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IL-13 has been implicated in the pathogenesis of ulcerative colitis (UC), and may have a role in animal models of gut fibrosis. We studied the involvement of IL-13 in inflammation and fibrosis in UC and Crohn's disease (CD). Intestinal biopsies and anti-CD3/CD28- or anti-CD2/CD28-stimulated lamina propria mononuclear cells from UC and CD patients and control subjects were cultured, and IL-13, IL-4, IL-5, IL-17A and IFN-γ production was measured. Mucosal IL-13-producing cells were characterised by flow cytometry. Gut explants from strictured CD, non-strictured CD and healthy donors were cultured ex vivo, and secreted IL-13, IL-1β and collagen were measured. IL-13 production by mucosal explants and activated lamina propria mononuclear cells did not differ between CD, UC and control subjects, and was at least a log lower than IFN-γ and IL-17A. IL-13-producing cells, and in particular natural killer T cells, were uniformly low in all groups. IL-4 and IL-5 were undetectable in culture supernatants. Explants of CD strictures produced low amounts of IL-13, whereas IL-1β and collagen were elevated. We could not confirm that UC or strictured CD are associated with elevated IL-13 production. These data suggest that an anti-IL-13 Ab would not be an appropriate therapeutic strategy in inflammatory bowel disease.

Background: Allergic rhinitis and asthma show many similarities in their epithelial and inflammatory responses to allergens. However, one notable difference is that disruption and desquamation of the epithelium is a characteristic feature of asthma, whereas in perennial allergic rhinitis the epithelium is intact and thickened. One reason for this might be differing expression of matrix metalloproteinases (MMPs) or their inhibitors (TIMPs). There are few published data on the presence of MMPs or TIMPs in the nasal mucosa in rhinitis. Objective: The purpose of this study was to investigate MMP and TIMP mRNA and protein in nasal mucosa from subjects with perennial allergic rhinitis and from nonrhinitic control subjects. Methods: Biopsy specimens of nasal mucosa were taken from 10 well-characterized subjects with perennial allergic rhinitis and 10 nonrhinitic control subjects. MMP and TIMP mRNA was quantified through use of competitive RT-PCR, and protein was detected by means of Western blotting and ELISA. Results: TIMP-1 mRNA and TIMP-2 mRNA were present in nasal samples, but there was no significant difference between the 2 groups. Only small amounts of MMP-1, -2, -3, and -9 mRNA were detected in the same samples. The corresponding proteins were detected by means of Western blotting. TIMP-1 protein and TIMP-2 protein were quantified in tissue homogenates; there was no significant difference between the 2 groups. Conclusion: Our studies have demonstrated the presence of large amounts of TIMP-1 and TIMP-2 mRNA and protein in nasal mucosa. There is no upregulation of MMPs or changes in TIMP expression in the nasal mucosa of patients with allergic rhinitis. (J Allergy Clin Immunol 2001;108:791-6.)

TGF-beta1 functions as a negative regulator of T cell immune responses, signaling to target cells using the Smad family of proteins. We show here that Smad7, an inhibitor of TGF-beta1 signaling, is overexpressed in inflammatory bowel disease (IBD) mucosa and purified mucosal T cells. Both whole tissue and isolated cells exhibit defective signaling through this pathway, as measured by phospho-Smad3 immunoreactivity. Specific antisense oligonucleotides for Smad7 reduce Smad7 protein expression in cells isolated from patients with IBD, permitting the cells to respond to exogenous TGF-beta1. TGF-beta1 cannot inhibit proinflammatory cytokine production in isolated lamina propria mononuclear cells from patients with Crohn disease (CD), but inhibition of Smad7 restores TGF-beta1 signaling and enables TGF-beta1 to inhibit cytokine production. In inflamed mucosal tissue explants from patients with CD, inhibition of Smad7 also restores p-Smad3 and decreases proinflammatory cytokine production, an effect that is partially blocked by anti-TGF-beta1. These results show that Smad7 blockade of TGF-beta1 signaling helps maintain the chronic production of proinflammatory cytokines that drives the inflammatory process in IBD and that inhibition of Smad7 enables endogenous TGF-beta to downregulate this response.

The ability of interferon (IFN)-alpha to induce autoimmunity and exacerbate Th1 diseases is well known. We have recently described enhanced expression of IFN-alpha in the mucosa of patients with celiac disease (CD), a gluten-sensitive Th1-mediated enteropathy, characterized by villous atrophy and crypt cell hyperplasia. Previous studies from this laboratory have shown that T cell activation in explant cultures of human fetal gut can also result in villous atrophy and crypt cell hyperplasia. We have, therefore, examined changes that take place in explant cultures of human fetal gut after activation of T cells with anti-CD3 and/or IFN-alpha. We show that activation of T cells with anti-CD3 alone elicits a small IFN-gamma and TNF-alpha response with no tissue injury. Similarly, no changes are seen in explants cultured with IFN-alpha alone. However, addition of IFN-alpha with anti-CD3 results in enhanced Th1 response and crypt cell hyperplasia. This is associated with enhanced phosphorylation of STAT1, STAT3, and Fyn, a Src homology tyrosine kinase, which interacts with both TCR and IFN-alpha signal components. Together these data indicate that IFN-alpha can facilitate activation of Th1-reactive cells in the gut and drive immunopathology.

