. 1990 Apr;58(4):868-73. 12. Miller HR. Gastrointestinal mucus, a medium for survival and for elimination of parasitic nematodes and protozoa. Parasitology. 1987;94 Suppl:S77-100. 13. Boyer B, Thiery JP. Epithelial cell adhesion mechanisms. J Membr Biol. 1989 Dec;112(2):97-108. 14. Keshav S, Lawson L, Chung LP, Stein M, Perry VH, Gordon S. Tumor necrosis factor mRNA localized to Paneth cells of normal murine intestinal epithelium by in situ hybridization. J Exp Med. 1990 Jan 1;171(1):327-32. 15. Ouellette AJ, Greco RM, James M, Frederick D, Naftilan J, Fallon JT. Developmental regulation of cryptdin, a corticostatin/defensin precursor mRNA in mouse small intestinal crypt epithelium. J Cell Biol. 1989 May;108(5):1687-95. 16. Rodning CB, Wilson ID, Erlandsen SL. Immunoglobulins within human small-intestinal Paneth cells. Lancet. 1976 May 8;1(7967):984-7. 17. Jarry A, Cerf-Bensussan N, Brousse N, Selz F, Guy-Grand D. Subsets of CD3+ (T cell receptor alpha/beta or gamma/delta) and CD3- lymphocytes isolated from normal human gut epithelium display

Capítulo 2

52

http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=Search&Term=%22Brandtzaeg%20P%22%5BAuthor%5D&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_DiscoveryPanel.Pubmed_RVAbstractPlus

http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=Search&Term=%22Farstad%20IN%22%5BAuthor%5D&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_DiscoveryPanel.Pubmed_RVAbstractPlus

http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=Search&Term=%22Haraldsen%20G%22%5BAuthor%5D&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_DiscoveryPanel.Pubmed_RVAbstractPlus

http://www.ncbi.nlm.nih.gov/pubmed/8020672?ordinalpos=4&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_DefaultReportPanel.Pubmed_RVDocSum

http://www.ncbi.nlm.nih.gov/pubmed/8020672?ordinalpos=4&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_DefaultReportPanel.Pubmed_RVDocSum

http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=Search&Term=%22Magnusson%20KE%22%5BAuthor%5D&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_DiscoveryPanel.Pubmed_RVAbstractPlus

http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=Search&Term=%22Stjernstr%C3%B6m%20I%22%5BAuthor%5D&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_DiscoveryPanel.Pubmed_RVAbstractPlus

phenotypical features different from their counterparts in peripheral blood. Eur J Immunol. 1990 May;20(5):1097-103. 18. London SD, Cebra JJ, Rubin DH. Intraepithelial lymphocytes contain virus-specific, MHC-restricted cytotoxic cell precursors after gut mucosal immunization with reovirus serotype 1/Lang. Reg Immunol. 1989 Mar-Apr;2(2):98-102. 19. Tagliabue A, Boraschi D, Villa L, Keren DF, Lowell GH, Rappuoli R, Nencioni L. IgA-dependent cell-mediated activity against enteropathogenic bacteria: distribution, specificity, and characterization of the effector cells. J Immunol. 1984 Aug;133(2):988-92. 20. Hirata I, Austin LL, Blackwell WH, Weber JR, Dobbins WO 3rd. Immunoelectron microscopic localization of HLA-DR antigen in control small intestine and colon and in inflammatory bowel disease. Dig Dis Sci. 1986 Dec;31(12):1317-30 21. Mayer L, Eisenhardt D, Salomon P, Bauer W, Plous R, Piccinini L. Expression of class II molecules on intestinal epithelial cells in humans. Differences between normal and inflammatory bowel disease. Gastroenterology. 1991 Jan; 100(1):3-12. 22. Cerf-Bensussan N, Quaroni A, Kurnick JT, Bhan AK. Intraepithelial lymphocytes modulate Ia expression by intestinal epithelial cells. J Immunol.1984 May; 132(5):2244-52. 23. Hirayama K, Matsushita S, Kikuchi I, Iuchi M, Ohta N, Sasazuki T. HLA-DQ is epistatic to HLA-DR in controlling the immune response to schistosomal antigen in humans. Nature. 1987 Jun 4-10; 327(6121):426-30. 24. Eckmann L, Jung HC, Schürer-Maly C, Panja A, Morzycka-Wroblewska E, Kagnoff MF.Differential cytokine expression by human intestinal epithelial cell lines: regulated expression of interleukin 8. Gastroenterology. 1993 Dec;105(6):1689-97. 25. Gebert A, Rothkötter HJ, Pabst R. M cells in Peyer's patches of the intestine. Int Rev Cytol. 1996;167:91-159. Review. 26. Kato T. A study of secretory immunoglobulin A on membranous epithelial cells (M cells) and adjacent absorptive cells of rabbit Peyer's patches. Gastroenterol Jpn. 1990 Feb;25(1):15-23. 27. Nagura H, Ohtani H, Masuda T, Kimura M, Nakamura S. HLA-DR expression on M cells overlying Peyer's patches is a common feature of human small intestine. Acta Pathol Jpn. 1991 Nov;41(11):818-23. 28. Ishikawa H, Kanamori Y, Hamada H, Kiyono H. Developtment and function of Organized Gut-Associated Lymphoid Tissues. In Ogra PL, Mestecky J, Lamm ME, Strober W, Bienenstock J, McGhee JR. Mucosal Immunology, 2nd edn. San Diego: Academic Press, 1999. 29. Radford-Smith GL, Edwards JE, Purdie DM, Pandeya N, Watson M, Martin NG, Green A, Newman B, Florin TH. Protective role of appendicectomy on onset and severity of ulcerative colitis and Crohn's disease. Gut. 2002 Dec;51(6):808-13. 30. Cosnes J, Carbonnel F, Beaugerie L, Blain A, Reijasse D, Gendre JP. Effects of appendicectomy on the course of ulcerative colitis. Gut. 2002 Dec;51(6):803-7. 31. Rock FL, Hardiman G, Timans JC, Kastelein RA, Bazan JF. A family of human receptors structurally related to Drosophila Toll. Proc Natl Acad Sci U S A. 1998 Jan 20;95(2):588-93. Links 32. Rugtveit J, Nilsen EM, Bakka A, Carlsen H, Brandtzaeg P, Scott H. Cytokine profiles differ in newly recruited and resident subsets of mucosal macrophages from inflammatory bowel disease. Gastroenterology. 1997 May;112(5):1493-505. 33. Wallace JL, Miller MJ. Nitric oxide in mucosal defense: a little goes a long way. Gastroenterology. 2000 Aug;119(2):512-20. 34. Ahrenstedt O, Knutson L, Nilsson B, Nilsson-Ekdahl K, Odlind B, Hällgren R. Enhanced local production of complement components in the small intestines of patients with Crohn's disease. N Engl J Med. 1990 May 10;322(19):1345-9. 35. Carbonare SB, Silva ML, Trabulsi LR, Carneiro-Sampaio MM. Inhibition of HEp-2 cell invasion by enteroinvasive Escherichia coli by human colostrum IgA. Int Arch Allergy Immunol. 1995 Oct;108(2):113-8. 36. Van Kerckhove C, Russell GJ, Deusch K, Reich K, Bhan AK, DerSimonian H, Brenner MB. Oligoclonality of human intestinal intraepithelial T cells. J Exp Med. 1992 Jan 1;175(1):57-63. 37. Ebert EC. Proliferative responses of human intraepithelial lymphocytes to various T-cell stimuli. Gastroenterology. 1989 Dec; 97(6):1372-81. 38. Ebert EC. Human intestinal intraepithelial lymphocytes have potent chemotactic activity. Gastroenterology. 1995 Oct;109(4):1154-9. 39. Fiocchi C, Battisto JR, Farmer RG. Studies on isolated gut mucosal lymphocytes in inflammatory bowel disease. Detection of activated T cells and enhanced proliferation to Staphylococcus aureus and lipopolysaccharides. Dig Dis Sci. 1981 Aug;26(8):728-36. 40. Fuss IJ, Neurath M, Boirivant M, Klein JS, de la Motte C, Strong SA, Fiocchi C, Strober W. Disparate CD4+ lamina propria (LP) lymphokine secretion profiles in inflammatory bowel disease. Crohn's disease LP cells manifest increased secretion of IFN-gamma, whereas ulcerative colitis LP cells manifest increased secretion of IL-5. J Immunol. 1996 Aug 1;157(3):1261-70.


53

41. Hattori M, Taylor TD. The human intestinal microbiome: a new frontier of human biology. DNA Res. 2009 Feb; 16(1):1-12. 42. Saulnier DM, Kolida S, Gibson GR. Microbiology of the human intestinal tract and approaches for its dietary modulation. Curr Pharm Des. 2009; 15(13):1403-14. 43. Hershberg R, Blumberg RS. The lymphocyte-epithelial-bacterial interface. In Targan SR, Shanahan F, Karp LC. Inflammatory Bowel Disease: From Bench to Bedside, 2nd edn. Springer, 2005. 44. Immunological tolerance. In Abbas, AK, Lichtman AH, Pillai S. Cellular and molecular immunology. 6th edn. Elseviers, 2007. 45. Thompson-Chagoyán OC, Maldonado J, Gil A. Aetiology of inflammatory bowel disease (IBD): role of intestinal microbiota and gut-associated lymphoid tissue immune response. Clin Nutr. 2005 Jun; 24(3):339-52. Epub 2005 Apr 9. 46. Guarner F, Malagelada JR. Gut flora in health and disease. Lancet. 2003 Feb 8; 361(9356):512-9. 47. Gordon JI, Hooper LV, McNevin MS, Wong M, Bry L. Epithelial cell growth and differentiation. III. Promoting diversity in the intestine: conversations between the microflora, epithelium, and diffuse GALT. Am J Physiol. 1997 Sep; 273(3 Pt 1):G565-70. 48. Bezirtzoglou E. The intestinal microflora during the first weeks of life. Anaerobe. 1997 Apr-Jun; 3(2-3):173-7. 49. Schwartz RH. T cell anergy. Annu Rev Immunol. 2003; 21:305-34. Epub 2001 Dec 19. 50. Fontenot JD, Rudensky AY. A well adapted regulatory contrivance: regulatory T cell development and the forkhead family transcription factor Foxp3. Nat Immunol. 2005 Apr; 6(4):331-7. 51. Cytokines. In Abbas, AK, Lichtman AH, Pillai S. Cellular and molecular immunology. 6th edn. Elseviers, 2007. 52. Locksley RM, Killeen N, Lenardo MJ. The TNF and TNF receptor superfamilies: integrating mammalian biology. Cell. 2001 Feb 23;104(4):487-501. 53. Platanias LC. Mechanisms of type-I- and type-II-interferon-mediated signalling. Nat Rev Immunol. 2005 May;5(5):375-86. 54. Neuman MG. Immune dysfunction in inflammatory bowel disease. Transl Res. 2007 Apr;149(4):173-86. 55. Papadakis KA. Chemokines in inflammatory bowel disease. Curr Allergy Asthma Rep. 2004 Jan; 4(1):83-9. 56. Malek TR, Bayer AL. Tolerance, not immunity, crucially depends on IL-2. Nat Rev Immunol. 2004 Sep;4(9):665-74. 57. Li MO, Wan YY, Sanjabi S, Roberston A-KL. Flavell RA. Transforming growth factor-β regulation of immune responses Annu Rev Immunol. 2006; 24: 99-146. 58. Kolls JK, Linden A: Interleukin-17 family members and inflammation. Immunity 2004, 21:467-476. 59. Kaplan MH, Sun YL, Hoey T, Grusby MJ. Impaired IL-12 responses and enhanced development of Th2 cells in Stat4-deficient mice. Nature. 1996 Jul 11;382(6587):174-7. 60. Zenewicz LA, Antov A, Flavell RA. CD4 T-cell differentiation and inflammatory bowel disease. Trends Mol Med. 2009 May;15(5):199-207. Epub 2009 Apr 8. 61. Mullen AC, High FA, Hutchins AS, Lee HW, Villarino AV, Livingston DM, Kung AL, Cereb N, Yao TP, Yang SY, Reiner SL. Role of T-bet in commitment of TH1 cells before IL-12-dependent selection. Science.2001. 292, 1907–1910. 62. Shimoda K, van Deursen J, Sangster MY, Sarawar SR, Carson RT, Tripp RA, Chu C, Quelle FW, Nosaka T, Vignali DA, Doherty PC, Grosveld G, Paul WE, Ihle JN. Lack of IL-4-induced Th2 response and IgE class switching in mice with disrupted Stat6 gene. Nature. 1996 Apr 18; 380(6575):630-3. 63. Dardalhon V, Awasthi A, Kwon H, Galileos G, Gao W, Sobel RA, Mitsdoerffer M, Strom TB, Elyaman W, Ho IC, Khoury S, Oukka M, Kuchroo VK. IL-4 inhibits TGF-beta-induced Foxp3+ T cells and, together with TGF-beta, generates IL-9+ IL-10+ Foxp3(-) effector T cells. Nat Immunol. 2008 Dec; 9(12):1347-55. Epub 2008 Nov 9. 64. Veldhoen M, Uyttenhove C, van Snick J, Helmby H, Westendorf A, Buer J, Martin B, Wilhelm C, Stockinger B. Transforming growth factor-beta 'reprograms' the differentiation of T helper 2 cells and promotes an interleukin 9-producing subset. Nat Immunol. 2008 Dec;9(12):1341-6. Epub 2008 Oct 19. 65. Yang XO, Pappu BP, Nurieva R, Akimzhanov A, Kang HS, Chung Y, Ma L, Shah B, Panopoulos AD, Schluns KS, Watowich SS, Tian Q, Jetten AM, Dong C. T helper 17 lineage differentiation is programmed by orphan nuclear receptors ROR alpha and ROR gamma. Immunity. 2008 Jan; 28(1):29-39. Epub 2007 Dec 27. 66. Zheng Y, Rudensky AY. Foxp3 in control of the regulatory T cell lineage. Nat Immunol. 2007 May;8(5):457-62. 67. Wan YY, Flavell RA. 'Yin-Yang' functions of transforming growth factor-beta and T regulatory cells in immune regulation. Immunol Rev. 2007 Dec;220:199-213. 68. Wan, Y.Y., Flavell, R.A. TGF-b and regulatory T cell in immunity and autoimmunity. J. Clin. Immunol. 2008. 28, 647–659

Capítulo 2

54

69. Zhou L, Lopes JE, Chong MM, Ivanov II, Min R, Victora GD, Shen Y, Du J, Rubtsov YP, Rudensky AY, Ziegler SF, Littman DR. TGF-beta-induced Foxp3 inhibits T(H)17 cell differentiation by antagonizing RORgammat function. Nature. 2008 May 8; 453(7192):236-40. Epub 2008 Mar 26. 70. Spehlmann ME, Eckmann L. Nuclear factor-kappa B in intestinal protection and destruction. Curr Opin Gastroenterol. 2009 Mar; 25(2):92-9. 71. Shaulian E, Karin M. AP-1 as a regulator of cell life and death. Nat Cell Biol. 2002 May;4(5):E131-6. 72. Shen G, Jeong WS, Hu R, Kong AN. Regulation of Nrf2, NF-kappaB, and AP-1 signaling pathways by chemopreventive agents. Antioxid Redox Signal. 2005 Nov-Dec; 7(11-12):1648-63. 73. Chang L, Karin M. Mammalian MAP kinase signaling cascades. Nature 410: 37–40, 2001. 74. Whitmarsh AJ, Davis RJ. Transcription factor AP-1 regulation by mitogen-activated protein kinase signal transduction pathways. J Mol Med 74: 589–607, 1996. 75. Im SH, Rao A. Activation and deactivation of gene expression by Ca2+/calcineurin-NFAT-mediated signaling. Mol Cells. 2004 Aug 31; 18(1):1-9. 76. Rao A, Luo C, Hogan PG. Transcription factors of the NFAT family: regulation and function. Annu Rev Immunol. 1997; 15:707-47. 77. Innate immunity. In Abbas, AK, Lichtman AH, Pillai S. Cellular and molecular immunology. 6th edn. Elseviers, 2007. 78. Oppmann B, Lesley R, Blom B, Timans JC, Xu Y, Hunte B, Vega F, Yu N, Wang J, Singh K, Zonin F, Vaisberg E, Churakova T, Liu M, Gorman D, Wagner J, Zurawski S, Liu Y, Abrams JS, Moore KW, Rennick D, de Waal-Malefyt R, Hannum C, Bazan JF, Kastelein RA. Novel p19 protein engages IL-12p40 to form a cytokine, IL-23, with biological activities similar as well as distinct from IL-12. Immunity. 2000 Nov; 13(5):715-25. 79. Aggarwal S, Ghilardi N, Xie MH, de Sauvage FJ, Gurney AL. Interleukin-23 promotes a distinct CD4 T cell activation state characterized by the production of interleukin-17. J Biol Chem. 2003 Jan 17; 278(3):1910-4. Epub 2002 Nov 3. 80. Watford WT, Hissong BD, Bream JH, Kanno Y, Muul L, O'Shea JJ. Signaling by IL-12 and IL-23 and the immunoregulatory roles of STAT4. Immunol Rev. 2004 Dec; 202:139-56. 81. Elewaut D, DiDonato JA, Kim JM, Truong F, Eckmann L, Kagnoff MF. NF-kB Is a Central Regulator of the Intestinal Epithelial Cell Innate Immune Response Induced by Infection with Enteroinvasive Bacteria. J Immunol. 1999 Aug 1;163(3):1457-66. 82. Shen YH, Godlewski J, Zhu J et al. Cross-talk between JNK/SAPK and ERK/MAPK pathways: Sustained activation of JNK blocks ERK activation by mitogenic factors. J Biol Chem 2003; 278: 26715–26721. 83. Szabo SJ, Sullivan BM, Stemmann C, Satoskar AR, Sleckman BP, Glimcher LH.Distinct effects of T-bet in TH1 lineage commitment and IFN-gamma production in CD4 and CD8 T cells. Science. 2002 Jan 11;295(5553):338-42. 84. Zarzuelo A; Gálvez J. Fibra dietética. En Tratado de Nutrición (2005). Edt. Gil A. 85. Rose JD, Roberts GM, Williams G, Mayberry JF, Rhodes J. Cardiff Crohn’s disease jubilee: the incidence over 50 years. Gut 1988; 29: 346-51. 86. Moum B, Vatn MH, Ekbom A, Aadland E, Fausa O, Lygren I, Sauar J, Schulz T, Stray N. Incidence of ulcerative colitis and indeterminate colitis in four countis in south-eastern Norway, 1990-1993. Scand J Gastroenterol 1996; 31: 362-6. 87. Ekbom A. The changing faces of Crohn’s disease and ulcerative colitis. In Targan SR, Shanahan F, Karp LC. Inflammatory Bowel Disease: From Bench to Bedside, 2nd edn. Springer, 2005. 88. Armitage E, Dummond H, Ghosh S, Ferguson A. Incidence of juvenile-onset Crohn’s disease in Stoctland. Lancet 1999, 353: 1496-7. 89. Askling J, Gahnquist L, Ekbom A, Finkel Y. Incidence of pediatric Crohn’s disease in Stockolm, Sweden. . Lancet 1999, 354: 1179. 90. Kull K, Salupere R,. Uibo R, Ots M, Salupere V. Antineutrophil cytoplasmic antibodies in Estonian patients with inflammatory bowel disease. Prevalence and diagnostic role. Hepatograstroenterology 1998; 45: 2132-7. 91. Vucelić B, Korać B, Sentić M, Milicić D, Hadzić N, Juresa V, Bozikov J, Rotkvić I, Buljevac M, Kovacević I, et al. Ulcerative colitis in Zageb, Yugoslavia: incidence and prevalence 1980-1989. Int J Epidemiol 1991; 20: 1043-7. 92. Acheson ED, Nefzger MD. Ulcerative colitis in the United States Army in 1944. Epidemiology: comparisons between patients and controls. Gastroenterology. 1963 Jan; 44:7-19. 93. Sonnenberg A. Disability from inflammatory bowel disease among employees in West Germany. Gut. 1989 Mar;30(3):367-70. 94. Gilat T, Hacohen D, Lilos P, Langman MJ. Childhood factors in ulcerative colitis and Crohn's disease. An international cooperative study. Scand J Gastroenterol. 1987 Oct; 22(8):1009-24. 95. Ekbom A, Adami HO, Helmick CG, Jonzon A, Zack MM. Perinatal risk factors for inflammatory bowel disease: a case-control study. Am J Epidemiol. 1990 Dec; 132(6):1111-9.


55

96. Truelove SC. Ulcerative colitis provoked by milk. Br Med J. 1961 Jan 21;1(5220):154-60. 97. Ghuty E. Morbus Crohn und Nahrungsfette- Hypothese zur Ätiologie der Enteritis regionalis. Dt Med Wochenschr 1982, 107: 71-3. 98. James AH. Breakfast and Crohn's disease. Br Med J. 1977 Apr 9; 1(6066):943-5 99. Persson PG, Ahlbom A, Hellers G. Diet and inflammatory bowel disease: a case-control study. Epidemiology. 1992 Jan; 3(1):47-52. 100. Russel MG, Engels LG, Muris JW, Limonard CB, Volovics A, Brummer RJ, Stockbrügger RW. Modern life' in the epidemiology of inflammatory bowel disease: a case-control study with special emphasis on nutritional factors. Eur J Gastroenterol Hepatol. 1998 Mar;10(3):243-9. 101. Baumgart DC, Carding SR. Inflammatory bowel disease: cause and immunobiology. Lancet. 2007 May 12;369(9573):1627-40. Review 102. Desreumaux P, Brandt E, Gambiez L, Emilie D, Geboes K, Klein O, Ectors N, Cortot A, Capron M, Colombel JF. Distinct cytokine patterns in early and chronic ileal lesions of Crohn's disease. Gastroenterology. 1997 Jul; 113(1):118-26. 103. Calkins BM. A meta-analysis of the role of smoking in inflammatory bowel disease. Dig Dis Sci. 1989 Dec; 34(12):1841-54. 104. Calkins BM, Lilienfeld AM, Garland CF, Mendeloff AI. Trends in incidence rates of ulcerative colitis and Crohn's disease. Dig Dis Sci. 1984 Oct;29(10):913-20. 105. Benoni C, Nilsson A. Smoking habits in patients with inflammatory bowel disease. A case-control study. Scand J Gastroenterol. 1987 Nov;22(9):1130-6. 106. Collins SM, Croitoru K. Pathophysiology of inflammatory bowel disease: the effect of inflammation on intestinal function. In Targan SR, Shanahan F, Karp LC. Inflammatory Bowel Disease: From Bench to Bedside, 2nd edn. Springer, 2005. 107. Radojevic N, McKay DM, Merger M, Vallance BA, Collins SM, Croitoru K. Characterization of enteric functional changes evoked by in vivo anti-CD3 T cell activation. Am J Physiol. 1999 Mar;276(3 Pt 2):R715-23. 108. Evans CM, Phillips AD, Walker-Smith JA, MacDonald TT. Activation of lamina propria T cells induces crypt epithelial proliferation and goblet cell depletion in cultured human fetal colon. Gut. 1992 Feb;33(2):230-5. 109. Merger M, Viney JL, Borojevic R, Steele-Norwood D, Zhou P, Clark DA, Riddell R, Maric R, Podack ER, Croitoru K. Defining the roles of perforin, Fas/FasL, and tumour necrosis factor alpha in T cell induced mucosal damage in the mouse intestine. Gut. 2002 Aug; 51(2):155-63. 110. Fish SM, Proujansky R, Reenstra WW. Synergistic effects of interferon gamma and tumour necrosis factor alpha on T84 cell function. Gut. 1999 Aug;45(2):191-8. 111. Martínez-Augustin O, Romero-Calvo I, Suárez MD, Zarzuelo A, de Medina FS.Molecular bases of impaired water and ion movements in inflammatory bowel diseases. Inflamm Bowel Dis. 2009 Jan;15(1):114-27. Review. 112. MacNaughton WK, Lowe SS, Cushing K. Role of nitric oxide in inflammation-induced suppression of secretion in a mouse model of acute colitis. Am J Physiol. 1998; 275:G1353–1360. 113. Asfaha S, Bell CJ, Wallace JL, MacNaughton WK. Prolonged colonic epithelial hyporesponsiveness after colitis: role of inducible nitric oxide synthase. Am J Physiol. 1999; 276:G703–710. 114. Sánchez de Medina F, Pérez R, Martínez-Augustin O, González R, Lorente MD, Gálvez J, Zarzuelo A. Disturbances of colonic ion secretion in inflammation: role of the enteric nervous system and cAMP. Pflugers Arch. 2002 Jun; 444(3):378-88. Epub 2002 Apr 4. 115. Mayer L, Eisenhardt D, Salomon P, Bauer W, Plous R, Piccinini L. Expression of class II molecules on intestinal epithelial cells in humans. Differences between normal and inflammatory bowel disease. Gastroenterology. 1991 Jan; 100(1):3-12. 116. Mayer L, Eisenhardt D. Lack of induction of suppressor T cells by intestinal epithelial cells from patients with inflammatory bowel disease. J Clin Invest. 1990 Oct;86(4):1255-60. 117. Snape WJ Jr, Matarazzo SA, Cohen S. Abnormal gastrocolonic response in patients with ulcerative colitis. Gut. 1980 May;21(5):392-6. 118. Reddy SN, Bazzocchi G, Chan S, Akashi K, Villanueva-Meyer J, Yanni G, Mena I, Snape WJ Jr. Colonic motility and transit in health and ulcerative colitis. Gastroenterology. 1991 Nov;101(5):1289-97. 119. Tomita R, Tanjoh K, Fujisaki S, Fukuzawa M. Peptidergic nerves in the colon of patients with ulcerative colitis. Hepatogastroenterology. 2000 Mar-Apr;47(32):400-4. 120. Boughton-Smith NK, Evans SM, Hawkey CJ, Cole AT, Balsitis M, Whittle BJ, Moncada S. Nitric oxide synthase activity in ulcerative colitis and Crohn's disease. Lancet. 1993 Aug 7; 342(8867):338-40. 121. Roberts PJ, Morgan K, Miller R, Hunter JO, Middleton SJ. Neuronal COX-2 expression in human myenteric plexus in active inflammatory bowel disease. Gut. 2001 Apr; 48(4):468-72. 122. Annese V, Bassotti G, Napolitano G, Usai P, Andriulli A, Vantrappen G. Gastrointestinal motility disorders in patients with inactive Crohn's disease. Scand J Gastroenterol. 1997 Nov;32(11):1107-17.

Capítulo 2

56

123. Dvorak AM, Onderdonk AB, McLeod RS, Monahan-Earley RA, Cullen J, Antonioli DA, Blair JE, Morgan ES, Cisneros RL, Estrella P, et al. Axonal necrosis of enteric autonomic nerves in continent ileal pouches. Possible implications for pathogenesis of Crohn's disease. Ann Surg. 1993 Mar; 217(3):260-71. 124. Kalff JC, Carlos TM, Schraut WH, Billiar TR, Simmons RL, Bauer AJ. Surgically induced leukocytic infiltrates within the rat intestinal muscularis mediate postoperative ileus. Gastroenterology. 1999 Aug;117(2):378-87. 125. Vermillion DL, Ernst PB, Scicchitano R, Collins SM. Antigen-induced contraction of jejunal smooth muscle in the sensitized rat. Am J Physiol. 1988 Dec;255 (6 Pt 1):G701-8. 126. Vermillion DL, Ernst PB, Collins SM. T-lymphocyte modulation of intestinal muscle function in the Trichinella-infected rat. Gastroenterology. 1991 Jul;101(1):31-8. 127. Khan WI, Vallance BA, Blennerhassett PA, Deng Y, Verdu EF, Matthaei KI, Collins SM. Critical role for signal transducer and activator of transcription factor 6 in mediating intestinal muscle hypercontractility and worm expulsion in Trichinella spiralis-infected mice. Infect Immun. 2001 Feb;69(2):838-44. 128. Muller MJ, Huizinga JD, Collins SM. Altered smooth muscle contraction and sodium pump activity in the inflamed rat intestine. Am J Physiol. 1989 Oct;257(4 Pt 1):G570-7. 129. Blennerhassett MG, Bovell FM, Lourenssen S, McHugh KM. Characteristics of inflammation-induced hypertrophy of rat intestinal smooth muscle cell. Dig Dis Sci. 1999 Jul;44(7):1265-72. 130. Khan I. Molecular basis of altered contractility in experimental colitis: expression of L-type calcium channel. Dig Dis Sci. 1999 Aug;44(8):1525-30. 131.Papadakis KA, Abreu MT.Systemic consequences of intestinal inflammation. In Targan SR, Shanahan F, Karp LC. Inflammatory Bowel Disease: From Bench to Bedside, 2nd edn. Springer, 2005. 132. Nielsen OH, Vainer B, Madsen SM, Seidelin JB, Heegaard NH. Established and emerging biological activity markers of inflammatory bowel disease. Am J Gastroenterol. 2000 Feb; 5(2):359-67. 133. Mahida YR, Ceska M, Effenberger F, Kurlak L, Lindley I, Hawkey CJ. Enhanced synthesis of neutrophil-activating peptide-1/interleukin-8 in active ulcerative colitis. Clin Sci (Lond). 1992 Mar; 82(3):273-5. 134. Kotler DP. Cachexia. Ann Intern Med. 2000 Oct 17; 133(8):622-34. 135. Sarraf P, Frederich RC, Turner EM, Ma G, Jaskowiak NT, Rivet DJ 3rd, Flier JS, Lowell BB, Fraker DL, Alexander HR. Multiple cytokines and acute inflammation raise mouse leptin levels: potential role in inflammatory anorexia. J Exp Med. 1997 Jan 6;185(1):171-5. 136. Plata-Salamán CR. Food intake suppression by growth factors and platelet peptides by direct action in the central nervous system. Neurosci Lett. 1988 Nov 22; 94(1-2):161-6. 137. Both H, Torp-Pedersen K, Kreiner S, Hendriksen C, Binder V. Clinical appearance at diagnosis of ulcerative colitis and Crohn's disease in a regional patient group. Scand J Gastroenterol. 1983 Oct;18(7):987-91. 138. Horina JH, Petritsch W, Schmid CR, Reicht G, Wenzl H, Silly H, Krejs GJ. Treatment of anemia in inflammatory bowel disease with recombinant human erythropoietin: results in three patients. Gastroenterology. 1993 Jun;104(6):1828-31. 139. Gasché C, Reinisch W, Lochs H, Parsaei B, Bakos S, Wyatt J, Fueger GF, Gangl A. Anemia in Crohn's disease. Importance of inadequate erythropoietin production and iron deficiency. Dig Dis Sci. 1994 Sep;39(9):1930-4. 140. Beeken WL. Remediable defects in Crohn disease: a prospective study of 63 patients. Arch Intern Med. 1975 May;135(5):686-90. 141. Gasché C, Dejaco C, Waldhoer T, Tillinger W, Reinisch W, Fueger GF, Gangl A, Lochs H. Intravenous iron and erythropoietin for anemia associated with Crohn disease. A randomized, controlled trial. Ann Intern Med. 1997 May 15; 126(10):782-7. 142. Schapira M, Henrion J, Ravoet C, Maisin JM, Ghilain JM, De Maeght S, Heller F. Thromboembolism in inflammatory bowel disease. Acta Gastroenterol Belg. 1999 Apr-Jun;62(2):182-6. 143. Jackson LM, O'Gorman PJ, O'Connell J, Cronin CC, Cotter KP, Shanahan F. Thrombosis in inflammatory bowel disease: clinical setting, procoagulant profile and factor V Leiden. Q J Med. 1997 Mar;90(3):183-8. 144. Heits F, Stahl M, Ludwig D, Stange EF, Jelkmann W. Elevated serum thrombopoietin and interleukin-6 concentrations in thrombocytosis associated with inflammatory bowel disease. J Interferon Cytokine Res. 1999 Jul; 19(7):757-60. 145. Greenstein AJ, Sachar DB, Panday AK, Dikman SH, Meyers S, Heimann T, Gumaste V, Werther JL, Janowitz HD. Amyloidosis and inflammatory bowel disease. A 50-year experience with 25 patients. Medicine (Baltimore). 1992 Sep; 71(5):261-70. 146. Lovat LB, Madhoo S, Pepys MB, Hawkins PN. Long-term survival in systemic amyloid A amyloidosis complicating Crohn's disease. Gastroenterology. 1997 Apr;112(4):1362-5.


57

147. Cronin CC, Shanahan F. Understanding symptoms and signs in inflammatory bowel disease. In Targan SR, Shanahan F, Karp LC. Inflammatory Bowel Disease: From Bench to Bedside, 2nd edn. Springer, 2005. 148. Bueno L, Fioramonti J, Delvaux M, Frexinos J. Mediators and pharmacology of visceral sensitivity: from basic to clinical investigations. Gastroenterology. 1997 May;112(5):1714-43 149. Bielefeldt K, Davis B, Binion DG. Pain and inflammatory bowel disease. Inflamm Bowel Dis. 2009 May; 15(5):778-88. 150. Cross RK, Wilson KT, Binion DG. Narcotic use in patients with Crohn's disease. Am J Gastroenterol. 2005 Oct;100(10):2225-9. 151. Strigo IA, Bushnell MC, Boivin M, Duncan GH. Psychophysical analysis of visceral and cutaneous pain in human subjects. Pain. 2002 Jun;97(3):235-46. 152. Matuk R, Crawford J, Abreu MT, Targan SR, Vasiliauskas EA, Papadakis KA. The spectrum of gastrointestinal toxicity and effect on disease activity of selective cyclooxygenase-2 inhibitors in patients with inflammatory bowel disease. Inflamm Bowel Dis. 2004 Jul; 10(4):352-6. 153. Takeuchi K, Smale S, Premchand P, Maiden L, Sherwood R, Thjodleifsson B, Bjornsson E, Bjarnason I. Prevalence and mechanism of nonsteroidal anti-inflammatory drug-induced clinical relapse in patients with inflammatory bowel disease. Clin Gastroenterol Hepatol. 2006 Feb; 4(2):196-202. 154. Wynn G, Ma B, Ruan HZ, Burnstock G. Purinergic component of mechanosensory transduction is increased in a rat model of colitis. Am J Physiol Gastrointest Liver Physiol. 2004 Sep;287(3):G647-57. 155. Linden DR, Chen JX, Gershon MD, Sharkey KA, Mawe GM. Serotonin availability is increased in mucosa of guinea pigs with TNBS-induced colitis. Am J Physiol Gastrointest Liver Physiol. 2003 Jul;285(1):G207-16. Epub 2003 Mar 19. 156. O'Hara JR, Ho W, Linden DR, Mawe GM, Sharkey KA. Enteroendocrine cells and 5-HT availability are altered in mucosa of guinea pigs with TNBS ileitis. Am J Physiol Gastrointest Liver Physiol. 2004 Nov;287(5):G998-1007. Epub 2004 Jul 1. 157. Miranda A, Peles S, Rudolph C, Shaker R, Sengupta JN. Altered visceral sensation in response to somatic pain in the rat. Gastroenterology. 2004 Apr;126(4):1082-9. 158. O’Keefe SJD, Rosser BG. Nutrition and inflammatory bowel disease. In Targan SR, Shanahan F, Karp LC. Inflammatory Bowel Disease: From Bench to Bedside. Baltimore: Williams & Wilkins, 1994, 461-77. 159. Yu L, Wang L, Chen S. Toll-like receptors, inflammation and tumor in the human female reproductive tract. Am J Reprod Immunol. 2009 Jul;62(1):1-8. 160. Uematsu S, Akira S. Toll-Like receptors (TLRs) and their ligands. Handb Exp Pharmacol. 2008;(183):1-20. In Bauer S, Hartmann G (eds). Toll-Like Receptors (TLRs) and Innate Immunity. Springer Berlin Heidelberg 161. Cario E. Therapeutic impact of toll-like receptors on inflammatory bowel diseases: a multiple-edged sword. Inflamm Bowel Dis. 2008 Mar;14(3):411-21. 162. Stenson WF. Toll-like receptors and intestinal epithelial repair. Curr Opin Gastroenterol. 2008 Mar; 24(2):103-7. 163. Takeuchi O, Kawai T, Sanjo H, Copeland NG, Gilbert DJ, Jenkins NA, Takeda K, Akira S.TLR6: A novel member of an expanding toll-like receptor family. Gene. 1999 Apr 29; 231(1-2):59-65. 164. Shimazu R, Akashi S, Ogata H, Nagai Y, Fukudome K, Miyake K, Kimoto M. MD-2, a molecule that confers lipopolysaccharide responsiveness on Toll-like receptor 4. J Exp Med. 1999 Jun 7;189(11):1777-82. Links 165. Hayashi F, Smith KD, Ozinsky A, Hawn TR, Yi EC, Goodlett DR, Eng JK, Akira S, Underhill DM, Aderem A. The innate immune response to bacterial flagellin is mediated by Toll-like receptor 5. Nature. 2001 Apr 26; 410(6832):1099-103. 166. Alexopoulou L, Holt AC, Medzhitov R, Flavell RA. Recognition of double-stranded RNA and activation of NF-kappaB by Toll-like receptor 3. Nature. 2001 Oct 18; 413(6857):732-8. 167. Diebold SS, Kaisho T, Hemmi H, Akira S, Reis e Sousa C. Innate antiviral responses by means of TLR7-mediated recognition of single-stranded RNA. Science. 2004 Mar 5;303(5663):1529-31. Epub 2004 Feb 19. 168. Heil F, Hemmi H, Hochrein H, Ampenberger F, Kirschning C, Akira S, Lipford G, Wagner H, Bauer S. Species-specific recognition of single-stranded RNA via toll-like receptor 7 and 8. Science. 2004 Mar 5;303(5663):1526-9. Epub 2004 Feb 19. 169. Hemmi H, Takeuchi O, Kawai T, Kaisho T, Sato S, Sanjo H, Matsumoto M, Hoshino K, Wagner H, Takeda K, Akira S. A Toll-like receptor recognizes bacterial DNA. Nature. 2000 Dec 7; 408(6813):740-5. 170. Lee J, Mo JH, Katakura K, Alkalay I, Rucker AN, Liu YT, Lee HK, Shen C, Cojocaru G, Shenouda S, Kagnoff M, Eckmann L, Ben-Neriah Y, Raz E. Maintenance of colonic homeostasis by distinctive apical TLR9 signalling in intestinal epithelial cells. Nat Cell Biol. 2006 Dec; 8(12):1327-36. Epub 2006 Nov 26.

Capítulo 2

58

171. Gewirtz AT, Navas TA, Lyons S, Godowski PJ, Madara JL. Cutting edge: bacterial flagellin activates basolaterally expressed TLR5 to induce epithelial proinflammatory gene expression. J Immunol. 2001 Aug 15;167(4):1882-5. 172. Cario E, Brown D, McKee M, Lynch-Devaney K, Gerken G, Podolsky DK. Commensal-associated molecular patterns induce selective toll-like receptor-trafficking from apical membrane to cytoplasmic compartments in polarized intestinal epithelium. Am J Pathol. 2002 Jan;160(1):165-73. 173. Cario E, Gerken G, Podolsky DK. Toll-like receptor 2 controls mucosal inflammation by regulating epithelial barrier function. Gastroenterology. 2007 Apr;132(4):1359-74. Epub 2007 Feb 25. 174. De Jager PL, Franchimont D, Waliszewska A, Bitton A, Cohen A, Langelier D, Belaiche J, Vermeire S, Farwell L, Goris A, Libioulle C, Jani N, Dassopoulos T, Bromfield GP, Dubois B, Cho JH, Brant SR, Duerr RH, Yang H, Rotter JI, Silverberg MS, Steinhart AH, Daly MJ, Podolsky DK, Louis E, Hafler DA, Rioux JD; Quebec IBD Genetics Consortium; NIDDK IBD Genetics Consortium. The role of the Toll receptor pathway in susceptibility to inflammatory bowel diseases. Genes Immun. 2007 Jul;8(5):387-97. Epub 2007 May 31. 175. Pierik M, Joossens S, Van Steen K, Van Schuerbeek N, Vlietinck R, Rutgeerts P, Vermeire S. Toll-like receptor-1, -2, and -6 polymorphisms influence disease extension in inflammatory bowel diseases. Inflamm Bowel Dis. 2006 Jan; 12(1):1-8. 176. Kabelitz D. Expression and function of Toll-like receptors in T lymphocytes. Curr Opin Immunol. 2007 Feb;19(1):39-45. Epub 2006 Nov 28. 177. Himmel ME, Hardenberg G, Piccirillo CA, Steiner TS, Levings MK. The role of T-regulatory cells and Toll-like receptors in the pathogenesis of human inflammatory bowel disease. Immunology. 2008 Oct;125(2):145-53. 178. van Maren WW, Jacobs JF, de Vries IJ, Nierkens S, Adema GJ. Toll-like receptor signalling on Tregs: to suppress or not to suppress?Immunology. 2008 Aug; 124(4):445-52. Epub 2008 Jun 28. 179. Taylor KD, Rotter JI, Yang H.Genetics of inflammatory bowel disease. In Targan SR, Shanahan F, Karp LC. Inflammatory Bowel Disease: From Bench to Bedside, 2nd edn. Springer, 2005. 180. Barrett JC, Hansoul S, Nicolae DL, et al. Genome-wide association defines more than 30 distinct susceptibility loci for Crohn’s disease. Nat Genet 2008 Aug; 40(8):955–62. 181. Bayless TM, Tokayer AZ, Polito JM 2nd, Quaskey SA, Mellits ED, Harris ML. Crohn’s disease: concordance for site and clinical type in affected family members–potential hereditary influences. Gastroenterology 1996 Sep; 111(3):573–9. 182. Monsen U, Bernell O, Johansson C, Hellers G. Prevalence of inflammatory bowel disease among relatives of patients withCrohn’s disease. Scand J Gastroenterol 1991 Mar; 26(3):302–6. 183. Pena AS. Genetics of inflammatory bowel disease. The candidate gene approach: susceptibility versus disease heterogeneity. Dig Dis 1998 Nov; 16(6):356–63. 184. Orholm M, Munkholm P, Langholz E, Nielsen OH, Sørensen TI, Binder V. Familial occurrence of inflammatory bowel disease. N Engl J Med 1991 Jan 10;324(2):84–8. 185. Noomen CG, Hommes DW, Fidder HH. Update on genetics in inflammatory disease. Best Pract Res Clin Gastroenterol. 2009; 23(2):233-43. 186. Tysk C, Lindberg E, Jarnerot G, Flodérus-Myrhed B. Ulcerative colitis and Crohn’s disease in an unselected population of monozygotic and dizygotic twins. A study of heritability and the influence of smoking. Gut 1988 Jul; 29(7):990–6. 187. Binder V. Genetic epidemiology in inflammatory bowel disease. Dig Dis 1998 Nov; 16(6):351–5. 188. Hugot JP, Chamaillard M, Zouali H, Lesage S, Cézard JP, Belaiche J, Almer S, Tysk C, O'Morain CA, Gassull M, Binder V, Finkel Y, Cortot A, Modigliani R, Laurent-Puig P, Gower-Rousseau C, Macry J, Colombel JF, Sahbatou M, Thomas G. Association of NOD2 leucine-rich repeat variants with susceptibility to Crohn’s disease. Nature 2001 May 31;411(6837):599–603. 189. Ogura Y, Bonen DK, Inohara N, Nicolae DL, Chen FF, Ramos R, Britton H, Moran T, Karaliuskas R, Duerr RH, Achkar JP, Brant SR, Bayless TM, Kirschner BS, Hanauer SB, Nuñez G, Cho JH. A frameshift mutation in NOD2 associated with susceptibility to Crohn’s disease. Nature 2001 May 31;411(6837):603–6. 189. Girardin SE, Boneca IG, Viala J, Chamaillard M, Labigne A, Thomas G, Philpott DJ, Sansonetti PJ. Nod2 is a general sensor of peptidoglycan through muramyl dipeptide (MDP) detection. J Biol Chem 2003 Mar 14; 278(11):8869–72. 191. Wehkamp J, Harder J, Weichenthal M, Schwab M, Schäffeler E, Schlee M, Herrlinger KR, Stallmach A, Noack F, Fritz P, Schröder JM, Bevins CL, Fellermann K, Stange EF. NOD2 (CARD15) mutations in Crohn’s disease are associated with diminished mucosal alpha-defensin expression. Gut 2004 Nov; 53(11):1658–64. 192. Schreiber S, Nikolaus S, Hampe J. Activation of nuclear factor kappa B inflammatory bowel disease. Gut 1998 Apr; 42(4):477–84.


