ovalbumin and Crohn-Disease

Topics: Administration, Oral; Animals; Antigens; B-Lymphocytes; Cell Differentiation; Crohn Disease; Gastric Mucosa; Humans; Immunoglobulin A; Immunoglobulin E; Intestinal Mucosa; Lymphoid Tissue; Mice; Mice, Inbred C3H; Ovalbumin; Peyer's Patches; T-Lymphocytes, Regulatory

New therapeutic strategies are needed for patients with refractory Crohn's disease (CD). We evaluated data from the Crohn's And Treg Cells Study (CATS1) to determine the safety and efficacy of antigen-specific T-regulatory (Treg) cells for treatment of patients with refractory CD.. We performed a 12-week, open-label, multicenter, single-injection, escalating-dose, phase 1/2a clinical study in 20 patients with refractory CD. Ovalbumin-specific Treg cells (ova-Tregs) were isolated from patients' peripheral blood mononuclear cells (PBMCs), exposed to ovalbumin, and administrated intravenously. Safety and efficacy were assessed using clinical and laboratory parameters. We evaluated proliferation of PBMCs in response to ovalbumin.. Injections of ova-Tregs were well tolerated, with 54 adverse events (2 related to the test reagent) and 11 serious adverse events (3 related to the test reagent, all recovered). Overall, a response, based on a reduction in Crohn's Disease Activity Index (CDAI) of 100 points, was observed in 40% of patients at weeks 5 and 8. Six of the 8 patients (75%) who received doses of 10(6) cells had a response at weeks 5 and 8, with a statistically significant reduction in CDAI. In this group, remission (based on CDAI ≤150) was observed in 3 of 8 patients (38%) at week 5 and 2 of 8 patients (25%) at week 8.. Administration of antigen-specific Tregs to patients with refractory CD (CATS1) was well tolerated and had dose-related efficacy. The ovalbumin-specific immune response correlated with clinical response, supporting immune-suppressive mechanisms of ova-Tregs. The consistency of results among different assessment methods supports the efficacy of ova-Tregs; this immune therapy approach warrants further clinical and mechanistic studies in refractory CD. Eudract, Number: 2006-004712-44.

Topics: Adult; Aged; C-Reactive Protein; Crohn Disease; Feces; Female; Humans; Immunotherapy; Leukocyte L1 Antigen Complex; Lymphocyte Count; Male; Middle Aged; Ovalbumin; Quality of Life; Severity of Illness Index; Surveys and Questionnaires; T-Lymphocytes, Regulatory; Young Adult

Inflammatory bowel disease may be due to failed tolerance to normal gut bacteria. We demonstrate that epicutaneous immunotherapy (ET) to ovalbumin can alleviate colitis in murine models. However, most people are tolerant to or have anergy to ovalbumin. Half of Crohn's disease (CD) patients have CBir1 antibodies that can be elevated years before CD development. We determined whether ET with a CBir1 multi-epitope peptide (MEP1) could alleviate colitis.. Wild type mice (C57BL/6) were transferred with CBir1 T cell receptor (TCR) T cells followed by epicutaneous application of MEP1. Proliferating Foxp3+ T cells were measured in mesenteric lymph nodes (LNs), spleen, small intestine, and colon by flow cytometry. Lymphocytes from MEP1 epicutaneously exposed and immunized C57BL/6 mice were cultured with MEP1. Interferon (IFN)-γ production was measured. Colitis was induced by transferring CD4+CD45Rbhi T cells from CBIR1 TCR or C57BL/6 mice into RAG1-/- mice. Mice were treated with ET. Body weight, colon length, colonic cytokine production, histological inflammation, inflammatory genes, and regulatory T cells (Tregs) from lamina propria were measured.. ET with 10 μg of MEP1 induced CBir1-specific Tregs that migrated to the small intestine and colon and suppressed MEP1-specific IFN-γ production. ET alleviated colitis when the model utilized CBir1 TCR T cells in mice colonized with CBir1 or A4Fla2 positive bacteria. Treated mice had improved colon length and histological inflammation and reduced colonic IFN-γ production.. Epicutaneous immunotherapy with MEP1 induced Tregs that migrate to intestines and suppress inflammation in mice with CBir1 or A4Fla2-positive bacterial colonization. This could be a potential strategy to treat CD and warrants further study.. Epicutaneous immunotherapy with a CBir1 multi-epitope peptide, the dominant flagellin for both murine and human, can induce Tregs that migrate to intestines and suppress inflammation in mice with CBir1 or A4Fla2-positive bacterial colonization.