Background: Antigen-specific responses can be detected in umbilical cord blood mononuclear cells. The fetal immune system must therefore attain a level of maturity compatible with the initiation of such responses as well as be exposed to antigen. Objective: We sought to assess the expression of costimulatory molecules in fetal gut and the presence of cytokines in amniotic fluid at this time as a preliminary analysis of the suitability of the fetal gut as a site of antigen priming during intrauterine life. Methods: Human fetal gut was analyzed for cells expressing costimulatory molecules through use of immunohistochemistry. Amniotic fluid was studied by ELISA, for cytokines regulating the nature of the response, and as a source of the common dietary antigen ovalbumin. Results: MHC class II–positive cells were abundant over the period examined (11-24 weeks of gestation), other surface antigens showing spatial and temporal variation in expression. From 11 to 14 weeks of gestation, CD68-positive and CD40-positive cells, like MHC class II–positive cells, were present throughout the lamina propria; few CD3-positive cells (T cells) were observed. With the emergence of lymphoid aggregates (14-16 weeks), CD83-positive cells (dendritic cells) and CD20-positive cells (B cells) could be detected in fetal gut; however, expression was restricted to the lymphoid aggregates. In contrast, MHC class II, CD40, and CD68 continued to be expressed in the lamina propria. CD28-positive cells were also evident from 14 weeks of gestation, occurring throughout the lamina propria and lymphoid aggregates; this corresponded to the increasing numbers of CD3-positive cells. The occasional CD86-positive, CD40L-positive, or CTLA4-positive cell could be seen in or around lymphoid aggregates after 14 weeks of gestation. Lymphoid follicles forming after 16 weeks of gestation contained MHC class II–positive, CD83-positive, CD20-positive, CD40-positive, CD86-positive, CD3-positive, CD28-positive, CD40L-positive, and CTLA4-positive cells. MHC class II–positive, CD40-positive, CD68-positive, CD3-positive, and CD28-positive cells continued to be present in the lamina propria at this time. At all times studied, CD14 was not expressed in the lamina propria or lymphoid follicles. Prostaglandin E 2 , TGFβ 1 , and IL-10 dominated the amniotic fluid cytokine milieu, and ovalbumin was also detectable in amniotic fluid from 3 of 26 women who had detectable circulating levels. Conclusion: Of the costimulatory molecules studied, CD40 was the most abundant. However, both of the ligand families studied (CD40-CD40L and CD86-CD28/CD152) could provide the costimulatory signals required for the initiation of antigenspecific reactivity in the gastrointestinal tract of the human fetus as early as 16 weeks of gestation. The cytokine milieu would favor the development of T H 2-type reactivity to antigens, such as ovalbumin, that are present at this time.(J Allergy Clin Immunol 2001;108:235-41.)

Oral tolerance is a well-characterized phenomenon in animals and is highly effective when induced as a treatment for experimental autoimmune disease. However, its use as a therapeutic modality for the treatment of autoimmune disease in humans has been disappointing. Much of the rationale for its use in humans is based on the finding that feeding antigen to rodents elicits regulatory T cells in Peyer's patches (PPs) that secrete immunosuppressive cytokines such as transforming growth factor (TGF)-β. By contrast, human antigen-specific PP T-cell responses, and mucosal T-cell responses in general, are strongly biased towards T helper 1 (Th1) cells, which are pro-inflammatory rather than immunosuppressive. This is caused by the high local levels of interleukin (IL)-12 in PPs.

The role of mucosal T-cell activation in HIV-associated enteropathy is uncertain. Twenty Zambian patients with AIDS and chronic diarrhea were studied, as were nine controls. Distal duodenal biopsies were taken at endoscopy. Morphometric analysis and dual color immunofluorescence staining were performed. Villous height was reduced [177 (118-228) vs 305 (244-358) microm P = 0.002] and crypt depth increased [220 (164-202) vs 194 (164-202) microm P = 0.008] in AIDS patients compared to controls. CD3+CD4+ T cells were reduced in AIDS patients compared to controls [12.9 (5.7-25.2) vs 47.6 (33.4-65.5)% P = 0.04]. There was no significant difference in expression of CD8, CD25, CD69, HML-1, or HLA-DR on T cells between the AIDS patients and controls, with the exception of CD3+HML-1+ cells, which were increased in AIDS patients (P = 0.05). Small intestinal T-cell activation was similar between AIDS patients and controls. We conclude, therefore, that this mechanism is not likely to be important in the pathogenesis of HIV-associated enteropathy.

Immunotherapy with biological agents or small molecules is revolutionising the treatment of chronic inflammatory disease in humans; however, a significant proportion of patients fail to respond or lose responsiveness. This is particularly evident in inflammatory bowel disease (IBD), a group of chronic, immune-mediated disorders of the gastrointestinal tract. Different responsiveness to treatment in IBD can be explained by substantial disease heterogeneity, which is being increasingly recognised by genetic and immunological studies. The current enthusiasm for stratified medicine suggests that it may become possible to identify clinical, immunological, biochemical or genetic biomarkers to target immunotherapy to patients more likely to respond. Here, we identify and highlight the opportunities and the challenges of this strategy in the context of IBD.

The carboxy-terminal 280 amino acids (Int280) of the bacterial adhesion molecule intimin include the receptor-binding domain. At least five different types of Int280, designated α, β, γ, δ, and ɛ, have been described based on sequence variation in this region. Importantly, the intimin types are associated with different evolutionary branches and contribute to distinct tissue tropism of intimin-positive bacterial pathogens. In this study we engineered a strain of Citrobacter rodentium , which normally displays intimin β, to express intimin γ from enterohemorrhagic Escherichia coli . We show that intimin γ binds to the translocated intimin receptor (Tir) from C. rodentium and has the ability to produce attaching and effacing lesions on HEp-2 cells. However, C. rodentium expressing intimin γ could not colonize orally infected mice or induce mouse colonic hyperplasia. These results suggest that intimin may contribute to host specificity, possibly through its interaction with a receptor on the host cell surface.

If we understand pathological processess within the alimentary tract, it is apparent that the fundamental aspects of microbe-host interactions need to be examined in greater detail. Pathogenic bacteria have evolved strategies to alter and subvert the function of T cels and phagocytes in the gut wall, and exploiting these molecules may lead to new treatments for chronic inflammatory bowel diseases. The adaptation of microbes to their host must involve microbe-mediated interference of the host innate immune response. The recent demonstration that nonpathogenic E. coli have a beneficial effect in ulcerative colitis further supports the notion that normal flora may alter the expression of the innate immune receptors or recognize alternative receptors compared with pathogenic variants. Such differences may conceivably lead to beneficial and protective alterations to the host through cytokine and antimicrobial peptide expression. Perhaps the contact point between microbes and host cells lies with the pattern-recognition receptors such as TLRs. However, although much light has been shed on the downstream consequences of TLR activation, many more questions remain unsolved. For example, little is known about the expression profiles of the different TLRs throughout the gastrointestinal tract. Additionally, ambiguities remain over the natural ligands for TLRs. The discovery that the Drosophila Toll receptor acts downstream of the pathogen recognition event suggests that there are many more twists and turns to be revealed in the story of host-microbe interactions in the gastrointestinal tract.