59

193. Nenci A, Becker C, Wullaert A, Gareus R, van Loo G, Danese S, Huth M, Nikolaev A, Neufert C, Madison B, Gumucio D, Neurath MF, Pasparakis M. Epithelial NEMO links innate immunity to chronic intestinal inflammation. Nature. 2007 Mar 29;446(7135):557-61. Epub 2007 Mar 14. 195. Franchimont D, Vermeire S, El Housni H, Pierik M, Van Steen K, Gustot T, Quertinmont E, Abramowicz M, Van Gossum A, Devière J, Rutgeerts P. Deficient host-bacteria interactions in inflammatory bowel disease? The toll-like receptor (TLR)-4 Asp299gly polymorphism is associated with Crohn’s disease and ulcerative colitis. Gut 2004 Jul; 53(7):987–92. 195. Rioux JD, Daly MJ, Silverberg MS, Lindblad K, Steinhart H, Cohen Z, Delmonte T, Kocher K, Miller K, Guschwan S, Kulbokas EJ, O'Leary S, Winchester E, Dewar K, Green T, Stone V, Chow C, Cohen A, Langelier D, Lapointe G, Gaudet D, Faith J, Branco N, Bull SB, McLeod RS, Griffiths AM, Bitton A, Greenberg GR, Lander ES, Siminovitch KA, Hudson TJ. Genetic variation in the 5q31 cytokine gene cluster confers susceptibility to Crohn disease. Nat Genet 2001 Oct; 29(2):223–8. 196. Peltekova VD, Wintle RF, Rubin LA, Amos CI, Huang Q, Gu X, Newman B, Van Oene M, Cescon D, Greenberg G, Griffiths AM, St George-Hyslop PH, Siminovitch KA. Functional variants of OCTN cation transporter genes are associated with Crohn disease. Nat Genet 2004 May; 36(5):471–5. 197. Vermeire S, Pierik M, Hlavaty T, Claessens G, van Schuerbeeck N, Joossens S, Ferrante M, Henckaerts L, Bueno de Mesquita M, Vlietinck R, Rutgeerts P. Association of organic cation transporter risk haplotype with perianal penetrating Crohn’s disease but not with susceptibility to IBD. Gastroenterology 2005 Dec;129(6):1845–53. 198. Armuzzi A, Ahmad T, Ling KL, de Silva A, Cullen S, van Heel D, Orchard TR, Welsh KI, Marshall SE, Jewell DP. Genotype-phenotype analysis of the Crohn’s disease susceptibility haplotype on chromosome 5q31. Gut 2003 Aug;52(8):1133–9. 199. Noble CL, Nimmo ER, Drummond H, Ho GT, Tenesa A, Smith L, Anderson N, Arnott ID, Satsangi J. The contribution of OCTN1/2 variants within the IBD5 locus to disease susceptibility and severity in Crohn’s disease. Gastroenterology 2005 Dec; 129(6):1854–64. 200. Newman B, Wintle RF, van Oene M, Yazdanpanah M, Owen J, Johnson B, Gu X, Amos CI, Keystone E, Rubin LA, Siminovitch KA. SLC22A4 polymorphisms implicated in rheumatoid arthritis and Crohn’s disease are not associated with rheumatoid arthritis in a Canadian Caucasian population. Arthritis Rheum 2005 Feb; 52(2): 425–9. 201. Annese V, Valvano MR, Palmieri O, Latiano A, Bossa F, Andriulli A. Multidrug resistance 1 gene in inflammatory bowel disease: a meta-analysis. World J Gastroenterol 2006 Jun 21;12(23):3636–44. 202. Onnie CM, Fisher SA, Pattni R, Sanderson J, Forbes A, Lewis CM, Mathew CG Associations of allelic variants of the multidrug resistance gene (ABCB1 or MDR1) and inflammatory bowel disease and their effects on disease behavior: a case-control and meta-analysis study. Inflamm Bowel Dis 2006 Apr;12(4):263–71. 203. Duerr RH, Taylor KD, Brant SR, Rioux JD, Silverberg MS, Daly MJ, Steinhart AH, Abraham C, Regueiro M, Griffiths A, Dassopoulos T, Bitton A, Yang H, Targan S, Datta LW, Kistner EO, Schumm LP, Lee AT, Gregersen PK, Barmada MM, Rotter JI, Nicolae DL, Cho JH. A genome-wide association study identifies IL23R as an inflammatory bowel isease gene. Science 2006 Dec 1; 314(5804):1461–3. 204. Hampe J, Franke A, Rosenstiel P, Till A, Teuber M, Huse K, Albrecht M, Mayr G, De La Vega FM, Briggs J, Günther S, Prescott NJ, Onnie CM, Häsler R, Sipos B, Fölsch UR, Lengauer T, Platzer M, Mathew CG, Krawczak M, Schreiber S. A genome-wide association scan of nonsynonymous SNPs identifies a susceptibility variant for Crohn disease in ATG16L1. Nat Genet 2007 Feb; 39(2):207–11. 205. Hershberg R, Blumberg RS. The lymphocyte-epithelial-bacterial interface. In Targan SR, Shanahan F, Karp LC. Inflammatory Bowel Disease: From Bench to Bedside, 2nd edn. Springer, 2005. 206. Sutherland L, Singleton J, Sessions J, Hanauer S, Krawitt E, Rankin G, Summers R, Mekhjian H, Greenberger N, Kelly M, Levine J, Thomson A, Alpert E, Prokipchuk E. Double blind, placebo controlled trial of metronidazole in Crohn's disease. Gut 1991 Sep; 32(9):1071-5. 207. Shanahan F. Probiotics and inflammatory bowel disease: is there a scientific rationale? Inflamm Bowel Dis. 2000 May;6(2):107-15. 208. Thompson-Chagoyán OC, Maldonado J, Gil A. Aetiology of inflammatory bowel disease (IBD): role of intestinal microbiota and gut-associated lymphoid tissue immune response. Clin Nutr. 2005 Jun; 24(3):339-52. Epub 2005 Apr 9. 209. Farrell RJ, LaMont JT. Microbial factors in inflammatory bowel disease. Gastroenterol Clin North Am. 2002 Mar; 31(1):41-62. 210. Casellas F, Borruel N, Papo M, Guarner F, Antolín M, Videla S, Malagelada JR. Antiinflammatory effects of enterically coated amoxicillin-clavulanic acid in active ulcerative colitis. Inflamm Bowel Dis. 1998 Feb; 4(1):1-5. 211. Swidsinski A, Ladhoff A, Pernthaler A, Swidsinski S, Loening-Baucke V, Ortner M, Weber J, Hoffmann U, Schreiber S, Dietel M, Lochs H. Mucosal flora in inflammatory bowel disease. Gastroenterology. 2002 Jan; 122(1):44-54.

Capítulo 2

60

212. Korzenik JR, Podolsky DK. Evolving knowledge and therapy of inflammatory bowel disease. Nat Rev Drug Discov. 2006 Mar; 5(3):197-209. 213. Zenewicz LA, Antov A, Flavell RA. CD4 T-cell differentiation and inflammatory bowel disease. Trends Mol Med. 2009 May;15(5):199-207. Epub 2009 Apr 8. 214.Yamamoto-Furusho JK, Korzenik JR. Crohn's disease: innate immunodeficiency? World J Gastroenterol. 2006 Nov 14; 12(42):6751-5. 215. Zhou L, Braat H, Faber KN, Dijkstra G, Peppelenbosch MP. Monocytes and their pathophysiological role in Crohn's disease. Cell Mol Life Sci. 2009 Jan; 66(2):192-202. 216. Marks DJ, Harbord MW, MacAllister R, Rahman FZ, Young J, Al-Lazikani B, Lees W, Novelli M, Bloom S, Segal AW. Defective acute inflammation in Crohn's disease: a clinical investigation. Lancet. 2006 Feb 25; 367(9511):668-78. 217. Korzenik JR, Dieckgraefe BK, Valentine JF, Hausman DF, Gilbert MJ; Sargramostim in Crohn's Disease Study Group. Sargramostim for active Crohn's disease. N Engl J Med. 2005 May 26; 352(21):2193-201. 218. Netea MG, Ferwerda G, de Jong DJ, Jansen T, Jacobs L, Kramer M, Naber TH, Drenth JP, Girardin SE, Kullberg BJ, Adema GJ, Van der Meer JW. Nucleotide-binding oligomerization domain-2 modulates specific TLR pathways for the induction of cytokine release. J Immunol. 2005 May 15; 174(10):6518-23. 219. ominelli F, Arseneau KO, Pizarro TT. The mucosal inflammatory response. Cytokines and chemokines. In Targan SR, Shanahan F, Karp LC. Inflammatory Bowel Disease: From Bench to Bedside, 2nd edn. Springer, 2005. 220. Neuman MG. Immune dysfunction in inflammatory bowel disease. Transl Res. 2007 Apr; 149(4):173-86. 221. Nielsen OH, Kirman I, Rudiger N, Hendel J, Vainer B. Upregulation of interleukin-12 and -17 in active inflammatory bowel disease. Scand J Gastroenterol 2003; 38: 180-185. 222. Kohno K, Kataoka J, Ohtsuki T, Suemoto Y, Okamoto I, Usui M, Ikeda M, Kurimoto M. IFN-gamma-inducing factor (IGIF) is a costimulatory factor on the activation of Th1 but not Th2 cells and exerts its effect independently of IL-12. J Immunol. 1997 Feb 15;158(4):1541-50. 223. Leach ST, Messina I, Lemberg DA, Novick D, Rubenstein M, Day AS. Local and systemic interleukin-18 and interleukin-18-binding protein in children with inflammatory bowel disease. Inflamm Bowel Dis 2008; 14: 68-74. 224. Pizarro TT, Michie MH, Bentz M, Woraratanadharm J, Smith MF Jr, Foley E, Moskaluk CA, Bickston SJ, Cominelli F. IL-18, a novel immunoregulatory cytokine, is up-regulated in Crohn's disease: expression and localization in intestinal mucosal cells. J Immunol. 1999 Jun 1; 162(11):6829-35. 225. Youngman KR, Simon PL, West GA, Cominelli F, Rachmilewitz D, Klein JS, Fiocchi C. Localization of intestinal interleukin 1 activity and protein and gene expression to lamina propria cells. Gastroenterology. 1993 Mar; 104(3):749-58. 226. Reinecker HC, Steffen M, Witthoeft T, Pflueger I, Schreiber S, MacDermott RP, Raedler A. Enhanced secretion of tumour necrosis factor-alpha, IL-6, and IL-1 beta by isolated lamina propria mononuclear cells from patients with ulcerative colitis and Crohn's disease. Clin Exp Immunol. 1993 Oct; 94(1):174-81. 227. Kallen KJ. The role of transsignalling via the agonistic soluble IL-6 receptor in human diseases. Biochim Biophys Acta. 2002 Nov 11; 1592(3):323-43. 228. Atreya R, Mudter J, Finotto S, Müllberg J, Jostock T, Wirtz S, Schütz M, Bartsch B, Holtmann M, Becker C, Strand D, Czaja J, Schlaak JF, Lehr HA, Autschbach F, Schürmann G, Nishimoto N, Yoshizaki K, Ito H, Kishimoto T, Galle PR, Rose-John S, Neurath MF. Blockade of interleukin 6 trans signaling suppresses T-cell resistance against apoptosis in chronic intestinal inflammation: evidence in crohn disease and experimental colitis in vivo. Nat Med. 2000 May; 6(5):583-8. 229. Kühn R, Löhler J, Rennick D, Rajewsky K, Müller W. Interleukin-10-deficient mice develop chronic enterocolitis. Cell. 1993 Oct 22; 75(2):263-74. 230. Autschbach F, Braunstein J, Helmke B, Zuna I, Schürmann G, Niemir ZI, Wallich R, Otto HF, Meuer SC. In situ expression of interleukin-10 in noninflamed human gut and in inflammatory bowel disease. Am J Pathol. 1998 Jul; 153(1):121-30. 231. Shih DQ, Targan SR. Immunopathogenesis of inflammatory bowel disease. World J Gastroenterol. 2008 Jan 21;14(3):390-400. 232. Migone TS, Zhang J, Luo X, Zhuang L, Chen C, Hu B, Hong JS, Perry JW, Chen SF, Zhou JX, Cho YH, Ullrich S, Kanakaraj P, Carrell J, Boyd E, Olsen HS, Hu G, Pukac L, Liu D, Ni J, Kim S, Gentz R, Feng P, Moore PA, Ruben SM, Wei P. TL1A is a TNF-like ligand for DR3 and TR6/DcR3 and functions as a T cell costimulator. Immunity 2002; 16: 479-492. 233. Wen L, Zhuang L, Luo X, Wei P. TL1A-induced NF-kappaB activation and c-IAP2 production prevent DR3-mediated apoptosis in TF-1 cells. J Biol Chem 2003; 278: 39251-39258. 234. Yamazaki K, McGovern D, Ragoussis J, Paolucci M, Butler H, Jewell D, Cardon L, Takazoe M, Tanaka T, Ichimori T, Saito S, Sekine A, Iida A, Takahashi A, Tsunoda T, Lathrop M, Nakamura Y.


61

Single nucleotide polymorphisms in TNFSF15 confer susceptibility to Crohn's disease. Hum Mol Genet 2005; 14: 3499-3506. 235. Fujino S, Andoh A, Bamba S, Ogawa A, Hata K, Araki Y, Bamba T, Fujiyama Y. Increased expression of interleukin 17 in inflammatory bowel disease. Gut 2003; 52: 65-70. 236. Schmidt C, Giese T, Ludwig B, Mueller-Molaian I, Marth T, Zeuzem S, Meuer SC, Stallmach A. Expression of interleukin-12-related cytokine transcripts in inflammatory bowel disease: elevated interleukin-23p19 and interleukin-27p28 in Crohn's disease but not in ulcerative colitis. Inflamm Bowel Dis 2005; 11: 16-23. 237. Fuss IJ, Heller F, Boirivant M, Leon F, Yoshida M, Fichtner-Feigl S, Yang Z, Exley M, Kitani A, Blumberg RS, Mannon P, Strober W. Nonclassical CD1d-restricted NK T cells that produce IL-13 characterize an atypical Th2 response in ulcerative colitis. J Clin Invest 2004; 113: 1490-1497. 238. Omata F, Birkenbach M, Matsuzaki S, Christ AD, Blumberg RS. The expression of IL-12 p40 and its homologue, Epstein-Barr virus-induced gene 3, in inflammatory bowel disease. Inflamm Bowel Dis 2001; 7: 215-220. 239. Daddaoua A, Puerta V, Zarzuelo A, Suárez MD, Sánchez de Medina F, Martínez-Augustin O. Bovine glycomacropeptide is anti-inflammatory in rats with hapten-induced colitis. J Nutr. 2005 May;135(5): 1164-70. 240. Panaccione R, Sutherland LR. Clinical course and complications of ulcerative colitis and ulcerative proctitis. In Targan SR, Shanahan F, Karp LC. Inflammatory Bowel Disease: From Bench to Bedside, 2nd edn. Springer, 2005. 241. Bouma G, Strober W. The immunological and genetic basis of inflammatory bowel disease. Nat Rev Immunol. 2003 Jul;3(7):521-33. 242. Langhans W, Hrupka B. Interleukins and tumor necrosis factor as inhibitors of food intake. Neuropeptides. 1999 Oct;33(5):415-24. 243. O’Keefe SJD, Rosser BG. Nutrition and inflammatory bowel disease. Clinical course and complications of ulcerative colitis and ulcerative proctitis. In Targan SR, Shanahan F, Karp LC. Inflammatory Bowel Disease: From Bench to Bedside. Baltimore: Williams & Wilkins. 1994, 461-77. 244. Boden, E.K., Snapper, S.B. Regulatory T cells in inflammatory bowel disease. 2008 Curr. Opin. Gastroenterol. 24, 733–741. 245. Functional CD4+ CD25high regulatory T cells are enriched in the colonic mucosa of patients with active ulcerative colitis and increase with disease activity. Holmen N, Lundgren A, Lundin S, Bergin AM, Rudin A, Sjovall H, Ohman L. Inflamm Bowel Dis 2006; 12:447–56. 246. Brandtzaeg P, Carlsen HS, Halstensen TS. The B-cell system in inflammatory bowel disease. Adv Exp Med Biol. 2006;579:149-67. 247. Jurjus AR, Khoury NN, Reimund JM. Animal models of inflammatory bowel disease. J Pharmacol Toxicol Methods. 2004 Sep-Oct; 50(2):81-92. 248. Elson CO, Weaver CT. Experimental mouse models of inflammatory bowel disease: new insights into pathogenic mechanisms. In Targan SR, Shanahan F, Karp LC. Inflammatory Bowel Disease: From Bench to Bedside, 2nd edn. Springer, 2005. 249. MacPherson BR, Pfeiffer CJ. Experimental production of diffuse colitis in rats. Digestion. 1978; 17(2):135-50. 250. Satoh H, Sato F, Takami K, Szabo S. New ulcerative colitis model induced by sulfhydryl blockers in rats and the effects of antiinflammatory drugs on the colitis. Jpn J Pharmacol. 1997 Apr; 73(4):299-309. 251. Boirivant M, Fuss IJ, Chu A, Strober W. Oxazolone colitis: A murine model of T helper cell type 2 colitis treatable with antibodies to interleukin 4. J Exp Med. 1998 Nov 16; 188(10):1929-39. 252. Sartor RB, Bond TM, Schwab JH. Systemic uptake and intestinal inflammatory effects of luminal bacterial cell wall polymers in rats with acute colonic injury. Infect Immun. 1988 Aug; 56(8):2101-8. 253. Okayasu I, Hatakeyama S, Yamada M, Ohkusa T, Inagaki Y, Nakaya R. A novel method in the induction of reliable experimental acute and chronic ulcerative colitis in mice. Gastroenterology. 1990 Mar; 98(3):694-702. 254. Dieleman LA, Ridwan BU, Tennyson GS, Beagley KW, Bucy RP, Elson CO. Dextran sulfate sodium-induced colitis occurs in severe combined immunodeficient mice. Gastroenterology. 1994 Dec; 107(6):1643-52. 255. Morris GP, Beck PL, Herridge MS, Depew WT, Szewczuk MR, Wallace JL. Hapten-induced model of chronic inflammation and ulceration in the rat colon. Gastroenterology. 1989 Mar; 96(3):795-803. 256. Neurath MF, Fuss I, Kelsall BL, Stüber E, Strober W. Antibodies to interleukin 12 abrogate established experimental colitis in mice. J Exp Med. 1995 Nov 1; 182(5):1281-90. 257. Elson CO, Beagley KW, Sharmanov AT, Fujihashi K, Kiyono H, Tennyson GS, Cong Y, Black CA, Ridwan BW, McGhee JR. Hapten-induced model of murine inflammatory bowel disease: mucosa immune responses and protection by tolerance. J Immunol. 1996 Sep 1; 157(5):2174-85. 258. Kelsall BL, Stüber E, Neurath M, Strober W. Interleukin-12 production by dendritic cells. The role of CD40-CD40L interactions in Th1 T-cell responses. Ann N Y Acad Sci. 1996 Oct 31; 795:116-26.

Capítulo 2

62

259. Duchmann R, Schmitt E, Knolle P, Meyer zum Büschenfelde KH, Neurath M. Tolerance towards resident intestinal flora in mice is abrogated in experimental colitis and restored by treatment with interleukin-10 or antibodies to interleukin-12. Eur J Immunol. 1996 Apr; 26(4):934-8. 260. Neurath MF, Pettersson S, Meyer zum Büschenfelde KH, Strober W. Local administration of antisense phosphorothioate oligonucleotides to the p65 subunit of NF-kappa B abrogates established experimental colitis in mice. Nat Med. 1996 Sep; 2(9):998-1004. 261. Baumgart DC. The diagnosis and treatment of Crohn's disease and ulcerative colitis. Dtsch Arztebl Int. 2009 Feb; 106(8):123-33. 262. Hanauer SB, Dassopoulos T. Evolving treatment strategies for inflammatory bowel disease. Annu Rev Med. 2001; 52:299-318. 263. Drugs for gastrointestinal tract disorders. In Brenner GM, Stevens CW. Pharmacology 3rd edn. Saunders (Elseviers) 2010. 264. Greenfield SM, Punchard NA, Teare JP, Thompson RP. Review article: the mode of action of the aminosalicylates in inflammatory bowel disease. Aliment. Pharmacol. Ther. 1993. 7:369–83. 265. Wahl C, Liptay S, Adler G, Schmid RM. Sulfasalazine: a potent and specific inhibitor of nuclear factor kappa B. J. Clin.Invest. 1998; 101:1163–74. 266. Farmacología de la secreción gastrointestinal y de la ulceración mucosa digestiva. In Jesús Florez, Juan Antonio Armijo, África Mediavilla. Farmacología Humana. 5 edn. 2008. Elsevier España. 267. Sandborn WJ, Löfberg R, Feagan BG, Hanauer SB, Campieri M, Greenberg GR. Budesonide for maintenance of remission in patients with Crohn's disease in medically induced remission: a predetermined pooled analysis of four randomized, double-blind, placebo-controlled trials. Am J Gastroenterol. 2005 Aug; 100(8):1780-7. 268. Pearson DC, May GR, Fick GH, Sutherland LR. Azathioprine and 6- mercaptopurine in Crohn disease. A metaanalysis. Ann. Intern. Med. 1995; 123:132–42. 269. Egan LJ, Sandborn WJ. Methotrexate for inflammatory bowel disease: pharmacology and preliminary results. Mayo Clin. Proc. 1996; 71:69–80. 270. Langford CA, Klippel JH, Balow JE, James SP, Sneller MC. Use of cytotoxic agents and cyclosporine in the treatment of autoimmune disease. Part 2: inflammatory bowel disease, systemic vasculitis, and therapeutic toxicity. Ann. Intern. Med. 1998 Jul; 129:49–58. 271. Fellermann K, Ludwig D, Stahl M, David-Walek T, Stange EF. Steroid-unresponsive acute attacks of inflammatory bowel disease: immunomodulation by tacrolimus (FK506). Am. J. Gastroenterol. 1998 Oct; 93:1860–66. 272. van Deventer SJ. Immunotherapy of Crohn’s disease. Scand. J. Immunol. 2000; 51:18–22. 273. Peyrin-Biroulet L, Deltenre P, de Suray N, Branche J, Sandborn WJ, Colombel JF. Efficacy and safety of tumor necrosis factor antagonists in Crohn's disease: meta-analysis of placebo-controlled trials. Clin Gastroenterol Hepatol. 2008 Jun; 6(6):644-53. 274. Ito H, Takazoe M, Fukuda Y, Hibi T, Kusugami K, Andoh A, Matsumoto T, Yamamura T, Azuma J, Nishimoto N, Yoshizaki K, Shimoyama T, Kishimoto T. A pilot randomized trial of a human anti-interleukin-6 receptor monoclonal antibody in active Crohn's disease. Gastroenterology. 2004 Apr; 126(4):989-96; discussion 947. 275. Hommes DW, Mikhajlova TL, Stoinov S, Stimac D, Vucelic B, Lonovics J, Zákuciová M, D'Haens G, Van Assche G, Ba S, Lee S, Pearce T. Fontolizumab, a humanised anti-interferon gamma antibody, demonstrates safety and clinical activity in patients with moderate to severe Crohn's disease. Gut. 2006 Aug; 55(8):1131-7. 276. Mannon PJ, Fuss IJ, Mayer L, Elson CO, Sandborn WJ, Present D, Dolin B, Goodman N, Groden C, Hornung RL, Quezado M, Yang Z, Neurath MF, Salfeld J, Veldman GM, Schwertschlag U, Strober W; Anti-IL-12 Crohn's Disease Study Group. Anti-interleukin-12 antibody for active Crohn's disease. N Engl J Med. 2004 Nov 11; 351(20):2069-79. 277. Requena P, Daddaoua A, Martinez-Plata E, Gonzalez M, Zarzuelo A, Suarez MD, Sanchez de Medina F, Martinez-Augustin O: Bovine glycomacropeptide ameliorates experimental rat ileitis by mechanisms involving downregulation of interleukin 17. Br J Pharmacol 2008;154:825-832. 279. Arbour NC, Lorenz E, Schutte BC, Zabner J, Kline JN, Jones M et al. TLR4 mutations are associated with endotoxin hyporesponsiveness in humans. Nat Genet 2000; 25: 187–191. 280. Cario E, Podolsky DK. Differential alteration in intestinal epithelial cell expression of toll-like receptor 3 (TLR3) and TLR4 in inflammatory bowel disease. Infect Immun. 2000 Dec;68(12):7010-7. 281. Söderholm JD, Olaison G, Peterson KH, Franzén LE, Lindmark T, Wirén M, Tagesson C, Sjödahl R. Augmented increase in tight junction permeability by luminal stimuli in the non-inflamed ileum of Crohn's disease. Gut. 2002 Mar;50(3):307-13. 282. Maynard CL, Weaver CT. Intestinal effector T cells in health and disease. Immunity. 2009 Sep 18;31(3):389-400.


63

http://www.ncbi.nlm.nih.gov/pubmed/19766082?itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum&ordinalpos=1

283. Yen, D., Cheung, J., Scheerens, H., Poulet, F., McClanahan, T., McKenzie, B., Kleinschek, M.A., Owyang, A., Mattson, J., Blumenschein, W., et al. (2006). IL-23 is essential for T cell-mediated colitis and promotes inflammation via IL-17 and IL-6. J. Clin. Invest. 116, 1310–1316. 284. Berg, D.J., Davidson, N., Kuhn, R., Muller, W., Menon, S., Holland, G., Thompson- Snipes, L., Leach, M.W., and Rennick, D. (1996). Enterocolitis and colon cancer in interleukin-10-deficient mice are associated with aberrant cytokine production and CD4(+) TH1-like responses. J. Clin. Invest. 98, 1010–1020. 285. Powrie, F., Leach, M.W., Mauze, S., Menon, S., Caddle, L.B., and Coffman, R.L. (1994). Inhibition of Th1 responses prevents inflammatory bowel disease in scid mice reconstituted with CD45RBhi CD4+ T cells. Immunity 1, 553–562. 286. Fuss, I.J., Neurath, M., Boirivant, M., Klein, J.S., de la Motte, C., Strong, S.A., Fiocchi, C., and Strober, W. (1996). Disparate CD4+ lamina propria (LP) lymphokine secretion profiles in inflammatory bowel disease. Crohn’s disease LP cells manifest increased secretion of IFN-gamma, whereas ulcerative colitis LP cells manifest increased secretion of IL-5. J. Immunol. 157, 1261–1270. 287. Fuss, I.J., Heller, F., Boirivant, M., Leon, F., Yoshida, M., Fichtner-Feigl, S., Yang, Z., Exley, M., Kitani, A., Blumberg, R.S., et al. (2004). Nonclassical CD1d-restricted NK T cells that produce IL-13 characterize an atypical Th2 response in ulcerative colitis. J. Clin. Invest. 113, 1490–1497. 288. Dieleman LA, Palmen MJ, Akol H, Bloemena E, Peña AS, Meuwissen SG, Van Rees EP. Chronic experimental colitis induced by dextran sulphate sodium (DSS) is characterized by Th1 and Th2 cytokines. Clin Exp Immunol. 1998 Dec;114(3):385-91. 289. Martínez-Augustin O, Merlos M, Zarzuelo A, Suárez MD, de Medina FS. Disturbances in metabolic, transport and structural genes in experimental colonic inflammation in the rat: a longitudinal genomic analysis. BMC Genomics. 2008 Oct 17;9:490. 290. Martínez-Augustin O, López-Posadas R, González R, Suárez MD, Zarzuelo A, Sánchez de Medina F. Genomic analysis of sulfasalazine effect in experimental colitis is consistent primarily with the modulation of NF-kappaB but not PPAR-gamma signaling. Pharmacogenet Genomics. 2009 May;19(5):363-72. 291. López Alvarez MJ. Proteins in human milk. Breastfeed Rev. 2007 Mar; 15(1):5-16. 292. Gill HS, Doull F, Rutherfurd KJ, Cross ML. Immunoregulatory peptides in bovine milk. Br J Nutr. 2000 Nov; 84 Suppl 1:S111-7. 293. Sánchez de Medina F, Daddaoua A, Requena P, Capitán-Cañadas F, Zarzuelo A, Suárez MD, Martínez-Augustin O. New insights into the immunological effects of food bioactive peptides in animal models of intestinal inflammation. 3rd International Immunonutrition Workshop Session IX. Food ingredients, immunity and inflammation: animal and in vitro 294. Bounous G, Kongshavn PA. Differential effect of dietary protein type on the B-cell and T-cell immune responses in mice. J Nutr. 1985 Nov; 115(11):1403-8. 295. Elitsur Y, Neace C, Liu X, Dosescu J, Moshier JA. Vitamin A and retinoic acids immunomodulation on human gut lymphocytes. Immunopharmacology. 1997 Jan; 35(3):247-53. 296. Mattsby-Baltzer I, Roseanu A, Motas C, Elverfors J, Engberg I, Hanson LA. Lactoferrin or a fragment thereof inhibits the endotoxin-induced interleukin-6 response in human monocytic cells. Pediatr Res. 1996 Aug; 40(2):257-62. 297. Shinoda I, Takase M, Fukuwatari Y, Shimamura S, Köller M, König W. Effects of lactoferrin and lactoferricin on the release of interleukin 8 from human polymorphonuclear leukocytes. Biosci Biotechnol Biochem. 1996 Mar; 60(3):521-3. 298. Otani H, Hata I. Inhibition of proliferative responses of mouse spleen lymphocytes and rabbit Peyer's patch cells by bovine milk caseins and their digests. J Dairy Res. 1995 May; 62(2):339-48. 299. Gattegno L, Migliore-Samour D, Saffar L, Jollès P. Enhancement of phagocytic activity of human monocytic-macrophagic cells by immunostimulating peptides from human casein. Immunol Lett. 1988 May; 18(1):27-31. 300. Brody, E.P., Biological activities of bovine glycomacropeptide. Br. J. Nutr. 2000. 84: S39-S46 301. Chabance, B., Jollès, P., Izquierdo, C., Mazoyer, E., Francoual, C., Drouet, L. and Fiat, AM., Characterization of an antithrombotic peptide from kappa-casein in newborn plasma after milk ingestion. Br J Nutr. 1995. 73: 583-90. 302. Chabance, B., Marteau, P., Rambaud, J.C., Migliore-Samour, D., Boynard, M., Perrotin, P., Guillet, R., et al., Casein peptide release and passage to the blood in humans during digestion of milk or yogurt. Biochimie. 1998. 80: 155-65. 303. Laclair CE, Ney DM, MacLeod EL, Etzel MR. Purification and use of glycomacropeptide for nutritional management of phenylketonuria. Journal of food science. 2009 May;74:E199-206. 304. van Calcar SC, MacLeod EL, Gleason ST, Etzel MR, Clayton MK, Wolff JA, Ney DM. Improved nutritional management of phenylketonuria by using a diet containing glycomacropeptide compared with amino acids. The American journal of clinical nutrition. 2009 Apr; 89:1068-77.

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http://www.ncbi.nlm.nih.gov/pubmed?term=%22L%C3%B3pez-Posadas%20R%22%5BAuthor%5D&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVAbstract

http://www.ncbi.nlm.nih.gov/pubmed?term=%22Gonz%C3%A1lez%20R%22%5BAuthor%5D&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVAbstract

http://www.ncbi.nlm.nih.gov/pubmed?term=%22Su%C3%A1rez%20MD%22%5BAuthor%5D&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVAbstract

http://www.ncbi.nlm.nih.gov/pubmed?term=%22Zarzuelo%20A%22%5BAuthor%5D&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVAbstract

http://www.ncbi.nlm.nih.gov/pubmed?term=%22S%C3%A1nchez%20de%20Medina%20F%22%5BAuthor%5D&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVAbstract

http://www.ncbi.nlm.nih.gov/pubmed?term=%22S%C3%A1nchez%20de%20Medina%20F%22%5BAuthor%5D&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVAbstract

305. Ney DM, Hull AK, van Calcar SC, Liu X, Etzel MR. Dietary glycomacropeptide supports growth and reduces the concentrations of phenylalanine in plasma and brain in a murine model of phenylketonuria. The Journal of nutrition. 2008 Feb;138:316-22. 306. Mikkelsen TL, Rasmussen E, Olsen A, Barkholt V, Frokiaer H. Immunogenicity of kappa-casein and glycomacropeptide. J Dairy Sci. 2006 Mar; 89: 824–830. 307. Aimutis WR. Bioactive properties of milk proteins with particular focus on anticariogenesis. J Nutr. 2004 Apr; 134(4):989S-95S. 308. Kawasaki Y, Isoda H, Shinmoto H, Tanimoto M, Dosako S, Idota T, Nakajima I. Inhibition by kappa-casein glycomacropeptide and lactoferrin of influenza virus hemagglutination. Biosci Biotechnol Biochem. 1993 Jul; 57(7):1214-5. 309. Kawasaki Y, Isoda H, Tanimoto M, Dosako S, Idota T, Ahiko K. Inhibition by lactoferrin and kappa-casein glycomacropeptide of binding of Cholera toxin to its receptor. Biosci Biotechnol Biochem. 1992 Feb; 56(2):195-8. 310. Bruck WM, Kelleher SL, Gibson GR, Nielsen KE, Chatterton DE, Lonnerdal B. rRNA probes used to quantify the effects of glycomacropeptide and alpha-lactalbumin supplementation on the predominant groups of intestinal bacteria of infant rhesus monkeys challenged with enteropathogenic Escherichia coli. Journal of pediatric gastroenterology and nutrition. 2003 Sep;37:273-80. 311. Bruck WM, Redgrave M, Tuohy KM, Lonnerdal B, Graverholt G, Hernell O, Gibson GR. Effects of bovine alpha-lactalbumin and casein glycomacropeptide-enriched infant formulae on faecal microbiota in healthy term infants. Journal of pediatric gastroenterology and nutrition. 2006 Nov; 43:673-9. 312. Monnai M, Otani H. Effect of bovine k-caseinoglycopeptide on secretion of interleukin-1 family cytokines by P388D1 cells, a line derived from mouse monocyte/macrophage. Milchwissenschaft 1997 52: 192–196. 313. Li EW, Mine Y. Immunoenhancing effects of bovine glycomacropeptide and its derivatives on the proliferative response and phagocytic activities of human macrophagelike cells, U937. J Agric Food Chem 2004 52: 2704–2708. 314 Daddaoua A, Puerta V, Zarzuelo A, Suarez MD, Sanchez de Medina F, Martinez-Augustin O. Bovine glycomacropeptide is anti-inflammatory in rats with hapten-induced colitis. The Journal of nutrition. 2005 May; 135:1164-70. 315. Farrell HM Jr, Jimenez-Flores R, Bleck GT, Brown EM, Butler JE, Creamer LK, Hicks CL, Hollar CM, Ng-Kwai-Hang KF, Swaisgood HE. Nomenclature of the proteins of cows' milk--sixth revision. J Dairy Sci. 2004 Jun;87(6):1641-74. 316. Fiat AM, Jollès P. Caseins of various origins and biologically active casein peptides and oligosaccharides: structural and physiological aspects. Mol Cell Biochem. 1989 May 4; 87(1):5-30. 317. Isoda H, Kawasaki Y, Tanimoto M, Dosako S, Idota T. Use of compounds containing or binding sialic acid to neutralize bacterial toxins. 1999. European Patent 385112. 318. Neeser JR, Chambaz A, Vedovo SD, Prigent MJ, Guggenheim B. Specific and nonspecific inhibition of adhesion of oral actinomyces and streptococci to erythrocytes and polystrene by caseinoglycopeptide derivatives. Infect Immun. 1988 Dec; 56(12):3201-8. 319. Kawasaki Y, Isoda K, Shinmoto H, Tanimoto M, Dosako S, Idota T, Nakajima I. Inhibition by k-casein glycomacropeptide and lactoferrin of influenza virus hemaglutination. Biosci Biotechnol Biochem. 1993 Jul; 57(7):1214-5. 320. Neeser JR, Chambaz A, Hoang KY, Link-Amster . Screening for complex carbohydrates inhibiting hemaggluatinations by CFA/I- and CFA/II-expressing enterotoxigenic Escherichia coli strains. FEMS Microbiology Letters. 1988; 49: 301-7. 321. Otani H, Monnai M. Inhibition of proliferative responses of mouse spleen lymphocytes by bovine milk k-casein digests. Food and Agricicultural Immunology. 1993; 5: 219-9. 322. Otani H, Monnai M, Kawasaki Y, Kawakami H, Tanimoto M. Inhibition of mitogen-induced proliferative responses of lymphocytes by bovine kappa-caseinoglycopeptides having different carbohydrate chains. J Dairy Res. 1995 May; 62(2):349-57. 323. Mikkelsen TL, Rasmussen E, Olsen A, Barkholt V, Frøkiaer H. Immunogenicity of kappa-casein and glycomacropeptide. J Dairy Sci. 2006 Mar; 89(3):824-30. 324. Otani H, Horimoto Y, Monnai M. Suppression of interleukin-2 receptor expression on mouse CD4+ T cells by bovine kappa-caseinoglycopeptide. Biosci Biotechnol Biochem. 1996 Jun; 60(6):1017-9. 325. Yun SS, Sugita-Konishi Y, Kumagai S, Yamauchi K. Glycomacropeptide from cheese whey protein concentrate enhances IgA production by lipopolysaccharide-stimulated spleen cells. Animal Science and Technology (Japan). 1996; 67: 458-62. 326. Snow Brand Milk Products Co (1996) Human normal B lymphocyte accelerating agent. Japanese Patent, 96018997 327. Martínez-Augustin O, Puerta V, Suárez MD. Nuevas fuentes de proteínas alimentarias. (2005)En Tratado de Nutrición. Edt. Gil A.


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328. Baró L, López-Huertas E, Boza JJ. Leche y derivados lácteos. (2005) En En Tratado de Nutrición. Edt. Gil A. 329. Massague J: The transforming growth factor-beta family. Annu Rev Cell Biol 1990;6:597-641. 330. Letterio JJ, Geiser AG, Kulkarni AB, Roche NS, Sporn MB, Roberts AB: Maternal rescue of transforming growth factor-beta 1 null mice. Science 1994;264:1936-1938. 331. Letterio JJ: Murine models define the role of TGF-beta as a master regulator of immune cell function. Cytokine Growth Factor Rev 2000;11:81-87. 332. Ozawa T, Miyata M, Nishimura M, Ando T, Ouyang Y, Ohba T, Shimokawa N, Ohnuma Y, Katoh R, Ogawa H, Nakao A: Transforming growth factor-beta activity in commercially available pasteurized cow milk provides protection against inflammation in mice. J Nutr 2009;139:69-75. 333. Schiffrin EJ, El Yousfi M, Faure M, Combaret L, Donnet A, Blum S, Obled C, Breuille D: Milk casein-based diet containing TGF-beta controls the inflammatory reaction in the HLA-B27 transgenic rat model. JPEN J Parenter Enteral Nutr 2005;29:S141-148; discussion S149-150, S184-148. 334. Beattie RM, Schiffrin EJ, Donnet-Hughes A, Huggett AC, Domizio P, MacDonald TT, Walker-Smith JA: Polymeric nutrition as the primary therapy in children with small bowel Crohn's disease. Aliment Pharmacol Ther 1994;8:609-615. 335. Fell JM, Paintin M, Arnaud-Battandier F, Beattie RM, Hollis A, Kitching P, Donnet-Hughes A, MacDonald TT, Walker-Smith JA: Mucosal healing and a fall in mucosal pro-inflammatory cytokine mRNA induced by a specific oral polymeric diet in paediatric Crohn's disease. Aliment Pharmacol Ther 2000;14:281-289. 336. Fell JM: Control of systemic and local inflammation with transforming growth factor beta containing formulas. JPEN J Parenter Enteral Nutr 2005;29:S126-128; discussion S129-133, S184-128.

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OBJETIVOS

OBJETIVOS DE ESTA TESIS DOCTORAL A pesar del gran número de fármacos que existe para el tratamiento de IBD, aún no existe un tratamiento ideal. Efectivamente, los medicamentos que producen remisión de manera eficaz sulen ir asociados a importantes efectos adversos. Además, la aparición de resistencia al tratamiento es también importante. Por esto, es necesaria la búsqueda de nuevos tratamientos farmacológicos con mejor pefil de efectos secundarios. Últimamente ha cobrado gran importancia la investigación en productos bioactivos, y a diario surgen nuevas aplicaciones para nutrientes cotidianos en nuestra alimentación. La búsqueda de fármacos en los alimentos (productos nutraceúticos) tiene una gran lógica, dada la seguridad que presentan en su uso. Efectivamente, uno de los principales factores limitantes en la comercialización de nuevos medicamentos es la aparición de efectos adversos durante las fases clínicas de los estudios. En cambio, los alimentos fuente de péptidos bioactivos, como la leche, han demostrado su inocuidad durante décadas o incluso siglos. Durante casi diez años, nuestro laboratorio ha estudiado el efecto inmunomodulador del glucomacropéptido bovino (BGMP). Un primer estudio realizado en ratas demostró su efecto anti-inflamatorio y protector frente a la colitis inducida por el TNBS (Daddaoua A. at al., 2005). Después de este estudio, nuestros objetivos fueron por un lado, comprobar el efecto anti-inflamatorio in vivo en otros modelos animales de IBD, y por otro, profundizar en los mecanismos de actuación. De esta forma:

- Estudiamos el efecto del BGMP en el modelo de colitis inducida por DSS en ratas (capítulo 2).