Topics: Animals; CD4-Positive T-Lymphocytes; Colitis; Crohn Disease; Disease Models, Animal; Immunotherapy; Inflammation; Mice; Mice, Inbred C57BL; Ovalbumin; Receptors, Antigen, T-Cell; T-Lymphocytes, Regulatory

IL-10 producing regulatory type 1 (Tr1) cells represents a subpopulation of CD4(+) regulatory cells able to prevent in vitro bystander T-cell proliferation and to cure ongoing chronic colitis in mice. In order to assess the efficacy and tolerance of Tr1 cell therapy in a Phase I/IIa clinical trial in patients displaying severe Crohn's disease, we set up a reproducible manufacturing process for the GMP production of human ovalbumin specific Tr1 cells. Procedures used for Tr1-cell production include the use of Drosophila derived artificial Antigen Presenting Cells transfected with specific stimulatory molecules. Characterization of the human cell therapy product shows an in vitro suppressive activity on T-cell proliferation dependent on the production of both IL-10 and TGF-beta. Manufactured Tr1 cells display a regulatory phenotype including Foxp3, GITR and CTLA-4 surface expression. In vitro toxicity studies of human Tr1 cell product show a safety profile compatible with the use of these regulatory Tr1 lymphocytes for cell therapy.

Topics: Animals; Antigens, CD; Cell Proliferation; Clinical Protocols; Clone Cells; Crohn Disease; Cytokines; Drosophila; Humans; Immunophenotyping; Immunotherapy, Adoptive; Karyotyping; Mice; Ovalbumin; T-Cell Antigen Receptor Specificity; T-Lymphocytes, Regulatory

In normal conditions intestinal epithelial cells (IECs) constitutively stimulate regulatory CD4(+) T cells. However, in Crohn's disease (CD), this major histocompatibility complex (MHC) class II-restricted antigen presentation results in stimulation of proinflammatory CD4(+) T cells. We hypothesized that these alternative functions might be mediated by differential sorting and processing of antigens into distinct MHC II-enriched compartments (MIICs). Accordingly, we analysed the endocytic pathways of lumenally applied ovalbumin (OVA) in IECs of the jejunum and ileum of wild-type (WT) and TNFDeltaARE/WT mice that develop a CD-resembling ileitis. Using quantitative reverse transcription polymerase chain reaction, we found that messenger RNA levels of interferon-gamma, tumour necrosis factor-alpha, interleukin-17 and interleukin-10 were significantly up-regulated in the inflamed ileum of TNFDeltaARE/WT mice, confirming CD-like inflammation. Fluorescence and immunoelectron microscopy revealed the presence of MHC II and invariant chain throughout the late endocytic compartments, with most molecules concentrated in the multivesicular bodies (MVB). OVA was targeted into MVB and, in contrast to other MIICs, accumulated in these structures within 120 min of exposure. The IEC-specific A33 antigen localized to internal vesicles of MVB and A33/class II-bearing exosomes were identified in intercellular spaces. Remarkably, the expression pattern of MHC II/invariant chain molecules and the trafficking of OVA were independent of mucosal inflammation and the specific region in the small intestine. MVB seem to be principally responsible for class II-associated antigen processing in IECs and to constitute the origin of MHC II-loaded exosomes. The distinctive functions of IECs in antigen presentation to CD4(+) T cells might arise as a result of differential processing within the MVB identified here.