Background/Aims: In our experience with the acute murine dextran sodium sulphate (DSS) model of experimental colitis, we noted both interstrain and interanimal variations in daily water consumption. One might critically question whether observed differences in injuries are just a dose dependency phenomenon reflecting variations in DSS intake. To clarify this important topic, we performed a dose and concentration dependency study of DSS in Balb/c mice. We also determined Th1 and Th2 cytokine levels to compare the cytokine profile to that from inflammatory bowel disease (IBD). Methods: In four groups (14 animals each group) different concentrations of DSS (0, 2.5, 5 and 7.5%) were given for 7 days ad libitum. Mucosal injury of the entire colon was histologically assessed and graded. Cytokine levels were determined by competitive quantitative RT-PCR. Results: A linear increase in the crypt damage score was noted with increasing concentrations (0, 4.9 ± 0.7, 11.9 ± 0.5 and 18.9 ± 1.3, respectively), but the total dose of DSS intake did not correlate with mucosal damage. Progressive upregulation in the transcripts for Th1 cytokines (IL-12, IFN-γ, IL-1, TNF-α) was observed with increasing dosage of DSS. Interestingly, an increase in IL-10, but not IL-4 mRNA transcripts was also noted. Discussion: Acute DSS-induced mucosal injury is dependent on the administered DSS water concentration but not on the consumed DSS dose. The cytokine profile is a classic Th1 response and is similar to that of various inflammatory conditions in the colon. Conclusions: Minor variations in fluid consumption do not affect the severity of DSS-induced injury in mice. The acute murine DSS colitis model is useful for studying the pathophysiological aspects of colonic inflammatory diseases as IBD and for evaluating new potential therapeutic agents

In recent years, new concepts have been formulated for the therapeutic management of the intractable forms of Crohn's disease and ulcerative colitis, the two major forms of inflammatory bowel disease. These advances are based largely on new insights into the immune-inflammatory events occurring in the gut of these patients. Analysis of the types of immune response ongoing in the inflamed intestine has revealed that in Crohn's disease there is predominantly a T-helper cell type 1 response, with exaggerated production of interleukin (IL)-12 and interferon (IFN)-gamma, whereas in ulcerative colitis the lesion seems more of an antibody-mediated hypersensitivity reaction. Despite these differences, downstream inflammatory events are the same in both conditions. In both Crohn's disease and ulcerative colitis mucosa, IL-1gamma, IL-6, IL-8 and tumour necrosis factor (TNF)-alpha are produced in excess, and the production of free radicals accompanying the influx of nonspecific inflammatory cells into the mucosa is above the normal range. Strategies aimed at inhibiting T-cell responses are therefore more relevant in Crohn's disease, whereas, in theory at least, inhibition of downstream inflammatory processes should be therapeutic in both Crohn's disease and ulcerative colitis. This review seeks to summarize studies in which anticytokine antibodies, cytokines or cytokine-modifying agents have been used in the treatment of either Crohn's disease or ulcerative colitis.

Crohn's disease and ulcerative colitis are caused by excessive immune reactivity in the gut wall. Analysis of the type of immune responses ongoing in diseased gut has revealed important features which suggest that these conditions are different. In Crohn's disease tissue there is considerable evidence for an ongoing T helper cell type 1 response, with excess interleukin-12, interferon-gamma and TNF-alpha. There is circumstantial evidence in patients that this response is directed against the normal bacterial flora and definitive evidence in mouse models that T cell responses to the flora cause gut disease. In ulcerative colitis, the role of tissue damaging T cell responses in the gut mucosa is much less clear and there is more evidence that the lesion is owing to antibody-mediated hypersensitivity. Although different types of immune reactions initiate tissue injury in both Crohn's disease and ulcerative colitis, the downstream events which actually damage the tissue are the same in each condition. Elevated cytokine concentrations in the mucosa lead to the production of excess matrix degrading enzymes by gut fibroblasts, loss of mucosal integrity and ulceration. The same process also leads to an increased production of epithelial growth factors such as KGF Keratinocyte Growth Factor by gut fibroblasts and produces the crypt cell hyperplasia characteristic of all gut inflammatory conditions.

T cells are crucial mediators of the skin damage in psoriasis. We here show that interleukin-21 (IL-21), a T cell-derived cytokine, is highly expressed in the skin of individuals with psoriasis, stimulates human keratinocytes to proliferate and causes epidermal hyperplasia when injected intradermally into mice. In the human psoriasis xenograft mouse model, blockade of IL-21 activity resolves inflammation and reduces keratinocyte proliferation. Blocking IL-21 may represent a new therapeutic strategy in psoriasis.

A recent symposium ∗ focused on immunological mechanisms of mucosal protection and disturbances of these that lead to inflammation and destruction in the gastrointestinal tract. Increased understanding of the molecular interactions involved is likely to have an important impact on new therapeutic strategies for autoimmune diseases, gastrointestinal infections and chronic inflammatory bowel disease.

The relationship between intestinal pathology and immune expulsion of gastrointestinal nematodes remains controversial. Parasite expulsion is associated with intestinal pathology in several model systems and both of these phenomena are T cell dependent. Immune expulsion of gastrointestinal helminth parasites is usually associated with Th2 responses, but the effector mechanisms directly responsible for parasite loss have not been elucidated. In contrast, the intestinal pathology observed in many other disease models closely resembles that seen in helminth infections, but has been attributed to Th1 cytokines. We have used infection with the nematode Trichinella spiralis in mice defective for cytokines or their receptors to investigate cytokine regulation of both immunopathlogy and parasite rejection. Consistent with previous findings, we found that parasite expulsion is IL-4 dependent. Contrary to expectations, however, the enteropathy is not regulated by IFN-γ but by IL-4. Moreover, abrogation of severe pathology in TNF receptor-defective animals does not prevent parasite expulsion. TNF is therefore involved in intestinal pathology in nematode infections, apparently under regulation by IL-4- and Th2-mediated responses. This work therefore not only reveals a novel interplay between IL-4 and TNF, but also that the IL-4-dependent protective response against the parasite operates by a mechanism other than merely the gross degradation of the parasite's environment brought about by the immune enteropathy.