- Comprobamos si el efecto anti-inflamatorio se producía también en íleon (ileitis inducida por TNBS), para demostrar si la actividad inmunomoduladora del BGMP era consecuencia del efecto “prebiótico” o de una acción directa sobre el sistema inmune (capítulo 3).

- Analizamos el efecto del BGMP en cada componente celular del sistema inmune gastrointestinal: monocitos/macrófagos (capítulo 5), esplenocitos enriquecidos en linfocitos (capítulo 6) y células epiteliales (capítulo 7).

- Y finalmente, realizamos un estudio genómico del efecto del BGMP en ratas colíticas, para obtener una información global del efecto inmunomodulador del péptido, así como determinar nuevas dianas en su acción (capítulo 8).

Objetivos

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Bovine glycomacropeptide has intestinal antiinflammatory effects in dextrane sulfate sodium rat colitis.

Rocío López-Posadas, Pilar Requena, Raquel González, María Dolores Suárez, Antonio Zarzuelo, Fermín Sánchez de Medina, Olga Martínez-Augustin.Submitted for publication.

CAPÍTULO 3

Background and purpose: Milk κ-casein derived bovine glycomacropeptide (GMP) has immunomodulatory and bacterial toxin binding effects it has been shown to exert intestinal antiinflammatory activity in the trinitrobenzenesulfonic acid model of colitis. However, its mechanism of action is not well characterized and it is not known whether GMP is effective in other experimental models. Experimental approach: The intestinal antiinflammatory activity of glycomacropeptide was assessed in the dextrane sulfate sodium model of rat colitis. Key results: GMP pretreatment (500 mg kg-1 day-1) reduced considerably the disease activity index and lowered the colonic damage score, as well as colonic myeloperoxidase activity. Also, GMP fully normalized the colonic expression of IL-1β, IL-17, IL-23, IL-6, TGF-β, IL-10 and Foxp3 as assessed by qRT-PCR. The production of IFN-γ by mesenteric lymph node cells ex vivo was also normalized by GMP treatment. In contrast, colonic thickening, COX2 and alkaline phosphatase were little affected or not affected at all. Conclusions and Implications: We conclude that GMP exerts intestinal antiinflammatory activity in this model, which may be primarily related to actions on Th1 and Th17 lymphocytes and perhaps macrophages. Keywords: glycomacropeptide, casein macropeptide, dextrane sulfate sodium, inflammatory bowel disease. Introduction Inflammatory bowel disease (IBD), comprising ulcerative colitis and Crohn’s disease, is a chronic relapsing condition of the intestine that causes a significant deterioration of the quality of life of patients and which has a substantial (and increasing) prevalence (Ekbom, 2004; Sands, 2000). Despite an intense investigative effort IBD etiology remains unknown, but it is believed to involve an interplay of genetic, enviromental, microbial and immunological factors. Because intestinal inflammation generally cannot be elicited in vivo in experimental animals reared in germ-free conditions (but it develops normally in SPF conditions) it is widely accepted that IBD probably represents an uncontrolled and exacerbated response to luminal antigens that are innocuous for the normal population. Thus intestinal inflammation would be the culmination of a cascade of events and processes initiated by antigens due to inadequate handling by the host’s immune system. These processes have been long thought to be related to augmented adaptive immunity responses, but it has also been proposed that a defect in innate immunity may paradoxically underlie the etiology of IBD (Nenci et al., 2007; Sainathan et al., 2008; Qualls et al., 2009; Qualls et al., 2006). The reasoning in the latter scenario is that if the initial response to a bacterial challenge fails to contain bacterial infiltration of the mucosa a subsequent, more robust reaction must come into play, giving rise to inflammation. Whatever the exact mechanism, IBD is regularly managed pharmacologically with drugs that downregulate the immune system such as corticoids, infliximab, aminosalicylates or azathioprine. All of these agents have a plethora of serious adverse effects which limit their application and they are not effective in all patients. Hence the search for new treatments with a low profile of adverse effects is much warranted (Sands, 2007). Bovine glycomacropeptide (GMP), also referred to as casein macropeptide, is a 64-aminoacid peptide that contains varying amounts (0 to 5 units) of N-acetylneuraminic (sialic) acid. This

BGMP has intestinal antiinflammatory effects in DSS rat colitis

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peptide results from the enzymatic hydrolysis of milk κ-casein in the bovine stomach due to the action of chymosin (pepsin in humans) (Brody, 2000). In addition, GMP is present at 10-15% in milk whey as a result of the action of the same enzyme during the cheese making process. Therefore, there is a substantial natural exposure to this peptide. GMP has nutritional interest because its aminoacid profile is high in branched chain aminoacids and lacks aromatic aminoacids, being therefore one of the few naturally occurring proteins safe for individuals with phenylketonuria and perhaps useful in the management of some liver diseases (Nakano et al., 2002; Nakay, S. and Modler, H. W., 1999). On the other hand, a number of biological activities have been ascribed to GMP. Thus there is some evidence that GMP may modulate colonic microflora, promoting the growth of bifidobacteria while inhibiting the proliferation of pathogens (Bruck et al., 2003c; Bruck et al., 2003a). Some authors have proposed that GMP may combat infection by binding to lectins, viruses and mycoplasma (Brody, 2000). In particular, we (Requena et al., 2009) and others (Fomon, S. J., 1993) have noted the stimulatory effect of GMP on macrophages, in terms of cytokine production and phagocytic activity. GMP may interfere with interleukin 1ß (IL-1) receptor binding (Monnai & Otani, 1997). On the other hand, GMP has been described to inhibit the proliferation of splenocytes and Peyer’s patch cells (Otani et al., 1995). We have recently observed that GMP inhibits IFN-γ release by rat splenocytes by blocking STAT4 activation (unpublished observations). Conversely, the production of IgG by mouse B-lymphocytes seems to be increased by GMP (Monnai et al., 1998). Taken together, these data suggest that GMP may constitute an excellent agent to treat IBD, in that it appears to boost innate immunity but block adaptive immunity. Indeed, we have established the intestinal antiinflammatory activity of GMP in TNBS colitis and ileitis (Requena et al., 2008; Daddaoua et al., 2005). We performed this study in order to (1) obtain confirmatory evidence in a separate model of colonic inflammation and (2) characterize the mechanism of action of GMP in depth. Methods Reagents Except where indicated, all reagents and primers were obtained from Sigma (Barcelona, Spain). Retrotranscription was achieved with the iScriptTM cDNA Synthesis Kit and iTM Sybr Green Supermix was used for amplification (Biorad, Alcobendas, Spain). Antibodies were purchased from Cayman Technologies (Pickerington, OH) and Sigma (Barcelona, Spain). GMP (BioPURE-GMPTM) was the kind gift of Davisco Foods International, Inc. (Eden Prairie, MN). According to the manufacturer the GMP content was 93% while fat and lactose contents accounted for 0.2% and less that 1%, respectively. Animals Female Wistar rats (175-225 g) obtained from Harlan (Barcelona, Spain) were used, housed in makrolon cages (up to 7 rats per cage) and maintained in our laboratory in air conditioned animal quarters with a 12 h light-dark cycle. Animals were provided with free access to tap water and food (Panlab A04, Panlab, Barcelona, Spain). This study was carried out in accordance with the Directive for the Protection of Vertebrate Animals used for Experimental and other Scientific Purposes of the European Union (86/609/EEC). Induction of colitis and experimental design Colitis was induced by adding dextrane sulfate sodium (DSS), obtained from ICN Biomedicals (Costa Mesa, CA), to drinking water for 10 days (Pérez-Navarro et al., 2005; Sanchez de Medina et al., 2004). We selected the conditions in order to achieve a mild to moderate degree of colitis by starting with 5% w/v of DSS and lowering the concentration to

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4 or 3% whenever the DAI (disease activity index) was increased disproportionally. The status of the animals was monitored by general examination and specifically by means of the DAI, a combined score for weight loss, diarrhea and hematochezia, which are 3 main signs of pathology in this model (Ito et al., 2008). Rats were randomly assigned to 3 different groups. The control (C) group (n=5) did not receive DSS and was administered water daily by means of a gastroesophageal catheter. The remainder rats drank DSS supplemented water, and received by gavage either 500 mg kg-1

day-1 of GMP (GMP group, n=7) or vehicle (1% methylcellulose, DSS group, n=7). Treatment started 2 days before DSS supplementation and were maintained until animals were sacrificed after 10 days by cervical dislocation. A small blood sample was drawn from the tail vein before euthanasia under halothane anaesthesia for subsequent determination of the leukocyte formula (Giemsa staining). Assessment of colonic damage The entire colon was removed, gently flushed with saline and placed on an ice-cold plate, cleaned of fat and mesentery, and blotted on filter paper. Each specimen was weighed and its length measured under a constant load (2 g). The large intestine was longitudinally opened and scored for visible damage by a blinded observer on a 0 to 25 scale according to the criterion detailed in Table 1. The score was assigned as follows: adhesions (0-3), obstruction (0-2), hyperaemia (0-3), fibrosis (rigidity and deformation, 0-5), necrosis (0-5), other features (proximal dilatation, fragility, scarring, 0-4). A small segment was dissected from the intestine and used for RNA isolation. The colon was subsequently divided longitudinally in several pieces for biochemical determinations. The fragments were immediately frozen in liquid nitrogen and kept at –80ºC until used. Myeloperoxidase and alkaline phoshatase (AP) activities were measured spectrophotometrically as described (Sanchez de Medina et al., 2004) and expressed as U g-1 and mU mg-1 protein, respectively. In addition, the sensitivity to the AP inhibitor levamisole was assessed. Analysis of gene expression by reverse transcriptase-PCR analysis Total RNA was obtained by the Trizol method (Invitrogen, Barcelona, Spain) and 1 µg retrotranscribed and specific RNA sequences amplified with a Stratagene MX3005P real time PCR device using the following primers: AAT GAC CTG TTC TTT GAG GCT G / CGA GAT GCT GCT GTG AGA TTT (IL-1β), TAC TGA ACT TCG GGG TGA TTG / CAG CCT TGT CCC TTG AAG AGA (tumor necrosis factor, TNF), CTG CTT GGC AGT GCT TGA GAA / CCC AGG AAA GAC AGC AAC CTT (Foxp3), AGT CAG CCA GAC CCA CAT G / TGC TCC ACT GCC TTG CTT TT (IL-10), TGG ACT CTG AGC CGC AAT GAG G / GAC GCA TGG CGG ACA ACA GAG G (IL-17), GCA CAC TAG CCT GGA GTG CA / AGA TGT CCG AGT CCA GCA GG (IL-23 p19), GCT CTG GTC TTC TGG AGT TCC G / TTG GAT GGT CTT GGT CCT TAG CC (IL-6), ACT GGC GAG CCT TAG TTT G / CGT GGC TTC TAG TGC TGA CG (TGF-β), and CCA TTG GAG GGC AAG TCT GGT G / CGC CGG TCC AAG AAT TTC ACC (18S). Western Blot The colonic levels of cyclooxygenase 2 (COX-2) were determined by immunoblotting. Colonic samples were homogenized in lysis buffer (0.1% w v-1 SDS, 0.1% w v-1 sodium deoxycholate, 1% v v-1 Triton X-100 in PBS) with protease and phosphatase inhibitors (1 mM 1,10-phenanthroline, 1 mM phenylmethylsulfonyl fluoride, 18 mg l-1 aprotinin, 2 mM sodium orthovanadate). The supernatants obtained after centrifugation (7000 X g, 10 min at 4ºC) were


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boiled for 4 min in Laemmli buffer, separated by SDS-PAGE (10%), electroblotted to PVDF membranes, and probed with the corresponding antibodies overnight at 4ºC. The bands were detected by enhanced chemiluminescence (PerkinElmer) and quantitated with NIH software (Scion Image). After the transference of the samples to PVDF membranes, equal loading was checked routinely by reversible Ponceau staining. The composition of the Laemmli buffer (5X) was: 312 mM SDS, 50% v v-1 glycerol, 1% v v-1 2-mercaptoethanol, 22.5 mM EDTA trisodium salt, 220 mM Tris, and traces of bromophenol blue (pH=6.8). Mesenteric lymph node cells Mesenteric lymph nodes (MLN) were extracted from the rats in the study using sterile technique and dissected mechanically. The cells were incubated in RPMI-1640 medium supplemented with 0.05 mM mercaptoethanol, 10% v v-1 fetal bovine serum (Boëhringer Mannheim, Barcelona, Spain), 100 mg l-1 streptomycin, 100000 U l-1 penicillin and 2.5 mg l-1 amphotericin B. The cells were cultured at 106 cells ml-1 and stimulated with concanavalin A. Cell culture medium was collected after 48 h and assayed for cytokine content by commercial ELISAs (Biosource Europe, Nivelles, Belgium and BD Biosciences, Erembodegem, Belgium). Statistical analysis The results are expressed as mean ± S.E.M. Differences among means were tested for statistical significance by one way ANOVA and a posteriori least significance tests on preselected pairs. All the analyses were carried out with the SigmaStat program (Jandel Corporation, San Rafael, CA, U.S.A.). Statistical significance was set at P<0.05. Results DSS colitis As expected, the supplementation of drinking water with DSS resulted in significant colonic damage, as reflected in the increased DAI, specially starting 5 days after DSS introduction (Fig. 1) and the augmented colonic damage score and weight to length ratio (Table 1). In addition, DSS treated rats exhibited anorexia, particularly after day 5 (9.1±0.4 vs. 17.3±0.5 g rat-1 day-1, P<0.05), and lower water intake (not shown), and the colonic tissue had increased MPO activity, indicating active neutrophil recruitment to the inflammatory site (Fig. 2). AP activity, a good marker of intestinal inflammation and epithelial stress, was also higher in the inflamed tissue, and its sensitivity to levamisole increased, consistent with the isoform shift previously described (Sanchez de Medina et al., 2004). This model has been described to be associated with a Th1 immune response, and accordingly mesenteric node cells had a higher IFN-γ and IL-2 secretion than the controls under ConA stimulation. Furthermore, these cells also displayed an increased production of TNF (without reaching significance), but no IL-4 (Fig. 3 and data not shown). The spleen was significantly enlarged compared to the controls (Table 1 and Fig. 4). DSS treated animals also tended to have a higher peripheral blood count of monocytes and a lower count of lymphocytes and neutrophils, although failing to reach statistical significance (Fig. 5). COX2 expression was generally higher in colitic animals, but the overall ANOVA was not significant (Fig. 6). Colonic mRNA levels of IL-1β, IL-23, IL-6, TGF-β, IL-10, Foxp3 and specially IL-17 were all significantly increased compared to the controls (Fig. 7). In contrast, there was no effect on TNF (not shown).

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Table 1 - Morphological parameters of inflammation

Colonic damage score Colon weight:length ratio (mg/cm)

Spleen weight (mg)

Control 0 (0-0) 73.4±5.7 539.8±34.5

DSS 5.9±0.8+ 105.9±7.1+ 807.7±85.0+

GMP 3.3±1.0+* 92.5±5.0 680.0±53.5 +P<0.05 vs. Control; *P<0.05 vs. DSS

Fig. 1. Evolution of the DAI in the control, DSS and GMP groups. DSS was started at day 0. +P<0.05 vs. control; *P<0.05 vs. DSS. Effect of GMP Pretreatment of rats with 500 mg kg-1 of GMP daily prevented DSS colitis partially, as evidenced early on by a lower DAI, which was significant from day 7 onward (Fig. 1). Food intake was slight but significantly improved in this group (11.7±0.4 g rat-1 day-1, P<0.05 vs. both the control and DSS groups). After the animals were sacrificed their large bowels exhibited a lower damage score (Table 1), derived mainly from less marked fibrotic features (adhesions, deformation). The colonic weight to length ratio and the spleen size were intermediate between those of the control and DSS groups, so that they were not significantly different from either one (Table 1).


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Fig.2. Myeloperoxidase (MPO) and alkaline phosphatase (AP) colonic activity in the control, DSS and GMP groups. Enzymatic activity and the sensitivity of AP to the specific inhibitor, levamisole, are shown. +P<0.05 vs. control; *P<0.05 vs. DSS.

Fig. 3. Cytokine secretion by mesenteric lymph node cells obtained from the control, DSS and GMP groups. Control levels were (pg/ml): 97.1±24.3 (TNF), 4803.3±1470.5 (IFN-γ) and 427.4±127.8 (IL-2). +P<0.05 vs. control; *P<0.05 vs. DSS.

Fig. 4. Splenomegaly induced by DSS colitis.

Biochemical parameters were in accordance with the macroscopic markers. Thus MPO activity was lower in the GMP group (Fig. 2). A similar result was obtained with colonic AP activity and enzyme sensitivity to levamisole (Fig. 2). On the other hand, mesenteric node cells from GMP treated animals had a fully normalized IFN-γ secretory response to ConA, and IL-2 was not significantly different from control values (Fig. 3). The leukocyte formula and the colonic levels of COX2 were similar to those of the DSS group (Figs. 5 and 6). However, GMP treatment had a major impact in the colonic expression of a variety of inflammatory markers, including IL-1β, IL-6, IL-23 and specially IL-17 (Fig. 7). The Treg

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marker Foxp3 and the antiinflammatory cytokines IL-10 and TGF-β were fully normalized also, thus correlating with the therapeutic response.

Fig. 5. Leukocyte counts in peripheric blood from the control, DSS and GMP groups. No significant differences were observed. Discussion and Conclusions Our data demonstrate that GMP pretreatment reduces the severity of DSS colitis in rats at the dose of 500 mg kg-1 day-1. This dose is roughly equivalent to 5 g for an adult human (on a body surface basis), an amount that cannot be easily achieved by milk consumption but that is easily attainable as a functional food or a drug. We had previously established the intestinal antiinflammatory activity of this peptide in the TNBS model of colitis (Daddaoua et al., 2005) and also in TNBS ileitis (Requena et al., 2008), a variation of the colitis model in which TNBS is injected into the ileum rather than administered intrarectally. In these experiments GMP was more effective as a pretreatment than as a postreatment and its effect was associated to a lower expression of IL-1β and iNOS, as well as of MPO and AP activities. In experimental ileitis GMP did not have any clear effect on cytokine production by mesenteric lymph node cells ex vivo, but it did decrease IL-17 and TNF, suggesting that GMP may act via modulation of Th17 but not Th1 cells. Thus the mechanism of action of GMP needs further clarification. On the other hand, the therapeutic effect of this peptide, combined with its very low toxicity, makes it a good candidate for clinical application either pharmacologically or nutritionally, but testing in humans requires extensive preclinical evidence of activity. Thus we set out to test GMP in other models of IBD. Because there is no ideal model of IBD (Jurjus et al., 2004), it follows that establishing therapeutic efficacy in different types of experimental intestinal inflammation represents a good approach to this problem. Furthermore, because there are substantial differences in the mechanism of colitis induction and in the pathology of the different models, relevant conclusions as to the mode of action of the agent tested may be reached. For instance, the flavonoid luteolin has completely different effects in DSS and IL-10 -/- colitis in mice, being deleterious in the former and protective in the latter (Karrasch et al., 2007). These disparate results might be explained by the inhibitory action of the flavonoid on the NF-κB pathway in the epithelium, which may compromise the defense of the mucosa toward epithelial disruption by DSS. Indeed, DSS is thought to elicit intestinal inflammation by slowly altering the epithelial integrity, augmenting permeability and ultimately resulting in an immune reaction against luminal antigens (Cooper et al., 1993). In contrast, TNBS acts as a hapten by a delayed hipersensitivity mechanism


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involving the reaction with mucosal proteins (Morris et al., 1989). Oxidative stress may also play a role (Grisham et al., 1991). Ethanol is routinely added to produce an initial, short lasting lesion in the epithelium that allows TNBS to gain access to the mucosa. The same result is obtained if TNBS without ethanol is injected directly into the mucosa (Allgayer et al., 1989). Therefore the mechanisms are quite different in both models, and the epithelium may play a more important role in DSS than in TNBS colitis. In this study we selected the conditions to produce a mild to moderate colitis compared to the extensive damage (including necrosis) associated with TNBS colitis (Morris et al., 1989; Ballester et al., 2007; Daddaoua et al., 2005). This in turn resulted in a relatively high variability in the magnitude of colitis. This is evident in the dispersion observed in MPO and COX2, as well as colonic cytokines, in the DSS group. The advantage of using a mild to moderate colitis is that it is more amenable to treatment, but the downside is that variability may make differences harder to reach statistical significance. Nevertheless, GMP had a dramatic impact in most inflammatory markers, including the DAI, colonic damage score, MPO activity, IL-17, IL-23, IL-1β, IL-6, TGF-β, IL-10 and Foxp3, most of which were fully normalized by the treatment. The impact on AP and specially COX2 was much less pronounced. AP is increased in the inflamed intestine as a result both of leukocyte infiltration and of an increase in epithelial enzyme expression, which is also associated to a change of isoform that can be detected by a higher sensitivity to inhibition by levamisole (Sanchez de Medina et al., 2004). COX2 on the other hand is expressed by various cell types, one of the most important of which are enterocytes (Longo et al., 1998; Singer et al., 1998). Because DSS primary effect is on the epithelium, as discussed above, these results are consistent with a mode of action of GMP that does not involve enterocytes, since AP and COX2 are downregulated in the TNBS models (Daddaoua et al., 2005; Requena et al., 2008). Indeed, preliminary data obtained with intestinal epithelial cell lines support this hypothesis (unpublished data). We have recently reported that GMP stimulates macrophage function via MAPK and NF-κB (Requena et al., 2009). Although the immediate consequence of this action would be to increase proinflammatory cytokines in the mucosal milieu, the pathology of IBD is more complex than that. It has been shown for instance that conditional suppression of NF-κB signaling in epithelial cells produces severe colonic inflammation spontaneously (Nenci et al., 2007). Similarly, GM-CSF appears to dampen rather than stimulate inflammation (Sainathan et al., 2008), and the absence of monocytes and dendritic cells aggravates rather than ameliorates experimental colitis (Qualls et al., 2009; Qualls et al., 2006). Thus recent evidence suggests that a defective response to proinflammatory stimuli may actually aggravate the outcome, possibly because it impairs a prompt resolution, triggering a more robust reaction to a normally trivial challenge. Hence macrophages may constitute a relevant target of GMP in its therapeutic effect. Lymphocytes are also affected by GMP ((Otani et al., 1995; Monnai et al., 1998) and paper in preparation). Our own unpublished results suggest that GMP may directly inhibit IFN-γ production by rat splenocytes. In the present study GMP treatment was associated with a dramatic effect on IFN-γ release by MLN cells, again supporting the hypothesis that GMP may act on Th1 cells. We could not measure IL-17 in these cells because of a lack of antibody, but its mRNA levels, along with those of IL-23, were also comparable to those of the control group in the colonic tissue, consistent with our previous observations (Requena et al., 2008). Of note, MLN cells exhibited also a lower IL-2 output, such that it was not significantly different from that of noncolitic rats, which would be expected if our hypothesis is correct. The fact that IL-10 and TGF-β, cytokines associated with Treg

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immunosuppression, were greatly diminished by GMP treatment, may simply represent the result of overall tissue improvement. Thus in a genomic study of TNBS colitis in rats by our group genes with established or presumed antiinflammatory or immunosuppresive function were generally downregulated as colitis resolved (Martinez-Augustin et al., 2008). Although this does not rule out a role of Treg cells in the GMP effect, this mechanism is certainly not supported by the data from this study.

Fig. 6. Colonic cyclooxygenase 2 expression in the control, DSS and GMP groups. COX2 was measured by Western blot and band density quantitated as described in Materials and methods. It is quite clear however that TNF was not upregulated in this experiment. Since this cytokine has been reported to be increased in this model by other authors (Obermeier et al., 2002; Carrier et al., 2006), and considering the relatively low increase of IFN-γ in MLN cells, it is likely that the mild degree of inflammation accounts for this discrepancy. However, our data argue against a major role of TNF in DSS colitis. In fact, TNF might be even protective in this model, since DSS colitis has been reported to be aggravated in TNF -/- mice (Naito et al., 2003). We have also performed a microarray study on the mechanism of action of GMP in the TNBS model, which suggests that the IL-6 pathway is a major target of GMP in the inflamed intestine (manuscript in preparation). In fact, since Th17 cell development is elicited by the combination of IL-6 and TGF-β, both of which are dramatically lowered by the peptide, the modulation of Th17 by GMP may be indirect. Consistent with this, IL-6 was completely normalized in the present study, and IL-6 has been unequivocally confirmed to be involved in DSS pathology (Naito et al., 2004). Because it has been suggested that GMP modulates white blood cell populations (Bruck et al., 2003b), we examined the percentages of leukocytes in all groups. However, there was no effect of either DSS colitis or GMP on this parameter, as there was no significant differences among groups. Thus this mechanism can be ruled out in the present experiment.


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Fig. 7. Colonic expression of inflammatory markers assessed by RT-qPCR in the control, DSS and GMP groups. The ratio over 18 S expression is shown. IL-17 is presented separately because of the difference in the magnitude of the DSS increase. +P<0.05 vs. control; *P<0.05 vs. DSS. In conclusion, GMP has substantial intestinal antiinflammatory effect in the DSS model of colitis. The mechanism is probably related to actions on macrophages and Th1/Th17 lymphocytes but not epithelial cells. More experiments are required to fully delineate the mechanistic aspects of GMP action and to introduce this nontoxic agent in clinical practice. Acknowledgements The authors are thankful to Dr. Mercedes González for technical assistance. We are also grateful to Davisco Foods International, Inc. (Eden Prairie, MN). CIBEREHD is funded by the Instituto de Salud Carlos III. Financial support: this work was supported by grants from the Instituto de Salud Carlos III (PI051625 and PI051651) and the Ministry of Science and Innovation (SAF2008-01432 and

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AGL2008-04332). The CIBER-EHD is funded by the Instituto de Salud Carlos III. RLP and PR are funded by the Ministry of Science and Innovation. RG is funded by CIBEREHD. The group is member of the Network for Cooperative Research on Membrane Transport Proteins (REIT), co-funded by the Ministerio de Educación y Ciencia, Spain and the European Regional Development Fund (Grant BFU2005-24983-E/BFI). References Allgayer H, Deschryver K, Stenson WF (1989). Treatment with 16,16'-dimethyl prostaglandin E2 before and

after induction of colitis with trinitrobenzenesulfonic acid in rats decreases inflammation. Gastroenterology 96: 1290-1300.

Ballester I, Daddaoua A, Lopez-Posadas R, Nieto A, Suarez MD, Zarzuelo A et al. (2007). The bisphosphonate alendronate improves the damage associated with trinitrobenzenesulfonic acid-induced colitis in rats. Br J Pharmacol 151: 206-215.

Brody EP (2000). Biological activities of bovine glycomacropeptide. Br J Nutr 84: S39-S46. Bruck WM, Graverholt G, Gibson GR (2003a). A two-stage continuous culture system to study the effect of

supplemental alpha-lactalbumin and glycomacropeptide on mixed cultures of human gut bacteria challenged with enteropathogenic Escherichia coli and Salmonella serotype typhimurium. J Appl Microbiol 95: 44-53.

Bruck WM, Kelleher SL, Gibson GR, Nielsen KE, Chatterton DE, Lonnerdal B (2003b). rRNA probes used to quantify the effects of glycomacropeptide and alpha-lactalbumin supplementation on the predominant groups of intestinal bacteria of infant rhesus monkeys challenged with enteropathogenic Escherichia coli. J Pediatr Gastroenterol Nutr 37: 273-280.

Bruck WM, Kelleher SL, Gibson GR, Nielsen KE, Chatterton DEW, Lonnerdal B (2003c). rRna probes used to quantify the effects of glycomacropeptide and alpha-lactalbumin supplementation on the predominant groups of intestinal bacteria of infant Rhesus monkeys challenged with enteropathogenic Escherichia coli. J Pediatr Gastroenterol Nutr 37: 273-280.

Carrier JC, Aghdassi E, Jeejeebhoy K, Allard JP (2006). Exacerbation of dextran sulfate sodium-induced colitis by dietary iron supplementation: role of NF-kappaB. Int J Colorectal Dis 21: 381-387.

Cooper HS, Murthy SN, Shah RS, Sedergran DJ (1993). Clinicopathologic study of dextran sulfate sodium experimental murine colitis. Lab Invest 69: 238-249.

Daddaoua A, Puerta V, Zarzuelo A, Suarez MD, Sanchez de Medina F, Martinez-Augustin O (2005). Bovine glycomacropeptide is anti-inflammatory in rats with hapten-induced colitis. J Nutr 135: 1164-1170.

Ekbom A (2004). The epidemiology of IBD: a lot of data but little knowledge. How shall we proceed? Inflamm Bowel Dis 10 Suppl 1: S32-S34.

Fomon, S. J. (1993). Nutrition of normal infants. Mosby Yearbook Inc., St. Louis. Grisham MB, Volkmer C, Tso P, Yamada T (1991). Metabolism of trinitrobenzene sulfonic acid by the rat colon

produces reactive oxygen species. Gastroenterology 101: 540-547. Ito R, Kita M, Shin-Ya M, Kishida T, Urano A, Takada R et al. (2008). Involvement of IL-17A in the

pathogenesis of DSS-induced colitis in mice. Biochem Biophys Res Commun 377: 12-16. Jurjus AR, Khoury NN, Reimund JM (2004). Animal models of inflammatory bowel disease. J Pharmacol

Toxicol Methods 50: 81-92. Karrasch T, Kim JS, Jang BI, Jobin C (2007). The flavonoid luteolin worsens chemical-induced colitis in NF-

kappaB(EGFP) transgenic mice through blockade of NF-kappaB-dependent protective molecules. PLoS One 2: e596.

Longo WE, Panesar N, Mazuski J, Kaminski DL (1998). Contribution of cyclooxygenase-1 and cyclooxygenase-2 to prostanoid formation by human enterocytes stimulated by calcium ionophore and inflammatory agents. Prostaglandins Other Lipid Mediat 56: 325-339.

Martinez-Augustin O, Merlos M, Zarzuelo A, Suarez MD, Sanchez de Medina F (2008). Disturbances in metabolic, transport and structural genes in experimental colonic inflammation in the rat: a longitudinal genomic analysis. BMC Genomics 9: 490.

Monnai M, Horimoto Y, Otani H (1998). Immunomodificatory Effect of Dietary Bovine Kappa-Caseinoglycopeptide on Serum Antibody Levels and Proliferative Responses of Lymphocytes in Mice. Milchwissenschaft-Milk Science International 53: 129-132.

Monnai M & Otani H (1997). Effect of bovine k-caseinoglycopeptide on secretion of interleukin-1 family cytokines by P388D1 cells, a line derived from mouse monocyte/macrophage. Milchwissenschaft 52: 192-196.

Morris GP, Beck PL, Herridge MS, Depew WT, Szewczuk MR, Wallace JL (1989). Hapten-induced model of chronic inflammation and ulceration in the rat colon. Gastroenterology 96: 795-803.

Naito Y, Takagi T, Handa O, Ishikawa T, Nakagawa S, Yamaguchi T et al. (2003). Enhanced intestinal


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inflammation induced by dextran sulfate sodium in tumor necrosis factor-alpha deficient mice. J Gastroenterol Hepatol 18: 560-569.

Naito Y, Takagi T, Uchiyama K, Kuroda M, Kokura S, Ichikawa H et al. (2004). Reduced intestinal inflammation induced by dextran sodium sulfate in interleukin-6-deficient mice. Int J Mol Med 14: 191-196.

Nakano T, Silva-Hernandez ER, Ikawa N, Ozimek L (2002). Purification of kappa-casein glycomacropeptide from sweet whey with undetectable level of phenylalanine. Biotechnol Prog 18: 409-412.

Nakay, S. and Modler, H. W. (1999). Food Proteins. Processsing applications. Wiley VCH, New York. Nenci A, Becker C, Wullaert A, Gareus R, van Loo G, Danese S et al. (2007). Epithelial NEMO links innate

immunity to chronic intestinal inflammation. Nature 446: 557-561. Obermeier F, Dunger N, Deml L, Herfarth H, Scholmerich J, Falk W (2002). CpG motifs of bacterial DNA

exacerbate colitis of dextran sulfate sodium-treated mice. Eur J Immunol 32: 2084-2092. Otani H, Monnai M, Kawasaki Y, Kawakami H, Tanimoto M (1995). Inhibition of mitogen-induced

proliferative responses of lymphocytes by bovine kappa-caseinoglycopeptides having different carbohydrate chains. J Dairy Res 62: 349-357.

Pérez-Navarro R, Ballester I, Zarzuelo A, Sánchez de Medina F (2005). Disturbances in epithelial ionic secretion in different experimental models of colitis. Life Sci 76: 1489-1501.

Qualls JE, Kaplan AM, van Rooijen N, Cohen DA (2006). Suppression of experimental colitis by intestinal mononuclear phagocytes. J Leukoc Biol 80: 802-815.

Qualls JE, Tuna H, Kaplan AM, Cohen DA (2009). Suppression of experimental colitis in mice by CD11c+ dendritic cells. Inflamm Bowel Dis 15: 236-247.

Requena P, Daddaoua A, Guadix E, Zarzuelo A, Suarez MD, Sanchez de Medina F et al. (2009). Bovine glycomacropeptide induces cytokine production in human monocytes through the stimulation of the MAPK and the NF-kappaB signal transduction pathways. Br J Pharmacol 157: 1232-1240.

Requena P, Daddaoua A, Martínez-Plata E, González M, Zarzuelo A, Suárez MD et al. (2008). Bovine glycomacropeptide ameliorates experimental rat ileitis by mechanisms involving downregulation of interleukin 17. Br J Pharmacol 154: 825-832.

Sainathan SK, Hanna EM, Gong Q, Bishnupuri KS, Luo Q, Colonna M et al. (2008). Granulocyte macrophage colony-stimulating factor ameliorates DSS-induced experimental colitis. Inflamm Bowel Dis 14: 88-99.

Sanchez de Medina F, Martinez-Augustin O, Gonzalez R, Ballester I, Nieto A, Galvez J et al. (2004). Induction of alkaline phosphatase in the inflamed intestine: a novel pharmacological target for inflammatory bowel disease. Biochem Pharmacol 68: 2317-2326.

Sands BE (2000). Therapy of inflammatory bowel disease. Gastroenterology 118: S68-S82. Sands BE (2007). Inflammatory bowel disease: past, present, and future. J Gastroenterol 42: 16-25. Singer II, Kawka DW, Schloemann S, Tessner T, Riehl T, Stenson WF (1998). Cyclooxygenase 2 is induced in

colonic epithelial cells in inflammatory bowel disease. Gastroenterology 115: 297-306.

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Bovine glycomacropeptide ameliorates experimental rat ileitis by mechanisms involving downregulation of interleukin 17.

Pilar Requena, Abdelali Daddaoua, Enrique Martínez-Plata, Mercedes González, Antonio Zarzuelo, María Dolores Suárez, Fermín Sánchez de Medina, Olga Martínez-Augustin.British Journal of Pharmacology (2008) 154, 825–832.

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Background and purpose: Bovine glycomacropeptide (BGMP) is an inexpensive, non-toxic milk peptide with antiinflammatory effects in rat experimental colitis but its mechanism of action is unclear. It is also unknown whether BGMP can ameliorate inflammation in proximal regions of the intestine. Our aim was therefore two-fold: first, to determine the antiinflammatory activity of BGMP in the ileum; second, to characterise its mechanism of action. Experimental approach: We used a model of ileitis induced by trinitrobenzenesulphonic acid in rats. Rats were treated orally with BGMP and its efficacy compared with that of oral 5-aminosalicylic acid or vehicle, starting 2 days before ileitis induction. Key results: BGMP pretreatment (500mgkg-1 day-1) resulted in marked reduction of inflammatory injury, as assessed by lower extension of necrosis and damage score, myeloperoxidase, alkaline phosphatase, inducible nitric oxide synthase, interleukin 1β, tumour necrosis factor and interleukin 17. These effects were generally comparable to those of 5-aminosalicylic acid (200mgkg-1 day-1). Neither compound affected the production of interferon γ, tumour necrosis factor and interleukin 2 by mesenteric lymph node cells isolated from animals with ileitis. The expression of Foxp3 was increased in ileitis and not reduced significantly by BGMP or aminosalicylate treatment. Conclusions and implications: These results demonstrate that BGMP has anti-inflammatory activity in the ileum with similar efficacy to 5-aminosalicylic acid. The mechanism of action may involve Th17 and regulatory T cells and perhaps macrophages but probably not Th1 lymphocytes. Patients with Crohn’s ileitis may benefit from treatment with BGMP. Keywords: glycomacropeptide; trinitrobenzenesulphonic acid; experimental ileitis; interleukin 17; inflammatory bowel disease; interleukin 1; regulatory T cells. Abbreviations: AP, alkaline phosphatase; IL-, interleukin; IL-1ra, interleukin 1 receptor antagonist; iNOS, inducible nitric oxide synthase; TFF3, trefoil factor 3; TNBS, trinitrobenzenesulphonic acid; TNF-α, tumour-necrosis factor; Treg, regulatory T cells. Introduction Bovine glycomacropeptide (BGMP) or casein macropeptide is a biologically active peptide derived from the hydrolysis of milk k-casein (Brody, 2000). This peptide is composed of 64 amino acids and contains varying units of N-acetylneuraminic (sialic) acid. BGMP is produced both physiologically as a result of the digestion of k-casein in the stomach of neonates, and in the industry as a part of the whey produced during the cheese-making process. As a nutrient, BGMP has an excellent safety record and in accordance with this, a recent study has described that it is not immunogenic, because it fails to induce T-cell-specific responses in mice, even when given in polymerised form, in contrast with the parent protein (Mikkelsen et al., 2006). As a consequence, this peptide is used in the production of infant formulas and due to its low content on aromatic amino acids, including phenylalanine, BGMP has been proposed to be useful in the making of products for individuals with phenylketonuria.Furthermore, BGMP is

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included in toothpastes because of its anticariogenic properties. In fact, it has antibacterial activity preventing dental caries and, at the same time, it prevents tooth demineralisation and promotes tooth enamel mineralisation (Aimutis, 2004). Prebiotic and immunoregulatory effects have been attributed to glycomacropeptide. Thus, BGMP has been shown to promote the growth of bifidobacteria in vitro (Idota et al., 1994; Yakabe et al., 1994), although there is no definitive proof so far in vivo because the studies carried out with Rhesus monkeys and human infants were hampered by a high initial level of bifidobacteria (Bruck et al., 2003, 2006). In addition, it may combat infection by binding to lectins, viruses and mycoplasma (Brody, 2000). On the other hand, BGMP inhibits mouse splenocyte proliferation induced by lipopolysaccharides (Otani et al., 1995), suppresses IL (interleukin)-2 receptor expression in mouse CD4+ T cells (Otani et al., 1996) and suppresses serum IgG antibody production by mouse lymphocytes (Monnai et al., 1998). Furthermore, it modulates the secretion of the IL-1 family of cytokines in a mouse monocytic cell line (Monnai and Otani, 1997) and enhances the proliferation and phagocytic activities of human macrophage-like cells (Li and Mine, 2004).

In a study carried out in our laboratory (Daddaoua et al., 2005), BGMP was found to be anti-inflammatory in a rat model of colitis induced by the administration of trinitrobenzenesulphonic acid (TNBS). This effect was comparable to that of sulfasalazine, a drug widely used to treat inflammatory bowel disease (Baumgart and Sandborn, 2007). This study indicated that BGMP could be beneficial in inflammatory bowel disease, a chronic and relapsing disease that significantly diminishes the quality of life of patients. Inflammatory bowel disease is comprised of two different but closely related conditions, namely ulcerative colitis and Crohn’s disease (Sands, 2007). Ulcerative colitis affects the large intestine at the mucosal level, whereas Crohn’s disease is characterised by transmural inflammation and may involve any segment of the gastrointestinal tract from the mouth to the colon, especially the ileum and colon (Baumgart and Carding, 2007). The immune response in Crohn’s disease has long been considered to be dominated by Th1 cells, based mainly on studies of colonic disease in humans and animal models. There is some evidence that both Th1 and Th2 cells may contribute to Crohn’s ileitis (Desreumaux et al., 1997). In SAMP1/Yit mice, which develop spontaneous ileitis, inflammation appears to be initiated by Th1 cells but Th2 cells also play a role in later stages (Bamias et al., 2005). Interestingly, luminal bacteria are not required for the development of the disease, whereas experimental colitis is highly dependent on the presence of viable flora (Bamias et al., 2007). Furthermore, in models of adoptive transfer of naive (CD4+CD45RBhigh) T cells into immunodeficient recipiente SCID or RAG KO mice, where naive T cells interact with antigen-presenting cells to become activated to a Th1 disease-producing phenotype, colitis with none or very mild ileal inflammation has been reported (Izcue et al., 2006). On the other hand, Dohi et al. (2003) transferred non interferonproducing Th cells (Th2) cells into T deficient mice and found that ileitis and not colitis was produced. Taken together, these results suggest that the immune cell responses involved in the inflammatory processes in ileitis and colitis may be different. Recently, the role of a novel T-cell subtype, Th17 cells, in a number of inflammatory diseases has been unravelled. At this point, the significance of Th17 cells in inflammatory bowel disease is suspected but unproven (Neurath, 2007).