Topics: Animals; Antigen Presentation; Antigens, Differentiation, B-Lymphocyte; Biological Transport; Crohn Disease; Cytokines; Disease Models, Animal; Endosomes; Epithelial Cells; Exosomes; Histocompatibility Antigens Class II; Ileum; Intestinal Mucosa; Mice; Mice, Knockout; Microscopy, Immunoelectron; Ovalbumin; Reverse Transcriptase Polymerase Chain Reaction; Tumor Necrosis Factor-alpha

There is no clear evidence that dietary proteins aggravate Crohn's disease (CD). We aimed to clarify the antibody response to dietary proteins in CD.. Antibody to porcine pancreatic amylase (PPA) a protease-resistant dietary protein (anti-PPA), was examined in CD patients (N = 104), ulcerative colitis (UC) patients (N = 85), and healthy controls (N = 83), and its relationship with the clinical characteristics of CD was investigated. Antibodies to casein and ovalbumin, anti-Saccharomyces cerevisiae antibodies (ASCA), and antibodies to I2 from Pseudomonas fluorescens (anti-I2) were also examined.. Thirty-eight percent (39/104) of the CD patients expressed anti-PPA antibodies, and this percentage was significantly higher as compared with the control group (5%, 4/83) and the UC group (9%, 8/85) (P < 0.001). A significantly higher level of anti-PPA antibodies was detected in patients with "small bowel disease-dominant" CD than in those with "colitis-dominant" CD (P < 0.05). Antibodies to casein and ovalbumin were not specifically expressed in CD patients. As ASCA was detected in 33% and anti-I2 in 46% of the CD patients, 72% of the CD patients were found positive for at least one of the three antibodies including anti-PPA antibodies.. CD patients showed a specific antibody response to PPA, as compared with UC patients and controls. There was a significantly higher level of anti-PPA antibody in patients with "small bowel disease-dominant" CD, suggesting that dietary proteins could play a role in the inflammatory response in CD patients with small bowel disease. Anti-PPA antibodies combined with ASCA/anti-I2 may be useful for the diagnosis of CD.

Topics: Adolescent; Adult; Aged; Amylases; Animals; Antibodies; Autoantibodies; Autoantigens; Caseins; Colitis, Ulcerative; Crohn Disease; Dietary Proteins; Female; Humans; Male; Middle Aged; Ovalbumin; Pancreas; Phenotype; Pseudomonas fluorescens; Saccharomyces cerevisiae Proteins; Superantigens; Sus scrofa

In Crohn's disease (CD), colonic epithelial cells (CECs) are suggested to stimulate pro-inflammatory CD4+ T cells. However, the endocytic pathways of luminal antigens involved in underlying MHC class II presentation by CECs remain unknown. Our aim was to elucidate antigen trafficking and associated MHC class II expression in CECs of CD patients in vivo. In CD patients (Crohn's colitis and remission) and healthy controls undergoing colonoscopy, ovalbumin (OVA) was sprayed onto inflamed or healthy mucosa. The subcellular localization of OVA and MHC class II was visualized in biopsies taken from OVA-incubated mucosa using fluorescence and cryoelectron microscopy. Targeting of OVA into late endosomes of CECs was found in healthy (controls and CD in remission) and inflamed mucosa (Crohn's colitis). MHC class II expression in CECs was not detected in healthy mucosa but strongly up-regulated during CD inflammation. Induced MHC class II in CECs was predominantly seen at basolateral membranes and in late endosomes, which were efficiently accessed by internalized OVA. Our data provide in vivo evidence that the endocytic pathway of luminal antigens in CECs of Crohn's colitis patients intersects MHC class II-enriched late endosomes and support the postulated role of CECs in MHC class II-associated antigen presentation during CD.