Anti-TNF-α Ab therapy has been shown to be of benefit in the treatment of active Crohn’s disease, but the tissue-injuring processes in the gut mediated by TNF-α that might be inhibited by neutralizing Ab are unknown. In this work, we have used a p55 TNF receptor-human IgG fusion protein (TNFR-IgG) to prevent the severe mucosal injury that ensues when lamina propria T cells in explant cultures of human fetal small intestine are directly activated with the lectin PWM. Following T cell activation and associated with mucosal injury, there is a marked elevation of soluble TNF-α in organ culture supernatants and a large increase in TNF-α mRNA transcripts. The addition of TNFR-IgG at the onset of cultures greatly reduced PWM-induced tissue injury, without inhibiting the increase in TNF-α and IFN-γ transcripts seen following T cell activation. Mucosal injury in this model is mediated by endogenously-produced matrix metalloproteinases (MMPs). When TNFR-IgG was added to PWM-stimulated explants, there was a reduction in MMPs in the explant culture supernatants, especially stromelysin-1. Recombinant TNF-α and IL-1β added directly to mucosal mesenchymal cell lines also caused an increase in MMP production, but only the former was inhibited by the TNFR-IgG. These results suggest that one of the ways in which TNF-α causes tissue injury in the gut is by stimulating mucosal mesenchymal cell to secrete matrix-degrading metalloproteinases. Neutralization of this activity should help maintain tissue integrity.

Enteral diets, both elemental and, more recently, polymeric (whole protein), are used as primary therapy in Crohn's disease and can induce disease remission without the concomitant use of immunosuppressive drugs. Controlled trials comparing enteral nutrition with corticosteroid therapy have given mixed results but suggest, at least in children, that they are as effective as corticosteroids in inducing remission. There is no clear consensus as to which dietary therapy is best. Elemental diets do not seem to be superior to polymeric whole protein-based diets, although further work is necessary. The effect of enteral diets does not seem to be related to the site of intestinal inflammation. Enteral nutrition is particularly appropriate in children and adolescents with Crohn's disease, improving nutrition and promoting growth and pubertal development, and avoiding the systemic toxicity of corticosteroid therapy. Most centers will use it as a first line of treatment. Supplementary enteral nutrition after primary therapy and remission induction may be associated with the prolongation of remission and promotion of linear growth. Pathways by which enteral diets may affect mucosal inflammation are discussed. Enteral diets may inhibit intestinal immune responses by reducing the number of cytokine-producing cells. Enteral nutrition may also boost immunosuppressive pathways, which then endogenously suppress ongoing inflammation. Enteral diets may promote epithelial healing and reepithelialization of Crohn's ulcers and may also reduce the bacterial load in the small bowel.

The major experimental and intellectual advance in gastroenterology in recent years has been the observation that mice with deletions in specific cytokine genes develop a spontaneous inflammatory bowel disease. Inflammatory bowel disease has also been seen in a number of other mouse strains with perturbed immune responses. The driving force for disease in a number of these models is the normal bacterial flora. These studies show that the gut immune system is in homeostasis with the antigens of the flora, but inappropriate T-helper 1 (Th1)-type responses to the flora cause severe gut injury and since the antigenic stimulus persists, the consequence is chronic inflammation.

RCD [refractory CD (coeliac disease)] is characterized by severe symptoms/signs of malabsorption and mucosal damage unresponsive to a GFD (gluten-free diet). The pathogenesis of RCD is not fully understood. In the present paper, we have characterized the mucosal profile of effector cytokines in RCD. Duodenal biopsies were taken from patients with RCD, patients with active CD and normal controls and were analysed for inflammatory cytokines by real-time PCR and ELISA. IFN (interferon)-γ and IL (interleukin)-21 transcripts were increased in active CD patients but not in RCD patients as compared with normal controls, whereas IL-17A RNA was up-regulated in both active CD and RCD. No significant increase in IL-15 transcripts was observed in both active CD and RCD, whereas IL-15 protein was increased in active CD. IL-6 and TNF (tumour necrosis factor)-α were up-regulated only in RCD. As a proof, we present the case of a woman affected by RCD who responded to anti-TNF-α treatment with improvement of malabsorptive symptoms/signs but no healing of mucosal lesions. The findings indicate that the profile of mucosal effector cytokines differs between RCD and active CD and suggest that TNF-α, IL-6 and IL-17A, but not Th1-type cytokines, could drive the detrimental response in this condition.

In inflammatory bowel disease (IBD), tissue damage is driven by an excessive immune response, poorly controlled by counter-regulatory mechanisms. SIRT1, a class III NAD+-dependent deacetylase, regulates negatively the expression of various proteins involved in the control of immune-inflammatory pathways, such as Stat3, Smad7, and NF-κB. Here we examined the expression, regulation, and function of SIRT1 in IBD. SIRT1 RNA and protein expression was less pronounced in whole biopsies and lamina propria mononuclear cells (LPMCs) of IBD patients in comparison with normal controls. SIRT1 expression was downregulated in control LPMC by tumor necrosis factor (TNF)-α and interleukin (IL)-21, and upregulated in IBD LPMC by neutralizing TNF-α and IL-21antibodies. Consistently, SIRT1 expression was increased in mucosal samples taken from IBD patients successfully treated with Infliximab. Treatment of IBD LPMC with Cay10591, a specific SIRT1 activator, reduced NF-κB activation and inhibited inflammatory cytokine synthesis, whereas Ex527, an inhibitor of SIRT1, increased interferon (IFN)-γ in control LPMC. SIRT1 was also reduced in mice with colitis induced by 2,4,6-trinitrobenzenesulphonic acid or oxazolone. Cay10591 prevented and cured experimental colitis whereas Ex527 exacerbated disease by modulating T cell-derived cytokine response. Data indicate that SIRT1 is downregulated in IBD patients and colitic mice and suggest that SIRT1 activation can help attenuate inflammatory signals in the gut.Mucosal Immunology advance online publication, 21 May 2014; doi:10.1038/mi.2014.35.

Transitional 2 B cells home to gut-associated lymphoid tissue and present an activated phenotype in healthy subjects, but gut immune compartments are depleted in SLE., We have tracked the fate of immature human B cells at a critical stage in their development when the mature B cell repertoire is shaped. We show that a major subset of bone marrow emigrant immature human B cells, the transitional 2 (T2) B cells, homes to gut-associated lymphoid tissue (GALT) and that most T2 B cells isolated from human GALT are activated. Activation in GALT is a previously unknown potential fate for immature human B cells. The process of maturation from immature transitional B cell through to mature naive B cell includes the removal of autoreactive cells from the developing repertoire, a process which is known to fail in systemic lupus erythematosus (SLE). We observe that immature B cells in SLE are poorly equipped to access the gut and that gut immune compartments are depleted in SLE. Thus, activation of immature B cells in GALT may function as a checkpoint that protects against autoimmunity. In healthy individuals, this pathway may be involved in generating the vast population of IgA plasma cells and also the enigmatic marginal zone B cell subset that is poorly understood in humans.