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Ileitis is not only observed in Crohn’s disease but can also result from intestinal manifestations of several diseases or from ileal bacterial or nematode infections (Sands, 2004; Navarro-Llavat et al., 2007). In addition, ileitis is a frequent complication of the ileal pouch-anal anastomosis interventions practiced to treat ulcerative colitis (Alexander, 2007). The pharmacological therapy of intestinal inflammation is influenced by the segmental localisation of the lesion, which determines the use of particular galenic release forms or different drugs altogether. For instance, sulfasalazine is efficacious in the large intestine, whereas its active moiety 5-aminosalicylic acid (5-ASA) is used as such in case of small intestinal involvement (Baumgart and Sandborn, 2007). In the present study, we have used a model of ileitis induced by the administration of TNBS in rats (Sanchez de Medina et al., 2004) to test the hypothesis that BGMP could be useful in the treatment of ileitis. Our objective was two fold: first, to elucidate whether the intestinal anti-inflammatory effects of BGMP are maintained in the ileum; second, to further explore the mechanism of action of this peptide. The results validate the intestinal anti-inflammatory activity of BGMP at the ileal level and suggest that it may work by downregulating Th17 cells, maintaining regulatory T cells (Tregs) and probably inhibiting macrophages, although additional mechanisms may be operative, such as prebiotic or systemic actions. Methods Animals All animal procedures in this study were carried out in accordance with the Directive for the Protection of Vertebrate Animals used for Experimental and other Scientific Purposes of the European Union (86/609/EEC). Female Wistar rats (175–225 g) obtained from the Laboratory Animal Service of the University of Granada were used, housed individually in makrolon cages and maintained in our laboratory in air-conditioned animal quarters with a 12-h light–dark cycle. Animals were provided with free access to tap water and food (Panlab A04, Panlab, Barcelona, Spain). Induction of ileitis Animals were fasted for 24 h and anaesthetised with halothane. Under these conditions, ileitis was induced, after a middle abdominal laparatomy, by the injection of TNBS in the ileal lumen, approximately 3 cm proximal to the caecum (Sanchez de Medina et al., 2004). Each ileitic rat received 10mg of TNBS dissolved in 0.25mL of 50% ethanol (v/v). Sham-operated rats received an equal volume of saline solution. Injection of ethanol by itself was shown to elicit only a short-lived inflammatory reaction in pilot experiments, as described for TNBS colitis (Morris et al., 1989) and was not explored further (data not shown). Experimental design Rats were randomly assigned to four different groups (n= 6–8): the control group (C), which did not receive the TNBS challenge and three more groups to which ileitis was induced (groups TNBS, 5-ASA and BGMP). The BGMP group received BGMP (500mgkg-1day-1 in 1% methylcellulose p.o.) starting 2 days before the TNBS challenge. The 5-ASA group received 5-ASA (200mgkg-1 day-1in 1% methylcellulose p.o.) in the same conditions, whereas group TNBS together with group C received the vehicle every day. The dose of 5-ASA is equivalent to 2.24 g day-1 for humans on a body surface


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basis, in the high end of normal dosing. Animals were killed 7 days after the induction of ileitis. Assessment of inflammation The distal ileum (approximately 10 cm) was removed and placed on an ice-cold plate, cleaned of fat and mesentery, and blotted on filter paper. Each specimen was weighed and its length measured under a constant load (2 g). The ileum was longitudinally opened and scored for macroscopically visible damage by an observer unaware of the treatment, according to the criterion previously proposed by us (Daddaoua et al., 2005), but slightly modified. The score was assigned as follows: adhesions (0–3), obstruction (0–2), hyperaemia (0–3), fibrosis (rigidity and deformation, 0–5), necrosis (0–5) and other features (proximal dilatation, fragility, scarring, 0–4). The ileum was subsequently divided in several longitudinal pieces for biochemical determinations. The fragments were immediately frozen in liquid nitrogen and kept at -80ºC until used. Biochemical determinations Alkaline phosphatase (AP) activity was measured spectrophotometrically, using disodium p-nitrophenylphosphate (5.5mM) as substrate in 50mM glycine buffer with 0.5mM MgCl2 (pH=10.5). Results are expressed as mU per mg of protein. Sensitivity to the AP inhibitor, levamisole (1mM), was measured and results are expressed as per cent inhibition of AP activity (Sanchez de Medina et al., 2004). Myeloperoxidase (MPO) activity was measured according to the technique described by Krawisz et al. (1984), using 0.5% hexadecyltrimethylammonium bromide in phosphatebuffered saline (pH=6.0) for tissue homogenisation and odianisidine dihydrochloride (0.5mM) as chromogen. The results are expressed as MPO units (μmol min-1) per gram of wet tissue.

Analysis of gene expression by reverse transcriptase-PCR analysis The expression of IL-1β, IL-1 receptor antagonist (IL-1ra), tumour-necrosis factor (TNF), IL-17 and trefoil factor 3 (TFF3) was examined by reverse transcriptase RT-PCR. For RT-PCR analysis, total RNA was extracted with Trizol (Invitrogen, Barcelona, Spain). Five micrograms of RNA per sample were subjected to reverse transcription using the First-strand cDNA synthesis kit (GE Healthcare, Madrid, Spain). PCR amplification was performed using 2 ml of cDNA for a final PCR reaction volume of 25 ml. TAq polymerase was purchased from GE Healthcare. The expression of the ribosomal 18S unit was examined as a standard of loading. The primers of the amplified fragments were: IL-1β (sense 50-AATGACCTGTTCTTTGAGGCTG-30; antisense 50-CGAGATGCTGCTGTGAGATTTGAAG-30); IL-1ra (sense 50-GAGTCAGCTGG CCACCCTG-30; antisense 50-CAGACTTGACACAAGACAGGCA-30); TNF (sense 50-TACTGAACTTCGGGGTGATTGGTCC-30; antisense 50-CAGCCTTGTCCCTT GAAGAGAACC-30); IL-17 (sense 50-TTCTCCAGAACGTGAAGGTC-30; antisense 50-GGACAATAGAGGAAACGCAG-30); TFF3 (sense 50-ATGGAGACCAGAGCC TTCTG-30; antisense 50-ACAGCCTTGTGCTGACTGTA-30); ribosomal 18S unit (sense 50-CCATTGGAGGGCAAGTCTGGTG-30; antisense 50-CGCCGGTCCAA GAATTTCACC-30). To set up the PCR conditions, different amounts of colonic RNA from a pool of samples and different Lumber of cycles were assayed (data not shown). The cycle numbers and hybridisation temperatures for each PCR were as follows: for IL-1β (32 cycles and 57 ºC), IL-1ra (40 cycles and 57 ºC), for TNF (32 cycles and 61 ºC), for IL-17 (35 cycles and 51 ºC), for TFF3 (25 cycles and 59 ºC) and for ribosomal 18S unit (15 cycles and 60 ºC). After the PCR amplification 10 ml of each reaction were

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resolved in 2.5% (w/v) agarose gels. Bands were quantitated with NIH software (Scion Image). Western blots The colonic levels of inducible oxide nitric synthase (iNOS), COX-2 and Foxp3 were determined by immunoblotting. Colonic samples were homogenised in lysis buffer (0.1% w/v SDS, 0.1% w/v sodium deoxycholate, 1% v/v Triton X-100 in phosphate-buffered saline) with protease inhibitors (1mM 1,10-phenanthroline, 1mM phenylmethylsulphonyl fluoride and 18mgl-1 aprotinin). The supernatants obtained after centrifugation (7000 g, 10 min at 4 ºC) were boiled for 4 min in Laemmli buffer, separated by SDS-PAGE (10%), electroblotted to nitrocellulose membranes and probed with the corresponding antibodies overnight at 4 ºC. The bands were detected by enhanced chemiluminescence (PerkinElmer) and quantitated with NIH software (Scion Image). After the transference of the samples to nitrocellulose membranes, equal loading was checked routinely by reversible Ponceau staining. The composition of the Laemmli buffer (5X) was: 312mM SDS, 50% v/v glycerol, 1% v/v 2-mercaptoethanol, 22.5mM EDTA trisodium salt, 220mM Tris and traces of bromophenol blue (pH¼6.8). Mesenteric lymph node cells Mesenteric lymph nodes were extracted from the rats in the study using sterile techniques and dissected mechanically. The cells were incubated in Dulbecco’s modified Eagle’s medium supplemented with 10% v/v fetal bovine serum (Boëhringer Mannheim, Barcelona, Spain), 100mg l-1 streptomycin, 100 000U l-1 penicillin and 2.5mg l-1 amphotericin B. The cells were cultured at 106 cells per mL and stimulated with concanavalin A. Cell culture medium was collected after 24 h and assayed for cytokine content by commercial ELISAs (Biosource Europe, Nivelles, Belgium and BD Biosciences, Erembodegem, Belgium). Statistical analysis The results are expressed as mean±s.e.m. Differences among means were tested for statistical significance by one-way analysis of variance and a posteriori least significance tests on preselected pairs. Nonparametric data (ileum damage score) were expressed as median (25–75% quartiles) and analysed by one-way analysis of variance on ranks followed by Dunn’s tests. All the analyses were carried out with the SigmaStat program (Jandel Corporation, San Rafael, CA, USA). Statistical significance was set at P<0.05.

Materials Except where indicated, all reagents and primers were obtained from Sigma (Barcelona, Spain). BGMP (BioPUREGMP) was a kind gift from Davisco Foods International, Inc. (Eden Prairie, MN, USA). Product certificates of análisis indicated that BGMP content was 93% whereas fat and lactose contents were 0.5% and less than 1% respectively. The BGMP product also contained small amounts of b-lactoglobulin and a-lactalbumin (0–4 and 1–2% of total protein, respectively) and 4.0% minerals. Results As expected, the induction of ileitis produced anorexia and a significant body weight loss in the TNBS group (Table 1). Furthermore, a high damage score was assigned to


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the ileal specimens owing mainly to the presence of epithelial necrosis, ileal adherences and fibrosis, producing rigidity and obstruction. Intestinal fibrosis resulted in an elevated weight to length ratio, which did not reach statistical significance, possibly because the segment of ileum taken for analysis was too long. The administration of either BGMP or 5-ASA failed to prevent the anorexia produced by ileitis (data not shown). Body weight loss in the rats treated with either BGMP or 5-ASA was intermediate between that of the control and TNBS groups, without reaching statistical significance (Table 1). The benefits of BGMP administration were appreciated when macroscopic damage parameters were studied. Thus, the administration of BGMP reduced significantly the extent of necrosis and the intestinal damage score (Table 1). The latter was attributable to a favourable effect on adherences, necrosis, obstruction and fibrosis (data not shown). The ileal weight to length ratio was not modified significantly. These effects were comparable to those of 5-ASA, although the damage score in this group was still significantly different from that of the control, unlike BGMP. Table 1. Effect of GMP on body weigh gain and intestinal macroscopic parameters.

Body weight gain (%)

Damage score

Extent of necrosis (cm)

Ileon weight:length ratio (mg cm-1)

C −0.9±0.6 0.0±0.0 0.0±0.0 64.8±3.4

TNBS −10.5±3.7+ 7.0 (3.5-9.5)+ 1.6±0.5+ 112.2±20.3

5-ASA −5.0±1.5 3.5 (2.0-6.0)+,* 0.3±0.1* 101.1±9.8

BGMP −5.6±1.8 4.0 (1.3-6.3) +,* 0.6±0.4* 106.1±27.9

Abbreviations: C, control; TNBS, trinitrobenzenesulfonic acid; 5-ASA, 5-aminosalicylic acid; BGMP, glycomacropeptide. The doses of 5-aminosalicylic acid and glycomacropeptide were 200 and 500 mg kg-1 day-1, respectively. Body weight gain is expressed as percent change from the start of the experiment. Values are means ±s.e.mean, n=6-8. +P<0.05 vs. control (C) group; *P<0.05 vs. TNBS group.

The biochemical characterisation of the ileal inflammatory response confirmed the expected features of increased AP (leukocyte infiltration and epithelial cell stress), MPO (neutrophil infiltration), COX-2 and iNOS (Figures 1 and 2). In accordance with their anti-inflammatory effect, the administration of either BGMP or 5-ASA resulted in a normalisation of AP and MPO activities and a marked reduction in the expression of COX-2 and iNOS (the effect of BGMP on COX-2 did not reach statistical significance).We also studied the effect on the expression of TFF3, a peptide with epithelial-regenerating properties. TFF3 was upregulated in ileitis and BGMP treatment normalised its expresión fully, whereas 5-ASA did not affect this parameter significantly (Figure 3). Thus TFF3 is unlikely to play a role in the mechanism of action of BGMP. From an immunological point of view, TNBS ileitis was characterised by an increase in the expression of the proinflammatory cytokines IL-1β and TNF-α, as well as of the

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anti-inflammatory IL-1ra, a natural antagonist that opposes both IL-1a and IL-1β activities. BGMP and 5-ASA reduced the IL-1β, TNF-α and IL-1ra mRNA levels to the levels found for control rats (Figure 3). In an attempt to elucidate whether the effect of BGMP is mediated by actions on lymphocytes, we examined the behaviour of mononuclear cells isolated from mesenteric lymph nodes. We focused on Th1 signature cytokines, namely TNF-α, IL-2 and interferon-γ. As expected, the mesenteric lymph node cells isolated from rats affected by ileitis produced higher levels of proinflammatory cytokines when stimulated in vitro; however, neither BGMP nor 5-ASA showed any effect at this level, suggesting that the mechanism of action in both cases does not involve Th1 cells (data not shown).

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Figure 1. Effect of BGMP (bovine glycomacropeptide) on ileal AP (alkaline phosphatase) and myeloperoxidase (MPO) activity. (A) Ileal AP activity. (B) AP sensitivity to the inhibitor levamisole (1 mM). (C) Ileal MPO activity. Values are means ± s.e.m., n=6-8. +P<0.05 vs. control (C) group; *P<0.05 vs. TNBS group.

Next we addressed the role of other T-cell subtypes, namely Tregs and Th17 cells. To this end, we measured Foxp3 levels by western blot and IL17 mRNA levels by RTPCR in whole ileal tissue (Figures 2 and 3). The expression of Foxp3, a transcription factor that drives Treg phenotypic differentiation, was markedly increased in rats with ileitis, compared to control rats, indicating that Treg number or function is augmented as part of the inflammatory response. Foxp3 expression was not reduced significantly by treatment with either BGMP or 5-ASA and remained elevated compared to the control group, although the levels were intermediate between those of the C and TNBS rats. On the other hand, IL-17 mRNA, which is characteristic of Th17 cells, was markedly increased in the inflamed ileum, suggesting an involvement of this cell type in TNBS


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ileitis. Interestingly, the administration of either 5-ASA or BGMP normalised these parameters, suggesting that Th17 cells may be a target of both therapies.

Figure 2. Effect of BGMP on COX2, inducible nitric oxide synthase (iNOS) and Foxp3 protein levels. Ileal samples were analyzed by Western blot and quantitated with Scion Image software. Representative blots are shown. Values are means ± s.e.m, n=6-8. +P<0.05 vs. control (C) group; *P<0.05 vs. TNBS group.

Figure 3. Effect of BGMP on the ileal mRNA levels of interleukin (IL)-1β, IL-1ra, trefoil factor 3 (TFF3), tumor necrosis factor (TNF) and IL-17. mRNA levels were measured by semiquantitative RT-PCR and densitometric analysis with Scion Image software. 18S mRNA was used as reference. Densitometric analysis represents the ratio over the 18S signal. Representative blots are shown. Values are means ± s.e.m., n=6-8. +P<0.05 vs. control (C) group; *P<0.05 vs. TNBS group.

Discussion Bovine glycomacropeptide has been described as an inexpensive, nontoxic natural product with a number of biological properties. We have previously established the intestinal anti-inflammatory activity of BGMP in an experimental model of colitis (Daddaoua et al., 2005). The beneficial effect of BGMP was found to be maximal when administered as a pretreatment at the dose of 500mg kg-1, that is the same condition used in the present study and was comparable to the protection afforded by oral sulfasalazine. BGMP had a significant impact on the upregulation of IL-1β and iNOS brought about by colonic inflammation, but its mechanism of action was not characterised further. Thus, we designed the present study to confirm that BGMP can act as an intestinal anti-inflammatory treatment at the ileal, rather than the colonic, segment and to explore its mechanism of action further. It should be noted that the BGMP product assayed was not completely pure but included some minor components,

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namely β-lactoglobulin, α-lactalbumin and unspecified fatty substances. β-lactoglobulin has no clearly identified biological function, although it seems to protect hydrophobic substances from gastric secretions (Madureira et al., 2007). α-lactalbumin appears to have some effects on cell division, which are concentration-dependent, and there is some evidence that it may influence B-cell response (Bounous and Kongshavn, 1985; Madureira et al., 2007). Because their relative concentration is rather low (equivalent to that in milk, actually), it is unlikely that they play a role in the anti-inflammatory effect observed, but it certainly cannot be ruled out based on existing evidence. On the other hand, while fat content is uncharacterised, it should be noted that after purification, the product is maintained at 4ºC, conditions under which any active eicoisanoid products are extremely unlikely to survive. Our data demonstrate that BGMP does have a significant therapeutic effect in the TNBS model of ileitis in the rat, as shown by the reduction in the extension of necrosis, damage score, MPO, AP and iNOS. We could not detect significant changes in the ileal weight to length ratio, possibly because we examined relatively long segments (~10 cm), which contained substantial amounts of normal tissue, thus ‘diluting’ the impact of inflammation. The anti-inflammatory effect of BGMP was generally comparable to that of 5-ASA, a drug used profusely in the treatment of ileal Crohn’s disease. Interestingly, BGMP failed to combat anorexia and only counteracted body weight loss to a limited extent, while it did have beneficial effects in the colitis model. The reason for this discrepancy is unknown, although it might be related to the fact that TNBS colitis, but not ileitis, is accompanied by diarrhoea. Thus, it is possible that BGMP improves food intake/weight gain in colitis by reducing the deleterious effects of diarrhoea. The effect of BGMP is linked to a marked reduction in TNF-α and IL-1β. These proinflammatory cytokines have a wide and largely overlapping array of biological effects, such as endothelial activation, monocyte stimulation, induction of acute phase proteins, etc. In particular, TNF-α is a known molecular target in human inflammatory bowel disease, although it is unclear whether the drugs that act by binding TNF-α work by cytokine neutralisation or by inducing apoptosis or targeted cell lysis in the cells expressing membrane-bound TNF-α (Gottlieb, 2007; Nesbitt et al., 2007). One of the main sources of both TNF-α and IL-1β are monocytes/macrophages, suggesting that this cell type may be affected directly or indirectly by BGMP. BGMP has been reported to stimulate the proliferation and phagocytic activity of U937 monocytic cells, but no cytokines were measured (Li and Mine, 2004). We have preliminary evidence that BGMP evokes cytokine secretion by THP-1 cells, another human monocyte cell line, but the effect may differ in primary monocytes depending on tissue of origin (data not shown). Inasmuch as BGMP appears to have a stimulatory effect on monocytes/macrophages, the inhibition of TNF-α and IL-1β observed in vivo is most likely to be indirect. Of note, BGMP has been reported to increase IL-1ra (Monnai and Otani, 1997), an action that has potential antiinflammatory impact. However, our data do not support that this is an important mechanism in this case, as IL-1ra levels are drastically reduced rather than increased. Because BGMP has been also reported to exert modulatory effects on T cells (Otani et al., 1995, 1996; Monnai et al., 1998), we examined the expression of cytokines by mesenteric node cells isolated from the different experimental groups. We focused on Th1 signature cytokines, that is, interferon-γ, TNF-α and IL-2. Although there was a significant increase in the release of all three cytokines by concanavalin A stimulated


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lymphocytes from ileitis animals, neither BGMP not 5-ASA had any effect. Hence it is unlikely that any of these compounds modulates ileal inflammation through an action on Th1 cells. Finally, we focused on other T-cell types, which may play a role in the pathophysiology of intestinal inflammation, namely Tregs and Th17 cells. Tregs can develop intrathymically or in peripheral tissues and are believed to be an essential safeguard to limit excessive inflammatory/immune responses (Izcue et al., 2006). In fact, the transcription factor which drives the phenotypic conversion of naive T cells toTregs, Foxp3, was originally identified as a defective gene in the so-called scurfy mice, which succumb early in life to dramatic autoimmune disease manifestations (Ochs et al., 2007). The role of Tregs in inflammatory bowel disease has been insufficiently investigated to date, but the evidence available from animal models suggests that an imbalance in T helper/Treg cell function is sufficient to cause chronic intestinal inflammation (Fantini et al., 2007). We have observed that TNBS ileitis is associated with a significant increase in Foxp3 immunoreactivity, suggesting that the inflammatory reaction is accompanied by augmented Treg cell function and/or number. Interestingly, Foxp3 levels remained significantly elevated after successful treatment with both BGMP and 5-ASA, that is, Treg cells are similarly present in treated and untreated rats despite the fact that intestinal inflammation is actually tempered. Thus, Tregs may be induced by BGMP and 5-ASA and participate in their therapeutic effect. Alternatively, it is possible that Tregs are induced early in the inflammatory process and are not affected promptly by amelioration of the disease. Further studies are required to clarify this issue. On the other hand, Th17 cells are emerging as a novel and important T-helper subtype that may be relevant to tissue damage in diseases formerly thought to depend on Th1 cells, like encephalomyelitis, rheumatoid arthritis and allergic airway hypersensitivity (Weaver et al., 2007). Th17 cells may be developed extrathymically by the exposure of T helper naive cells to IL-6/transforming growth factor-β (Bettelli et al., 2007). Mature Th17 cells are characterised by the production of IL-17, which in turn can activate signalling pathways that result in activation of NF-kB, induction of iNOS, COX-2, etc. Although IL-23 (involved in Th17 development/maintenance) and IL-17 are both increased in inflammatory bowel disease and IL23R appears to be a susceptibility gene, interventional studies that confirm its relevance are limited so far to animal Studies (Neurath, 2007). Our data confirmed that IL-17 mRNA was increased in TNBS ileitis, consistent with a role of Th17 cells in this model. Interestingly, both BGMP and 5-ASA reduced IL-17 expression to control levels, indicating that the mechanism of action may involve interference with Th17 cells. Further experiments are required to test this hypothesis specifically. In addition to the aforementioned direct mechanisms, BGMP may ameliorate ileal inflammation by prebiotic effects, as suggested by other investigators (Idota et al., 1994; Yakabe et al., 1994; Brody, 2000). We could not obtain suitable samples for bacterial measurements because of the localisation of intestinal inflammation in this particular study, but we plan to assess this hypothesis in future experiments using faecal samples in a colitis model. Previous animal and human studies in vivo have failed to show a substantial impact of BGMP on intestinal flora, but they were hampered by high bifidobacterial counts before dietary intervention (Bruck et al., 2003, 2006). Interestingly, in one of these studies, BGMP was shown to augment circulating

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neutrophils and to partially prevent diarrhoea induced by enteropathogenic Escherichia coli (Bruck et al., 2003). This observation is consistent with Zimecki et al. (2006), who recently reported that parenteral BGMP has protective effects against experimental bacteremia in mice, which were associated to an increase in circulating granulocytes. Thus it is possible that the anti-inflammatory effect of BGMP involves systemic actions. Little is known about the pharmacokinetics of BGMP, but two studies have previously shown that it reaches the bloodstream in significant amounts (~1 μg mL-1) and the concentration remains relatively stable for at least 8 h, in addition to being detected in the duodenum shortly after oral intake (Chabance et al., 1995, 1998). Therefore, the intestinal anti-inflammatory effect observed may be due to luminal and/or systemic actions. In summary, we have demonstrated that BGMP is an intestinal anti-inflammatory agent in the ileal segment with an efficacy similar to that of 5-ASA, using a preclinical model. The mechanism of action is related to inhibition of IL-1β and TNF and possibly a downregulation of Th17 cells. Treg cells may be also involved. Additionally, BGMP may exert prebiotic and/or systemic actions. BGMP may be a valuable tool in the therapy of Crohn’s ileitis. Acknowledgements

CIBEREHD is funded by the Instituto de Salud Carlos III. This study was supported by grants of the Instituto de Investigación Carlos III (PI051651 and PI051625) and funds from the Junta de Andalucía. Olga Martínez Augustin was funded by the I3 program of the Ministry of Education and Science. Pilar Requena was funded by the Spanish Ministry of Science and Technology. Conflict of interest The authors state no conflict of interest. References Aimutis WR (2004). Bioactive properties of milk proteins with particular focus on anticariogenesis. J

Nutr 134: 989S–995S. Alexander F (2007). Complications of ileal pouch anal anastomosis. Semin Pediatr Surg 16: 200–204. Bamias G, Martin C, Mishina M, Ross WG, Rivera-Nieves J, Marini M et al. (2005). Proinflammatory

effects of TH2 cytokines in a murine model of chronic small intestinal inflammation. Gastroenterology128: 654–666.

Bamias G, Okazawa A, Rivera-Nieves J, Arseneau KO, De La Rue SA, Pizarro TT et al. (2007). Commensal bacteria exacerbate intestinal inflammation but are not essential for the development of murine ileitis. J Immunol 178: 1809–1818.

Baumgart DC, Carding SR (2007). Inflammatory bowel disease: cause and immunobiology. Lancet 369: 1627–1640.

Baumgart DC, Sandborn WJ (2007). Inflammatory bowel disease: clinical aspects and established and evolving therapies. Lancet 369:1641–1657.

Bettelli E, Korn T, Kuchroo VK (2007). Th17: the third member of the effector T cell trilogy. Curr Opin Immunol 19: 652–657.

Bounous G, Kongshavn PA (1985). Differential effect of dietary protein type on the B-cell and T-cell immune responses in mice. J Nutr 115: 1403–1408.

Brody EP (2000). Biological activities of bovine glycomacropeptide. Br J Nutr 84: S39–S46. Bruck WM, Kelleher SL, Gibson GR, Nielsen KE, Chatterton DEW, Lonnerdal B (2003). rRna probes

used to quantify the effects of glycomacropeptide and alpha-lactalbumin supplementation on the


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predominant groups of intestinal bacteria of infant Rhesus monkeys challenged with enteropathogenic Escherichia coli. J Pediatr Gastroenterol Nutr 37: 273–280.

Bruck WM, Redgrave M, Tuohy KM, Lonnerdal B, Graverholt G, Hernell O et al. (2006). Effects of bovine alpha-lactalbumin and casein glycomacropeptide-enriched infant formulae on faecal microbiota in healthy term infants. J Pediatr Gastroenterol Nutr 43: 673–679.

Chabance B, Jolles P, Izquierdo C, Mazoyer E, Francoual C, Drouet L et al. (1995). Characterization of an antithrombotic peptide from kappa-casein in newborn plasma after milk ingestion. Br J Nutr 73: 583–590.

Chabance B, Marteau P, Rambaud JC, Migliore-Samour D, Boynard M, Perrotin P et al. (1998). Casein peptide release and passage to the blood in humans during digestion of milk or yogurt. Biochimie 80: 155–165.

Daddaoua A, Puerta V, Zarzuelo A, Suarez MD, Sanchez de Medina F, Martinez-Augustin O (2005). Bovine glycomacropeptide is antiinflammatory in rats with hapten-induced colitis. J Nutr 135: 1164–1170.

Desreumaux P, Brandt E, Gambiez L, Emilie D, Geboes K, Klein O et al. (1997). Distinct cytokine patterns in early and chronic ileal lesions of Crohn’s disease. Gastroenterology 113: 118–126.

Dohi T, Fujihashi K, Koga T, Shirai Y, Kawamura YI, Ejima C et al.(2003). T helper type-2 cells induce ileal villus atrophy, goblet cell metaplasia, and wasting disease in T cell-deficient mice. Gastroenterology 124: 672–682.

Fantini MC, Rizzo A, Fina D, Caruso R, Becker C, Neurath MF et al. (2007). IL-21 regulates experimental colitis by modulating the balance between T(reg) and Th17 cells. Eur J Immunol 37: 3155–3163.

Gottlieb AB (2007). Tumor necrosis factor blockade: mechanism of action. J Investig Dermatol Symp Proc 12: 1–4.

Idota T, Kawakami H, Nakajima I (1994). Growth-promoting effects of N-acetylneuraminic acid-containing substances on Bifidobacteria. Biosci Biotech Biochem 58: 1720–1722.

Izcue A, Coombes JL, Powrie F (2006). Regulatory T cells suppress systemic and mucosal immune activation to control intestinal inflammation. Immunol Rev 212: 256–271.

Krawisz JE, Sharon P, StensonWF (1984). Quantitative assay for acute intestinal inflammation based on myeloperoxidase activity. Assessment of inflammation in rat and hamster models. Gastroenterology 87: 1344–1350.

Li EW, Mine Y (2004). Immunoenhancing effects of bovine glycomacropeptide and its derivatives on the proliferative response and phagocytic activities of human macrophagelike cells, U937. J Agric Food Chem 52: 2704–2708.

Madureira AR, Pereira CI, Gomes AMP, Pintado ME, Malcata FX (2007). Bovine whey proteins—overview on their main biological properties. Food Res Intern 40: 1197–1211.

Mikkelsen TL, Rasmussen E, Olsen A, Barkholt V, Frokiaer H (2006). Immunogenicity of kappa-casein and glycomacropeptide. J Dairy Sci 89: 824–830.

Monnai M, Horimoto Y, Otani H (1998). Immunomodificatory effect of dietary bovine kappa-caseinoglycopeptide on serum antibody levels and proliferative responses of lymphocytes in mice. Milchwissenschaft 53: 129–132.

Monnai M, Otani H (1997). Effect of bovine k-caseinoglycopeptide on secretion of interleukin-1 family cytokines by P388D1 cells, a line derived from mouse monocyte/macrophage. Milchwissenschaft 52: 192–196.

Morris GP, Beck PL, Herridge MS, Depew WT, Szewczuk MR, Wallace JL (1989). Hapten-induced model of chronic inflammation and ulceration in the rat colon. Gastroenterology 96: 795–803.

Nakai S, Modler HW (1999). Food Proteins. Processsing applications. Wiley-VCH Inc.: New York. Navarro-Llavat M, Domenech E, Masnou H, Ojanguren I, Manosa M, Lorenzo-Zuniga V et al. (2007).

Collagenous duodeno-ileo-colitis with transient IgG deficiency preceded by Yersinia enterocolitica intestinal infection: case report and review of literature. Gastroenterol Hepatol 30: 219–221.

Nesbitt A, Fossati G, Bergin M, Stephens P, Stephens S, Foulkes R et al. (2007). Mechanism of action of certolizumab pegol (CDP870): in vitro comparison with other anti-tumor necrosis factor alpha agents. Inflamm Bowel Dis 13: 1323–1332.

Neurath MF (2007). IL-23: a master regulator in Crohn disease. Nat Med 13: 26–28. Ochs HD, Gambineri E, Torgerson TR (2007). IPEX, FOXP3 and regulatory T-cells: a model for

autoimmunity. Immunol Res 38: 112–121. Otani H, Horimoto Y, Monnai M (1996). Suppression of interleukin-2 receptor expression on mouse

CD4þ T cells by bovine kappacaseinoglycopeptide. Biosci Biotechnol Biochem 60: 1017–1019.

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Otani H, Monnai M, Kawasaki Y, Kawakami H, Tanimoto M (1995). Inhibition of mitogen-induced proliferative responses of lymphocytes by bovine kappa-caseinoglycopeptides having different carbohydrate chains. J Dairy Res 62: 349–357.

Sanchez de Medina F, Martinez-Augustin O, Gonzalez R, Ballester I, Nieto A, Galvez J et al. (2004). Induction of alkaline phosphatase in the inflamed intestine: a novel pharmacological target for inflammatory bowel disease. Biochem Pharmacol 68: 2317–2326.

Sands BE (2004). From symptom to diagnosis: clinical distinctions among various forms of intestinal inflammation. Gastroenterology 126: 1518–1532.

Sands BE (2007). Inflammatory bowel disease: past, present, and future. J Gastroenterol 42: 16–25. Weaver CT, Hatton RD, Mangan PR, Harrington LE (2007). IL-17 family cytokines and the expanding

diversity of effector T cell lineages. Annu Rev Immunol 25: 821–852. Yakabe T, Kawakami H, Idota T (1994). Growth stimulation agent for bifidus and lactobacillus. Japanese

patent 07-267866. Zimecki M, Artym J, Chodaczek G, Kocieba M, Rybka J, Skorska A et al. (2006). Glycomacropeptide

protects against endotoxemia and bacteremia in mice. Electr J Polish Agricult Univ 9 (online).


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Bovine glycomacropeptide induces cytokine production in human monocytes through the stimulation of the MAPK and the NF-kB signal transduction pathways .

Pilar Requena, Abdelali Daddaoua, Emilia Guadix, Antonio Zarzuelo, María Dolores Suárez, Fermín Sánchez de Medina, Olga Martínez-Augustin. British Journal of Pharmacology (2009), 157,1232–1240

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Background and purpose: Bovine glycomacropeptide (BGMP) is a natural milk peptide that is produced naturally in the gastrointestinal tract during digestion. Glycomacropepide has intestinal anti-inflammatory activity, but the mechanism of action is unknown. Here we have characterized the effects of BGMP on monocytes. Experimental approach: We have used human THP-1 cells as an in vitro monocyte model. The effect of BGMP on the secretion of tumour necrosis factor (TNF), interleukin (IL)-1β and IL-8 was assessed, as well as the involvement of the NF-κB and MAP kinase signalling pathways. The stimulatory effect of BGMP was also tested in human peripheral blood monocytes. Key results: BGMP up-regulated the secretion of TNF, IL-1β and IL-8 in a concentration-dependent fashion. The biological activity was exerted by the intact peptide, because cytokine secretion was not affected by protease inhibitors. The secretion of IL-8 and specially TNF and IL-1β was blocked by PD98059, SP600125, SB203580 and Bay11-7082, suggesting the involvement of the MAP kinases p38, c-Jun N-terminal kinase and ERK and particularly the NF-κB pathway, although IL-8 secretion was independent of p38. BGMP was shown to elicit the phosphorylation of IκB-α and the nuclear translocation of the NF-κB subunits p50 and p65. The effect of BGMP on cytokine secretion was validated in human primary blood monocytes. Conclusions and implications: BGMP stimulates human monocytes, operating via MAP kinase and NF-κB pathways. BGMP may exert an indirect intestinal anti-inflammatory effect by potentiating host defences against invading microorganisms. Keywords: bovine glycomacropeptide; THP-1 cells; NF-κB Abbreviations: BGMP, bovine glycomacropeptide; FBS, foetal bovine serum; IKK, IκB kinase Introduction The concept of milk as a biologically active fluid was established a long time ago (Newby et al., 1982), and in the last decades the therapeutic value of milk proteins and peptides has been widely described (Brody, 2000; Cross and Gill, 2000; Clare et al., 2003; Florisa et al., 2003; Zimecki and Kruzel, 2007). Bovine glycomacropeptide (BGMP) or casein macropeptide is one of the biologically active components of milk (Brody, 2000; Zimecki and Kruzel, 2007). BGMP is a 64-amino-acid peptide, corresponding to amino acids 106– 169 of κ-casein, which contains N-acetylneuraminic (sialic) acid and is physiologically obtained in the stomach of neonates (and adults) by the chymosin digestion of native protein (Brody, 2000; Zimecki and Kruzel, 2007). The industrial source of BGMP is milk whey, produced during the cheese-making process, in which BGMP is the result of the action of the rennet chymosin (Brody, 2000). BGMP is currently added to infant formulas and, due to its low content of aromatic amino acids including phenylalanine, it has been proposed to be useful in the elaboration of products for individuals with phenylketonuria (Lim et al., 2007). In addition, BGMP is included in toothpaste because of its anti-cariogenic properties; it

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has antibacterial activity, preventing dental caries and tooth demineralization, while promoting enamel mineralization (Aimutis, 2004). A wide array of additional BGMP activities have been described, including its ability to bind cholera and Escherichia coli enterotoxins, the inhibition of bacterial and viral adhesions, the promotion of bifidobacterial growth and the modulation of immune system responses (Brody, 2000; Nakajima et al., 2005; Bruck et al., 2006). In this regard, we have established the anti-inflammatory effect of BGMP in a rat model of colitis (Daddaoua et al., 2005) and recently also in experimental ileitis (Requena et al., 2008). Interestingly, it has been recently reported that BGMP does not induce T cell -mediated immune responses in vivo, unlike the native protein κ-casein, even when administered as a polymer (Mikkelsen et al., 2006).There are some studies available on the effect of BGMP on cells of the immune system in vitro, most of which have focused on lymphocytes. Thus, BGMP has been claimed to inhibit lipopolysaccharide (LPS)-induced splenocyte proliferation (Otani and Monnai, 1993; Mikkelsen et al., 2005), to suppress interleukin (IL)-2 receptor expression in mouse CD4+ T cells (Otani et al., 1996) and to block serum IgG antibody production by mouse lymphocytes (Monnai et al., 1998). There are only two studies focused on the effect of BGMP on macrophages showing that BGMP augments the secretion of IL-1receptor antagonist (IL-1ra), without affecting IL-1β, in a mouse monocytic cell line (Monnai and Otani, 1997), and that it enhances the proliferation and phagocytic activity of human macrophage-like cells (Li and Mine, 2004). Of note, the intestinal anti-inflammatory effect of BGMP is associated with a marked decrease in intestinal tumour necrosis factor (TNF) and IL-1β, cytokines mainly derived from monocytes/macrophages, suggesting that this cell type may be affected directly or indirectly (i.e. via hydrolytic peptide fragments) by BGMP (Daddaoua et al., 2005; Requena et al., 2008). Thus, we designed the present study to characterize the immunoregulatory effect of BGMP in the innate immune response mediated by monocyte/macrophages and to determine the intracellular signalling pathways that could be regulated by this peptide. Specifically, we focused on the pro-inflammatory cytokines expressed by monocytes/macrophages, which play an important role in intestinal inflammation, expecting a down-regulation by BGMP. We used the human monocyte THP-1 cell line as a model and validated the main results in human primary blood monocytes. This study demonstrates that BGMP did not inhibit and actually enhanced the expression of TNF, IL-1β and IL-8 in monocytes and that its mechanism of action is related to the stimulation of the MAP kinase (MAPK) and the IκB/NF-κB signal transduction pathways. Methods Cell culture The human monocytic cell line THP1 (ECACC 88081201) was cultured in RPMI-1640 medium supplemented with 10% (v·v-1) heat-inactivated foetal bovine serum (FBS), 2 mmol·L-1 glutamine, 100 U·L-1 penicillin, 0.1 mg·mL-1 streptomycin, 2.5 mg·mL-1 amphotericin B and 0.05 mmol·L-1 mercaptoethanol. Cells were incubated under humidified 5% CO2 atmosphere at 37°C. The cells were cultured at a concentration of 106 cells·mL-1. Monocytes were isolated from human blood obtained from volunteers. All the

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participants provided an informed consent and the protocol was approved by the Research Ethics Committee of the University of Granada. In brief, heparinized peripheral blood was diluted 1:2 with sterile saline and separated using Ficoll (GE Healthcare, Barcelona, Spain). The mononuclear cell preparation was washed once in sterile Hank’s balanced salt solution and placed on a culture dish for 1 h. Adherent cells were retrieved and plated at a density of 106 cells·mL-1 for assay. Human primary monocytes were cultured in DMEM medium supplemented with as above but without mercaptoethanol. Effect on cytokine secretion Cytokine secretions were determined in THP-1 cells cultured in the presence of BGMP, bovine serum albumin or casoplatelin for 24 h. After 24 h cells were collected and cytokine concentration was measured by ELISA (Biosource Europe, Nivelles, Belgium and BD Biosciences, Erembodegem, Belgium), following the protocols recommended by the manufacturer. In some experiments the serine protease inhibitor Pefabloc® SC [4-(2-aminoethyl)-benzenesulphonyl fluoride, hydrochloride, 0.1 mmol·L-1, Roche Applied Science, Mannheim, Germany] or a protease inhibitor cocktail (1:200 v·v-1, Sigma) – containing 4-(2-aminoethyl)-benzenesulphonyl fluoride, aprotinin, leupeptin, bestatin, pepstatin A and E-64 – was added to the cell medium together with BGMP(1 mg·mL-1), the cells were incubated for 24 h and cytokine concentration was measured as described above. Cell proliferation assay THP-1 cells were incubated for 24 h with different concentrations of BGMP and then cells were pulsed for 6 h with [3H] thymidine (0.6 μCi·mL-1; GE Healthcare, Spain). The cells were subsequently harvested, washed three times with trichloroacetic acid (10% v·v-1), resuspended in lysis buffer (1% w·v-1 SDS, 0.3 N NaOH) for 30 min at room temperature and collected into plastic vials. Then 4 mL of scintillation liquid (Beckman Coulter, Madrid, Spain) per vial was added and the amount of incorporated [3H] thymidine was determined on a Tri-Carb liquid scintillation analyser (Packard Instrument, Meriden, CT). Lactate dehydrogenase assay Cellular toxicity was measured as the release of lactate dehydrogenase. Cells were cultured with different concentrations of BGMP for 24 h and lactate dehydrogenase activity in supernatants was measured spectrophotometrically using sodium pyruvate (25 mmol·L-1) as substrate in 50 mmol·L-1 sodium phosphate buffer (pH = 7.5) (Halprin and Ohkawara, 1966). NF-κB and MAPK inhibitors assay In order to explore signalling pathways, the kinase inhibitors [PD98059 for the mitogen-activated protein kinase MAPK ERK1/2, SB203580 for p38 MAPK, SP600125 for c-Jun N-terminal kinase (JNK) and Bay11-7082 for NF-κB] were added to the cell culture medium (10 μmol·L-1 in all cases) 1 h before the addition of the BGMP (1 mg·mL-1). Then the cells were incubated for 24 h and the supernatants were used to determine cytokine concentrations as described above. Western blot For the detection of phosphorylated IκB-α, cells were homogenized in lysis buffer


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(0.1% w·v-1 SDS, 0.1% w·v-1 sodium deoxycholate, 1% v·v-1 Triton X-100 in PBS) with protease inhibitor cocktail 1:100 (v·v-1). Then homogenates were sonicated and centrifuged at 7000xg for 5 min at 4°C. For the detection of nuclear NF-κB p50 and p65 subunits, nuclear extracts were obtained using the Nuclear Extract kit (Active Motif Europe, Rixensart, Belgium) following the kit instructions. Protein concentrations in cell and nuclear extracts were determined by the bicinchoninic acid assay (Smith et al., 1985). Samples were boiled for 5 min in Laemmli buffer, separated by SDS-PAGE, electroblotted to PVDF membranes (Millipore, Madrid, Spain), and probed with the corresponding antibodies. The bands were detected by enhanced chemiluminescence (PerkinElmer, Waltham, MA) and quantified with NIH software (Scion Image). The composition of the Laemmli buffer (5x) was: 312 nmol·L-1 SDS, 50% v·v-1 glycerol, 1% v·v-1 2-mercaptoethanol, 22.5 mmol·L-1 EDTA trisodium salt, 220 mmol·L-1 Tris and traces of bromphenol blue (pH = 6.8). Statistical analysis All results are expressed as mean ± SEM. Differences among means were tested for statistical significance by one-way ANOVA and a posteriori least significance tests. All analyses were carried out with the SigmaStat 2.03 program (Jandel Corporation, San Rafael, CA). Concentration–response curves were fitted to a logistic curve when possible with Origin 7.0 (OriginLab Corporation, Northampton, MA). Differences were considered significant at P < 0.05. Materials Except where indicated, all reagents were obtained from Sigma (Barcelona, Spain). The NF-κB p65 and p60 antibodies were purchased from Santa Cruz Biotechnology, Inc. (Heidelberg, Germany); the phospho-IκB-α (Ser32) antibody was purchased from Cell Signaling Technology (Boston, MA, USA); the JLA20 antibody against actin developed by Dr Lin (Lin, 1981) was obtained from the Development Studies Hybridoma Bank developed under the auspices of the National Institute of Child Health and Human Development and maintained by the University of Iowa, Department of Biological Sciences (Iowa City, IA). BGMP (BioPURE-GMPTM) was the kind gift of Davisco Foods International (Eden Prairie, MN). Product certificate of analysis indicated that BGMP content was 93% (97% of dry weight) while fat and lactose contents were 0.5% and less than 1% respectively. The BGMP product also contained small amounts of β-lactoglobulin and α-lactalbumin, which were <1% based on Western blot analysis (not shown), and 4.0% minerals. Casoplatelin was synthesized with a purity >95% by Innovagen (Lund, Sweden). Results Effect of BGMP on cytokine secretion in THP-1 cells To test the hypothesis that BGMP modifies the secretion of cytokines in monocytes/macrophages, THP-1 cells were cultured with different concentrations of BGMP for 24 h and TNF, IL-1β and IL-8 concentrations were determined in the cell culture medium. The addition of BGMP to THP-1 cells increased the concentration of TNF, IL-1β and IL-8 in the cell culture medium in a concentration-dependent fashion (Figure 1). This effect was obtained consistently at concentrations of 1 mg ml-1 or higher. The resulting curves appear sigmoidal, but they could not be completed because of the solubility limits of BGMP and thus an EC50 could not be

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calculated. The effect of bovine serum albumin was also studied to determine whether the action of the BGMP was specific or simply the consequence of the addition of protein (Figure 2). Bovine serum albumin had no effect on cytokine secretion at 1 mg·mL-1, although a certain tendency for increase was noted. However, these experiments were all carried out with complete culture medium, which contains FBS and therefore bovine serum albumin. Thus, we repeated the experiments in FBS-free medium, finding in this case a robust induction of TNF, IL-1β and IL-8 that was comparable to that evoked by BGMP at the same concentration.