Topics: Adult; Aged; Antigen Presentation; Antigens; Colon; Crohn Disease; Endosomes; Epithelial Cells; Female; Gene Expression Regulation; Histocompatibility Antigens Class II; Humans; Intestinal Mucosa; Male; Middle Aged; Ovalbumin; Protein Transport

Alimentary antigens may play a role in the perpetuation of inflammation in Crohn's disease (CD). Yeast antigens are widespread components of food. A proportion of CD patients develop antibodies against the yeast Saccharomyces cerevisiae (ASCA), but little is known about the cellular immune reactivity against food antigens in antibody-positive and -negative patients.. Lymphocytes from patients with CD, ulcerative colitis, and healthy controls were tested for their proliferative response after stimulation with the yeast antigen mannan and ovalbumin. The cellular phenotypes and activation markers were analyzed via FACS. Cytokine concentrations and antibody titers were determined by ELISA.. Only lymphocytes of ASCA-positive patients with CD proliferated after stimulation with mannan. These lymphocytes expressed increased activation markers (CD25, CD69). Activation of T cells was mediated by antigen-presenting cells and was associated with increased tumor necrosis factor-alpha (TNF-alpha) levels. The immune reactivity to ovalbumin was predominantly found in CD patients. It was weaker compared with mannan, independent of ASCA status, and also present in healthy controls.. A disturbed humoral and cellular response to the yeast antigen mannan is specifically seen in a subgroup of CD patients. This phenomenon may be due to a loss of tolerance toward yeast and is possibly genetically determined.

Topics: Antibodies, Fungal; Antigens, Fungal; Case-Control Studies; Cell Division; Cells, Cultured; Crohn Disease; Cytokines; Enzyme-Linked Immunosorbent Assay; Female; Humans; Immunization; Male; Mannans; Ovalbumin; Reference Values; Saccharomyces cerevisiae; Sensitivity and Specificity; T-Lymphocytes

T helper type 1 (Th1) cells secreting interferon-gamma (IFN-gamma) have been closely associated with Crohn's disease (CD). Monokine-induced by IFN-gamma (MIG), IFN-gamma-inducible T cell alpha chemoattractant (I-TAC), and IFN-gamma-inducible protein-10 (IP-10), are chemokines that bind CXCR3 and mediate the chemotaxis of leukocytes. IP-10, MIG, and CXCR3 have been shown to be expressed at sites of CD. The current study stems from our recent findings that IP-10, MIG, and I-TAC significantly contribute to the development of Th1-mediated inflammatory responses. To better understand the role of CXCR3 interactions during CD, we characterized the effects of IP-10, MIG, I-TAC, and CXCR3+ T cells on mucosal immune responses. IP-10, MIG, and I-TAC significantly enhanced antigen-specific serum and mucosal antibodies through Th1-mediated events and CD28 modulation. Additionally, the adoptive transfer of naive CXCR3+ T cells and CD4+CD45RB(HI) to T cell receptor beta (TCRbeta) x delta(-/-) mice resulted in the onset of murine colitis. Taken together, these studies suggest that IP-10, MIG, I-TAC, and CXCR3 interactions are involved in mucosal immune responses required for the induction of CD.

Topics: Adjuvants, Immunologic; Adoptive Transfer; Animals; Antibody Specificity; CD28 Antigens; CD4-Positive T-Lymphocytes; Cell Division; Chemokines; Colitis; Crohn Disease; Female; Gene Deletion; Immunity, Mucosal; Interferon-gamma; Ligands; Mice; Mice, Knockout; Ovalbumin; Receptors, Antigen, T-Cell; Receptors, Chemokine; Receptors, CXCR3