Tumor necrosis factor α (TNF-α) plays a major role in the tissue-damaging immune response in inflammatory bowel diseases (IBDs). The tissue concentration of TNF-α is related to the activity of "A Disintegrin And Metalloprotease" (ADAMs), enzymes that process membrane-bound TNF-α and liberate the TNF-α trimer into the extracellular environment. Although IBD-related inflammation is associated with high ADAM17 levels, the contribution of other members of the ADAMs family is not known. In this study, we characterized the expression of other TNF-α convertases (i.e., ADAM9, ADAM10, and ADAM19) in IBD.Normal and IBD biopsies were examined for the content of ADAMs by real-time polymerase chain reaction, Western blotting and immunohistochemistry. ADAM19 was also analyzed in intestinal epithelial cells and normal colonic explants stimulated with inflammatory cytokines and in ex vivo biopsies taken from IBD patients before and after a successful infliximab treatment.ADAM19 RNA transcripts and protein were upregulated in patients with ulcerative colitis and, to a lesser extent, in patients with Crohn's disease compared with normal controls. In contrast, ADAM9 and ADAM10 expression did not differ between patients with IBD and controls. Immunohistochemical analysis showed that epithelial cells were the major source of ADAM19 in IBD. ADAM19 expression was increased in colonic epithelial cell lines and normal colonic explants by TNF-α, interleukin 21 and interleukin 6, and was downregulated in IBD tissue by infliximab.These findings suggest the existence of a positive feedback mechanism involving cytokines and ADAM19 that can amplify cytokine production in IBD.

Many of the clinicopathologic features of neonatal respiratory distress syndrome (RDS) may be related to the inflammatory response mounted by the affected infant, although little is known about the interstitial component of this response. We have thus studied the local inflammatory response in this condition by immunohistochemical analysis of whole lung lobes, obtained at postmortem from 40 infants who died from acute RDS in the first week of life. All had demonstrated classical clinical history and histologic features. An archival subgroup from the early 1970s had never received ventilatory support. Immunohistochemical analysis demonstrated rapid temporal increase from birth in the mucosal density of CD68+ macrophages, MAC-387+ monocytes/macrophages, polymorphonuclear neutrophils, and tumor necrosis factor-alpha-immunoreactive cells, maximal in those dying at or after 72 h. Using a cationic probe specific for sulfated glycosaminoglycans (GAGs), the inflammatory infiltration was seen to be associated with striking loss of endothelial, basement membrane, and interstitial GAGs, which was almost complete by 48-72 h. GAG degradation products were found within hyaline membranes in all infants dying after 48 h. This study confirms that neonatal RDS is characterized by intense interstitial inflammation, significantly underestimated on routine staining. This begins within hours of birth and is maximal by 72 h of age. Breakdown of sulfated GAGs within the extracellular matrix follows the same time course and may explain much of the physiologic derangement characteristic of this condition.

Cholera toxin (CT) or its subunits were given orally to mice and division of intestinal intraepithelial lymphocytes (IEL) in vivo measured by double immunofluorescence using 5-bromo-2'-deoxyuridine (BRdU) and membrane alpha beta T-cell receptors (TCR) or gamma delta TCR staining in frozen sections. Cholera toxin (10 micrograms) produced a two- to eightfold-increase in the uptake of BRdU in alpha beta TCR+ IEL in the duodenum and a two-to fivefold increase in gamma delta TCR IEL in the ileum. Increased uptake of BRdU was also seen after a dose of 100 micrograms of CT but this dose was also associated with the loss of alpha beta TCR+ IEL and gamma delta TCR+ IEL in the duodenum. CT-A and CT-B subunit produced increased BRdU incorporation by alpha beta TCR in the duodenum and by gamma delta TCR IEL in the ileum. Cholera toxin therefore appears to be mitogenic for IEL probably due to an indirect mechanism.

Intraepithelial lymphocytes have been shown to be oligoclonal and to be disseminated widely along the human intestine. However, studies using monoclonal antibodies have suggested that superimposed on the widespread clones, there is local variability in the mucosal T cell population. We have investigated the possibility that local dominant clones of T cells are present in the colonic mucosa by polymerase chain reaction (PCR) amplification of T cell receptor beta chain junctional regions using DNA extracted from microdissected fragments of tissue sections. Colon from two right hemicolectomy specimens was sampled at 7-cm intervals. Adjacent areas of mucosa were microdissected from sections from each colon sample. When the PCR products were separated according to size on polyacrylamide gels, bands of identical size were often observed when DNA extracted from adjacent fragments of mucosa had been used. Different bands were present when the different samples of colon had been studied. Sequencing of the PCR products confirmed that clonally related T cells were present in adjacent areas of mucosa, whereas different clones dominated at distant sites. DNA extracted from cells microdissected from the T cell zone of Peyer's patch was treated identically. The sequences obtained from the Peyer's patch, as expected, were diverse. However, one of the sequences identified was identical to that of one of the clones in the colon, implying that this clone was either trafficking through the Peyer's patch or possibly originated from the Peyer's patch. In this study, we also identified the Peyer's patches as a site of proliferation of CD4+ T cells. No T cell division was observed in the lamina propria. The molecular and immunohistochemical observations together support the hypothesis that the Peyer's patches are a source of mucosal T cells.

The development of the mucosal immune system in the human fetus has been studied in some detail. Aggregates of T and B cells form early Peyer's patches by 16 weeks gestation and by 19 weeks organised Peyer's patches with T and B cell zones are seen. T cells populate the mucosal lamina propria and epithelium from 11 weeks gestation and increase in number thereafter. As in the adult, most intraepithelial lymphocytes are CD8+ and most lamina propria T cells are CD4+. By 20 weeks, villus epithelial cells are HLA-DR+ and secretory component is also expressed. In the absence of lumenal stimulation in the fetus there is no intestinal secretory IgA antibody response. A few IgA plasma cells are present in fetal salivary glands but the number does not dramatically increase until after birth.