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Effect of BGMP on THP-1 cell viability and proliferation As a routine procedure we assessed the possible effect of BGMP on cell viability and proliferation. The level of lactate dehydrogenase activity in the culture medium was similar in the presence and absence of the peptide, indicating that BGMP was not toxic to the cells (data not shown). Cell proliferation (studied by [3H] thymidine incorporation) was slightly (~22%), but significantly lowered in the presence of BGMP at 1 mg·mL-1, but not at lower concentrations (data not shown). Effect of protein hydrolysis on BGMP activity As several peptides derived from milk proteins by digestive proteolysis have been shown to exert biological actions, we next studied whether intact BGMP or hydrolytic peptide fragments account for the observed activity. We used a serine protease inhibitor and a cocktail of protease inhibitors to block the possible hydrolysis of BGMP by cell proteases. As shown in Figure 3, blocking protease activity in THP-1 cells did not alter BGMP-stimulated cytokine production. In addition, as casoplatelin is a bioactive peptide (Bal dit Sollier et al., 1996) whose sequence comprises the first 11 amino acids of BGMP (106–116 of the κ-casein), we tested the hypothesis that casoplatelin could be responsible for the effects of BGMP. The addition of casoplatelin to the cell culture medium in a concentration equivalent, after proteolysis, to 1 mg·mL-1 of BGMP produced no effect on cytokine production (not shown). Signal transduction pathways Both the MAPK and the NF-κB signalling pathways have been shown to be implicated in the production of TNF, IL-1β and IL-8 in monocytes/macrophages (Beinke and Ley, 2004). The stimulation of the MAPK signal transduction pathways by BGMP was studied using inhibitors for ERK1/2 (PD98059), p38 MAPK (SB203580) and JNK (SP600125) that were added to the culture medium of the cells for 1 h. The BGMP was then added, cells were incubated for 24 h and cytokines were measured in the culture medium by ELISA. The addition of either PD98059 or SP600125 to the cell culture medium prevented the increase in the expression of TNF and IL-1β almost completely, while a substantial but significantly lower inhibitory effect was achieved with SB203580 (Figure 4). Inhibition of IL-8 secretion was less effective, and it only reached statistical significance in the case of PD98059 or SP600125 (Figure 4). The role of the NF-κB signalling pathway in the stimulation of THP-1 by BGMP was also studied. One of the ways to activate NF-κB by extracellular stimuli involves the rapid degradation of IκB-α following phosphorylation of Ser32 of IκB-α by IκB kinase (IKK, the so-called canonical pathway). Bay11-7082 inhibits this phosphorylation step and therefore blocks the NF-κB canonical activation pathway. Complete inhibition of the secretion of all the cytokines assayed was observed when cells were cultured in the presence of Bay11-7082, indicating that this pathway plays a pivotal role in the response to BGMP (Figure 5). To further confirm this point, we studied the effect of BGMP on the phosphorylation of IκB-α, adding BGMP to cells for 5, 10 or 20 min and 1, 6 or 24 h and carrying out a Western blot to detect Ser32-phosphorylated IκB-α. As expected, an increase in specific Ser32- phosphorylated IκB-α immunoreactivity was observed, with a maximum expression 1 h after BGMP addition, confirming that BGMP operates chiefly via the canonical pathway (Figure 5A). As the phosphorylation of IκB-α is necessary but not sufficient for the

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activation of the NF-κB pathway, nuclear levels of NF-κB p65 and p50 were also determined by Western blot in nuclear extracts from THP-1 cells cultured with BGMP for 1 and 3 h. Both p65 and p50 were present in the cell nucleus 1 h after the addition of BGMP. As a control, this effect was fully prevented by Bay11-7082 (Figure 5B).

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Figure 4. Effect of MAP kinase and NF-κB inhibitors on tumor necrosis factor (TNF), interleukin (IL)-1β and IL-8 secretion by THP-1 cells stimulated bovine glycomacropeptide (BGMP). Cytokine concentrations were determined by ELISA in the supernatants of cells pre-incubated for 1 h with the signal transduction inhibitors [MAPK inhibitors PD98059 (ERK1/2 inhibitor), SB203580 (ERK1/2 inhibitor), SP600125 (JNK inhibitor) or the NF-κB inhibitor Bay 11-7082] followed by a 24 h incubation with GMP. Data are mean ± SEM. Results are representative of at least three independent experiments. n=3. Means for a variable without a common letter differ, P<0.05. C: control group.


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BGMP activates cytokine secretion in primary monocytes Finally, to validate the data obtained and to make sure that the effect described was not specific for this cell line, human monocytes were obtained from blood and cultured with BGMP. As shown in Figure 6, our results indicate that BGMP also induced the production of TNF, IL-1β and IL-8 in human blood monocytes while casoplatelin had no effect on the production of these cytokines (not shown). Of note, the potency of BGMP for IL-8 induction was higher than that for TNF or IL-1β in primary monocytes but not in THP-1 cells. Discussion The present study was undertaken with the aim to assess the biological activity of BGMP on monocytes, on the basis of its immunomodulatory effects, including intestinal antiinflammatory activity. BGMP has been previously shown to increase IL-1ra but not IL-1β secretion in the P388D1 mouse monocyte cell line (Monnai and Otani, 1997) at a maximal concentration of 0.1 mg·mL-1, which in our hands had no effect in THP-1 cells and only evoked IL-8 secretion in primary monocytes. IL-1ra is a cytokine that behaves as an antagonist of IL-1 receptors, thereby limiting IL-1a and IL-1β effects. From a physiological point of view, IL-1ra production is a normal counterpart of IL-1α/β induction and its role is to limit the pro-inflammatory response of this cytokine. Similar mechanisms operate for TNF. To the best of our knowledge, IL-1ra is not normally induced on its own, that is, without parallel IL-1α/β increase.

Figure 5. Effect of bovine glycomacropeptide (BGMP) on the phosphorylation of IκB-α (A) and on the translocation of p50 and p65 to the nucleus of THP-1 cells (B). A. Cells were cultured with BGMP for different periods of time and Western blots experiments were carried out with cell extracts. α-Actin was used as a loading control. B. Cells were cultured for different periods with BGMP or BGMP plus the NF-κB inhibitor Bay 11-7082 (BAY). Western blot experiments were carried out with nuclear extracts.

Thus, the predominant effect of BGMP on monocytes seems to be an activating one, as supported by the induction of pro-inflammatory cytokines and inhibited proliferation in BGMP-treated THP-1 cells (activated monocytes/macrophages are characterized by slowed rather than hastened proliferation). This is also consistent with the previous study by Li and Mine (2004), showing increased phagocytic activity of U937 cells after BGMP treatment. However, we cannot exclude a possible disproportionate increase in IL-1ra levels by BGMP. The concentration–response curves obtained in THP-1 cells indicate that the potency of BGMP for the induction of the three cytokines was comparable, with reliable

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increase of production being observed starting at 1 mg·mL-1 (lower amounts were active, although slightly, in some experiments). Because of the limits in solubility, the curves could not be completed; that is, the secretory response was not saturated even at 5 mg·mL-1 of BGMP. Naturally, this circumstance prevents us from calculating EC50 values, at least in THP-1 cells. Interestingly, while these effects were largely reproduced in primary human monocytes, used for validation purposes here, the potency for IL-8 induction was clearly higher than that for TNF or IL-1β in these cells. This discrepancy may be explained by the differences of the two cell populations (tumour vs. normal cells), but it should be noted that the magnitude of cytokine secretion was increased dramatically in primary monocytes compared with THP-1 cells despite the fact that identical cell culture conditions were used. This robust response of primary cells may explain why maximal secretion was achieved only in these cells (with the exception of IL-1β).In addition, these data may be interpreted to indicate a predominant effect of BGMP on the signalling pathways leading to IL-8 versus TNF/IL-1β production, probably NF-κB versus MAPK activation. The biological implications of this preference are unclear, but the effects of IL-8 are generally considered more restricted and less pronounced than those of TNF/ IL-1β. Thus, this may represent a ‘scalated’ or gradual biological response of monocytes to BGMP.

Next we addressed the issue of whether BGMP exerts these effects itself or by way of hydrolytic peptide fragments. Our data strongly indicate that native rather than hydrolysed BGMP is required to affect monocyte activity, based on the fact that BGMP effects are unchanged in the presence of protease inhibitors and that casoplatelin, a biologically active undecapeptide comprising the BGMP terminal sequence that features a trypsin cleaving site, does not elicit cytokine production. We have additionally delineated the signalling pathway involved in BGMP effects. All the cytokines studied were clearly induced via JNK and ERK, while they were less sensitive to the inhibition of p38 MAPK. In addition, IL-8 secretion was relatively resistant to inhibition compared with that of TNF or IL-1β (specially with SB203580), indicating that it is less dependent on MAPK signalling.

On the other hand, NF-κB was essential for the induction of the three cytokines, as Bay 11-7082 showed the highest degree of inhibition, resulting in abolition of TNF, IL-1β and IL-8 release. Furthermore, p50/p65 nuclear translocation was shown to be elicited by BGMP and blocked by Bay 11-7082. The dual involvement of MAPK and NF-κB is not unexpected because they are both activated by the IKK complex in monocytes, for instance, in response to LPS (Beinke and Ley, 2004). Our data show that the NF-κB pathway was absolutely required for the induction of TNF, IL-1β and IL-8, while MAPK pathways appeared to play a secondary role, possibly enhancing the effect of NF-κB. It is likely also that the MAPK may exert additional regulatory functions with regard to other genes such as cyclooxygenase 2, as previously suggested (Beinke and Ley, 2004). It should be noted that these conclusions are based largely on pharmacological assays, which have their well-recognized limitations in terms of specificity, and therefore they should be considered with caution. An additional question is how BGMP activates these signalling pathways. Although we initially used bovine serum albumin as a control in order to assess the specificity of BGMP effects, finding that it did not elicit cytokine secretion, this was later found to be due to continuous exposure to the protein, which is contained in cell culture medium as part of FBS. The question of whether similar tolerance to BGMP


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develops cannot be answered with our present data, but clearly there is no cross-tolerance with bovine serum albumin because BGMP stimulates cytokine secretion in the presence or absence of FBS. Previous evidence suggests that BGMP is not immunogenic by itself (Mikkelsen et al., 2006). Hence, BGMP may exert its effects by interaction with cell receptors. In this regard, it is interesting to note that bovine β-casein has been shown recently to act as a TLR4 ligand in mouse splenocytes (Tobita et al., 2006). It is possible that human monocytes do not respond to human albumin, but if they do tolerance makes sense because albumin is present in plasma at concentrations well over those activating monocytes in the present study. We are currently exploring the receptor responsible for BGMP effects and the ligand specificity compared with human and bovine albumin and other peptides.

Figure 6. Concentration response curves for the production of tumor necrosis factor (TNF), interleukin (IL)-1β and IL-8 by primary human monocytes in the presence of bovine glycomacropeptide bGMP. After a 24 h incubation in the presence of the product, secretion of cytokines was measured in the culture medium by ELISA. Logistic sigmoidal curves could be fitted in the cases of TNF and IL-8 (EC50 0.92±0.09 mg ml-1 and 0.72±0.40 µg ml-1, respectively). Results are expressed as mean ± s.e.mean of two different experiments (n=3 in each experiment). *P<0.05 vs. control.

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We have previously demonstrated that BGMP has intestinal anti-inflammatory effects in preclinical models of colitis and ileitis, displaying a degree of efficacy similar to that of sulphasalazine, a drug widely used in the therapy of inflammatory bowel disease (Daddaoua et al., 2005; Baumgart and Sandborn, 2007; Requena et al., 2008). Of note, in both cases BGMP treatment resulted in normalization of the expression of IL-1β, whose main cellular sources are monocytes/macrophages. Clearly, BGMP does not reproduce this effect in vitro. There are two possible explanations for this discrepancy. First, it is possible that BGMP may act by other mechanisms, for instance, via effects on epithelial cells or lymphocytes (studies are underway to test these hypotheses), and that the activation of monocytes does not play a role in its intestinal anti-inflammatory activity or even opposes it. Second, monocyte activation may be involved in anti-inflammatory activity, a mechanism that certainly appears in the context of intestinal inflammation. However, the immune regulation of the intestine is especially complex in this regard. For instance, one of the few genes positively identified to affect the incidence of inflammatory bowel disease is NOD2, a member of the Nod1/Apaf-1 family (Peyrin-Biroulet and Chamaillard, 2007). The protein product is expressed intracellularly and recognizes LPS-derived muramyl dipeptide. The polymorphic alleles linked to increased risk of inflammatory bowel disease have been reported to be associated with reduced rather than augmented activation of leukocytes via NF-κB mechanisms, although this issue remains controversial (Bamias and Cominelli, 2007; Cho and Weaver, 2007; Peyrin-Biroulet and Chamaillard, 2007). In line with this concept, Nenci et al. (2007) used a smart in vivo mouse model in which the intestinal epithelial expression of IKKγ (also known as NEMO), IKKα and IKΚβ was suppressed conditionally, resulting in reduced activation of the NF-κB pathway. The consequence was spontaneous severe colonic inflammation. Cytosine-phosphate-guanosine oligonucleotides, which are ligands for TLR9 and produce epithelial and immune cell activation, have anti- inflammatory activity when administered as a pre-treatment in experimental colitis, but exacerbate the inflammatory response when given as a post-treatment (Obermeier et al., 2003). Similarly, granulocyte-macrophage-colony stimulating factor (GM-CSF) has anti-inflammatory effects, which have been linked to the expansion of dendritic cells (Sainathan et al., 2008). Other studies have shown that the absence of monocytes and dendritic cells aggravates rather than ameliorates experimental colitis (Qualls et al., 2006). Taken together, these data suggest that a defective response to proinflammatory stimuli may actually worsen the outcome, at least in some cases. It has been well established that experimental colitis is strongly dependent on the presence of non-pathogenic bacteria, probably acting as a source of antigens that fuel the intestinal immune reaction, ultimately potentiating inflammation (Seksik et al., 2006). Thus, defects in immune function may impair a prompt resolution of intestinal injury, triggering a more robust reaction to a normally trivial challenge. If so, monocyte stimulation would result in a more efficient and prompt response to luminal antigens as they gain access to the subepithelial milieu. In this regard, it is tempting to speculate that BGMP may exert monocyte/ macrophage-stimulating functions in breastfed infants. It is well known that the intestine of newborns is immature, exhibiting an increased permeability to macromolecules, and it is also prone to developing intense inflammatory responses (necrotizing enterocolitis) (Martinez-Augustin et al., 1997; Goldman, 2000). Additional experiments will be required to confirm the exact role of monocyte activation by BGMP in intestinal inflammation.


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Acknowledgements CIBEREHD is funded by the Instituto de Salud Carlos III. This study was supported by grants from the Instituto de Investigación Carlos III to F.S.M.L.H. and O.M.A. (PI051651 and PI051625). Additional funding was provided by the Junta de Andalucía. Pilar Requena was funded by the Spanish Ministry of Science and Technology. Conflicts of interest None. References Aimutis WR (2004). Bioactive properties of milk proteins with particular focus on anticariogenesis. J

Nutr 134: 989S–995S. Bal dit Sollier C, Drouet L, Pignaud G, Chevallier C, Caen J, Fiat AM et al. (1996). Effect of kappa-

casein split peptides on platelet aggregation and on thrombus formation in the guinea-pig. Thromb Res 81: 427–437.

Bamias G, Cominelli F (2007). Immunopathogenesis of inflammatory bowel disease: current concepts. Curr Opin Gastroenterol 23: 365– 359.

Baumgart DC, Sandborn WJ (2007). Inflammatory bowel disease: clinical aspects and established and evolving therapies. Lancet 369: 1641–1657.

Beinke S, Ley SC (2004). Functions of NF-kappaB1 and NF-kappaB2 in immune cell biology. Biochem J 382: 393–409.

Brody EP (2000). Biological activities of bovine glycomacropeptide. Br J Nutr 84: S39–S46. Bruck WM, Kelleher SL, Gibson GR, Graverholt G, Lonnerdal BL (2006). The effects of alpha-

lactalbumin and glycomacropeptide on the association of CaCo-2 cells by enteropathogenic Escherichia coli, Salmonella typhimurium and Shigella flexneri. FEMS Microbiol Lett 259: 158–162.

Cho JH, Weaver CT (2007). The genetics of inflammatory bowel disease. Gastroenterology 133: 1327–1339.

Clare DA, Catignani GL, Swaisgood HE (2003). Biodefense properties of milk: the role of antimicrobial proteins and peptides. Curr Pharm Des 9: 1239–1255.

Cross ML, Gill HS (2000). Immunomodulatory properties of milk. Br J Nutr 84 (Suppl. 1): S81–S89. Daddaoua A, Puerta V, Zarzuelo A, Suarez MD, Sanchez de Medina F, Martinez-Augustin O (2005).

Bovine glycomacropeptide is anti-inflammatory in rats with hapten-induced colitis. J Nutr 135: 1164– 1170.

Florisa R, Recio I, Berkhout B, Visser S (2003). Antibacterial and antiviral effects of milk proteins and derivatives thereof. Curr Pharm Des 9: 1257–1275.

Goldman AS (2000). Modulation of the gastrointestinal tract of infants by human milk. Interfaces and interactions. An evolutionary perspective. J Nutr 130: 426S–431S.

Halprin KM, Ohkawara A (1966). Lactate production and lactate dehydrogenase in the human epidermis. J Invest Dermatol 47: 222–229.

Li EW, Mine Y (2004). Immunoenhancing effects of bovine glycomacropeptide and its derivatives on the proliferative response and phagocytic activities of human macrophagelike cells, U937. J Agric Food Chem 52: 2704–2708.

Lim K, van Calcar SC, Nelson KL, Gleason ST, Ney DM (2007). Acceptable low-phenylalanine foods and beverages can be made with glycomacropeptide from cheese whey for individuals with PKU. Mol Genet Metab 92: 176–178.

Lin JJ (1981). Monoclonal antibodies against myofibrillar components of rat skeletal muscle decorate the intermediate filaments of cultured cells. Proc Natl Acad Sci U S A 78: 2335–2339.

Martinez-Augustin O, Boza JJ, Navarro J, Martinez-Valverde A, Araya M, Gil A (1997). Dietary nucleotides may influence the humoral immunity in immunocompromised children. Nutrition 13: 465– 469.

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Mikkelsen TL, Bakman S, Sorensen ES, Barkholt V, Frokiaer H (2005). Sialic acid-containing milk proteins show differential immunomodulatory activities independent of sialic acid. J Agric Food Chem 53: 7673–7680.

Mikkelsen TL, Rasmussen E, Olsen A, Barkholt V, Frokiaer H (2006). Immunogenicity of kappa-casein and glycomacropeptide. J Dairy Sci 89: 824–830.

Monnai M, Otani H (1997). Effect of bovine κ-caseinoglycopeptide on secretion of interleukin-1 family cytokines by P388D1 cells, a line derived from Mouse monocyte/macrophage. Milchwissenschaft 52: 192–196.

Monnai M, Horimoto Y, Otani H (1998). Immunomodificatory effect of dietary bovine kappa-caseinoglycopeptide on serum antibody levels and proliferative responses of lymphocytes in mice. Milchwissenschaft 53: 129–132.

Nakajima K, Tamura N, Kobayashi-Hattori K, Yoshida T, Hara-Kudo Y, Ikedo M et al. (2005). Prevention of intestinal infection by glycomacropeptide. Biosci Biotechnol Biochem 69: 2294–2301.

Nenci A, Becker C, Wullaert A, Gareus R, van Loo G, Danese S et al. (2007). Epithelial NEMO links innate immunity to chronic intestinal inflammation. Nature 446: 557–561.

Newby TJ, Stokes CR, Bourne FJ (1982). Immunological activity of milk. Vet Immunol Immunopathol 3: 67–94.

Obermeier F, Dunger N, Strauch UG, Grunwald N, Herfarth H, Scholmerich J et al. (2003). Contrasting activity of cytosin-guanosin dinucleotide oligonucleotides in mice with experimental colitis. Clin Exp Immunol 134: 217–224.

Otani H, Monnai M (1993). Inhibition of proliferative responses of mouse spleen lymphocytes by bovine milk κ-casein digests. Food Agric Immunol 5: 219–229.

Otani H, Horimoto Y, Monnai M (1996). Suppression of interleukin-2 receptor expression on mouse CD4+ T cells by bovine kappa-caseinoglycopeptide. Biosci Biotechnol Biochem 60: 1017–1019.

Peyrin-Biroulet L, Chamaillard M (2007). NOD2 and defensins: translating innate to adaptive immunity in Crohn’s disease. J Endotoxin Res 13: 135–139.

Qualls JE, Kaplan AM, van Rooijen N, Cohen DA (2006). Suppression of experimental colitis by intestinal mononuclear phagocytes. J Leukoc Biol 80: 802–815.

Requena P, Daddaoua A, Martínez-Plata E, González M, Zarzuelo A, Suárez MD et al. (2008). Bovine glycomacropeptide ameliorates experimental rat ileitis by mechanisms involving downregulation of interleukin 17. Br J Pharmacol 154: 825–832.

Sainathan SK, Hanna EM, Gong Q, Bishnupuri KS, Luo Q, Colonna M et al. (2008). Granulocyte macrophage colony-stimulating factor ameliorates DSS-induced experimental colitis. Inflamm Bowel Dis 14: 88–99.

Seksik P, Sokol H, Lepage P, Vasquez N, Manichanh C, Mangin I et al. (2006). Review article: the role of bacteria in onset and perpetuation of inflammatory bowel disease. Aliment Pharmacol Ther 24 (Suppl. 3): 11–18.

Smith PK, Krohn RI, Hermanson GT, Mallia AK, Gartner FH, Provenzano MD et al. (1985). Measurement of protein using bicinchoninic acid. Anal Biochem 150: 76–85.

Tobita K, Kawahara T, Otani H (2006). Bovine beta-casein (1-28), a casein phosphopeptide, enhances proliferation and IL-6 expression of mouse CD19+ cells via Toll-like receptor 4. J Agric Food Chem 54: 8013–8017.

Zimecki M, Kruzel ML (2007). Milk-derived proteins and peptides of potential therapeutic and nutritive value. J Exp Ther Oncol 6: 89–106.


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The intestinal antiinflammatory agent glycomacropeptide has immunomodulatory actions on rat splenocytes.

Pilar Requena, Raquel González, Rocío López-Posadas, Ana Abadía-Molina, María Dolores Suárez, Antonio Zarzuelo, Fermín Sánchez de Medina and Olga Martínez-Augustin.Submitted for publication.

CAPÍTULO 6

Abstract: Bovine glycomacropeptide (GMP) is an immunologically active milkpeptide that is a part of the normal human diet. GMP has therapeutic value inpreclinical models of intestinal inflammation, and its mechanism may be related toeffects on lymphocytes. This study focuses on the actions of GMP on rat splenocytes invitro and in vivo. Bovine serum albumin and lactoferrin were used for comparativepurposes. GMP (0.01-0.1 mg ml-1) enhanced concanavalin A (ConA) evoked but notbasal splenocyte proliferation. At 1 mg ml-1 GMP lost this effect but augmented basalTNF-α secretion and also iNOS and COX2 expression. IFN-γ, IL-2 and IL-17 were notaffected by GMP in quiescent splenocytes, but IL-10 was augmented at allconcentrations tested. On the other hand, GMP produced a marked inhibitory effect(70%) on IFN-γ secretion and to a lower extent (50%) also on TNF-α. GMP was shownto block STAT4 but not IκB-α phosphorylation. The Treg marker Foxp3 was markedlyupregulated by GMP. Bovine serum albumin had some effects on splenocyte functionwhich were of lower magnitude and not entirely coincidental, while lactoferrin had astrong antiproliferative effect, as expected, indicating a specific effect of GMP. Whenadministered for 3 days to normal Wistar rats, GMP reproduced the Foxp3 inductioneffect observed previously in vitro. This was observed in splenocytes but not inthymocytes, and only when administered by the oral rather than the intraperitonealroute. Thus our results support the hypothesis that GMP may limit intestinalinflammation acting at least in part on lymphocytes.

Keywords: glycomacropeptide, STAT4, inflammatory bowel disease, Th1, Treg,Foxp3

Introduction

Glycomacropeptide (GMP), also known as κ-caseinglycopeptide, is one of thebiologically active components of milk [1, 2]. GMP is the N-acetylneuraminic (sialic)acid containing, N-terminal 64-aminoacid peptide of κ-casein. GMP is a nativecomponent of milk, but it is produced mainly by chymosin/pepsin mediated proteolysisof the parent protein during the digestion of milk. Thus GMP is normally released inthe newborn and adult human gastrointestinal tract after milk ingestion [3, 4]. Inaddition, GMP is obtained in large quantities as a byproduct of the cheese makingprocess, as part of the resulting milk whey [2]. GMP (of bovine origin) has nutritionaland industrial value, as it is currently added to infant formulas and, due to its lowcontent on aromatic aminoacids, including an absolute absence of phenylalanine, it hasbeen proposed to be useful in the elaboration of products for individuals withphenylketonuria [22]. In addition, GMP is included in tooth pastes because of itsanticariogenic properties [23].

In the last decades it has become clear that milk proteins have more than just nutritionalvalue, and the biological effects of GMP have been studied by several research groups[5]. GMP has been reported to bind cholera and Escherichia coli enterotoxins, to inhibitbacterial and viral epithelial adhesion, to promote bifidobacterial growth and tomodulate the immune system response [2, 6, 7]. We have found that oral administrationof GMP results in substantial antiinflammatory effects in experimental colitis and ileitis[8, 9]. The mechanism of action has not been unequivocally established, althoughinhibition of IL-17 expression was found in the ileitis study [9]. Some in vitro studieshave described that GMP inhibits mouse splenocyte proliferation induced by LPS andphytohemagglutinin [11], suppresses the IL-2 receptor expression on mouse CD4+ Tcells [5], induces the expression of an interleukin-1 receptor antagonist-like component

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in mouse spleen cells [12] and suppresses serum IgG antibody production in mouselymphocytes [13]. In macrophages, it has been shown that GMP modulates thesecretion of IL-1 family cytokines in a mouse monocytic cell line [14] and that itenhances proliferation and phagocytic activities of human macrophagelike cells [15].Also we recently found that GMP enhances TNF-α, IL-8 and IL-1β production in thehuman monocytic cell line THP-1 and in primary monocytes, via activation of the NF-κB and MAP kinase pathways [16]. The contribution of these actions on macrophagesto the intestinal antiinflammatory effect of GMP is unclear at the moment. Conversely,our in vivo data and previous results obtained in vitro by other groups suggest thatlymphocytes may be involved. Thus we set out to further explore this possiblemechanism of action by studying in vitro and in vivo effects of GMP on ratsplenocytes.

This study demonstrates that GMP inhibits the expression of TNF-α and IFN-γ inconcanavalin A (ConA) stimulated splenocytes while enhancing the expression ofFoxp3 and IL-10 secretion in quiescent cells.

Material and methods

ReagentsExcept where indicated, all reagents were obtained from Sigma (Barcelona, Spain). Thephosho-IκB-α (Ser32) antibody was purchased from Cell Signaling Technology(Boston, MA, USA); the JLA20 antibody against actin developed by Dr. Lin [17] wasobtained from the Development Studies Hybridoma Bank developed under the auspicesof the National Institute of Child Health and Human Development and maintained bythe University of Iowa, Department of Biological Sciences (Iowa City, IA); the COX-2antibody was purchased from Cayman Chemical Company (Ann Arbor, MI, USA); theiNOS and phosphospecific STAT4 antibodies were purchased from BD BiosciencesPharmingen (San Jose, CA, USA). GMP (BioPURE-GMPTM) was the kind gift ofDavisco Foods International (Eden Prairie, MN). According to the manufacturer, theGMP content is 93% while fat and lactose contents comprise 0.2 and <1%,respectively. Casoplateline (CAS, NH2-MAIPPKKNQDK-COOH) was synthesizedwith a purity >95% by Innovagen (Lund, Sweden).

Spleen and thymus mononuclear cell isolationFemale Wistar rats were sacrificed by cervical dislocation and the spleen and thymuswere extracted aseptically. Cell suspensions were obtained by disrupting the tissuesbetween dissecting forceps in medium. After centrifuging, cells were cleared oferythrocytes (spleen only) by suspension on hypotonic lysis buffer (0.15 M NH4Cl, 10mM KHCO3, 0.1 mM Na2EDTA·2H2O, pH=7.3) for 30 min on ice. Mononuclear cellswere washed and suspended in RPMI medium supplemented with 10% FBS, 100 mg l-1

streptomycin, 100000 U l-1 penicillin, 2.5 mg l-1 amphotericin B and 0.05 mM ofmercaptoethanol.

Trypan blue assayCell viability was quantitated with the Trypan blue exclusion assay. Briefly, cells wereanalyzed as suspensions of 106 cells ml-1 in RPMI medium in the presence or absenceof GMP, bovine serum albumin (BSA) or lactoferrin (LF) 0.01, 0.1 or 1 mg ml-1 orequivalent CAS concentrations for 48 h. Cell suspensions were then diluted 1:2 in 0.4%Trypan blue in PBS, incubated 2 min while shaking, and viable (not blue) and totalcells were counted.

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Cell proliferation assayFor the thymidine uptake experiments, 2x106 cells ml-1 were incubated in RPMImedium for 48 h with GMP, BSA or LF 0.01, 0.1 or 1 mg ml-1 or equivalent CASconcentrations and [3H]-thymidine (1 µCi mL-1; GE Healthcare, Spain) in the absenceor presence of ConA (5 µg mL-1). The cells were then harvested, washed 3 times withtrichloroacetic acid (10% v v-1), suspended in lysis buffer (1% w v-1 SDS, 0.3 N NaOH)for 30 min at room temperature and collected into plastic vials. Then 4 mL ofscintillation liquid (Beckman Coulter, Madrid, Spain) per vial were added and theamount of [3H]-thymidine incorporated was measured with a Tri-Carb liquidscintillation analyzer (Packard Instrument, Meriden, CT).

Cytokine determinationFor cytokine determinations cell suspensions (106 cells ml-1 in RPMI medium) werecultured as above (without [3H]-thymidine) for 48 h. The supernatants collected aftergentle centrifugation (3000 g, 5 min, 4ºC) were kept at −80°C until cytokineconcentration was measured by ELISA kits (Biosource Europe, Nivelles, Belgium andBD Biosciences, Erembodegem, Belgium), following the protocols recommended bythe manufacturers.

Western blotFor cytoplasmic protein expression experiments, 5x106 cells per mL were harvested,washed once with PBS and total protein content was extracted with RIPA buffer (0.1%sodium dodecylsulfate, 0.1% sodium deoxycholate, 1% Triton X-100 in phosphatebuffered saline, with freshly added protease inhibitors −phenylmethylsulfonyl fluoride,aprotinin, leupeptin, 1,10-phenanthroline−). For STAT4 detection, nuclear proteinswere extracted using the Nuclear Extract kit (Active Motif Europe, Rixensart, Belgium)following the kit instructions. Protein was quantitated with the bincinchoninic acidmethod using bovine serum albumin as standard, and samples were boiled for 5 min inLaemmli buffer, separated by SDS-PAGE, electroblotted to PVDF membranes(Millipore, Madrid, Spain), and probed with the corresponding antibodies. The bandswere detected by enhanced chemiluminescence (PerkinElmer, Waltham, MA) andquantitated with NIH software (Scion Image). The composition of the Laemmli buffer(5X) was: 312 nM SDS, 50% v/v glycerol, 1% v/v 2-mercaptoethanol, 22.5 mM EDTAtrisodium salt, 220 mM Tris and traces of bromphenol blue (pH=6.8).

RT-qPCRThe expression of IL-17, IFN-γ and TNF-α was examined by reverse transcriptase(RT)-qPCR. For the RT-qPCR analysis total RNA was extracted with Trizol(Invitrogen, Barcelona, Spain). 1 µg of RNA per sample was subjected to reversetranscription using the First-strand cDNA synthesis kit (GE Healthcare, Barcelona,Spain). Real time qPCR was performed using 2 µl of cDNA for a final PCR reactionvolume of 25 µl (Sybr-green, Biorad). The expression of the ribosomal 18 S unit wasexamined as a loading standard. The primers used were: IL-17 (sense 5’-TTC TCCAGA ACG TGA AGG TC-3’; antisense 5’-GGA CAA TAG AGG AAA CGC AG-3’);TNF-α [sense 5’-TAC TGA ACT TCG GGG TGA TTG GTC C-3’; antisense 5’-CAGCCT TGT CCC TTG AAG AGA ACC-3’); IFNγ (sense 5’-TTC ATT GAC AGC TTTGTG CTG G-3’; antisense 5’ AAC AGT AAA GCA AAA AAG GAT GCA TT-3’);ribosomal 18 S unit (sense 5’-CCA TTG GAG GGC AAG TCT GGT G-3’; antisense5’-CGC CGG TCC AAG AAT TTC ACC-3’).

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Female Wistar rats (185-215 g) obtained from the Laboratory Animal Service of theUniversity of Granada were used, housed in makrolon cages and maintained in ourlaboratory in air conditioned animal quarters with a 12 h light-dark cycle. Animals wereprovided with free access to tap water and food (Panlab A04, Panlab, Barcelona,Spain). Rats were randomly assigned to 4 different groups (n=3): the control oral (Cor)and intraperitoneal (Cip) groups, and the oral (Gor) and intraperitoneal (Gip) GMPgroups. The GMP groups received GMP (500 mg kg-1 day-1) either in 1%methylcellulose p.o. or in saline solution i.p., while the control groups received thevehicle every day. Animals received the treatments for 3 days and then were sacrificedby cervical dislocation. Then their splenic and thymus cells were examined for cytokinesecretion (after 48 h) and Foxp3 expression (without incubation). In a separateexperiment, rats were treated as above and splenocytes examined for cytokine/Foxp3expression at the mRNA level after only 5 h of incubation. Cells were studied in thepresence or absence of ConA (5 µg mL-1). This study was carried out in accordancewith the Directive for the Protection of Vertebrate Animals used for Experimental andother Scientific Purposes of the European Union (86/609/EEC).

Statistical analysis All results are expressed as mean ± SEM. Differences among means were tested forstatistical significance by one way ANOVA and a posteriori least significance tests onpreselected pairs. All analyses were carried out with the SigmaStat 2.03 program(Jandel Corporation, San Rafael, CA). Differences were considered significant atp<0.05.

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Fig 1. Effect of glycomacropeptide (GMP), casoplateline (CAS) and bovine serum albumin (BSA) on splenocyteproliferation, assessed by the thymidine uptake assay. Cells were incubated with GMP 0.01, 0.1 and 1 mg mL-1, CAS1 (equivalent molar concentration to GMP 1mg/mL) or BSA 1 mg mL-1 in absence or presence of Concanavalin A(Con A, 5 µg ml-1). [3H]-Thymidine was added at the same time and uptake was measured 48 h after. Data areexpressed as mean ± S.E.M.; all Con A-stimulated cells were different from the corresponding ConA free cells.*p<0.05 vs. ConA-stimulated cells. The pooled (normalized) results from 2 different experiments are shown.

Results

GMP has proliferative effects on ConA-stimulated but not unstimulated splenocytes

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GMP was initially tested for effects on cell viability and proliferation in the ratsplenocyte population. As shown in Fig. 1, GMP (0.01-1 mg mL-1) had no effect onsplenocyte thymidine uptake in basal conditions. As expected, ConA evoked a robustproliferative response, which was significantly enhanced by GMP at 0.01-0.1 mg mL-1

but not with 1 mg mL-1. As reported before [27], LF strongly inhibited ConA-stimulated proliferation, but it also had no effect in basal conditions. BSA, used as acontrol, was inactive. In addition, GMP had no significant impact on splenocyteviability (not shown).

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Fig. 2. Effect of glycomacropeptide (GMP),casoplateline (CAS), bovine serum albumin (BSA)and lactoferrin (LF) on IFN-γ secretion. Splenocyteswere plated in 24-well plates (5x105 cells per well)and cultured with GMP or BSA 0.01, 0.1 and 1mg/mL, or CAS 0.01, 0.1 and 1 (equivalent molarconcentration) in absence (A) or presence (B) ofConcanavalin A (ConA, 5 µg/ml). After 48 h ofincubation, culture medium was collected and frozenat -80ºC until ELISA analysis. Results are expressedas mean ± S.E.M. of cytokine concentration (pg/ml).+p < 0.05 vs. control, *p < 0.05 vs. ConA-stimulatedcells. The pooled (normalized) results from 3 differentexperiments are shown.

Fig. 3 Effect of glycomacropeptide (GMP),casoplateline (CAS), bovine serum albumin (BSA)and lactoferrin (LF) on TNF-α secretion. Splenocyteswere plated in 24-well plates (5x105 cells per well)and cultured with GMP or BSA 0.01, 0.1 and 1mg/mL, or CAS 0.01, 0.1 and 1 (equivalent molarconcentration) in absence (A) or presence (B) ofConcanavalin A (ConA, 5 µg/ml). After 48 h ofincubation, culture medium was collected and frozenat -80ºC until ELISA analysis. Results are expressedas mean ± S.E.M. of cytokine concentration (pg/ml).+p < 0.05 vs. control, *p < 0.05 vs. ConA-stimulatedcells. The pooled (normalized) results from 3different experiments are shown

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GMP modulates cytokine production by primary splenocytesGMP did not change basal IL-2 or IFN-γ release in naive splenocytes, but it increasedTNF-α production markedly at the highest concentration assayed, 1 mg mL-1 and,interestingly, it also augmented IL-10 production at all concentrations tested (Figs. 2-5). There was no significant effect on IL-17 expression at the mRNA level (data notshown). The effect of GMP was higher than that of BSA, which enhanced IL-10production weakly, and differed from that of LF, which increased TNF-α and IL-2release at the concentration of 0.01 and 1 mg mL-1, respectively. The levels of the 4cytokines were upregulated markedly under ConA stimulation, as expected (Figs. 2-5).GMP had a 50/80% inhibitory effect on TNF-α and IFN-γ release, respectively, whileIL-2 and IL-10 were unchanged. Inhibition was achieved mostly at the concentrationsof 0.01 and 0.1 mg mL-1, and it was greatly diminished (IFN-γ) or completely gone(TNF-α) at 1 mg mL-1. BSA was not inactive, as it lowered IFN-γ at the highestconcentration assayed. LF inhibited all proinflammatory cytokines markedly, althoughIL-2 and IFN-γ were somewhat less sensitive than TNF-α. The latter was an expectedresult, since LF is a strong inhibitor of lymphocyte proliferation [30].

Effect of GMP on COX2 and iNOS expression in primary splenocytesWestern blot analysis of rat naive splenocytes treated with 1 or 0.1 mg mL-1 of GMP invitro showed a marked stimulatory effect on the expression of both COX2 and iNOS atthe highest concentration assayed (Fig. 6). BSA had a less marked effect. In keepingwith these effects, GMP augmented the phosphorylation of IκB-α at the sameconcentrations assayed, indicating the activation of the NF-κB canonical pathway (Fig.6).

Effect of GMP on STAT4 phosphorylation in primary splenocytesThe results obtained so far suggest a possible inhibition of Th1 lymphocytes by GMP.In order to examine this effect with more detail we tested the hypothesis that GMP actsat the level of STAT4 activation, a key step in IFN-γ production [19]. The results,shown in Fig. 7, confirm that this step of the pathway is inhibited in ConA-stimulatedspleen cells by GMP 0.1 mg mL-1 by approximately 75%. However, GMP did notabolish the phosphorylation of IκB-α when cells were stimulated with ConA (Fig. 7).

Effect of casoplatelinCasoplatelin (CAS), a 11-aminoacid subpeptide of GMP (aminoacids 106-116 ofbovine k-casein), has been described to exert various biological effects [28, 29] andthus it might act as the active moiety of GMP. In order to test this hypothesis weassessed the possible effects of CAS on primary rat splenocytes, using GMP equimolarconcentrations. In these conditions CAS did not affect splenocyte proliferation (Fig. 1)or cytokine production (Figs. 2-5), with two exceptions: CAS enhanced IL-2 productionand showed an increase in ConA-stimulated TNF-α secretion at low concentrations.Thus while this peptide may retain some of GMP activity, it cannot account for itseffects in our experimental system.