Immune function in the gut mucosa is tightly regulated to prevent deleterious tissue damaging responses to the indigenous microbiota. In animal models, negative regulatory molecules such as transforming growth factor β1 (TGFβ1) and interleukin (IL)-10 seem to be particularly important. Although IL-10 is made by macrophages, T cells and B cells, TGFβ1 is made by many non-lymphoid/myeloid cells, especially epithelial cells. We have been able to show that in the human gut, neutralization of TGFβ1 increases Th1 and Th17 responses. In IBD, however, especially Crohn’s disease, exogenous TGFβ1 cannot inhibit inflammation because of a block in intracellular signalling mediated by Smad7. Knockdown of Smad7 allows endogenous TGFβ1 to dampen inflammation in mucosal tissues from Crohn’s patients. We have also generated a mouse which over-expresses Smad7 in T cells. This animal develops more severe colitis in a number of different models, but the inflammation helps protect against colon cancer.

Interleukin (IL)-21, a cytokine produced by activated conventional CD4+ T lymphocytes and Natural Killer T cells, drives anti-tumor immunity in the skin and kidney. However IL-21 is also pro-inflammatory in many tissues and promotes colitis-associated colon cancer. Understanding the biology of IL-21 in these different situations is needed to ensure maximal therapeutic benefit.

In the gut of patients with inflammatory bowel disease (IBD), immune and nonimmune cells produce large amounts of cytokines that drive the inflammatory process leading to the tissue damage. Cytokine blockers, such as anti-tumor necrosis factor alpha (TNF-α), have been used with some success in IBD. However, not all patients respond, and the therapeutic effects wane with time, demonstrating the need for more effective and long-lasting antiinflammatory strategies. A key question is whether neutralizing other proinflammatory cytokines such as interleukin (IL)-12, IL-21, IL-27, or IL-33 will lead to a better clinical response than with anti-TNF-α antibodies. Equally, we now know that IBD-related inflammation is marked by defective production/activity of antiinflammatory cytokines, and there are strategies to correct these defects. An alternative approach is to try to target individual therapies to individual patients, to improve clinical efficacy in subsets of patients, but this has proven difficult. Here we try to evaluate the potential of each of these choices.

The human mucosal immune system is structurally mature and has all the necessary cellular components to generate an immune response at birth. However, in the absence of dietary antigens and bacterial flora, there are no secondary follicles in the Peyer's patches and virtually no immunoglobulin A plasma cells in the lamina propria. Reactive follicle centers develop after birth but it takes 2 years for mucosal IgA plasma cell density to reach adult levels. T cells are present in the epithelium and lamina propria at birth, albeit at a lower frequency than later in life and there are major differences in phenotype between T cells in fetal intestine and postnatal intestine. There is no information on the impact of the massive antigenic challenge at birth on the mucosal immune system. Well-documented deficiencies in the ability of the blood T cells of the neonate to produce interleukin-4 and interferon-gamma may also occur in the intestine. It is still an open question whether it is better to try to prevent immunological sensitization of the newborn by avoiding potential allergens (i.e. cow's milk), or whether early exposure (as happens when premature infants are given formula feeds) might tolerize the infant. Hydrolysed cow's milk formulae are probably less antigenic than whole cow's milk and have been widely used in the treatment of cow's milk allergy. Some thought is now being given as to whether the prophylactic use of hydrolysates can reduce cow's milk allergy in ‘at-risk’ infants.

BACKGROUND & AIMS: Many patients with inflammatory bowel disease (IBD) fail to respond to anti-tumor necrosis factor (TNF) agents such as infliximab and adalimumab, and etanercept is not effective for treatment of Crohn's disease. Activated matrix metalloproteinase 3 (MMP3) and MMP12, which are increased in inflamed mucosa of patients with IBD, have a wide range of substrates, including IgG1. TNF-neutralizing agents act in inflamed tissues; we investigated the effects of MMP3, MMP12, and mucosal proteins from IBD patients on these drugs. METHODS: Biopsy specimens from inflamed colon of 8 patients with Crohn's disease and 8 patients with ulcerative colitis, and from normal colon of 8 healthy individuals (controls), were analyzed histologically, or homogenized and proteins were extracted. We also analyzed sera from 29 patients with active Crohn's disease and 33 patients with active ulcerative colitis who were candidates to receive infliximab treatment. Infliximab, adalimumab, and etanercept were incubated with mucosal homogenates from patients with IBD or activated recombinant human MMP3 or MMP12 and analyzed on immunoblots or in luciferase reporter assays designed to measure TNF activity. IgG cleaved by MMP3 or MMP12 and antihinge autoantibodies against neo-epitopes on cleaved IgG were measured in sera from IBD patients who subsequently responded (clinical remission and complete mucosal healing) or did not respond to infliximab. RESULTS: MMP3 and MMP12 cleaved infliximab, adalimumab, and etanercept, releasing a 32-kilodalton Fc monomer. After MMP degradation, infliximab and adalimumab functioned as F(ab')2 fragments, whereas cleaved etanercept lost its ability to neutralize TNF. Proteins from the mucosa of patients with IBD reduced the integrity and function of infliximab, adalimumab, and etanercept. TNF-neutralizing function was restored after incubation of the drugs with MMP inhibitors. Serum levels of endogenous IgG cleaved by MMP3 and MMP12, and antihinge autoantibodies against neo-epitopes of cleaved IgG, were higher in patients who did not respond to treatment vs responders. CONCLUSIONS: Proteolytic degradation may contribute to the nonresponsiveness of patients with IBD to anti-TNF agents. publisher: Elsevier articletitle: Proteolytic Cleavage and Loss of Function of Biologic Agents That Neutralize Tumor Necrosis Factor in the Mucosa of Patients With Inflammatory Bowel Disease journaltitle: Gastroenterology articlelink: http://dx.doi.org/10.1053/j.gastro.2015.07.002 content_type: article ispartof: Gastroenterology vol:149 issue:6 pages:1564-1574 ispartof: location:United States status: published

Summary While Crohn's disease and ulcerative colitis are both conditions characterized by intestinal inflammation, with some overlap in their clinical and histological features, they are essentially different in pathogenesis. Crohn's disease appears to be primarily a condition of chronic T-lymphocyte activation, with tissue damage induced by secondary macrophage activation. What activates the T-cells is unknown. In this chapter we look at the evidence for and against cell-wall deficient mycobacteria species, viral infection of vascular endothelium and luminal contents as potential mechanisms of chronic activation. In ulcerative colitis, by contrast, there is no strong evidence for T-cell activation, and humoral mechanisms predominate. While the finding of atypical anti-neutrophil cytoplasmic antibodies (P-ANCAs) may be useful in screening, the only novel pathogenetic discovery is the co-localization of a 40 kD colonic autoantibody with immunoglobulins and complement on the apical enterocyte surface. Despite the fundamental differences in initiating mechanisms, the twoconditions have many ‘downstream’ inflammatory processes in common. We discuss the evidence for local production of cytokines, arachidonic acid metabolites and reactive oxygen and nitrogen radicals, highlighting the potential adverse consequences for intestinal vascular integrity.