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Fig. 4. Effect of glycomacropeptide (GMP),casoplateline (CAS), bovine serum albumin(BSA) and lactoferrin (LF) on IL-2 secretion.Splenocytes were plated in 24-well plates (5x105cells per well) and cultured with GMP or BSA0.01, 0.1 and 1 mg/mL, or CAS 0.01, 0.1 and 1(equivalent molar concentration) in absence (A)or presence (B) of Concanavalin A (Con A, 5µg/ml). After 48 h of incubation, culture mediumwas collected and frozen at -80ºC until ELISAanalysis. Results are expressed as mean ± S.E.M.of cytokine concentration (pg/ml). +p < 0.05 vs.control, *p < 0.05 vs. ConA-stimulated cells. Thepooled (normalized) results from 3 differentexperiments are shown.

Fig. 5. Effect of glycomacropeptide (GMP),casoplateline (CAS) and bovine serum albumin(BSA) on IL-10 secretion. Splenocytes wereplated in 24-well plates (5x105 cells per well)and cultured with GMP or BSA 0.01, 0.1 and 1mg/mL, or CAS 0.01, 0.1 and 1 (equivalentmolar concentration) in absence (A) or presence(B) of Concanavalin A (Con A, 5 µg/ml). After48 h of incubation, culture medium wascollected and frozen at -80ºC until ELISAanalysis. Results are expressed as mean ± S.E.M.of cytokine concentration (pg/ml). +p < 0.05 vs.control, all ConA-stimulated were different vs C.The pooled (normalized) results from 3 differentexperiments are shown.

Effect of GMP on FoxP3.Finally, we set out to verify whether GMP has an effect on Treg cells. The expressionof Foxp3 measured by Western blotting was used as a suitable marker of this cellpopulation. Fig. 8 shows that in vitro treatment of primary rat splenocytes with 1 mgmL-1 of GMP resulted in a 3-fold increase in Foxp3 expression. Of note, BSA alsodisplayed a significant although lower effect at the same concentration.

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Fig. 6. Effect of GMP on COX2, iNOS and IL-17expression and NF-κB activation. A) Cells wereincubated with GMP 1 and 0.1 mg/mL and BSA 1mg/mL for 24 h. Protein samples were analyzed byWestern blot, and COX2 and iNOS were determined.B) Cells were incubated with GMP 1 and 0.1 mg/mLfor 16 h. Total RNA was extracted and RT-PCR wasperformed for IL-17 and 18s. Results are expressedas geometric media +SD of three differentexperiments. C) Cells were incubated with GMP 1mg/mL and D) GMP 0.1 mg/mL up to 24 h. Totalprotein samples were analyzed by Western blot andpIkB was determined. Blots are representative of 3different experiments.

Fig 7. Effect of GMP on NFκB and STAT-4activation. A) Cells were incubated with ConA (5µg/ml) in the absence or presence of GMP 0.1 mg/mLduring 15 and 60 minutes. Total protein samples wereanalyzed by Western blot and pIkB was determined.B) Cells were incubated with ConA (5 µg/ml) in theabsence or presence of GMP 0.1 mg/mL up to 24h.Nuclei protein samples were analyzed by Westernblot and total STAT-4 protein was determined. Blotsare representative of 2 different experiments.

Effect of GMP in vivoSince our ultimate aim was to elucidate the mechanism of the intestinalantiinflammatory effects of GMP in vivo, we carried out an experiment in normal ratsin which GMP was administered at the previously established effective dose inexperimental intestinal inflammation either by oral gavage or by intraperitonealinjection. The animals were treated daily with GMP or vehicle for 3 days and then thebehaviour of primary splenocytes and thymocytes assessed as above. In a firstexperiment cytokine and Foxp3 expression was measured (Figs. 8B and 9). Oral GMPincreased Foxp3 expression in splenocytes, as determined by Western blot. Themagnitude of the effect was comparable to that of the in vitro experiments.Interestingly, intraperitoneal GMP administration failed to produce this effect (data notshown). Furthermore, no effect was observed in the thymus by either route, indicating aspecific effect on the spleen cells (Fig. 8C). The same results were observed in twosuch different experiments. GMP had no effect however on the ex vivo splenocyteproduction of TNF-α, IL-2 or IFN-γ after treatment by either route (Fig. 9), althoughIFN-γ was generally higher with oral GMP. We performed 2 additional experiments inwhich splenocytes were incubated for only 5 h, in order to be able to detect any effectthat may have been missed after long deprivation of GMP (because of the 48 hincubation applied for cytokine measurement). Since protein levels may not be alteredat this early time point, mRNA was measured in this case. The results obtained are

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shown in Fig. 10, and confirm that cytokines are not significantly modulated by GMP in vivo treatment.

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Fig. 8. Effect of GMP on FoxP3 expression. A) Spleen cells were incubated with GMP 1 and 0.1 mg/mL and BSA 1 mg/mL for 24 h. Total protein samples were analyzed by Western blot, and FoxP3 were determined. B-C) Female Wistar rats were fed for three days with GMP (500 mg/kg/d) or vehicle and then killed and spleen (B) and thymus cells (C) were obtained and analysed for Foxp3 expression. Total protein samples were analyzed by Western blot and FoxP3 was determined. Blots are representative of 3 different experiments.

Fig. 9. Effect of in vivo oral or intraperitoneal GMP treatment on ex vivo splenocyte production of IFN-γ, TNF-α and IL-2. The cells were stimulated with ConA as described in Methods. The production of cytokines in the Cor group was: 4777.8±1375.6 ng mL-1. No significant differences were observed. The pooled (normalized) results from 2 different experiments are shown.

Discussion The aim of this study was to evaluate the possible role of lymphocytes in the intestinal antiinflammatory activity of GMP, using primary rat splenocytes as an initial approach. Our results confirm that GMP modulates the activity of these cells and, specifically, they suggest that GMP hampers the activation of Th1 cells while favoring the differentiation of Treg cells, although this may be a direct or an indirect effect (via macrophages). Thus the predominant effect of GMP on primary splenocytes in vitro was: (1) a substantial increase in Foxp3 and IL-10 expression in basal conditions; (2) a marked inhibition of IFN-γ and, to a lower extent, TNF-α, in ConA stimulated cells, with no changes in IL-2 release. However, GMP also produced a mild (compare the top and bottom panel of Fig. 3) stimulation of TNF-α production in quiescent splenocytes, increased cell proliferation under ConA activation, and even upregulated COX2 and iNOS in basal conditions. It should be noted that these latter effects were observed mainly at the highest concentration assayed, i.e. 1 mg mL-1 of GMP, at which the inhibitory effects mentioned are already greatly diminished. Because we have previously established that GMP activates monocytes at 1 mg mL-1 [16], it is likely that these represent actions on spleen macrophages. Moreover, we have shown an activation


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of the NF-κB pathway in unstimulated spleen cells after culturing with GMP 0.1 and 1 mg mL-1, a pathway reported previously to be activated by GMP in macrophages [16]. Thus the inhibitory effect would be exerted probably on lymphocytes. It is also possible that the proliferation of Treg lymphocytes balances the putative antiproliferative effect on Th1 cells to a certain extent, but this action is clearly overwhelmed under ConA stimulation, since GMP cannot increase Foxp3 (or IL-10) expression any further in these conditions. This question cannot be clarified until further experiments with isolated cell populations (which are underway) are performed. Thus our conclusions must be considered with caution. A likely source of IL-10 in this study was Treg cells, since both IL-10 and Foxp3 were maximally increased at 1 mg mL-1 of GMP, but it could be macrophages or even Th2 cells as well, since we did not measure Th2 cytokines.

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Fig. 10. Effect of in vivo oral or intraperitoneal GMP treatment on ex vivo splenocyte mRNA expression of IFN-γ and TNF-α after short incubation. No significant differences were observed. The pooled (normalized) results from 2 different experiments are shown. Cor: Control, oral route; Gor: GMP, oral route; Cip: Control, i.p. route; Gip: GMP, i.p. route. It appears therefore that actions on lymphocytes predominate at concentrations below 1 mg mL-1. The mechanism of action of GMP is not known but clearly is located at, or upstream of, STAT4 translocation, which is exquisitely inhibited by the peptide. The effect on TNF-α production could be just the consequence of the impaired IFN-γ production, as macrophages lacked this stimulus. There are two main pathways to stimulate IFN-γ production in lymphocytes: IL-12/IL-18 and T Cell Receptor (TCR). STAT4 has been found to be essential for IFN-γ production in CD4+ T cells, and to account for CD8+ T cell IFN-γ production. Both lineages require STAT4 activation for IFN-γ production induced by IL-12/IL-18 signaling, but only CD4+ T cells require STAT4 for IFN-γ induction via the TCR pathway [18, 19]. ConA acts by binding TCR independently of clone specificity and thereby producing a steady and powerful stimulation, bypassing the interaction with the MHC-peptide complex. STAT4 is phosphorylated as part of the signaling cascade that is thus activated, forming homodimers that regulate IFN-γ expression at the transcriptional level in the nucleus [24]. Of course, this is only one of STAT4 regulated genes, which include CD25 (IL-2 receptor alpha), among others [25, 26]. In addition, STAT4 has a role in chromatin remodeling in the context of CD25 regulation [21]. Interestingly, Otani et al. showed that GMP suppresses IL-2 receptor expression on mouse CD4+ T cells [5]. Thus we

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have at least two STAT4 dependent genes that are down-regulated by GMP. Otani et al. found also that bovine GMP binds the mouse CD4+ T cell membrane, suggesting a surface site of action. Unfortunately our own efforts to locate the site of GMP actions have been hampered so far by the lack of quality antibodies, but obviously this is an important point to pursue. Certainly the effect of GMP is not due to casoplatelin, as this subpeptide shows a greatly diminished activity.

BSA was used in our study as a control for any nonspecific effects of protein on splenocytes. Unexpectedly, BSA had some effects on splenocytes, which were in part similar to those of GMP, since they both reduce IFN-γ in ConA stimulated splenocytes, but with several important differences. At any rate, our data suggest that BSA has immunological effects that may have been overlooked, or that protein exerts some effect at this level in a non-specific fashion [31]. On the other hand, lactoferrin, included here for comparison purposes, has well characterized effects on lymphocyte proliferation and, accordingly, reduced dramatically thymidine uptake as well as TNF-α, IFN-γ and IL-2 production in ConA stimulated splenocytes. These effects are thus substantially different from those of GMP and indicate a different mechanism of action.

The other important issue in our study is the observation that GMP retains the ability to increase Foxp3 expression in splenocytes, but not thymocytes, in vivo, suggesting a peripheral Treg differentiation effect of GMP in which the oral route of administration plays a major role. Perhaps the reported prebiotic properties of GMP may partially account for this dependence of the oral route of administration [32, 33]. It should be noted that although it was believed originally that Foxp3+ Treg cells were generated solely in the thymus, it has now become clear that peripheral CD4+ cells can differentiate into Foxp3+ Treg cells as well under selective conditions. These appear to involve the inhibition of Th1/Th2 differentiation; thus neutralizing IFN-γ and IL-4 not only potentiates TGF-β-mediated Foxp3 induction in vitro but can also enhance antigen-specific Foxp3+ Treg differentiation in vivo [20]. However, in the in vivo experiments IFN-γ was not inhibited, but rather showed a certain tendency to increase after oral treatment. We speculated that the effect on Th1 cells/IFN-γ may require continuous GMP contact, and that cell isolation would therefore cancel it. In order to check this possibility, the same experiment was repeated (twice) but examining cells after only 5 h after ConA stimulation, a short incubation time yet one that ought to reveal any existing differences. Indeed, IFN-γ mRNA was induced robustly in the cultured splenocytes, but no effect of GMP could be detected. The reason for this important discrepancy between the in vitro and in vivo settings cannot be explained at this time. It is likely that GMP exerts direct inhibitory effects on Th1 lymphocytes and long term effects on Tregs. However, it suggests that IFN-γ downregulation may not be the primary mechanism responsible for Treg induction. Further experiments will be needed to elucidate the mechanism of these effects. Nevertheless, our results are consistent with a relevant role of lymphocyte actions in the intestinal antiinflammatory effect of GMP. The balancing effects of Treg (or Th2) cells and/or the direct inhibition of Th1 cells is expected not only to reduce inflammation in the intestine, but also to contribute to homeostasis and perhaps even intestinal maturation in the neonate. Experiments are underway to examine this possibility.


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Acknowledgements The authors are thankful to Dr. Mercedes González for technical assistance. We are also grateful to Davisco Foods International, Inc. (Eden Prairie, MN). CIBEREHD is funded by the Instituto de Salud Carlos III. References [1]. Zimecki M, Kruzel ML. Milk-derived proteins and peptides of potential therapeutic and nutritive

value. J Exp Ther Oncol 2007;6:89-106. [2]. Brody EP. Biological activities of bovine glycomacropeptide. Br J Nutr 2000;84:S39-S46. [3]. Chabance B, Jollès P, Izquierdo C, Mazoyer E, Francoual C, Drouet L, Fiat AM. Characterization of

an antithrombotic peptide from kappa-casein in newborn plasma after milk ingestion. Br J Nutr 1995;73:583-90.

[4]. Chabance B, Marteau P, Rambaud JC, Migliore-Samour D, Boynard M, Perrotin P, et al. Casein peptide release and passage to the blood in humans during digestion of milk or yogurt. Biochimie 1998;80:155-65.

[5]. Otani H, Horimoto Y, Monnai M. Suppression of interleukin-2 receptor expression on mouse CD4+ T cells by bovine kappacaseinoglycopeptide. Biosci Biotechnol Biochem 1996;60:1017-1019.

[6]. Bruck WM, Kelleher SL, Gibson GR, Graverholt G, Lonnerdal BL. The effects of alpha-lactalbumin and glycomacropeptide on the association of CaCo-2 cells by enteropathogenic Escherichia coli, Salmonella typhimurium and Shigella flexneri. FEMS Microbiol Lett 2006;259:158-62.

[7]. Nakajima K, Tamura N, Kobayashi-Hattori K, Yoshida T, Hara-Kudo Y, Ikedo M, et al. Prevention of intestinal infection by glycomacropeptide. Biosci Biotechnol Biochem 2005;69:2294-301.

[8]. Daddaoua A, Puerta V, Zarzuelo A, Suárez MD, Sánchez de Medina F, Martínez-Augustin O. Bovine Glycomacropeptide Is Anti-Inflammatory in Rats with Hapten-Induced Colitis. J Nutr 2005;135:1164-70.

[9]. Requena P, Daddaoua A, Martínez-Plata E, González M, Zarzuelo A, Suárez MD, et al. Bovine glycomacropeptide ameliorates experimental rat ileitis by mechanisms involving downregulation of interleukin 17. Br J Pharmacol. 2008;154:825-32.

[10]. Cross ML, Gill HS. Immunomodulatory properties of milk. Brit J Nutr 2000;84:S81-S89. [11]. Otani H, Monnai M, Kawasaki Y, Kawakami H, Tanimoto M. Inhibition of mitogen-induced

proliferative responses of lymphocytes by bovine κ-caseinoglycopeptides having different carbohydrate chains. J Dairy Res 1995;62:349-57.

[12]. Otani H, Monnai M. Induction of an interleukin-1 receptor antagonist-like component produced from mouse spleen cells by bovine kappa-caseinoglycopeptide. Biosci Biotechnol Biochem 1995;59:1166-8.

[13]. Monnai M, Horimoto Y, Otani H. Immunomodificatory effect of dietary bovine κ-caseinoglycopeptide on serum antibody levels and proliferative responses of lymphocytes in mice. Milchwissenschaft 1998;53:129-132.

[14]. Monnai M, Otani H. Effect of bovine κ-caseinoglycopeptide on secretion of interleukin-1 family cytokines by P3888D1 cells, a line derived from mouse monocyte/macrophage. Milchwissenschaft 1997;52:192-196.

[15]. Li EW, Mine Y. Immunoenhancing effects of bovine glycomacropeptide and its derivatives on the proliferative response and phagocytic activities of human macrophagelike cells, U937. J Agric Food Chem 2004;52:2704-2708.

[16]. Requena P, Daddaoua A, Guadix E, Zarzuelo A, Suarez MD, Sanchez de Medina F, et al. Bovine glycomacropeptide induces cytokine production in human monocytes through the stimulation of the MAPK and the NF-kappaB signal transduction pathways. Br J Pharmacol 2009;157:1232-1240.

[17] Lin JJ. Monoclonal antibodies against myofibrillar components of rat skeletal muscle decorate the intermediate filaments of cultured cells. Proc Natl Acad Sci U S A 1981;78:2335-9.

[18]. Thierfelder WE, van Deursen JM, Yamamoto K, Tripp RA, Sarawar SR, Carson RT, et al. Requirement for Stat4 in interleukin-12-mediated responses of natural killer and T cells. Nature 1996;382:171-4.

[19]. Carter LL, Murphy KM. Lineage-specific requirement for signal transducer and activator of transcription (Stat)4 in interferon gamma production from CD4(+) versus CD8(+) T cells. J Exp Med 1999;189:1355-60.

Capítulo 6

130

Capítulo 6

130

http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=Search&Term=%22Zimecki%20M%22%5BAuthor%5D&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVAbstractPlus

http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=Search&Term=%22Kruzel%20ML%22%5BAuthor%5D&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVAbstractPlus

http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=Search&Term=%22Chabance%20B%22%5BAuthor%5D&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVAbstractPlus

http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=Search&Term=%22Joll%C3%A8s%20P%22%5BAuthor%5D&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVAbstractPlus

http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=Search&Term=%22Izquierdo%20C%22%5BAuthor%5D&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVAbstractPlus

http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=Search&Term=%22Mazoyer%20E%22%5BAuthor%5D&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVAbstractPlus

http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=Search&Term=%22Francoual%20C%22%5BAuthor%5D&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVAbstractPlus

http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=Search&Term=%22Drouet%20L%22%5BAuthor%5D&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVAbstractPlus

http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=Search&Term=%22Fiat%20AM%22%5BAuthor%5D&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVAbstractPlus

http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=Search&Term=%22Chabance%20B%22%5BAuthor%5D&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVAbstractPlus

http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=Search&Term=%22Marteau%20P%22%5BAuthor%5D&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVAbstractPlus

http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=Search&Term=%22Rambaud%20JC%22%5BAuthor%5D&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVAbstractPlus

http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=Search&Term=%22Migliore-Samour%20D%22%5BAuthor%5D&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVAbstractPlus

http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=Search&Term=%22Boynard%20M%22%5BAuthor%5D&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVAbstractPlus

http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=Search&Term=%22Perrotin%20P%22%5BAuthor%5D&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVAbstractPlus

http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16684117&ordinalpos=8&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum


http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=Search&Term=%22Requena%20P%22%5BAuthor%5D&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_DiscoveryPanel.Pubmed_RVAbstractPlus

http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=Search&Term=%22Daddaoua%20A%22%5BAuthor%5D&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_DiscoveryPanel.Pubmed_RVAbstractPlus

http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=Search&Term=%22Mart%C3%ADnez-Plata%20E%22%5BAuthor%5D&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_DiscoveryPanel.Pubmed_RVAbstractPlus

http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=Search&Term=%22Gonz%C3%A1lez%20M%22%5BAuthor%5D&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_DiscoveryPanel.Pubmed_RVAbstractPlus

http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=Search&Term=%22Zarzuelo%20A%22%5BAuthor%5D&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_DiscoveryPanel.Pubmed_RVAbstractPlus

http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=Search&Term=%22Su%C3%A1rez%20MD%22%5BAuthor%5D&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_DiscoveryPanel.Pubmed_RVAbstractPlus

http://www.ncbi.nlm.nih.gov/pubmed/8700208?ordinalpos=12&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum

http://www.ncbi.nlm.nih.gov/pubmed/10209051?ordinalpos=9&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum

[20]. Wei J, Duramad O, Perng OA, Reiner SL, Liu YJ, Qin FX. Antagonistic nature of T helper 1/2 developmental programs in opposing peripheral induction of Foxp3+ regulatory T cells. Proc Natl Acad Sci U S A 2007;104:18169-74.

[21]. O'Sullivan A, Chang HC, Yu Q, Kaplan MH. STAT4 is required for interleukin-12-induced chromatin remodeling of the CD25 locus. J Biol Chem 2004;279:7339–7345.

[22]. Lim K, van Calcar SC, Nelson KL, Gleason ST, Ney DM. Acceptable low-phenylalanine foods and beverages can be made with glycomacropeptide from cheese whey for individuals with PKU. Mol Genet Metab 2007;92:176-8.

[23]. Aimutis WR. Bioactive properties of milk proteins with particular focus on anticariogenesis. J Nutr 2004;134:989S-95S.

[24]. Watford WT, Hissong BD, Bream JH, Kanno Y, Muul L, O'Shea JJ. Signaling by IL-12 and IL-23 and the immunoregulatory roles of STAT4. Immunol Rev 2004;202:139-56

[25]. Maritano D, Sugrue ML, Tininini S, Dewilde S, Strobl B, Fu X, et al. The STAT3 isoforms alpha and beta have unique and specific functions. Nat Immunol 2004;5:401-9.

[26]. Hoey T, Zhang S, Schmidt N, Yu Q, Ramchandani S, Xu X, et al. Distinct requirements for the naturally occurring splice forms Stat4alpha and Stat4beta in IL-12 responses. EMBO J 2003;22:4237-48.

[27]. Miyauchi H, Kaino A, Shinoda I, Fukuwatari Y, Hayasawa H. Immunomodulatory Effect of Bovine Lactoferrin Pepsin Hydrolysate on Murine Splenocytes and Peyer’s Patch Cells. J Dairy Sci 1997;80:2330–2339.

[28]. Bal dit Sollier C, Drouet L, Pignaud G, Chevallier C, Caen J, Fiat AM, et al. Effect of kappa-casein split peptides on platelet aggregation and on thrombus formation in the guinea-pig. Thromb Res 1996;81:427-37.

[29]. Jollès P, Lévy-Toledano S, Fiat AM, Soria C, Gillessen D, Thomaidis A, et al. Analogy between fibrinogen and casein. Effect of an undecapeptide isolated from kappa-casein on platelet function. Eur J Biochem 1986;158:379-82.

[30]. Caccavo D, Pellegrino NM, Altamura M, Rigon A, Amati L, Amoroso A, et al. Antimicrobial and immunoregulatory functions of lactoferrin and its potential therapeutic application. J Endotoxin Res 2002;8:403-17.

[30]. Wakabayashi H, Takakura N, Yamauchi K, Tamura Y. Modulation of immunity-related gene expression in small intestines of mice by oral administration of lactoferrin. Clin Vaccine Immunol 2006;13:239-45.

[32]. Bruck WM, Kelleher SL, Gibson GR, Nielsen KE, Chatterton DE, Lonnerdal B. rRNA probes used to quantify the effects of glycomacropeptide and alpha-lactalbumin supplementation on the predominant groups of intestinal bacteria of infant rhesus monkeys challenged with enteropathogenic Escherichia coli. J Pediatr Gastroenterol Nutr 2003;37:273-80.

[33]. Bruck WM, Redgrave M, Tuohy KM, Lonnerdal B, Graverholt G, Hernell O, et al. Effects of bovine alpha-lactalbumin and casein glycomacropeptide-enriched infant formulae on faecal microbiota in healthy term infants. J Pediatr Gastroenterol Nutr 2006;43:673-9.


131

http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=Search&Term=%22Wei%20J%22%5BAuthor%5D&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_DiscoveryPanel.Pubmed_RVAbstractPlus

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http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=Search&Term=%22Muul%20L%22%5BAuthor%5D&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_DiscoveryPanel.Pubmed_RVAbstractPlus

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Effect of BGMP on intestinal epithelial cells as a part of the immune system.

CAPÍTULO 7

Introduction Bovine glycomacropeptide (BGMP) is a milk peptide which is produced naturally in the gastrointestinal tract during digestion. Glycomacropeptide has several immune related effects, including intestinal anti-inflammatory activity in rats (1,2,10). The immunomodulatory activity of BGMP has been largely studied and ratified in vitro, suggesting stimulatory actions in monocytes (3, 4) and inhibitory effects in splenocytes/lymphocytes (5, 6). However, to our knowledge there is not information available about the immune effect of BGMP in intestinal epithelial cells (IECs). Therefore, we set up to investigate these actions and using two different cell lines, we demonstrated that BGMP does not exert a clear immune effect on IECs. Material and methods Reagents Except where indicated, all reagents were obtained from Sigma (Barcelona, Spain). GMP (BioPURE-GMPTM) was the kind gift of Davisco Foods International (Eden Prairie, MN). According to the manufacturer, the GMP content was 93% while fat and lactose contents comprised 0.2 and <1%, respectively. Casoplatelin (CAS) was synthesized with a purity >95% by Innovagen (Lund, Sweden). HT29 cells and Caco2 cells were cultured in D-MEM medium supplemented with 10% (v·v-1) heat-inactivated foetal bovine serum (FBS), 2 mmol·L-1glutamine, 100 U·L-1 penicillin, 0.1 mg·mL-1 streptomycin and 2.5 mg·mL-1 amphotericin B. Cytokine determination Cells were cultured in 24-well plates. After reaching confluence, BGMP or CAS were added to the medium during 24h. The supernatants collected after gentle centrifugation (4.5 g, 5 min, 4ºC) were kept at −80°C until IL-8 concentration was measured by ELISA kits (Biosource Europe, Nivelles, Belgium), following the protocols recommended by the manufacturers. Statistical analysis All results are expressed as mean ± SEM. Differences among means were tested for statistical significance by one way ANOVA and a posteriori least significance tests on preselected pairs. All analyses were carried out with the SigmaStat 2.03 program (Jandel Corporation, San Rafael, CA). Differences were considered significant at p<0.05. Results BGMP did not change IL-8 release by human intestinal epithelial cells HT-29 (Fig.1) and Caco-2 (Fig.2), with one exception: the highest concentration, 1 mg·ml-1, it slightly enhanced IL-8 production on HT29, reaching statistical significance. The addition of CAS, the undecapeptide produced after BGMP hydrolysis, to the medium did not modify IL-8 production either.

Effect of BGMP in intestinal epithelial cells

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0

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C BGMP CAS

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Fig.1 Effect of bovine glycomacropeptide (BGMP) and casoplateline (CAS) on the production of interleukin (IL)-8 by HT29 cells. After a 24h incubation with either peptide (0.001, 0.1 and 1 mg·mL-1) the secretion of the cytokine was measured by ELISA. Results are expressed as mean±SEM of six different experiments (n=3 in each experiment). + p<0.001 vs. control (C).

0

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Fig.2 Effect of bovine glycomacropeptide (BGMP) on the production of interleukin (IL)-8 by Caco2 cells. After a 24h incubation with either peptide (0.001, 0.1 and 1 mg·mL-1) the secretion of the cytokine was measured by ELISA. Results are expressed as mean±SEM of two different experiments (n=3 in each experiment). Discussion After having shown the bioactivity of BGMP on macrophages (4) and spleen cells (6), the aim of this study was to evaluate the possible role of IECs, the third type of cell important for the gastrointestinal (GI) immune system, in the intestinal anti-inflammatory activity of BGMP. Digestion and absorption of nutrients are the primary functions of IECs; however in the last years it has become clear that they act

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significantly against the entrance of pathogens by way of several mechanisms. Tight junctions, for example, form a physical barrier to the entrance of pathogens. Also, human enterocytes express in their basolateral membrane MHC-I as well as MHC-II, suggesting an antigen presenting-like function (7,8). And colonic epithelial cells produce a variety of interleukins (IL) including IL6, IL8, IL10, TNF, and TFG-β, which work as signals of mucosal barrier disruption to the lamina propria cells (9). To test whether BGMP exerts an effect on the immunological functions of IECs, we used the production of IL-8 as a parameter. Two different cell lines were used: HT29 and Caco. In HT29 cells, BGMP did not modify IL-8 production at the concentration of 0.01 and 0.1 mg·ml-1 and produced a less than 1.5 fold increase at the highest concentration assayed (1 mg·ml-1). The effect was none in Caco cells. As expected, the addition of casoplatelin to the cell culture medium in a concentration equivalent, after proteolysis, to 1 mg·mL-1 of BGMP produced no effect on cytokine production by HT29 cells. These results suggest that BGMP does not modify IL-8 production in IECs and therefore, probably it is not active on these cells from an immunological point of view. The concentration of 1 mg·ml-1 showed statistical differences versus control, at least in HT29 cells. However, we don’t consider this statistical effect as a biological effect. First of all, the discrepancy among experiments was very big and probably the statistical result is the consequence of an exaggerated number of samples (n=25). In addition, even if significant BGMP produced a less than 1.5-fold increase in IL-8 production, whereas the effects observed before in other cell types like macrophages and splenocytes were around 5-10 fold change. From our present data, we can not ascertain that BGMP does not regulate immune functions in IECs, since other parameters such as IL-6 or COX-2 could be modify. However these results are consistent with a mode of action of BGMP that does not involve enterocytes. Actually, the fact that BGMP did not affect to the production of AP or COX-2 in DSS-induced colitic rats despite its clear anti-inflammatory effect (10), strongly support that hypothesis. References 1. Daddaoua, A., Puerta, V., Zarzuelo, A., Suárez, M.D., Sánchez de Medina, F. and Martínez-Augustín,

O., Bovine Glycomacropeptide Is Anti-Inflammatory in Rats with Hapten-Induced Colitis. J. Nutr. 2005. 135: 1164-70.

2. Requena, P., Daddaoua, A., Martínez-Plata, E., González, M., Zarzuelo, A., Suárez, MD., Sánchez de Medina, F., Martínez-Augustin, O., Bovine glycomacropeptide ameliorates experimental rat ileitis by mechanisms involving downregulation of interleukin 17. Br J Pharmacol. 2008 154 (4): 825-32.

3. Requena P, Daddaoua A, Guadix E, Zarzuelo A, Suarez MD, Sanchez de Medina F et al. (2009). Bovine glycomacropeptide induces cytokine production in human monocytes through the stimulation of the MAPK and the NF-kappaB signal transduction pathways. Br J Pharmacol 157: 1232-1240.

4. Li EW, Mine Y (2004). Immunoenhancing effects of bovine glycomacropeptide and its derivatives on the proliferative response and phagocytic activities of human macrophagelike cells, U937. J Agric Food Chem 52: 2704–2708.

5. Otani H, Monnai M, Kawasaki Y, Kawakami H, Tanimoto M (1995). Inhibition of mitogen-induced proliferative responses of lymphocytes by bovine kappa-caseinoglycopeptides having different carbohydrate chains. J Dairy Res 62: 349-357.

6. Requena P, González R, López-Posadas R, Abadía-Molina A, Suárez MD, Zarzuelo A, Sánchez de Medina F, Martínez-Augustin O. The intestinal antiinflammatory agent glycomacropeptide has

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immunomodulatory actions on rat splenocytes. Sumitted for publication. 7. Hirata I, Austin LL, Blackwell WH, Weber JR, Dobbins WO 3rd. Immunoelectron microscopic

localization of HLA-DR antigen in control small intestine and colon and in inflammatory bowel disease. Dig Dis Sci. 1986 Dec; 31(12):1317-30

8. Mayer L, Eisenhardt D, Salomon P, Bauer W, Plous R, Piccinini L. Expression of class II molecules on intestinal epithelial cells in humans. Differences between normal and inflammatory bowel disease. Gastroenterology. 1991 Jan; 100(1):3-12.

9. Eckmann L, Jung HC, Schürer-Maly C, Panja A, Morzycka-Wroblewska E, Kagnoff MF.Differential cytokine expression by human intestinal epithelial cell lines: regulated expression of interleukin 8. Gastroenterology. 1993 Dec;105(6):1689-97.

10. López-Posadas R, Requena P, González R, Suárez MD, Zarzuelo A, Sánchez de Medina F, Martínez-Augustin O. Bovine glycomacropeptide has intestinal antiinflammatory effects in dextrane sulfate sodium rat colitis. Sibmitted for publication.

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Pathways mediating the anti-inflammatory effect of bovine glycomacropeptide in hapten-induced colitis in the rat involve the upregulation of IL-10 expression.

Fermín Sánchez de Medina, Abdelali Daddaoua, Raquel González, Pilar Requena, Antonio Zarzuelo, María Dolores Suárez, Olga Martínez-Augustin.Submitted for publication.

CAPÍTULO 8

Background and purpose: Bovine glycomacropeptide (BGMP) is a bioactive peptide derived from milk κ-casein. Previous studies have shown that it has anti-inflammatory effects in rat models of colitis and ileitis, but its mechanism of action is incompletely understood. This study was carried out to further elucidate the anti-inflammatory mechanism of action of BGMP. Experimental approach: A model of colitis induced by the administration of trinitrobenzene sulfonic acid was used. Affymetrix microarrays, and postgenomic ReT-PCR on 93 selected genes were performed. An in vitro model of primary splenocytes and isolated spleenic macrophages and T lymphocytes was also used to profile BGMP actions on specific cell types. Key results: Our results indicate that the anti-inflammatory effect of BGMP is associated with an inflammatory infiltrate in which lymphocytes rather than neutrophils or macrophages predominate, consistent with a more advanced state of recovery. Ingenuity analysis of the colonic transcriptome indicates that IL-6 is a pivotal cytokine in BGMP effect. In vitro BGMP elicits IFN-γ release by concanavalin A stimulated T lymphocytes but inhibits it in splenocytes. IL-10 production is enhanced by the peptide in basal and stimulated conditions in unfractionated spleen cells and in isolated macrophages. Our results therefore suggest that BGMP acts at least in part increasing IL-10 in the colonic mucosa. Conclusion and implications: The anti-inflammatory mechanism of action of BGMP involves upregulation of IL-10 expression by macrophages and downregulation of IL-6 expression. Our data support the use of BGMP as an anti-inflammatory peptide in the treatment of inflammatory bowel disease. Key words: inflammatory bowel disease, glycomacropeptide, genomic analysis, interleukin 10, interleukin 6, interleukin 17 Alphabetical list of non-standard abbreviations: AP: alkaline phosphatase; CD: Crohn’s disease; BGMP: bovine glycomacropeptide; IBD: inflammatory bowel disease; IL: interleukin; MHC: major histocompatibility complex; MMP: matrix metalloproteinase; MPO: myeloperoxidase; ReT-PCR: Real time polymerase chain reaction; RQ: Relative quantitation; TGF: transforming growth factor; TNBS: trinitrobenzene sulphonic acid; UC: ulcerative colitis. Introduction The concept of milk as a biologically active fluid was established a long time ago (Newby et al., 1982), and in the last decades the therapeutic value of milk proteins and peptides has been described (Brody, 2000; Zimecki et al., 2007). Bovine glycomacropeptide (BGMP) is a 64 amino acid glycosylated peptide derived from milk κ-casein (amino acids 106 to 169). This peptide is produced both physiologically by digestion of the native protein (Brody, 2000; Zimecki et al., 2007), and in the cheese making process as a result of the action of rennet chymosin (Brody, 2000). Therefore, it is present in relatively high amounts in milk whey, from which it can be readily purified. Because of its low content in aromatic amino acids (including phenylalanine) and its high content in branched chain amino acids, BGMP has been proposed as an ideal protein source to elaborate functional formulations for individuals with liver disorders or phenylketonuria

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(Lim et al., 2007; M. H. Abd El-Salama, 1996). In addition, BGMP is a functional peptide that has been shown to reduce dental plaque and caries and therefore is currently being added to tooth pastes (Aimutis, 2004). Inflammatory bowel disease (IBD) is comprised of two different but closely related diseases, namely ulcerative colitis (UC) and Crohn’s disease (CD). Both are chronic and relapsing conditions characterized by the inflammation of the gastrointestinal tract, although there are differences with regard to their pathophysiology and treatment. Thus, while UC affects only the mucosal layer of the colon, CD may involve any segment of the gastrointestinal tract and is characterized by transmural inflammation. The importance of inflammatory bowel disease lies in its increasing incidence and prevalence in developed countries and in its impact on the patients’ quality of life (Dubinsky, 2008; Koloski et al., 2008). IBD is caused by multifactorial environmental agents in genetically predisposed hosts, involving specially the mucosal immune system and the intestinal microbiota (Torres et al., 2008). Although there are treatments available for inflammatory bowel disease, the risk of adverse effects is high, particularly considering the chronic and relapsing nature of this disease. Therefore, several nutritional strategies have been developed in the last few years (Clarke et al., 2008). In this regard, studies carried out by our research group have established the anti-inflammatory effect of BGMP in models of ileitis and colitis in rats (Daddaoua et al., 2005; Requena et al., 2009). In these experiments BGMP was shown to decrease a wide range of inflammatory markers like alkaline phosphatase (AP), myeloperoxidase (MPO), nitric oxide synthase and interleukin-1β (IL-1β). Furthermore, in the model of ileitis we found that BGMP decreased IL-17 and TNF-α. In a recent in vivo study performed in the DSS model of colitis in the rat (manuscript submitted), the anti-inflammatory effect of BGMP was additionally associated with a reduction in the production of IFN-γ by mesenteric lymph node cells ex vivo. Thus the mechanism of action of BGMP may be related, at least in part, to actions on Th1 and Th17 cells. The aim of the present study was to further elucidate the intestinal anti-inflammatory mechanism of action of BGMP. We have used the model of colitis induced by the administration of trinitrobenzene sulfonic acid (TNBS) to rats and have characterized colonic gene expression using microarrays and ReT-PCR techniques. In addition, splenocytes and macrophage and T lymphocyte populations from normal rat spleens were obtained and cultured with BGMP in basal and LPS or ConA stimulated conditions. The main findings derived from our study are: (1) BGMP administration increases B and T cells at the site of inflammation while it reduces macrophages and neutrophils, suggesting that inflammation in BGMP treated animals is closer to resolution. (2) Upregulation of IL-10 could be a key feature in the mechanism of action of BGMP. (3) Macrophages seem to be the main source of BGMP-evoked IL-10 production, although other cells might also contribute. (4) IL-6 is a key cytokine in the effect of BGMP, and it is likely involved in the decrease of IL-17, in the reduction of neutrophil infiltration and the diminished matrix metalloproteinase gene expression and tissue damage. (5) The decrease in IL-17 expression, and therefore arguably also in Th17 cells, is correlated with a downregulation of IL-6 without alteration of the expression of IL-23.

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Methods Except where indicated, all reagents and primers were obtained from Sigma (Barcelona, Spain). Taq polymerase was purchased from Amersham Biosciences (Barcelona, Spain). Antibodies were purchased from Santa Cruz Biotechnology (Heidelberg, Germany) and Sigma (Barcelona, Spain). BGMP (BioPURE-BGMPTM) was the kind gift of Davisco Foods International, Inc. (Eden Prairie, MN). According to the manufacturer the BGMP content was 93% while fat and lactose contents accounted for 0.2% and less that 1%, respectively. Animals Female Wistar rats (175-225 g) obtained from the Laboratory Animal Service of the University of Granada were used, housed in makrolon cages and maintained in our laboratory in air conditioned animal quarters with a 12 h light-dark cycle. Animals were provided with free access to tap water and food (Panlab A04, Panlab, Barcelona, Spain). This study was carried out in accordance with the Directive for the Protection of Vertebrate Animals used for Experimental and other Scientific Purposes of the European Union (86/609/EEC). Induction of colitis Colitis was induced by the method of Morris et al. with minor modifications (Martinez-Augustin et al., 2008). Briefly, animals were fasted overnight and anaesthetized with halothane. Under these conditions, animals were given 10 mg of TNBS dissolved in 0.25 ml of 50% ethanol (v·v-1) by means of a Teflon cannula inserted 8 cm through the anus. Animals were kept in a head-down position for an additional 30 s and returned to their cage. Experimental design Rats were randomly assigned to three different groups. A control group C (n=3) that did not receive the TNBS challenge but 0.25 ml of phosphate buffered saline (PBS) intrarectally, and two groups (group T (n=3) and group BGMP (n=5)) that received the TNBS challenge as described above. The rats of the BGMP group received 500 mg·kg-1·day-1 of BGMP in 1% methylcellulose p.o, starting two days before the TNBS challenge, i.e. the dose previously reported as active in rats (Daddaoua et al., 2005; Requena et al., 2008). Treatment was administered to rats for five days after the TNBS challenge using an oesophageal catheter. Because we have previously characterized the TNBS model using microarrays and ReT-PCR in a large number of samples, n=3 for TNBS and control groups was considered and adequate number to carry out these experiments (Martinez-Augustin et al., 2009; Martinez-Augustin et al., 2008). The T and C groups received the vehicle. In all cases body weight as well as group water and food intake were recorded daily. Assessment of colonic damage Animals were sacrificed by cervical dislocation and the entire colon was removed and placed on an ice-cold plate, cleaned of fat and mesentery, and blotted on filter paper. Each specimen was weighed and its length measured under a constant load (2 g). The large intestine was longitudinally opened and scored for visible damage on a 0 to 25 scale according to the following criterion: adhesions (0-3), obstruction (0-2), thickening (0-2), hyperemia (0-3), fibrosis (0-3), necrosis (0-5), scarring and deformation (0-7). The colon was subsequently divided longitudinally in several pieces for biochemical determinations. All the samples were kept at –80ºC until used. AP activity was measured spectrophotometrically, using disodium nitrophenylphosphate (5.5 mM) as substrate in 50 mM glycine buffer with 0.5 mM MgCl2 (pH=10.5) (Sanchez de Medina et al., 2004). MPO activity was measured according to the technique described by Krawisz et al. (1984), using 0.5% hexadecyltrimethylammonium bromide in phosphate-buffered saline (pH 6.0) for tissue homogenization and o-dianisidine


143

dihydrochloride (0.5 mM) as chromogen. The results are expressed as MPO units (µmol·min-

1) per gram of wet tissue. RNA extraction, microarray hybridization and data analysis RNA was extracted from homogenized full-thickness colonic tissues in Trizol® reagent (Invitrogen, Barcelona, Spain) and purified with RNeasy affinity columns (Qiagen, Madrid, Spain). Quantity and integrity of RNA were assessed by spectrophotometry and 0.8% agarose gel electrophoresis, respectively. Equal amounts of RNA from each sample were pooled to obtain one final sample per group. Therefore three microarrays were analyzed. Sample labeling, hybridization, staining and scanning procedures were carried out using Affymetrix standard protocols (www.affymetrix.com). The microarray analysis was performed by Progenika Biopharma (Bilbao, Spain) on GeneChip® Rat Expression Array 230 2.0 microchips (Affymetrix), containing sequences for approximately 31000 rat genes. Normalization was carried out using GeneSpring v7.1 (Agilent) while Ingenuity Pathway Analysis software (Monuntain View, CA, USA) was used to perform the functional analysis. The analysis produced three categories of results: functions, molecular networks and global canonical pathways affected by the treatment. MIAME recommendations (Killion et al., 2003) were followed to ensure that all information needed to understand, interpret, reproduce and compare our results is given in detail. The data are accessible at the European Bioinformatics Institute Arrayexpress database (http://www.ebi.ac.uk, reference E-MEXP-1631). Postgenomic validation Postgenomic validation was carried out by measuring 93 genes in fresh samples using ReT-PCR with TaqMan® Low Density Arrays (Applied Biosystems). The relative Ct values of each gene compared to that of the reference gene (18 S) were used to calculate the RQ (relative quantitation) parameter, which represents the change in mRNA expression compared to a control sample. The RQs were then used to calculate fold change ratios. Results are expressed as mean ± SEM. In addition, we applied real time PCR to assess IL-17A mRNA levels. This gene was not available either in the microarray or in the TaqMan low density array; therefore we used the following primers and conditions: sense 5’-TTC TCC AGA ACG TGA AGG TC-3’; antisense 5’-GGA CAA TAG AGG AAA CGC AG-3’. Hybridization temperature was set at 51ºC. The ribosomal 18 S unit was also assessed as a cDNA loading control using the following primers and conditions: sense 5’-CCA TTG GAG GGC AAG TCT GGT G-3’; antisense 5’-CGC CGG TCC AAG AAT TTC ACC-3’. Hybridization temperature was set at 60ºC. Fold change ratios for IL-17A were calculated as above. Cell subsets isolation and culture Female Wistar rats were sacrificed by cervical dislocation and the spleen was extracted aseptically. Cell suspensions were obtained disrupting the splenic tissue between dissecting forceps in medium. After centrifuging, cells were cleared of erythrocytes by resuspension on hypotonic lysis buffer (0.15 M NH4Cl, 10 mM KHCO3, 0.1 mM Na2EDTA·2H2O, pH=7.3) for 30 min on ice. For splenocyte experiments, mononuclear cells were washed and suspended in RPMI medium supplemented with 10% FBS, 100 mg l-1 streptomycin, 100000 U l-1 penicillin, 2.5 mg l-1 amphotericin B and 0.05 mM of mercaptoethanol. To purify lymphocytes, mononuclear cells were resuspended in rinse buffer (phosphate buffer saline pH=7.2, 0.5% BSA, 2 mmol L-1 EDTA) and single-cell suspensions were obtained using a 70 μm cell strainer (BD Falcon™, Madrid, Spain). For lymphocyte isolation, non-target cells were magnetically labeled and retained in a MACS® column placed in a MACS® separator.

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http://www.affymetrix.com/

http://www.ebi.ac.uk/

Target cells passed through the column were collected, rinsed and suspended in RPMI medium supplemented as above. Magnetic labeling was indirect, using biotin- or phycoeritrin-labeled antibodies against CD11b, CD161.a and CD45RA (BD Biosciences, Erembodegem, Belgium) and specific MACS® microbeads (Miltenyi Biotec, Madrid, España). To isolate macrophages, we used the same protocol as for lymphocytes with the following changes. Target cells were suspended in DMEM medium supplemented with 10% FBS, 100 mg l-1 streptomycin, 100000 U l-1 penicillin, 2.5 mg l-1 and amphotericin B; and the labeling antibodies were anti-CD161.a-biotin, CD45RA-PE and anti-CD3-biotin. Separation protocols were set up and validated by flow citometry. Cytokine determination Splenocytes and lymphocytes (106 cells mL-1 in RPMI medium) were cultured for 48 h with BGMP 0.01, 0.1 or 1 mg ml-1, in the absence or presence of concanavalin A (ConA, 5 µg mL-

1). Macrophages (106 cells mL-1 in DMEM) were cultured for 24 h with the same concentrations of BGMP and when needed were stimulated with lipopolysaccharide (LPS, 1 μg mL-1). The supernatants collected after gentle centrifugation (4.5 g, 5 min, 4ºC) were kept at 80ºC until cytokine concentrations (IL-6, IL-10, IFN-γ) were measured using ELISA kits (Biosource Europe, Nivelles, Belgium and BD Biosciences, Madrtid, Spain). Statistical analysis A cutoff value of 2.0/0.5 was applied for both fold change vs. control and fold change vs. the T group. In general, differences among means were tested for statistical significance by one way ANOVA and a posteriori least significance tests. All the regular analyses were carried out with the SigmaStat program (Jandel Corporation, San Rafael, CA, U.S.A.). Statistical significance was set at P<0.05. Table 1. Food intake, body weight gain and macroscopic indexes of rat colonic inflammation in the different experimental groups.

C T BGMP

Food intake (g) 16.13 ± 2.33a 3.90 ± 3.18b 10.43±2.65a

Body weight gain (g) 7.36 ± 1.71a -13.45 ±2.13b 3.01 ±3.87a

Colonic weight (g) 1.11 ± 0.06a 2.18 ± 0.12b 1.41 ± 0.16a

Colonic length (cm) 16.33 ± 0.25a 13.00 ± 0.00b 16.5 ± 0.76a

Colonic weight/length ratio (mg/cm)

68 ± 3.8a 168 ± 8.7.7b 86 ±11.7c

Damage Score 11.3 ± 0.6a 3.0 ± 0.6 b

Damage length (cm) 3.3 ± 0.6a 0.3 ± 0.3 b

1Values are means ± s.e.m. Means for a row without a common letter differ, P<0.05.


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Results BGMP has anti-inflammatory effect on TNBS treated rats. The morphological and biochemical features of TNBS colitis were consistent with previous results (Daddaoua et al., 2005). Induction of colitis produced anorexia and body weight loss, and resulted in extensive mucosal damage, edema, fibrosis and epithelial necrosis (Table 1). An increase in colonic MPO activity was also observed, indicating leukocyte infiltration. Accordingly, colonic AP activity, a marker of inflammation, was also increased after TNBS exposure (Figure 1). As expected, and in agreement with previous results (Daddaoua et al., 2005), the administration of BGMP modified the above mentioned parameters and generally reduced intestinal inflammation (Table 1, Figure 1).

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performed using TaqMan Low density arrays with 93 selected genes. All the samples (n=3 for C and T groups and n=5 for the BGMP group) were assayed separately. There was an excellent correlation between microarray and ReT-PCR values (regression coefficient 0.98231), indicating the validity of microarray data (Figure 2). Furthermore, the data obtained for the T and C groups were largely consistent with previous results (Martinez-Augustin et al., 2009; Martinez-Augustin et al., 2008). Tables 2 and 3 show fold change values for genes selected for the ReT-PCR analysis whose expression was affected by TNBS colitis (downregulated or upregulated, respectively), and the effect of BGMP (unchanged, upregulated or downregulated). Microarray and ReT-PCR analysis evidenced the impact of colonic inflammation (T group) in the form of a dramatic increase in classical proinflammatory genes (Il1β, IL-6, Il23 or Nos, for instance) and in genes related to tissue remodeling (Mmp3, 2, 9 or 12). Table 2. Genes downregulated by the administration of TNBS and not altered, increased or normalized by the administration of BGMP. qRT.PCR results expressed as fold change. Gene Description TNBS vs C BGMP vs C Atp1a1 ATPase, Na+/K+ transporting, alpha 1 polypeptide 0.598 ± 0.053* 0.627 ± 0.024*Cftr Cystic fibrosis transmembrane conductance

regulator 0.643 ± 0.030* 0.670 ± 0.045*

Ckm Creatine kinase, muscle 0.433 ± 0.015* 0.602 ± 0.051*Cnr1 Cannabinoid receptor 1 0.383 ± 0.065* 0.453 ± 0.133*Dnase1l3 Desoxirribonuclease I 0.592 ± 0.054* 0.705 ± 0.029*Epim Epimorphin 0.669 ± 0.012* 0.715 ± 0.036*Gpd1 Glycerol-3-phosphate dehydrogenase 1 0.661 ± 0.057* 0.689 ± 0.044*Idh3g Isocitrate dehydrogenase 3 (NAD+) gamma 0.657 ± 0.024* 0.631 ± 0.010*Pdhb Pyruvate dehydrogenase (lipoamide) B 0.563 ± 0.037* 0.696 ± 0.034*Ppara Peroxisome proliferator activated receptor alpha 0.302 ± 0.018* 0.489 ± 0.053*Scnn1a Sodium channel, nonvoltage-gated 1 alpha 0.446 ± 0.019* 0.624 ± 0.046*Scd1 Stearoyl-Coenzyme A desaturase 1 0.072 ± 0.009* 0.110 ± 0.009*Slc12a2 NKCC 0.671 ± 0.054* 0.727 ± 0.030*Slc9a2 NHE2 0.458 ± 0.030* 0.507 ± 0.008*Slc9a3 NHE3 0.471 ± 0.028* 0.495 ± 0.019*Tacr2 Tachykinin receptor 2 0.505 ± 0.026* 0.719 ± 0.023*Alox5 Arachidonate 5-lipoxygenase 0.477 ± 0.055*# 0.782 ± 0.056*Cd3d CD3d molecule, delta (CD3-TCR complex) 0.163 ± 0.018*# 0.645 ± 0.058*Cd3z CD3d molecule, zeta (CD3-TCR complex) 0.130 ± 0.021*# 0.772 ± 0.055*Slc26a3 Down-regulated in adenoma 0.534 ± 0.036*# 0.698 ± 0.033*Aldob Aldolase b 0.185 ± 0.004*# 0.425 ± 0.031 Cd74 Cd74 (invariant polypeptide of major

histocompatibility complex, class II antigen-associated)

0.550 ± 0.017*# 0.984 ± 0.037

Cd79b B-cell antigen receptor complex-associated protein beta chain; B-cell-specific glycoprotein B29

0.025 ± 0.003*# 0.812 ± 0.041

Cd8a CD8 antigen alpha polypeptide; OKT8 T-cell antigen 0.577 ± 0.036*# 0.810 ± 0.048 Cxcr4 Chemokine (C-X-C) receptor 4 0.501 ± 0.024*# 0.900 ± 0.040 Il12a IL-12, subunit p35 0.745 ± 0.036*# 1.060 ± 0.036 Il2rg Interleukin 2 receptor gamma chain 0.400 ± 0.026*# 0.828 ± 0.029 Lat Linker for activation of T cells 0.298 ± 0.032*# 0.880 ± 0.070 Lck T-lymphocyte specific protein tyrosine kinase p56lck;

p56(LSTRA) protein-tyrosine kinase 0.207 ± 0.010*# 0.791 ± 0.065

Lnk SH2B adaptor protein 3, lymphocyte adaptor protein 0.493 ± 0.049*# 0.845 ± 0.063 Means±SEM are shown. *: vs C, p<0.05; # vs BGMP, p<0.05.


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Table 3. Genes upregulared by the administration of TNBS and not altered, decreased or normalized by the administration of BGMP. qRT.PCR results expressed as fold change. Gene Description TNBS vs C BGMP vs C Col1a1 Alpha 1 type I collagen 3.856 ± 0.252* 3.298 ± 0.190*Cybb Cytochrome b-245, beta polypeptide 1.752 ± 0.189* 1.462 ± 0.089*Il23a Interleukin 23, alpha subunit p19 2.567 ± 0.294* 2.112 ± 0.304*Tff2 Trefoil factor 2 2.544 ± 0.373* 3.321 ± 0.284*Aqp9 Aquiaporin 9 18.086 ± 2.811*# 8.760 ± 1.471*C3 Complement component 3 22.083 ± 1.819*# 10.337 ± 0.510*Ccl2 Monocyte chemoattractant protein-1 6.166 ± 0.412*# 2.438 ± 0.314*Cxcl1 GRO1 oncogene 48.772 ± 1.887*# 23.821 ± 0.542*Fcgr3 Fc fragment of IgG low affinity IIa receptor

for (CD32) 7.619 ± 0.626*# 3.439 ± 0.170*

Gpr109a G protein-coupled receptor 109B; interferon-gamma inducible gene, Puma-g

12.920 ± 0.659*# 8.802 ± 0.618*

Igf1 Insulin-like growth factor 1; somatomedin C 2.061 ± 0.166*# 1.622 ± 0.090*Il1b Intrerleukin 1 beta 15.965 ± 0.355*# 10.881 ± 0.290*Il1r2 CD121b; type II interleukin-1 receptor, beta 16.797 ± 0.751*# 11.876 ± 0.511*Il1rn IL1RN (IL1F3); intracellular IL-1 receptor

antagonist type II 10.352 ± 0.716*# 7.194 ± 0.595*

Il6 Interleukin 6 (interferon, beta 2) 17.135 ± 2.254*# 8.098 ± 1.669*Il17a Interleukin 17a 10.50 ± 0.86*# 6.15 ± 0.54*

Itgam Integrin, alpha-M (complement component receptor-3, alpha; antigen CD11B (p170)

4.338 ± 0.127*# 2.252 ± 0.067*

Lox Lysyl oxidase 6.505 ± 0.179*# 4.466 ± 0.136*Mmp9 Matrix metalloproteinase 9 19.527 ± 0.431*# 6.571 ± 1.001*Mmp12 Matrix metalloprotease 12 2.442 ± 0.134*# 1.817 ± 0.126*Nos2 Nitric oxide synthase2, inducible 912.012 ± 15.388*# 521.714 ± 40.226*Ptges Prostaglandin E synthase 2.882 ± 0.094*# 1.954 ± 0.233*Ptgs2 Cox2 prostaglandin-endoperoxide synthase 2 7.366 ± 0.352*# 5.149 ± 0.249*S100a8 S100 calcium binding protein A8; calgranulin A 555.694 ± 37.117*# 165.114 ± 9.092*Selp Selectin P, CD62 13.949 ± 2.401*# 8.720 ± 1.220*Spp1 Secreted phosphoprotein 1; osteopontin 21.602 ± 2.021*# 9.929 ± 0.458*Tgm Transglutaminase 1 81.688 ± 3.377*# 45.927 ± 3.354*Mgp Down-regulated in adenoma 3.134 ± 0.117*# 1.093 ± 0.089 Mmp2 Matrix metalloproteinase 2 1.804 ± 0.076*# 1.249 ± 0.046 Mmp3 Matriz metalloproteinase 3 19.357 ± 1.557*# 2.453 ± 0.245 Pla2g2a Phospholipase A2 12.169 ± 1.700*# 3.218 ± 0.137 Nox1 NADH/NADPH mitogenic oxidase subunit p65-

mox 3.948 ± 0.726*# 2.028 ± 0.497

Cebpb CCAAT/enhancer binding protein beta; interleukin 6-dependent DNA-binding protein nuclear factor of interleukin 6

2.345 ± 0.152*# 1.261 ± 0.041

F2r Coagulation factor II receptor; thrombin receptor

1.425 ± 0.043*# 0.738 ± 0.026

Fcgr2b Fc fragment of IgG, low affinity II 7.402 ± 0.488*# 2.620 ± 0.389 Means±SEM are shown. *: vs C, p<0.05; # vs BGMP, p<0.05. BGMP decreases colonic neutrophil/macrophage and increases B and T cell infiltration. To rationalize microarray data, an analysis using the Ingenuity Pathway Analysis software was carried out with the list of 650 genes (400 upregulated and 250 downregulated) that resulted when the BGMP and T groups were compared. This approach showed that the main global functions affected by BGMP treatment were: the immune response (134 genes, selected specific functions altered in the immune response are shown in Table 4), the immune and lymphatic system development and function (126 genes), tissue morphology (98 genes),

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inflammatory disease (108 genes) and hematological system development and function (141 genes). The study of functions altered in the immune response showed that the administration of BGMP downregulated a wide array of genes specifically expressed in macrophages and/or neutrophils (Table 4), indicating a decrease in the activation, movement, recruitment and/or accumulation of these cell types. These results were further confirmed by the ReT-PCR data (Table 3). Our study also indicated a downregulation of genes involved in tissue remodeling, such as Mmp2, Mmp3, Mmp19, Mmp12, Timp and Lcn (Table 3).

Table 4. Immune response specific functions altered by BGMP administration in several cell types.

Function Cell type Downregulated genes Upregulated genes

Innate immune response

C3, C541, Ccl2, Ccl3, Ccl7, Ccr1, Cebpb, Csf3, Cxcl2, Cxcl3, Eln, Fcgr3a, Gna11, Il1a, Il1b, Il1rn, Il6, Il8rb, Itgam, Mmp9, Mx1, Nos2a, Orm1, Plau, Plaur, Reg-III, S100a8, S100a9, Serpina3, Slc11a1, Spp1, Tnfrsf1b

Activation Neutrophils C3, C5r1, Csf3, Cxcl3, Il1a, Il1b, Il1rn, Il6, Itgam

Macrophages Ccl2, Ccl3, Ccl7, Csf3, Il1b, Nos2a, RegIIII, Slc11a1

T Lymphocytes Ccl2, Ccl3, Ccl7, Cdkn1a, Il1rl1, Il6, Pla2g2a, Slc11a1, Tnfrsf1b, Tyrobp

Cd3d, Cd3e, Cd3z, Cd8a, Lat, Lck, Plp1, Tprc, Trb@, Vav2

B lymphocytes Lilrb3 Btk, Cd8a, Cr2, Lilrb3, Pprc, Vav2

Proliferation T lynphocytes Cdkn1a, Hmox1, Il1a, Il1b, Il2rl, Il6, Itgam, Mmp9, Spp1, Tnfrsf1b

Art2b, Cd37, Cd3e, Cd3z, Cd6, Cd8a, Cxcr4, Hla-drb1, Il2rg, Lck, Plp1, Ptpn6, Ptprc, Rasgrp1, cd37

B lymphocytes Cdkn1a, Cebpb, Il6, Slp1 Btk, Cd3e, Cd8a, Cr2, Il2rg,Lef1, Pik3r1, Plcg2, Ptpn6, Ptprc, Vav2

Movement neutrophils

C3, Ccl2, Ccl3, Cxcl2, Cxcl3, Fcgra3, Il1b, Il6, Il8rb, Itgam, Mmp9, Nos2a, Plau, S100a8, S100a9, Slp1, Socs3, Tnfrsf1b

Il16, Ptpn6

macrophages C3, Ccl2, Ccl3, Ccl7, Cxcl2, Eln, Il6, Plau, Plaur, Serpina3

Cxcr4, Il16

T lymphocytes C3, Ccl2, Ccl3, Ccr1, Cxcr4, Plau, Spp1

Cd69, Clk, Cxcr4, Il16

Recruitment Granulocytes/ neutrophils/ eosinophils

C3, C5r1, CclL2, Ccl3, Ccl7, Ccr1, Csf3, Cxcl2, Cxcl3, Il1b, Il1r1l, Il6, Il6rb, Il8rb, Itgam, Nos2a, Slp1, Socs3, Tnfrsf1b

CR2, Scgb1a1

Mononuclear leukocites/ macrophages

Apob, C3, Ccl2, Ccl3, Ccl7, Ccr1, Il6, Plau,

Il16

Accumulation Granulocytes/ neutrophils

Ccl2, Ccl7, Csf3, Cxcl3, Mmp8, Mmp9, S100a8, S100a9, Slp1

Macrophages Ccl2, Ccr1, Cxcl3, Il1rn, Mmp3

Degranulation Granulocytes Ccl2, Cxcl2, Cxcl3, Fcgra3, Itgam, Plaur


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On the other hand, BGMP increased the expression of genes involved in B cell activation and proliferation, and of genes expressed by B cells, including Btk, Cd79b, Cd8a, Cebpb, Lat, Ebf, Plcg2 and Ptpn6 (Tables 2 and 4). In addition, an upregulation of genes involved in both B and T cell receptor signaling (e.g. Cd3d, Cd3e, Cd3z, Cd79b, Cd8a, Lat, Lck, Pik3r1, Plcg2, Ppp3cc, Ptpn7, Ptpn6, Ptprc or Rasgrp1) and in major histocompatibility complex (MHC) molecules (Hla-dcb2, Hla-dqa1, Hla-dmb, Mhc1-b, Hla-drb1, Hmc2α) was observed (Figure 3, Tables 2 and 4).

Figure 3. IL-6 dependent biological network downregulated and lymphocyte network upregulated by the administration of BGMP to colitic rats. The Ingenuity Pathway Analysis software was used to identify biologically relevant networks affected by the treatment with BGMP of colitic rats. Red: Upregulation of gene expression in the BGMP group compared to the T group. Green: Downregulation of gene expression in the BGMP group compared to the T group. IL-6, IL-17 and IL-10 gene expression is decreased in the colon of BGMP treated rats. A further study of molecular networks affected by BGMP identified two interesting ones: an Il-6 (Figure 3) and an Il1b-dependent biological network (Figure 4), pointing at these genes as key regulators in the anti-inflammatory effect of BGMP. Interestingly, the expression of IL-10, an anti-inflammatory cytokine, was decreased by colitis and augmented (normalized) by BGMP (fold changes 0.19 (TNBS vs C) and 4.92 (BGMP vs C)).

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Figure 4. IL-1 dependent biological network downregulated by the administration of BGMP to colitic rats. The Ingenuity Pathway Analysis software was used to identify biologically relevant networks affected by the treatment with BGMP of colitic rats. Red: Upregulation of gene expression in the BGMP group compared to the T group. Green: Downregulation of gene expression in the BGMP group compared to the T group. Taking into account that previous experiments indicated that Th17 cells could be involved in the mechanism of action of BGMP, we studied the expression of IL-17 and related genes in the microarrays. Our results indicate that the expression of both IL-17f (FC 4.0 T vs C) and IL-17 (also called IL-17A, Table 3) was increased after the administration of TNBS, and decreased by BGMP treatment (IL-17f FC 0.20 BGMP vs T, see Table 3 for IL-17a). Differentiation of activated T cells into Th17 cells is potently induced by the simultaneous exposure to IL-6 and TGF-β, while IL-23 (composed of Il23a and Il12b) has been shown to be important for their proliferation and survival. TNBS colitic rats showed an increased expression of Il6 and Il23, while Il12b was unchanged (Table 2 and data not shown). Of note, BGMP administration specifically downregulated the expression of IL-6 without affecting Il23 or Il12b. Finally, it is interesting to note that Ifng was not changed by the administration of TNBS or BGMP (0.66±0.38 (TNBS vs C) and 0.31±0.18 (BGMP vs C)). The high


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standard deviation of the control group probably prevented this difference from reaching statistical significance. BGMP increases the production of IL-10 in spenocytes and spleen-derived macrophages and T-lymphocytes. To further elucidate the mechanism of action of BGMP primary splenocytes as well as splenic isolated macrophages and T lymphocytes were obtained. The production of IL-6, IL-10 and IFN-γ was studied in the appropriate isolated cells in basal and stimulated conditions. Our results indicate that BGMP increased IL-10 expression in all three cell preparations in basal conditions. In addition, it augmented IL-10 in LPS-treated macrophages and ConA-treated splenocytes, but not in stimulated T lymphocytes (Figure 5). On the other hand, BGMP had no effect on IL-6 production by macrophages or splenocytes in basal conditions, although an increase was observed when macrophages were cultured in the presence of LPS (Figure 6). Finally, BGMP caused an augmented release of IFN-γ in ConA-treated T lymphocytes, while it produced the opposite effect in the mixed splenocyte population (Figure 6). Discussion and conclusions The present study was undertaken in order to delineate the mechanism of action of BGMP as an intestinal anti-inflammatory agent. Using semiquantitative RT-PCR we showed previously that BGMP decreases the expression of IL-1β, IL-1ra, TNF, and IL-17 in ileitis/colitis (Daddaoua et al., 2005; Requena et al., 2008). BGMP also activates macrophages in vitro, while it inhibits IFN-γ production and nuclear STAT4 translocation in splenocytes ((Requena et al., 2009) and manuscript submitted). In addition, Foxp3 expression is increased in splenocytes of rats given BGMP for 3 days (manuscript submitted). Based on these and other results we anticipated that the mechanism of action of BGMP probably involves actions on lymphocytes (Th1, Th17 and Treg cells) and macrophages (Requena et al., 2009). The use of powerful genomic and postgenomic techniques has allowed us to gain further insight into the mechanism of action of this peptide. We have carried out the present study in the model of colitis, a more frequent condition than ileitis. In the above mentioned studies the dose of 500 mg/kg·day was effective administered as a pretreatment. Furthermore, its effect was comparable to that of sulfasalazine, a widely used drug in the treatment of IBD. We have used the same dose and our results are consistent with the described anti-inflammatory effect. It is known that in acute inflammation, and in particular in the TNBS model of colitis, leukocyte recruitment to the inflammatory site is characterized by an initial infiltration of neutrophils, which are later replaced by a more sustained influx of mononuclear cells, initially macrophages and then lymphocytes (Topley et al., 1996). Therefore, the presence of increased amounts of lymphocytes, together with a decrease in neutrophils and macrophages, may be interpreted as indicative of an advanced stage of recovery from inflammation. Indeed, data processing with the Ingenuity Pathway Analysis software indicated just that, namely a decrease in the expression of genes related to macrophage and granulocyte activation, movement, recruitment and/or accumulation with BGMP treatment (Table 4), together with a general upregulation of both B and T cell related genes (Table 4, Figure 4), suggesting that the inflammatory state of BGMP treated animals was closer to resolution. These results are further sustained by the finding that downregulation of Il1b (mainly produced by macrophages) (di Giovine et al., 1990), and Il6 (a neutrophil recruitment cytokine) (Mitsuyama et al., 2006) are identified by Ingenuity as key genes in the effect of BGMP (Figures 3 and 4, see below). Because of the nature of our study we cannot ascertain whether BGMP prevented the inflammatory damage induced by TNBS administration or accelerated

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recovery from injury. Nevertheless, in a previous study we examined the effect of BGMP administered before (pretreatment) or after (postreatment) the TNBS challenge and found that pretreatment was much more effective (Daddaoua et al., 2005). Thus the action of BGMP is probably of the preventive type. Ifng expression in the colon was neither affected by inflammation nor by BGMP treatment. The TNBS model of colitis is considered a Th1 driven type of inflammation in mice, and IFN-γ is increased in mesenteric lymph node cells isolated from TNBS treated rats (unpublished data). However, expression of IFN-γ in the colon is only weakly increased at best, specially compared with other cytokines (Martinez-Augustin et al., 2008; Perez-Navarro et al., 2005), and therefore the lack of significant changes in IFN-γ in our study is not surprising.

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In order to clarify this issue we performed in vitro experiments with BGMP in isolated cell populations (T lymphocytes and macrophages) obtained from the spleen of normal (uninflamed) rats, and compared them with those performed with the mixed splenocyte population. Our results show that, while BGMP reduces IFN-γ secretion by ConA-stimulated splenocytes, it does not inhibit but actually increases IFN-γ production in T lymphocytes. Therefore we hypothesized that the macrophages present in the splenocyte population may account for the global inhibitory effect observed in these cells. Indeed, BGMP evokes IL-10 production both in basal conditions and under LPS stimulation, suggesting that IL-10 mediates the inhibitory effects attributed to direct actions on lymphocytes in our previous study. Furthermore, the effect on IL-10 is more potent (significant effect at 0.1 and 1 g/l) than that exerted on T lymphocytes, which appears only at 1 g/l. In contrast, BGMP does not enhance ConA-evoked IL-10 release in T lymphocytes, consistent with macrophages as the main anti-inflammatory target of BGMP. Consistent also with a major role of IL-10 in the

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mechanism of BGMP, this cytokine was shown to be downregulated by the induction of colitis in our study, and upregulated back to control levels by treatment with the peptide. Of note, an increase in IL-10 is expected to augment Treg differentiation and Foxp3 expression, as observed in previous studies with BGMP (O'Connor et al., 2009; Requena et al., 2008). As alluded to before, IL-6 was singled out by Ingenuity as a pivotal cytokine in the effect of BGMP. IL-6 is a key factor in the development of inflammation and specifically in the uncontrolled inflammatory process in IBD (Carey et al., 2008; Mitsuyama et al., 2006; Mudter et al., 2007). Increased blood and mucosal levels of this cytokine, related to the severity of the disease, have been described in UC and CD patients (Mudter et al., 2007). Accordingly, antibodies against IL-6 or its soluble receptor have been shown to be useful in the treatment of experimental colitis and/or IBD (Mitsuyama et al., 2006). There is growing evidence indicating that this cytokine, produced mainly by macrophages and CD4+ T cells, is one of the main ones in the chronic phase of colitis (Mudter et al., 2007). IL-6 has three main roles in inflammation: (1) the recruitment of neutrophils to the inflammation site and their activation to produce matrix metalloproteinases, contributing to severe tissue damage. (2) The regulation of CD4+ leukocyte apoptosis: IL-6, together with IL-12 and TNF, is an important antiapoptotic cytokine involved in the pathogenesis of CD, whose inflammatory response, like that of the TNBS model of colitis, is driven by Th1 (and possibly Th17) cells. And, (3) the induction of Th17 cell differentiation, a newly described subpopulation of helper T cells, considered to be involved in the pathogenesis of inflammatory bowel disease (Carey et al., 2008; Mitsuyama et al., 2006; Mudter et al., 2007). Th17 cell differentiation and survival depends on TGF-β, IL-6 and IL-23 (Mizoguchi et al., 2008; Mudter et al., 2007). Our data indicate that while the induction of colitis increased the colonic expression of the Il6 and Il23 genes, BGMP only dowregulated IL-6, in a specific fashion. However, the fact that our in vitro studies showed no effect of BGMP on the production of IL-6 in isolated splenocytes either in basal or stimulated conditions suggests that BGMP may not have a direct effect on the inhibition of IL-6 expression in colitis. It should be noted in this regard that the expression of IL-10 and IL-6 is correlated. In fact, several studies have shown upregulation of IL-6 production in IL-10 knock out mice under several conditions (Robertson et al., 2007; Stoffels et al., 2009). On the other hand, a number of studies in animal models of colitis have demonstrated a link between IL-6 inhibition or suppression and IL-10 production, indicating that the former could be of great importance in the resolution of colitis (Gay et al., 2006; Naito et al., 2004). Thus, while we cannot ascertain from our in vivo data whether the primary mechanism of action of BGMP is the induction of IL-10 or the downregulation of IL-6, our in vitro data point to the downregulation of IL-10 as one of the key actions of BGMP in inflammatory conditions. The subsequent downregulation of IL-6 would (see Fig. 7): (1) decrease Th17 cells, (2) reduce neutrophil infiltration at the site of inflammation, (3) lower the expression of tissue remodelation genes, probably as a consequence of the decrease neutrophil infiltration. Additional studies will be required to assess the mechanism of IL-10 enhanced production and its relation with IL-6 modulation in vivo.


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Figure 7. New insights into the antiinflammatory mechanism of bovine glycomacropeptide (BGMP) in the intestinal inflammation. BGMP increases IL-10 production mainly in macrophages, while specifically downregulates IL-6. This in turn could downregulate IL-17 expression.

Acknowledgements The authors are grateful to Davisco Foods International (Eden Prairie, MN) for kindly providing us with the BGMP. This study was supported by grants of the Instituto de Investigación Carlos III (PI051651 and PI051625), grants of the Ministry of Science and Education (AGL2008-04332 and SAF2008-01432) and funds from Junta de Andalucía and the CIBEREHD. OMA was funded by the I3 program of the Ministry of Education and Science. RG is funded by the CIBEREHD. PR is funded by Ministery of Science and Innovation. The CIBER-EHD is funded by the Instituto de Salud Carlos III. The group is member of the Network for Cooperative Research on Membrane Transport Proteins (REIT), co-funded by the Ministery of Education and Science, Spain and the European Regional Development Fund (Grant BFU2005-24983-E/BFI).

Conflicts of interest None.

Il23

Il17aIL17f

Il12b

Il10 Il6

Il10

Il10 Il6 IL‐10 IL‐6 Il17a

IL17f

Macrophages

Lymphocytes

Macrophages

Th 17

Macrophages Macrophages

Th 17

Effect of TNBS on colonic gene expression (TNBS vs control)

Effect of BGMP on gene expression in TNBS induced colitis (BGMP vs TNBS)

Downregulated gene

Upregulated gene

Unchanged gene expression

Il6CD4+Tcells

Il23Il12b

CD4+TcellsIl6

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References Aimutis, WR (2004). Bioactive properties of milk proteins with particular focus on anticariogenesis. J Nutr 134:

989S-95S. Brody, EP (2000). Biological activities of bovine glycomacropeptide. Br J Nutr 84 Suppl 1: S39-46. Carey, R, Jurickova, I, Ballard, E, Bonkowski, E, Han, X, Xu, H, Denson, LA (2008). Activation of an IL-

6:STAT3-dependent transcriptome in pediatric-onset inflammatory bowel disease. Inflamm Bowel Dis 14: 446-57.

Clarke, JO, Mullin, GE (2008). A review of complementary and alternative approaches to immunomodulation. Nutr Clin Pract 23: 49-62.

Daddaoua, A, Puerta, V, Zarzuelo, A, Suarez, MD, Sanchez de Medina, F, Martinez-Augustin, O (2005). Bovine glycomacropeptide is anti-inflammatory in rats with hapten-induced colitis. J Nutr 135: 1164-70.

di Giovine, FS, Duff, GW (1990). Interleukin 1: the first interleukin. Immunol Today 11: 13-20. Dubinsky, M (2008). Special issues in pediatric inflammatory bowel disease. World J Gastroenterol 14: 413-20. Gay, J, Kokkotou, E, O'Brien, M, Pothoulakis, C, Karalis, KP (2006). Interleukin-6 genetic ablation protects

from trinitrobenzene sulfonic acid-induced colitis in mice. Putative effect of antiinflammatory cytokines. Neuroimmunomodulation 13: 114-21.

Killion, PJ, Sherlock, G, Iyer, VR (2003). The Longhorn Array Database (LAD): an open-source, MIAME compliant implementation of the Stanford Microarray Database (SMD). BMC Bioinformatics 4: 32.

Koloski, NA, Bret, L, Radford-Smith, G (2008). Hygiene hypothesis in inflammatory bowel disease: a critical review of the literature. World J Gastroenterol 14: 165-73.

Lim, K, van Calcar, SC, Nelson, KL, Gleason, ST, Ney, DM (2007). Acceptable low-phenylalanine foods and beverages can be made with glycomacropeptide from cheese whey for individuals with PKU. Mol Genet Metab 92: 176-8.

M. H. Abd El-Salama, SE-SaWB (1996). Characteristics and potential uses of the casein macropeptide International Dairy Journal 6: 15.

Martinez-Augustin, O, Lopez-Posadas, R, Gonzalez, R, Suarez, MD, Zarzuelo, A, Sanchez de Medina, F (2009). Genomic analysis of sulfasalazine effect in experimental colitis is consistent primarily with the modulation of NF-kappaB but not PPAR-gamma signaling. Pharmacogenet Genomics 19: 363-72.

Martinez-Augustin, O, Merlos, M, Zarzuelo, A, Suarez, MD, de Medina, FS (2008). Disturbances in metabolic, transport and structural genes in experimental colonic inflammation in the rat: a longitudinal genomic analysis. BMC Genomics 9: 490.

Mitsuyama, K, Sata, M, Rose-John, S (2006). Interleukin-6 trans-signaling in inflammatory bowel disease. Cytokine Growth Factor Rev 17: 451-61.

Mizoguchi, A, Mizoguchi, E (2008). Inflammatory bowel disease, past, present and future: lessons from animal models. J Gastroenterol 43: 1-17.

Mudter, J, Neurath, MF (2007). Il-6 signaling in inflammatory bowel disease: pathophysiological role and clinical relevance. Inflamm Bowel Dis 13: 1016-23.

Naito, Y, Takagi, T, Uchiyama, K, Kuroda, M, Kokura, S, Ichikawa, H, Yanagisawa, R, Inoue, K, Takano, H, Satoh, M, Yoshida, N, Okanoue, T, Yoshikawa, T (2004). Reduced intestinal inflammation induced by dextran sodium sulfate in interleukin-6-deficient mice. Int J Mol Med 14: 191-6.

Newby, TJ, Stokes, CR, Bourne, FJ (1982). Immunological activities of milk. Vet Immunol Immunopathol 3: 67-94.

O'Connor, W, Jr., Kamanaka, M, Booth, CJ, Town, T, Nakae, S, Iwakura, Y, Kolls, JK, Flavell, RA (2009). A protective function for interleukin 17A in T cell-mediated intestinal inflammation. Nat Immunol 10: 603-9.

Perez-Navarro, R, Ballester, I, Zarzuelo, A, Sanchez de Medina, F (2005). Disturbances in epithelial ionic secretion in different experimental models of colitis. Life Sci 76: 1489-501.

Requena, P, Daddaoua, A, Guadix, E, Zarzuelo, A, Suarez, MD, Sanchez de Medina, F, Martinez-Augustin, O (2009). Bovine glycomacropeptide induces cytokine production in human monocytes through the stimulation of the MAPK and the NF-kappaB signal transduction pathways. Br J Pharmacol 157: 1232-40.

Requena, P, Daddaoua, A, Martinez-Plata, E, Gonzalez, M, Zarzuelo, A, Suarez, MD, Sanchez de Medina, F, Martinez-Augustin, O (2008). Bovine glycomacropeptide ameliorates experimental rat ileitis by mechanisms involving downregulation of interleukin 17. Br J Pharmacol 154: 825-32.

Robertson, SA, Care, AS, Skinner, RJ (2007). Interleukin 10 regulates inflammatory cytokine synthesis to protect against lipopolysaccharide-induced abortion and fetal growth restriction in mice. Biol Reprod 76: 738-48.

Sanchez de Medina, F, Martinez-Augustin, O, Gonzalez, R, Ballester, I, Nieto, A, Galvez, J, Zarzuelo, A (2004). Induction of alkaline phosphatase in the inflamed intestine: a novel pharmacological target for inflammatory bowel disease. Biochem Pharmacol 68: 2317-26.

Stoffels, B, Schmidt, J, Nakao, A, Nazir, A, Chanthaphavong, RS, Bauer, AJ (2009). Role of interleukin 10 in murine postoperative ileus. Gut 58: 648-60.


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Topley, N, Liberek, T, Davenport, A, Li, FK, Fear, H, Williams, JD (1996). Activation of inflammation and leukocyte recruitment into the peritoneal cavity. Kidney Int Suppl 56: S17-21.

Torres, MI, Rios, A (2008). Current view of the immunopathogenesis in inflammatory bowel disease and its implications for therapy. World J Gastroenterol 14: 1972-80.

Zimecki, M, Kruzel, ML (2007). Milk-derived proteins and peptides of potential therapeutic and nutritive value. J Exp Ther Oncol 6: 89-106.

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Discusión.

CAPÍTULO 9

DISCUSIÓN La expresión “enfermedad inflamatoria intestinal” (IBD) se refiere a varias afecciones del tracto gastrointestinal de carácter inflamatorio crónico, de las cuales destacan la colitis ulcerosa (UC) y la enfermedad de Crohn (CD). Ambas consisten en estados inflamatorios crónicos y recurrentes del intestino, cuyos síntomas principales son la diarrea y el dolor intestinal, si bien existen síntomas y signos propios de cada una, e incluso algunos extra-intestinales. La IBD causa un deterioro significativo de la calidad de vida del paciente y tiene una incidencia y prevalencia relativamente elevadas, que además están en aumento (1, 2). A pesar de un intenso esfuerzo investigador realizado durante los últimos años, se desconoce la etiología exacta de la IBD, aunque actualmente se considera que la enfermedad representa una respuesta exacerbada e incontrolada frente a antígenos luminales intestinales en individuos genéticamente predispuestos. Aunque existe tratamiento farmacológico para la IBD, el cual produce mejoría en la mayoría de los pacientes, a menudo los fármacos usados, como los corticoides, aminosalicilatos o azatioprina, tienen efectos adversos importantes que limitan su uso. Además, los fármacos no son eficaces en todos los casos. Por lo tanto, la búsqueda de nuevos tratamientos con un mejor perfil de efectos secundarios está plenamente justificada (3). Es un hecho bien conocido que la leche es un alimento biológicamente activo, ya que su consumo produce beneficios más allá de los meramente nutricionales (4). Entre los componentes activos de la leche se encuentran tanto algunas de sus proteínas como fragmentos (péptidos) derivados de las mismas, que poseen, además de valor nutricional, capacidad moduladora de procesos fisiológicos. El glucomacropéptido bovino (BGMP), también denominado macropéptido o caseinmacropéptido, es uno de estos péptidos bioactivos de la leche de vaca (5). Entre las actividades que se han atribuido al BGMP se encuentra la unión a enterotoxinas de Vibrio cholerae y de Escherichia coli, la inhibición de la adhesión de bacterias y virus, la supresión de las secreciones gástricas, la promoción del crecimiento de bifidobacterias y la inhibición de la trombosis (5). Además, se ha demostrado que el BGMP modula la respuesta inmune, produciendo en general una activación de las respuestas del sistema inmune innato, mientras que posee un efecto inhibitorio o bloqueante sobre los esplenocitos de ratón (6-12). Estas propiedades biológicas convierten al BGMP en un péptido potencialmente útil en el tratamiento de la inflamación intestinal, especialmente considerando su baja toxicidad, puesta de manifiesto no sólo por formar parte de la dieta normal sino por la constatación de su baja inmunogenicidad (13). Sin embargo, existen muy pocos estudios sobre los efectos del BGMP sobre el sistema inmune in vivo. Entre ellos se encuentra uno realizado por nuestro grupo de investigación, el cual puso de manifiesto la actividad antiinflamatoria intestinal del BGMP en el modelo de colitis inducida por el ácido trinitrobenceno sulfónico (TNBS) en ratas (14). El modelo del TNBS es uno de los más usados a nivel preclínico para probar nuevos tratamientos, así como para estudiar la fisiopatología de la IBD. El TNBS es un hapteno y, por tanto, al unirse a las proteínas de la mucosa, desencadena una respuesta inflamatoria que se extiende durante varias semanas (26, 27). Tras 5-7 días, la inflamación inducida por TNBS se caracteriza por una respuesta CD4+, con inflamación transmural tipo Th1, fibrosis, infiltración de monocitos y linfocitos, y alteraciones de la motilidad y del transporte iónico intestinal.