OBJECTIVE The concept that HIV infection per se alters small intestinal mucosal structure and function (HIV enteropathy) remains controversial and in this study we report in vitro experiments designed to elucidate the matter. METHODS Twenty pairs of human fetal intestinal tissue explants were maintained in vitro for up to 14 days; one explant of each pair was incubated and infected with HIV, and the other served as a matched uninfected control. At various times after infection, explant culture fluid and tissue were removed, p24 concentration was measured and tissue formalin fixed. Explant tissue was embedded in paraffin wax and sections stained by an immunoperoxidase method directed against proliferating cell nuclear antigen (PCNA). The percentage of proliferating crypt and villous epithelial cells, stained by PCNA, was calculated in paired samples. The difference between the percentage for paired samples was designated delta crypt proliferation (delta CP) and delta villous proliferation (delta VP), respectively. Epithelial cell proliferation was deemed to be enhanced if the percentage of PCNA-stained cells was greater in the HIV-infected than in the control tissue. RESULTS Explant culture fluid from tissue exposed to HIV showed a progressive rise in p24 antigen (Ag) level, indicating HIV infection of these explants. Fifteen pairs of explants showed progressively positive delta CP with time (P < 0.01) indicating crypt hyperplasia and all 20 pairs of explants showed positive delta VP, indicating hyperplasia of villous epithelial cells. CONCLUSIONS This study provides direct evidence that HIV stimulates epithelial cell proliferation in intestinal mucosa. HIV-infected human intestinal explants provide a model of crypt hyperplastic villous atrophy previously described as HIV enteropathy and detected in clinical biopsy specimens from HIV-infected patients.

We performed genetic and immunohistochemical studies in a sister and brother with autosomal recessive neonatal inflammatory skin and bowel lesions. The girl died suddenly at 12 years of age from parvovirus B19-associated myocarditis; her brother had mild cardiomyopathy. We identified a loss-of-function mutation in ADAM17, which encodes a disintegrin and metalloproteinase 17 (also called tumor necrosis factor α [TNF-α]-converting enzyme, or TACE), as the probable cause of this syndrome. Peripheral-blood mononuclear cells (PBMCs) obtained from the brother at 17 years of age showed high levels of lipopolysaccharide-induced production of interleukin-1β and interleukin-6 but impaired release of TNF-α. Despite repeated skin infections, this young man has led a relatively normal life. (Funded by Barts and the London Charity and the European Commission Seventh Framework Programme.).

The gastrointestinal tract is the largest immune interface with the environment. Exposure to large numbers of dietary and microbial antigens requires complex and highly regulated immune responses by different mucosal cell types, which result in the induction and maintenance of intestinal homeostasis. Defects in this equilibrium can disrupt the homeostatic mechanisms and lead to chronic intestinal inflammation. We review the cell populations and mechanisms involved in the control of intestinal homeostasis and inflammation, focusing on inflammatory bowel diseases. We describe some aspects of gut immunity that could alter the delicate balance between inflammatory and tolerogenic responses and result in chronic gastrointestinal tract inflammation in patients.

Tissues exposed to ischemia and reperfusion develop an inflammatory response. We investigate the morphological and immunological changes occurring in the mucosa of a jejunal loop transplanted in the oropharynx of a man undergoing circular pharyngolaryngectomy. Jejunal biopsies were collected during the transplantation procedures (cold and warm ischemia, reperfusion), during the 7 post-operative days through an exteriorized jejunal segment for flap monitoring, and 45 days after transplantation through an upper endoscopy. Matrix metalloproteinase (MMP)-3 and MMP-12 increase was accompanied by a parallel rise in apoptotic enterocytes, and by a concomitant reduction of surface area to volume ratio and enterocyte height. Goblet cell hyperplasia is coupled with Paneth cell disappearance at the crypt base. CD8-positive intraepithelial lymphocytes initially decrease, then they increase in accordance with the peak of enterocyte apoptosis. We identified alterations in lymphocyte infiltration, mucosal architecture and epithelial cell turnover, which may give a window to mechanisms of small bowel ischemia–reperfusion in humans.

Recent progresses in basic science have opened new pathogenic scenarios in inflammatory bowel disease. The T helper cell type (Th)1/Th2 paradigm has been outdated thanks to the advances in understanding the function of Th17 cells. Innate immunity, nonimmune cells, and defective tolerogenic mechanisms play a no less crucial role than do adaptive immunity, immune cells, and hyperactivation of effector mechanisms. These new paradigms, together with the no longer "static" but "dynamic" vision of intestinal inflammation, highlight new possible therapeutic targets in inflammatory bowel disease.

IL-21 expression is increased in the gut of patients with colitis-associated colon cancer, and genetic ablation or antibody neutralization of IL-21 reduces tumor size and inflammation in mice treated with dextran sulfate sodium and azoxymethane., Chronic inflammation is a major driving force in the development of cancer in many tissues, but the array of factors involved in this neoplastic transformation are not well understood. We have investigated the role of interleukin (IL)-21 in colitis-associated colon cancer (CAC), as this cytokine is overexpressed in the gut mucosa of patients with ulcerative colitis (UC), a chronic inflammatory disease associated with colon cancer. IL-21 was increased in the gut of patients with UC-associated colon cancer, and in mice with CAC induced by azoxymethane (AOM) and dextran sulfate sodium (DSS). After AOM+DSS treatment, IL-21 KO mice showed reduced mucosal damage, reduced infiltration of T cells, and diminished production of IL-6 and IL-17A. IL-21–deficient mice also developed fewer and smaller tumors compared with wild-type (WT) mice. Absence of IL-21 reduced signal transducer and activator of transcription 3 activation in tumor and stromal cells. Administration of a neutralizing IL-21 antibody to WT mice after the last DSS cycle decreased the colonic T cell infiltrate and the production of IL-6 and IL-17A and reduced the number of tumors. These observations indicate that IL-21 amplifies an inflammatory milieu that promotes CAC, and suggest that IL-21 blockade may be useful in reducing the risk of UC-associated colon cancer.