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Así pues, el modelo reproduce hasta cierto punto las características de la enfermedad de Crohn. En el citado experimento, se administró por vía oral BGMP diariamente a las ratas empezando 2 días antes (pre-tratamiento) o 3 horas después (post-tratamiento) de la inducción de la colitis. El efecto del BGMP (500 mg/kg · día) fue comparado con el de la sulfasalazina, un medicamento utilizado comúnmente en el tratamiento de la IBD. Los resultados mostraron que el pre-tratamiento con BGMP tenía un efecto antiinflamatorio dosis-dependiente, que se caracterizó por una menor pérdida de peso, una disminución de la anorexia (57%), del daño en el colon (65%), y de la proporción peso/longitud del colon (32%), así como por una reducción de la actividad fosfatasa alcalina (42%) y los niveles de mRNA de IL-1β, TFF3 e iNOS en colon (p < 0.05) (14). En principio, cabe pensar que, al tratarse de un péptido, la administración oral de BGMP carece de sentido, dado el alto grado de hidrólisis proteica que se produce en el tracto gastrointestinal. Pero Chabance et al. demostraron que, aunque efectivamente tras la ingestión de leche de vaca (y humana) se detectan péptidos pequeños derivados del BGMP en el estómago y en el duodeno de humanos adultos, el BGMP tal cual o al menos una fracción del péptido completo se observa también en el estómago y en el plasma. Cabe destacar que los niveles de BGMP son detectados en el plasma hasta 8 h después de la ingestión de leche. Los autores de este estudio proponen que la continua producción de BGMP en el tracto gastrointestinal (al ser hidrolizada la κ-caseína) y los glúcidos del BGMP (que pueden proteger de la hidrólisis plasmática) son responsables de la presencia del BGMP en plasma durante tanto tiempo (29). Por otro lado, dado que alguno de los péptidos derivados del BGMP son bioactivos también (28), no podía descartarse que la hidrólisis fuese necesaria para la actividad biológica. La administración de BGMP a las ratas con colitis inducida por TNBS demostró por primera vez la actividad terapéutica del BGMP en la inflamación intestinal. La dosis usada equivale a 5 g en un humano adulto (equivalencia establecida en función del área corporal), una cantidad que no se obtiene por el consumo de leche pero que es fácilmente alcanzable si el BGMP se administra como un alimento funcional o un medicamento. Sin embargo, ese experimento constituía una aproximación insuficiente para poder conseguir la incorporación de este agente al tratamiento de la IBD, bien a nivel farmacológico, bien como nutracéutico. Se hace necesario en este caso realizar estudios adicionales, de carácter en buena medida mecanístico, al objeto de acumular evidencia suficiente para constituir una “prueba de concepto” que posibilite la realización eventual de estudios clínicos. Por tanto, planteamos extender nuestras observaciones a otros modelos animales y distintas poblaciones celulares. En consecuencia, este trabajo que integra la presente Tesis Doctoral, tiene por objeto la caracterización exhaustiva del efecto antiinflamatorio intestinal del BGMP. Para ello, en primer lugar verificamos la eficacia terapéutica en otro modelo de colitis animal, inducido por DSS en ratas. A continuación, determinamos el efecto del BGMP en un modelo animal de ileítis inducida por TNBS en ratas, para comprobar si la actividad antiinflamatoria del BGMP se limitaba al colon, lo que podría implicar la existencia de características farmacocinéticas que restringieran su actuación a determinados segmentos intestinales o bien un mecanismo prebiótico. Por otra parte, procedimos al estudio del efecto del BGMP sobre los principales tipos celulares implicados en IBD, para lo que usamos distintos modelos in vitro de linfocitos, macrófagos y enterocitos. Así, estudiamos la actividad del péptido sobre las líneas celulares epiteliales HT29 y

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Caco 2 (epiteliales), y THP1 (monocitos), así como sobre cultivos primarios de macrófagos humanos y esplenocitos de rata. En último término, hemos podido abordar el estudio de los efectos del BGMP sobre poblaciones aisladas de macrófagos y linfocitos T esplénicos murinos. Finalmente, una vez demostrada la bioactividad del BGMP sobre el sistema inmune, un estudio genómico en ratas con colitis inducida por TNBS nos ha permitido encontrar nuevas rutas inmunológicas modificadas por el BGMP, así como comprobar el funcionamiento in vivo de los mecanismos de acción caracterizados in vitro. Puesto que el BGMP resultó ser más eficaz en las ratas colíticas cuando fue administrado como pretratamiento que como postratamiento, se sugirió que el BGMP podría tener un efecto inmunomodulador previo, que mantuviera al sistema inmune “preparado” pero no “inflamado” ante cualquier ataque o daño que pudiera producirse posteriormente. En cualquier caso, parece más probable que el efecto del BGMP tenga más que ver con la prevención de la respuesta inmunológica que con su terminación y los procesos de reparación tisular, si bien este modo de acción es lo habitual en este tipo de ensayos preclínicos. Con base en este estudio, en los ensayos in vivo posteriores incluidos en la presente Tesis Doctoral se utilizó el mismo protocolo de pretratamiento con la misma dosis. Nuestros datos demuestran que el pretratamiento con BGMP (500 mg/kg•día) también reduce la gravedad de la colitis inducida por el DSS y de la ileítis inducida por el TNBS en ratas, con una eficacia similar a la que había demostrado en la colitis inducida por TNBS. En el modelo del DSS se escogieron las condiciones para inducir una colitis leve a moderada, mediante la utilización de concentraciones relativamente bajas de este compuesto y el seguimiento de la respuesta inflamatoria. Pese a la variabilidad que esto produjo en el grupo DSS, el BGMP tuvo un efecto sustancial sobre la mayoría de los marcadores inflamatorios, incluidos el índice de actividad de la enfermedad (DAI), la valoración macroscópica del daño colónico, la actividad MPO, las citoquinas TGFβ, IL23, IL1β, IL6, IL17 e IL10, así como el factor de transcripción Foxp3, la mayoría de los cuales fueron totalmente normalizados por el tratamiento, un efecto notable. Sin embargo, el tratamiento no consiguió modificar la actividad AP colónica ni la expresión de COX2 (ver más adelante). En la ileítis, el BGMP produjo una reducción de la extensión de la necrosis, el índice de daño macroscópico, la mieloperoxidasa (MPO), la AP y la iNOS, pero no tuvo efectos significativos sobre parámetros como la anorexia o la ratio peso:longitud del colon, al contrario de lo que había sucedido en la colitis. Teniendo en cuenta que en este modelo se extrae una muestra fija (y arbitraria) de unos 10 cm de intestino, es muy posible que, al hacerlo, incorporásemos una gran parte de tejido no inflamado que “diluyese” el impacto sobre este parámetro. Por su parte, la ausencia de efecto sobre la anorexia puede achacarse a una baja potencia estadística, dado que el consumo de comida fue mayor en el grupo tratado que en el TNBS desde el tercer día de ileítis hasta el fin del experimento. El BGMP también produjo la normalización de algunos mediadores de la inflamación, como el TNF y la IL1β. Por tanto, no existen discrepancias insalvables con respecto al modelo de colitis por TNBS. El hecho de que el BGMP muestre en la inflamación ileal un efecto antiinflamatorio similar al que mostró en la inflamación colónica sugiere que su mecanismo de acción está relacionado con un efecto directo sobre el sistema inmune, ya que la cantidad y variedad de bacterias en el íleon es mucho menor que en el colon. Aunque en nuestros estudios no

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hemos abordado la caracterización de la flora bacteriana intestinal, otros autores han puesto de manifiesto que el BGMP favorece el crecimiento de bifidobacterias in vitro. Existen también dos estudios realizados in vivo que son coherentes con el comportamiento del BGMP como prebiótico, pero cuyos resultados se ven comprometidos por un nivel muy alto de bifidobacterias previo al tratamiento (25,30,31,33). Por tanto, la posibilidad de que el BGMP actúe en parte como prebiótico no es descartable, aunque a la luz de nuestros resultados no parezca muy probable. Teniendo en cuenta la patogénesis de la IBD en general, y de los modelos animales utilizados en particular, planteamos la hipótesis de que el BGMP podría actuar a tres niveles:

1. Epitelio intestinal 2. Macrófagos 3. Linfocitos

1. Efecto del BGMP sobre el epitelio intestinal. En el estudio sobre el efecto del BGMP en la colitis inducida por el TNBS, el grupo BGMP había mostrado valores menores de fosfatasa alcalina (AP), TFF3, MUC2 y MUC4, moléculas expresadas fundamental o exclusivamente por el epitelio intestinal. En aquel momento, y dado que el aumento de todos estos factores en el grupo TNBS y su disminución en los grupos tratados era coherente con el mayor o menor estado inflamatorio de la mucosa, se concluyó que la inhibición asociada al BGMP no era probablemente mecanística sino consecuencia de una mejoría global, aunque no podía descartarse un efecto directo del BGMP sobre las células del epitelio intestinal. De la misma manera, en la ileítis inducida por el TNBS se produjo una disminución de la expresión de COX-2 (otra enzima expresada abundantemente en el epitelio (16)), AP y TFF3 en el grupo BGMP. Curiosamente, en el modelo de colitis del DSS, el BGMP no tuvo efecto alguno sobre la fosfatasa alcalina (AP) y la COX-2. En este modelo el primer signo de la colitis es la depleción progresiva de criptas intestinales, sugiriendo un primer efecto directo tóxico sobre las células epiteliales, seguido de una respuesta aguda del sistema inmune innato y una fase crónica en la que son activados linfocitos de tipo Th1 y Th2 (34, 35). Asumiendo por tanto que el daño primario del DSS se produce en el epitelio, nuestros resultados sugieren que el efecto del BGMP no está mediado por acciones sobre el epitelio. El hecho de que AP y COX-2 sí resultaran inhibidas por el péptido en el modelo del TNBS se debería en este caso a una acción indirecta, esto es, a la mejoría global del intestino inflamado (14,15). Por otra parte, los experimentos realizados en líneas celulares de epitelio intestinal (HT-29 y Caco-2) han mostrado que el BGMP no modifica la producción de IL-8 en estas células. Además, nuestro grupo de investigación ha constatado recientemente que el BGMP carece de efectos sobre el transporte iónico en colon de rata montado en cámaras de Ussing, así como sobre la reparación epitelial en células IEC18 (datos no mostrados). En consecuencia, podemos concluir que el mecanismo de acción del BGMP no está mediado por las células epiteliales intestinales.

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2. Efecto del BGMP sobre macrófagos En el estudio sobre el efecto del BGMP en la colitis inducida por el TNBS, el grupo BGMP había mostrado valores reducidos de IL1β e IL1ra, citoquinas producidas por los macrófagos. El BGMP redujo también la IL1β en los otros dos modelos animales (colitis inducida por DSS e ileitis inducida por TNBS). Además, otras citoquinas características del sistema inmune innato, como el TNF y la IL6, fueron inhibidas tras el tratamiento con BGMP. Por tanto, los macrófagos constituían una diana celular plausible del BGMP. En consecuencia, diseñamos un estudio in vitro para caracterizar los efectos del BGMP a este nivel. Además, algunos datos publicados por otros autores sugerían que los macrófagos se veían afectados por BGMP aunque, paradójicamente, al contrario de lo esperado, los estudios correspondientes indicaban un papel activador sobre los macrófagos. Así, anteriormente se había descrito que el BGMP aumentaba la producción de IL-1ra (pero no IL-1β) en la línea celular de monocitos de ratón P388D1 (11) y que aumentaba la capacidad fagocítica de la línea celular U937 (macrófagos humanos) (12). Para nuestro diseño experimental usamos una línea celular de monocitos humanos (THP1) y un cultivo primario de macrófagos humanos de sangre periférica, utilizados fundamentalmente con fines de validación. El BGMP indujo la producción de IL1β, IL8 y TNF en ambos modelos celulares, aunque con distinta potencia, siendo en general la concentración mínima eficaz la de 1 mg·mL-1. La activación de NF-κB fue esencial para la inducción de las 3 citoquinas, ya que Bay 11-7082, el inhibidor de esta ruta de señalización, inhibió completamente la secreción de las tres citoquinas inducida por BGMP. Además, el péptido indujo la traslocación de p50/p65 al núcleo y provocó la fosforilación de IκB (17). Las MAPK parecen desempeñar un papel accesorio en estos efectos, según los ensayos de inhibición farmacológica. Por tanto, nuestros resultados corroboran y amplían los anteriormente obtenidos por otros autores, y están en contradicción aparente con el efecto antiinflamatorio intestinal del BGMP. Existen varias explicaciones posibles para la discrepancia que existe entre los datos in vivo e in vitro. Por un lado, puede ocurrir que el BGMP actúe por otros mecanismos in vivo, como la inhibición de linfocitos Th1 o la activación de linfocitos T reguladores (Treg), y que la inhibición de las citoquinas del sistema innato que se observa sea indirecta, es decir, debida a la propia mejoría clínica. Por otro lado, conviene recordar que el aumento de citoquinas producido in vitro, aunque significativo y relevante, no es comparable al efecto proinflamatorio de estímulos como el LPS y, de hecho, el BGMP no estimula la secreción de citoquinas en células previamente estimuladas con LPS (datos no mostrados). Por tanto, no resulta extraño que el BGMP no aumente in vivo los valores de citoquinas previamente incrementados por la inflamación. En ambos casos, las acciones sobre los macrófagos no estarían implicadas en el efecto terapéutico del BGMP. Sin embargo, existe una interpretación alternativa, según la cual la activación monocítica sí que estaría implicada en el efecto terapéutico. En efecto, hace algunos años un ensayo clínico destacó la falta de neutrófilos y la baja producción de IL8 e IL1β en pacientes con CD (23), así como disfunciones en la actividad neutrofílica (22). Otro estudio demostró que sargramostim, el factor estimulante de colonias de granulocitos-macrófagos (GM-CSF), produce mejoría en la CD (19). Además, la represión condicional de NEMO y, por tanto, de NF-κB en células epiteliales produce inflamación

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colónica de manera espontánea (18). También se ha descrito que la ausencia de monocitos y células dendríticas empeora la colitis experimental (20, 21). Algunos otros datos apoyan esta teoría. Las mutaciones en el gen nod2 asociadas a CD, muestran una capacidad disminuida para activar NFκB en respuesta a MDP (32). Además las mutaciones NOD2 están asociadas con una capacidad disminuida de las mucosas para producir α-defensinas. Por tanto, puede plantearse la hipótesis de que la inflamación intestinal pueda desencadenarse como consecuencia no de una respuesta especialmente robusta del sistema inmunológico innato, sino por todo lo contrario. Así que las respuestas innatas débiles pueden estár asociadas a la aparición de la CD, y estimular dichas respuestas, parece beneficioso. Puede resultar lógico, ya que un sistema innato imperfecto es incapaz de eliminar rápidamente las bacterias que hayan sobrepasado la barrera epitelial, permitiendo la activación del sistema inmune adaptativo, el cual es el responsable último de la inflamación crónica (36, 37). Por lo tanto, puede que los macrófagos constituyan una diana del BGMP en su efecto terapéutico, ya que al estar en estado activo, podrían prevenir la aparición de la IBD. Esta hipótesis es coherente asimismo con el hecho de que el BGMP sea más eficaz en el protocolo de pretratamiento que en postratamiento.

Sin perjuicio de lo anteriormente expuesto, los últimos experimentos realizados en esta Tesis Doctoral revelaron un aspecto adicional de extraordinaria relevancia relacionado con las acciones del BGMP sobre los macrófagos. En concreto, el análisis genómico del transcriptoma colónico en animales tratados con BGMP y los resultados obtenidos en linfocitos T aislados (ver más adelante) indicaron que la IL-10 podría estar implicada en el mecanismo de acción del BGMP. De hecho, los experimentos realizados en último término demostraron que la activación de macrófagos también implica una mayor producción de la citoquina inmunosupresora IL-10, tanto en macrófagos humanos como de rata, la cual se manifiesta tanto en condiciones basales como cuando son previamente activados por LPS, lo cual parece ser el factor clave del efecto del BGMP. Estos resultados serán discutidos más adelante. En cualquier caso, aunque la hipótesis de la activación monocítica explica muchos de los efectos del BGMP, éste puede tener otros mecanismos de acción, ya que la administración como postratamiento en ratas colíticas también produce mejoría en la enfermedad. Además, existen estudios que han demostrado un aumento de la actividad NF-κB en IBD (50), y según nuestro modelo de macrófagos in vitro, el BGMP activaría más aún NF-κB. 3. Efecto del BGMP sobre los linfocitos T Con anterioridad a la realización de esta Tesis Doctoral se habían publicado algunos estudios sobre las acciones del BGMP en linfocitos de ratón. Dichos estudios aportaban datos concluyentes sobre las propiedades antiproliferativas de este péptido en esta población celular (10, 12, 24). Por tanto, pensamos que dichos efectos podrían contribuir o ser esenciales para su efecto antiinflamatorio intestinal. Sin embargo, no había datos disponibles sobre el efecto del BGMP en otros aspectos de la biología linfocitaria. Por tanto, realizamos un estudio específico en cultivos primarios obtenidos del bazo de rata. A continuación discutimos globalmente los resultados obtenidos, diferenciando los posibles efectos sobre los diferentes subtipos de linfocitos T.

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Th1

En una primera aproximación utilizamos esplenocitos no fraccionados como material de estudio. Los esplenocitos son una mezcla de leucocitos mononucleares, principalmente linfocitos B y T y monocitos. La adición del mitógeno concanavalina A (ConA) produce una activación global (no clonal) de linfocitos T y un enriquecimiento relativo en linfocitos, debido a la proliferación estimulada por la activación linfocitaria. En estos estudios el BGMP inhibió las células Th1, ya que produjo una marcada inhibición de IFNγ, STAT4 y TNF (pero no IL-2) en esplenocitos de rata estimulados con ConA. Además, favoreció la diferenciación de células Treg, ya que produjo un aumento sustancial de Foxp3 e IL-10 en condiciones basales. Estos efectos se manifestaron de forma simultánea al incremento significativo de la expresión de TNF, COX2, e iNOS, así como de la activación de la vía canónica de NF-κB, en condiciones basales. Teniendo en cuenta que dichos efectos se observaron únicamente a la concentración más alta usada (1 mg mL-1), que corresponde con el umbral de activación en macrófagos, la explicación más sencilla es un efecto estimulante sobre los macrófagos del bazo. Estos efectos, además, desaparecían cuando los esplenocitos eran activados por ConA (que, indirectamente, produce una cierta estimulación de los macrófagos), al igual que ocurría con los macrófagos humanos.

Nuestra conclusión inicial fue, por tanto, que el BGMP ejercía un doble efecto en esplenocitos: estimulador, sobre macrófagos, e inhibidor, sobre linfocitos, y muy concretamente sobre linfocitos Th1, dando lugar a una marcada disminución de la secreción de IFN-γ y de la translocación nuclear de STAT4, implicado en la vía de señalización correspondiente en estas células. Sin embargo, este efecto no se reprodujo in vivo cuando el BGMP fue administrado por vía oral a ratas normales. El objetivo de este experimento era verificar la relevancia de los resultados obtenidos in vitro, y en lo que respecta al IFNγ, no cabe duda de que la extrapolación quedo invalidada. Sin embargo, el BGMP sí produjo un efecto constatable sobre Foxp3 en las mismas condiciones, por lo que la ausencia de efecto sobre esta citoquina no puede achacarse a una falta global de efectos farmacológicos in vivo.

Por otra parte, en los modelos animales de inflamación en los que se midió la

expresión de IFN-γ se observaron resultados dispares: en el modelo de la colitis por DSS el BGMP redujo significativamente la producción de IFNγ en células mononucleares de ganglios mesentéricos (MNMC) ex vivo, pero no se observó efecto alguno en la ileítis por TNBS. No obstante, en este último caso la variabilidad fue considerable, por lo que cualquier efecto real podría haber quedado enmascarado. El análisis genómico de las ratas con colitis inducida por TNBS mostró, por su parte, una falta de efecto sobre la expresión colónica de IFN-γ (a nivel de mRNA – Ifng), aunque la tendencia era hacia la disminución. Estas discrepancias se explican en parte por las diferencias existentes entre las poblaciones linfocitarias presentes en la mucosa colónica y los órganos linfoides periféricos. Así, aunque el modelo de TNBS está considerado como esencialmente de tipo Th1 en ratones, y a pesar de que la producción de IFNγ está aumentada en MNMC de ratas con colitis por TNBS, la propia expresión de esta citoquina en el tejido colónico apenas se ve modificada, o se incrementa muy ligeramente (38, 39). Por tanto, cabe esperar que los efectos del BGMP sean más pronunciados a este respecto en las MNMC que en intestino. Como, además, el BGMP carece de efecto sobre el IFNγ en esplenocitos no estimulados con ConA, nuestros

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resultados eran en principio coherentes con un mecanismo inhibidor de linfocitos Th1, el cual se manifestaría únicamente en condiciones de inflamación.

Sin embargo, cuando pudimos estudiar linfocitos T aislados, mediante técnicas

de separación por magnetismo, lo que se observó no fue una inhibición sino una estimulación de los mismos con el BGMP. Al mismo tiempo, el efecto sobre los esplenocitos (no fraccionados) siguió siendo de inhibición de la secreción de IFN-γ, lo que indica un mecanismo modulador de Th1 pero de carácter indirecto. En esplenocitos hay, además de linfocitos T, macrófagos y linfocitos B, fundamentalmente. Como se ha indicado anteriormente, el BGMP estimula la secreción de IL-10 en macrófagos aislados, y a concentraciones menores que las que originan la liberación de otras citoquinas proinflamatorias. Por tanto, nuestros datos indican que los macrófagos son las células mediadoras del efecto inhibidor de IFN-γ en esplenocitos y, presumiblemente, también en el intestino inflamado. Th2

En los experimentos de puesta a punto de la técnica de separación linfocitaria, así como en diversos experimentos con esplenocitos no fraccionados, la IL-4, citoquina característica de los linfocitos Th2, apenas se expresa en niveles apreciables. Por este motivo carecemos de datos sobre los posibles efectos del BGMP a este nivel. Th17 Los datos que tenemos sobre el efecto del BGMP en esta población linfocitaria son escasos, debido en buena medida a la falta de anticuerpos frente a la IL-17 de rata. En cualquier caso, la expresión colónica de Il6 e Il17 fue inhibida por el BGMP en la colitis inducida por DSS y TNBS, así como en la ileítis inducida por TNBS. Además, el estudio genómico (ver más adelante) indicó que la inhibición de IL-6 desempeñaba un papel central en el efecto del BGMP, al menos en la colitis por TNBS. Una de las funciones primordiales de la IL-6 es precisamente la diferenciación de linfocitos Th0 a Th17, actuando conjuntamente con TGF-β. Por el contrario, en ausencia de IL-6 (u otras citoquinas proinflamatorias activadas por agonistas TLR), el TGFβ conduce a la formación de células Treg. Nuestros resultados no apoyan la hipótesis de un efecto inhibitorio directo del BGMP sobre la producción de IL-6, y por tanto, de IL-17, ya que los experimentos in vitro mostraron que carece de efecto sobre linfocitos T, en tanto que potencia la secreción de esta citoquina por macrófagos tratados con LPS. Es posible que el BGMP pueda inhibir la expresión de Il6 en otras células in vivo, por lo que la posibilidad de un efecto directo no puede descartarse. Sin embargo, la modulación de la producción de IL-10 en macrófagos es suficiente, en principio, para justificar la inhibición de la IL-6. Además, la IL-10 es una citoquina clave en la función de las células Treg (ver apartado siguiente). Treg Como se ha indicado anteriormente, la expresión de Foxp3 aumenta significativamente en esplenocitos expuestos a BGMP, lo que puede interpretarse como indicio de un incremento de diferenciación de células Treg. La otra observación importante del estudio sobre los esplenocitos fue que el BGMP, administrado por vía oral a ratas normales, retiene la capacidad de aumentar la expresión de FoxP3 en estas

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células. De hecho, este efecto requiere la administración oral del péptido, y se ejerce específicamente sobre el bazo, ya que no se registró efecto alguno en timocitos. Por tanto, los linfocitos Treg podrían ser otra diana del BGMP en su efecto antiinflamatorio intestinal. El hecho de que el efecto dependa de la administración oral podría ser indicativo de un mecanismo de tipo prebiótico, o bien de la implicación de centros inmunológicos locales. Los datos disponibles en los modelos de inflamación no son concluyentes en cuanto a la implicación de linfocitos Treg en el mecanismo, porque los niveles de Foxp3 aumentan en el tejido inflamado como parte de la respuesta (aumentan también in vitro en linfocitos estimulados con ConA), y disminuyen con la mejoría clínica. Desafortunadamente, carecemos de datos de la expresión de FoxP3 en esplenocitos en dichos modelos animales. Por tanto, no podemos afirmar categóricamente que esta acción esté implicada en el mecanismo antiinflamatorio del BGMP. No obstante, es algo que parece probable, dado que la expresión de Foxp3 aumenta in vitro e in vivo con el tratamiento con BGMP, y es coherente con el efecto de este péptido sobre la IL-10. La IL-10 y la IL-6 como factores clave en el mecanismo de acción del BGMP Una de las herramientas básicas utilizadas en la presente Tesis Doctoral para el esclarecimiento del mecanismo de acción del BGMP ha sido el análisis genómico. Mediante la utilización de microarrays de Affymetrix podemos examinar la expresión de más de 30000 genes del genoma de la rata, lo que constituye un porcentaje muy elevado del total. Nuestro grupo de investigación ha aplicado con éxito esta técnica para examinar, en primer lugar, los cambios que se producen en el transcriptoma colónico en el modelo de colitis por TNBS en ratas (39). Disponemos, por tanto, de una base de datos muy potente en la que basar el análisis comparativo de los efectos de tratamientos experimentales. Así, nuestro grupo ha publicado recientemente un estudio genómico sobre el mecanismo de acción de la sulfasalazina, un agente ampliamente utilizado en la IBD (40), y se halla inmerso actualmente en un estudio similar con el flavonoide rutina (41) y con el bisfosfonato pamidronato (datos no publicados), además del que se expone aquí. Cabe destacar que el impacto de todos estos tratamientos sobre el transcriptoma colónico es cuantitativa y, sobre todo, cualitativamente distinto, de forma que existen diferencias sustanciales en los cambios de expresión génica asociados al efecto antiinflamatorio dependiendo del agente antiinflamatorio de que se trate. No se trata, por tanto, de probar el efecto terapéutico, sino de generar hipótesis de carácter mecanístico sobre el modo de actuación del fármaco o agente terapéutico. El procedimiento aceptado internacionalmente consiste en la aplicación del análisis genómico (microarrays) seguida de un proceso de validación de los hallazgos más relevantes mediante otra técnica, típicamente PCR en tiempo real. Finalmente, se intentan validar dichos hallazgos en términos de función biológica, lo que comúnmente se denomina genómica funcional. Éste ha sido el procedimiento aplicado en esta Tesis Doctoral: el análisis de los cambios en el transcriptoma utilizando la plataforma Ingenuity indicó cambios sustanciales y significativos sobre familias de genes relacionadas con la inflamación y la respuesta inmunológica y, lo que es más importante, seleccionó la IL-6 y la IL-1 como elementos clave en la respuesta. La PCR en tiempo real confirmó que el tratamiento con BGMP se tradujo en una bajada marcada de Il6 y una subida de Il10 en el intestino inflamado, mientras que la Il23a permanecía inalterada, esto es, insensible al tratamiento. Como hemos comentado anteriormente, no hemos podido constatar que el GMP inhiba la producción de IL6 en linfocitos T in

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vitro, pero no es descartable que éste ejerza acciones directas en la mucosa colónica, lo que requerirá nuevos experimentos. En lo que respecta a la validación funcional, ya hemos indicado que el BGMP aumentó la producción de IL-10 en todos los modelos probados. Así, la inducción de colitis mediante el TNBS inhibió la expresión colónica de Il10 mientras que la administración de BGMP aumentó su expresión hasta los niveles del grupo control. En esplenocitos de rata, el BGMP aumentó la producción de IL-10 en condiciones basales y, levemente, cuando los esplenocitos fueron estimulados con ConA. En linfocitos T de rata el BGMP produjo también un aumento de la producción de IL-10, si bien éste aumento fue muy pequeño y se produjo sólo a la concentración más alta ensayada y únicamente en condiciones basales. Finalmente, el BGMP aumento la producción de IL-10 en macrófagos humanos de sangre periférica (datos no mostrados), así como en macrófagos aislados de rata, incluso cuando éstos fueron estimulados con LPS. La mayor potencia de inducción de la IL-10 observada en macrófagos sugiere que estas células sean la fuente primordial de IL-10 in vivo y que, por tanto, los macrófagos y la IL-10 sean las dianas más importantes del BGMP como agente antiinflamatorio intestinal. En cualquier caso, teniendo en cuenta el efecto del BGMP sobre Foxp3, y presumiblemente sobre la diferenciación de linfocitos Treg, estas células podrían contribuir al aumento de IL-10 observado en el grupo BGMP. Es interesante considerar, en este sentido, el artículo reciente del grupo de Mitchell Kronenberg (Nature Immunol 2009;10:1178) que sugiere que la IL-10 (producida por células mieloides CD11b+) es esencial para el mantenimiento de las células Treg en la inflamación crónica. En la medida en que esta hipótesis sea aplicable al ámbito clínico, el efecto del BGMP puede ser especialmente interesante. En cualquier caso, la consecuencia de la modulación del eje IL-10/IL-6/Treg sería la normalización total o parcial de los parámetros inflamatorios, como la AP, la IL-1β o el TNF y una disminución de la infiltración de neutrófilos, como efectivamente se constata en nuestros estudios in vivo. En particular, el análisis genómico indica una disminución de la expresión de genes relacionados con la activación, reclutamiento y acumulación de macrófagos y neutrófilos, así como un aumento de la expresión de genes relacionados con el número de células B y T, en comparación con el grupo TNBS, en consonancia con un estadío avanzado de recuperación MODELO DESCRIPTIVO DEL MECANISMO DE ACCIÓN DEL BGMP Resulta arriesgado proponer un modelo sobre el mecanismo de acción de cualquier sustancia. Sin embargo, con base en los datos disponibles, creemos que podemos proponer el modelo siguiente con cierto grado de certeza. Cuando el BGMP (500 mg/kg·día) es administrado a ratas sin inflamación (pretratamiento) se estimula la diferenciación o proliferación de linfocitos Treg presentes en la lamina propria, a la vez que se produce una cierta activación de los macrófagos residentes, los cuales producirían diversas citoquinas proinflamatorias y adquirirían una mayor capacidad fagocítica, pero también más IL-10. El estado de activación en los macrófagos es, en cualquier caso, insuficiente para provocar una respuesta inflamatoria per se, pero suficiente como para crear un “estado de alerta”.

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En las ratas que no han recibido el BGMP, la administración de TNBS o DSS produce una alteración del epitelio que provoca la entrada en contacto de los antígenos luminales con el sistema inmune. Según nuestro modelo, el sistema de “alerta” inducido por el BGMP mantendría a raya la entrada de antígenos luminales a la lamina propria. De esta forma se produciría una cierta contención de la invasión, lo que se traduciría en una menor respuesta inmune adaptativa. Más adelante, incluyendo cuando el BGMP es administrado como postratamiento, la IL-10, cuya expresión está elevada gracias al efecto activador del BGMP sobre los macrófagos (y tal vez sobre los linfocitos Treg), actuaría inhibiendo las respuestas inmunes de tipo Th1 y las propias respuestas de los macrófagos, como la producción de IL6, características de la inflamación inducida por DSS y TNBS. Como se ha indicado, la IL-10 de origen no linfocitario es esencial en este sentido, al menos en el modelo de colitis por transferencia. Por último, queda la cuestión de cómo opera el BGMP en su interacción con las células diana. En este sentido, la falta de un buen anticuerpo frente a BGMP ha dificultado enormemente nuestro trabajo, y a día de hoy no podemos afirmar nada al respecto, si bien otros autores han sugerido un sitio de acción extracelular (8). De nuestros estudios sólo podemos deducir que el BGMP promueve la activación de NF-κB en macrófagos y esplenocitos (17), pero existen muchas rutas de señalización que convergen en este factor de transcripción. La inhibición de STAT4 parece ser, como hemos indicado, indirecta. Al menos en macrófagos, el efecto parece depender de la presencia del péptido intacto. Serán necesarios experimentos adicionales para resolver estas cuestiones pendientes. Bibliografía 1. Ekbom, A. (2004). The epidemiology of IBD: a lot of data but little knowledge. How shall we proceed? Inflamm Bowel Dis. 10 (suppl.1 ) S32-S34. 2. Sands, B.E. (2000). Therapy of inflammatory bowel disease. Gastroenterol. 118: S68-S82. 3. Neurath, M., Fuss, I., Strober, W. (2000) TNBS-colitis. Int Rev Immunol. 19: 51-62. 4.Newby, TJ., Stokes, C.R., Bourne, F.J. (1982) Immunological activity of milk. Vet Immunol Immunopatho. 3, 67-94. 5. Brody, E.P. (2000) Biological activities of bovine glycomacropeptide. Br J Nutr. 84: S39-S46. 6. Otani, H., Monnai, M. (1993) Inhibition of proliferative responses of mouse spleen lymphocytes by bovine milk k-casein digests. Food Agric Immunol. 5, 219-229. 7. Mikkelsen, T.L., Bakman, S., Sørensen E.S., Barkholt V., Frøkiær H. (2005) Sialic acid-containing milk proteins show differential immunomodulatory activities independent of sialic acid. J Agric Food Chem. 53, 7673-7680 8. Otani, H., Horimoto, Y., Monnai, M. Suppression of interleukin-2 receptor expression on mouse CD4+ T cells by bovine kappacaseinoglycopeptide. (1996) Biosci, Biotechnol, Biochem. 60, 1017-1019. 9. Otani, H., Monnai, M. (1995) Induction of an interleukin-1 receptor antagonist-like component produced from mouse spleen cells by bovine kappa-caseinoglycopeptide. Biosci, Biotechnol, Biochem. 59, 1166-1168. 10. Monnai, M., Horimoto, Y., Otani, H. (1998) Immunomodificatory effect of dietary bovine κ-caseinoglycopeptide on serum antibody levels and proliferative responses of lymphocytes in mice. Milchwissenschaft 53 (3), 129-132. 11. Monnai, M., Otani, H. (1997) Effect of bovine κ-caseinoglycopeptide on secretion of interleukin-1 family cytokines by P3888D1 cells, a line derived from mouse monocyte/macrophage. Milchwissenschaft 52 (4), 192-196. 12. Li, E. W., Mine, Y.(2004) Immunoenhancing effects of bovine glycomacropeptide and its derivatives on the proliferative response and phagocytic activities of human macrophagelike cells, U937. J Agric Food Chem. 52, 2704-2708.

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13. Mikkelsen TL, Rasmussen E, Olsen A, Barkholt V, Frokiaer H. Immunogenicity of kappa-casein and glycomacropeptide. J Dairy Sci. 2006 Mar; 89: 824–830. 14. Daddaoua, A., Puerta, V., Zarzuelo, A., Suárez, M.D., Sánchez de Medina, F., Martínez-Augustín, O. (2005) Bovine Glycomacropeptide Is Anti-Inflammatory in Rats with Hapten-Induced Colitis. J Nutr. 135(5): 1164-70. 15. Requena P, Daddaoua A, Martínez-Plata E, González M, Zarzuelo A, Suárez MD, Sánchez de Medina F, Martínez-Augustin O. Bovine glycomacropeptide ameliorates experimental rat ileitis by mechanisms involving downregulation of interleukin 17. British Journal of Pharmacology 2008; 154:825-32. 16. Singer II, Kawka DW, Schloemann S, Tessner T, Riehl T, Stenson WF. Cyclooxygenase 2 is induced in colonic epithelial cells in inflammatory bowel disease. Gastroenterology 1998; 115:297-306. 17. Requena P, Daddaoua A, Guadix E, Zarzuelo A, Suarez MD, Sanchez de Medina F, Martínez-Augustin O. Bovine glycomacropeptide induces cytokine production in human monocytes through the stimulation of the MAPK and the NF-kappaB signal transduction pathways. Br J Pharmacol 2009; 157:1232-40. 18. Nenci A, Becker C, Wullaert A, Gareus R, van Loo G, Danese S, Huth M, Nikolaev A, Neufert C, Madison B, Gumucio D, Neurath MF, Pasparakis M. Epithelial NEMO links innate immunity to chronic intestinal inflammation. Nature. 2007 Mar 29;446(7135):557-61. Epub 2007 Mar 14. 19. Korzenik JR, Dieckgraefe BK, Valentine JF, Hausman DF, Gilbert MJ; Sargramostim in Crohn's Disease Study Group. Sargramostim for active Crohn's disease. N Engl J Med. 2005 May 26; 352(21):2193-201. 20. Qualls JE, Tuna H, Kaplan AM, Cohen DA. Suppression of experimental colitis in mice by CD11c+ dendritic cells. Inflamm Bowel Dis 2009; 15:236-47. 21. Qualls JE, Kaplan AM, van Rooijen N, Cohen DA. Suppression of experimental colitis by intestinal mononuclear phagocytes. J Leukoc Biol 2006; 80:802-15. 22. Zhou L, Braat H, Faber KN, Dijkstra G, Peppelenbosch MP. Monocytes and their pathophysiological role in Crohn's disease. Cell Mol Life Sci. 2009 Jan; 66(2):192-202. 23. Marks DJ, Harbord MW, MacAllister R, Rahman FZ, Young J, Al-Lazikani B, Lees W, Novelli M, Bloom S, Segal AW. Defective acute inflammation in Crohn's disease: a clinical investigation. Lancet. 2006 Feb 25; 367(9511):668-78. 24. Otani H, Monnai M, Kawasaki Y, Kawakami H, Tanimoto M. Inhibition of mitogen-induced proliferative responses of lymphocytes by bovine kappa-caseinoglycopeptides having different carbohydrate chains. J Dairy Res 1995;62:349-57. 25. Bruck WM, Kelleher SL, Gibson GR, Nielsen KE, Chatterton DE, Lonnerdal B. rRNA probes used to quantify the effects of glycomacropeptide and alpha-lactalbumin supplementation on the predominant groups of intestinal bacteria of infant rhesus monkeys challenged with enteropathogenic Escherichia coli. J Pediatr Gastroenterol Nutr 2003;37:273-80. 26. Jurjus, AR., Khoury, NN., Reimund ,JM. (2004) Animal models of inflammatory bowel disease. J Pharmacol Toxicol Methods. 50 (2):81-92. 27. Morris, G.P., Beck, P.L., Herridge, M.S., Depew, W.T., Szewczuk, M.R., Wallace, J.L. (1989) Hapten-induced model of chronic inflammation and ulceration in the rat colon. Gastroenterol. 96: 795-803. 28. Bal dit Sollier C, Drouet L, Pignaud G, Chevallier C, Caen J, Fiat AM, Izquierdo C, Jolles P. Effect of kappa-casein split peptides on platelet aggregation and on thrombus formation in the guinea-pig. Thromb Res. 1996 Feb 15;81(4):427-37. 29. Chabance, B., Marteau, P., Rambaud, J.C., Migliore-Samour, D., Boynard, M., Perrotin, P., Guillet, R., et al., Casein peptide release and passage to the blood in humans during digestion of milk or yogurt. Biochimie. 1998. 80: 155-65. 30. Idota T KH, Nakajima I. : Growth-promoting effects of N-acetylneuraminic acid-containing substances on Bifidobacteria. Biosci Biotech Biochem 1994;58:1720-1722. 31. Yakabe T KH, Idota T. Japanese patent.: Growth stimulation agent for bifidus and lactobacillus. 1994:07-267866. 32. Yamamoto-Furusho JK, Korzenik JR. Crohn's disease: innate immunodeficiency? World J Gastroenterol. 2006 Nov 14; 12(42):6751-5. 33. Bruck WM, Redgrave M, Tuohy KM, Lonnerdal B, Graverholt G, Hernell O, Gibson GR: Effects of bovine alpha-lactalbumin and casein glycomacropeptide-enriched infant formulae on faecal microbiota in healthy term infants. J Pediatr Gastroenterol Nutr 2006;43:673-679. 34. Okayasu I, Hatakeyama S, Yamada M, Ohkusa T, Inagaki Y, Nakaya R. A novel method in the induction of reliable experimental acute and chronic ulcerative colitis in mice. Gastroenterology. 1990 Mar; 98(3):694-702.

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35. Dieleman LA, Palmen MJ, Akol H, Bloemena E, Peña AS, Meuwissen SG, Van Rees EP. Chronic experimental colitis induced by dextran sulphate sodium (DSS) is characterized by Th1 and Th2 cytokines. Clin Exp Immunol. 1998 Dec;114(3):385-91. 36. Khan I. Molecular basis of altered contractility in experimental colitis: expression of L-type calcium channel. Dig Dis Sci. 1999 Aug;44(8):1525-30. 37.Papadakis KA, Abreu MT.Systemic consequences of intestinal inflammation. In Targan SR, Shanahan F, Karp LC. Inflammatory Bowel Disease: From Bench to Bedside, 2nd edn. Springer, 2005. 38. Pérez-Navarro R, Ballester I, Zarzuelo A, Sánchez de Medina F. Disturbances in epithelial ionic secretion in different experimental models of colitis. Life Sci. 2005 Feb 11;76(13):1489 501. Epub 2004 Dec 21. 39. Martínez-Augustin O, Merlos M, Zarzuelo A, Suárez MD, de Medina FS. Disturbances in metabolic, transport and structural genes in experimental colonic inflammation in the rat: a longitudinal genomic analysis. BMC Genomics. 2008 Oct 17;9:490. 40. Martínez-Augustin O, López-Posadas R, González R, Suárez MD, Zarzuelo A, Sánchez de Medina F. Genomic analysis of sulfasalazine effect in experimental colitis is consistent primarily with the modulation of NF-kappaB but not PPAR-gamma signaling. Pharmacogenet Genomics. 2009 May;19(5):363-72. 41. Sánchez de Medina F, López-Posadas R, Romero I, Mascaraque C, Daddaoua A, González R, González M, Martínez-Plata E, Suárez MD, Zarzuelo A, Martínez-Augustin O. Clinical Nutrition. 2009; 4 (suppl. 2), p.49, P057.

Discusión

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CONCLUSIONES

CONCLUSIONES

1. El glucomacropéptido bovino (BGMP) tiene efecto antiinflamatorio intestinal en ratas, ya que produce una notable mejoría en la colitis inducida por el DSS y por el TNBS, así como en la ileítis inducida por el TNBS.

2. El BGMP estimula la producción de citoquinas proinflamatorias en monocitos y macrófagos humanos in vitro, mediante la activación de NFκB.

3. El BGMP estimula la expresión de FoxP3 e IL10 tanto in vivo como in vitro. La potencia de inducción de IL10 in vitro es mayor sobre los macrófagos que sobre los linfocitos, y se mantiene tras la estimulación con LPS.

4. El BGMP estimula la producción de IFN-γ en linfocitos T in vitro, pero la inhibe en presencia de macrófagos, acción achacable a la secreción de IL-10 por éstos.

5. El BGMP produce, de manera directa o indirecta, una inhibición de la expresión de IL-6 in vivo que parece ser esencial en el efecto antiinflamatorio intestinal del BGMP.

Conclusiones

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