Crohn's disease (CD) and ulcerative colitis (UC) are the major forms of inflammatory bowel diseases (IBD) in man. They are caused by damage to the lining of the intestine and deeper layers, due to an excessive immune response directed against components of the gut microflora and poorly controlled by counter-regulatory mechanisms. CD and UC are however immunologically distinct. CD-related inflammation is characterized by a marked mucosal infiltration of T lymphocytes secreting T helper type (Th) 1 and Th17 cytokines. In UC, the local immune response is less polarized but may show enhanced production of IL-5, IL-13 and Th17 cytokines. Downstream however CD and UC share important end-stage effector pathways of intestinal injury, mediated by an active cross-talk between immune and non-immune cells. The clarification of the complex networks of immune-inflammatory mediators operating in the gut of IBD patients has led to the identification of new targets that should facilitate the development of novel biological therapies.

An altered balance between effector and regulatory factors is supposed to sustain the tissue-damaging immune response in inflammatory bowel disease (IBD). We have recently shown that in IBD, there is a defective synthesis of the counter-regulatory cytokine, interleukin (IL)-25. In this study we investigated factors that control IL-25 production in the gut. IBD patients produced less IL-25 when compared with normal controls. Stimulation of normal intestinal explants with tumor necrosis factor-α (TNF-α), but not interferon-γ (IFN-γ) or IL-21, reduced IL-25 synthesis. Consistently, IL-25 production was enhanced by anti-TNF-α both in vitro and in vivo. Upregulation of IL-25 was also seen in normal colonic explants stimulated with transforming growth factor-β1 (TGF-β1). As in IBD, TGF-β1 activity is abrogated by Smad7, we next assessed whether inhibition of Smad7 with an antisense oligonucleotide enhanced IL-25 expression. Knockdown of Smad7 was accompanied by an increase in IL-25 production. Data show that IL-25 production is differently regulated by TNF-α and TGF-β1 in the human gut.

Gut inflammation in patients with inflammatory bowel disease (IBD) has traditionally been considered to be mediated by an exaggerated TH1 response (Crohn’s disease) or an atypical TH2 response (ulcerative colitis). Recent work however, has also shown that in both types of IBD there is enhanced synthesis of cytokines made by a distinct lineage of T helper cells, termed TH17 cells. Targeting these TH17 cells may be a new avenue for the development of therapeutic strategies in IBD. Interleukin (IL)-23, a heterodimeric cytokine produced by activated antigen presenting cells, is necessary for amplifying TH17 cell responses. Paradoxically, recent studies have also shown that TH17-related cytokines have protective effects in the gut. In this chapter, we review the available data regarding the role of TH17 cells in chronic intestinal inflammation in an attempt to resolve this conundrum.

Patients with inflammatory bowel disease (IBD) have an increased risk of developing colorectal cancer due to chronic inflammation. In IBD, chronic inflammation relies upon a TGFβ signaling blockade, but its precise mechanistic relationship to colitis-associated colorectal cancer (CAC) remains unclear. In this study, we investigated the role of the TGFβ signaling inhibitor Smad7 in CAC pathogenesis. In human colonic specimens, Smad7 was downregulated in CD4+ T cells located in the lamina propria of patients with complicated IBD compared with uncomplicated IBD. Therefore, we assessed CAC susceptibility in a transgenic mouse model where Smad7 was overexpressed specifically in T cells. In this model, Smad7 overexpression increased colitis severity, but the mice nevertheless developed fewer tumors than nontransgenic mice. Protection was associated with increased expression of IFNγ and increased accumulation of cytotoxic CD8+ and natural killer T cells in the tumors and peritumoral areas. Moreover, genetic deficiency in IFNγ abolished the Smad7-dependent protection against CAC. Taken together, our findings defined a novel and unexpected role for Smad7 in promoting a heightened inflammatory response that protects against CAC. Cancer Res; 71(24); 7423–32. ©2011 AACR.

In the intestine large numbers of bacteria and their products are separated by a single epithelial layer from resident inflammatory cells (macrophages and lymphocytes). Many of these bacterial products, such as lipopolysaccharides and peptidoglycans, are potent stimulators of free radical and inflammatory cytokine production by macrophages. This can occur in vivo in response to mucosal invasion by enteropathogenic bacteria or because of inappropriate activation of these cells, as in chronic inflammatory bowel disease. In this review we present evidence for production of cytokines in normal intestine and in intestinal inflammatory conditions. The adverse effects of cytokine production upon intestinal homeostasis, in particular disruption of epithelial integrity and prothrombotic changes in the vascular endothelium, are also discussed.

This study determined the location and tissue density of cells immunoreactive for tumour necrosis factor alpha (TNF alpha) in intestinal specimens from 24 patients with chronic inflammatory bowel disease (15 with Crohn's disease, nine with ulcerative colitis) and 11 controls. There was significantly increased density of TNF alpha immunoreactive cells in the lamina propria of both ulcerative colitis and Crohn's disease specimens, although the distribution of these cells differed in the two conditions. In ulcerative colitis most of the TNF alpha immunoreactivity was seen in the subepithelial macrophages, with comparatively less in the deep lamina propria, while in Crohn's disease immunoreactive cells were distributed evenly throughout the lamina propria. Increased submucosal immunoreactivity was found only in Crohn's disease, in which TNF alpha positive macrophages tended to cluster around arterioles and venules, often infiltrating and disrupting vascular endothelium. It is suggested that this degree of TNF alpha production probably contributes significantly to the pathogenesis of both Crohn's disease and ulcerative colitis, by impairing the integrity of epithelial and endothelial membranes, increasing inflammatory cell recruitment, and by prothrombotic effects on the vascular endothelium.