To investigate the humoral and cellular immune response against cow's milk proteins in Behçet's disease and to distinguish any behaviour during active or inactive disease.. Peripheral blood mononuclear cells from 16 patients and from eight normal controls were cultured in the presence of phytohaemagglutinin (PHA), beta-casein, beta-lactoglobulin, or chicken egg albumin. Interferon gamma (IFNgamma) and interleukin 4 (IL4) were measured in the culture supernatants by enzyme linked immunosorbent assay (ELISA). Serum samples from 46 patients with Behçet's disease and from 37 healthy subjects were also studied for antibody detection. Antibodies to beta-casein, beta-lactoglobulin, and chicken egg albumin were determined by ELISA.. High IFNgamma but not IL4 levels were found in the supernatants of lymphocytes from patients with active disease cultured in the presence of cow's milk proteins. Levels were comparable with those obtained in cultures stimulated with PHA. A significantly higher level of anti-beta-casein and anti-beta-lactoglobulin IgG and IgA antibodies was found in patients with active Behçet's disease. No relation was found between their occurrence and the age of the patients, the duration of disease, or the presence of gastrointestinal abnormalities. Antibodies to chicken albumin were detected at low levels and with a prevalence similar to that of healthy subjects.. The results indicate that an active immune response occurs in Behçet's disease. This response involves an increased frequency of antibodies to cow's milk protein and a strong Th1 polarisation after exposure to these antigens. The occurrence of these abnormalities supports a putative role for cow's milk proteins immune response in the pathogenesis of Behçet's disease.

Topics: Acute Disease; Adolescent; Adult; Aged; Animals; Antibodies; Behcet Syndrome; Case-Control Studies; Caseins; Cattle; Celiac Disease; Chickens; Crohn Disease; Female; Humans; Immunity, Cellular; Immunoglobulin A; Immunoglobulin G; Interferon-gamma; Interleukin-4; Lactoglobulins; Leukocytes, Mononuclear; Male; Middle Aged; Milk Proteins; Ovalbumin

Crohn's disease (CD) and ulcerative colitis (UC) are inflammatory bowel diseases (IBD) of unknown etiology. Oral absorption studies have shown an increased intestinal permeability for various sugar molecules in patients with IBD and their healthy relatives as a possible pathogenetic factor. However, the various transport pathways through the mucosal barrier have not yet been examined. This study therefore investigated whether antigens pass the epithelial barrier by a transcellular or a paracellular pathway. Mucosa of freshly resected specimens from CD (n = 10) or UC (n = 10) patients was investigated by immunoelectron microscopy and compared with healthy mucosa. Epithelial transport was studied with the antigens ovalbumin and horseradish peroxidase after defined incubation. Labeling density of subunit c of ATP synthetase was determined in mitochondria of enterocytes of all specimens. In all specimens epithelial transport of OVA and HRP was principally transcellular through enterocytes with normal ultrastructure, although some tight junctions in CD and UC were dilated. Antigens were transported within vesicles to the basolateral membrane 2.5 min after incubation. The level of enterocytes with electron-lucent cytoplasm containing a high amount of antigens was higher in CD and UC than in healthy mucosa, depending on the grade of inflammation. ATP synthetase was significantly decreased in electron-lucent cytoplasm of CD and UC to normal ultrastructure of healthy mucosa. Our study shows that ovalbumin and horseradish peroxidase taken up by the apical membrane reach the paracellular space by vesicular transport in healthy and IBD enterocytes within a few minutes. Transcellular pathway is affected in both CD and UC, which is indicated by a high level of antigens within the cytosol. We speculate that increased intestinal permeability in IBD results substantially from enhanced transcellular transport.

Topics: Adult; Aged; Antigens; Biological Transport; Colitis, Ulcerative; Crohn Disease; Female; Horseradish Peroxidase; Humans; Intestinal Mucosa; Male; Middle Aged; Ovalbumin; Permeability

Crohn's disease (CD) is associated with a disturbed intestinal barrier. Permeability studies have focused on inert molecules, but little is known about transepithelial transport of macromolecules with antigenic potential in humans. The aim of this study was to quantify permeation and to characterize passage routes for macromolecules in ileal mucosa in CD.. Noninflamed and inflamed ileal mucosa specimens from patients with CD (n = 12) and ileal specimens from patients with colon cancer (n = 7) were studied regarding transmucosal permeation of ovalbumin, dextran (mol wt, 40,000), and 51Cr-EDTA for 90 minutes in vitro in Ussing chambers. Transepithelial passage routes for fluorescent ovalbumin and dextran 40,000 were investigated by confocal microscopy.. Noninflamed ileum from CD patients showed increased permeation of ovalbumin compared with ileum from colon cancer patients (P < 0.05). Dextran permeation was equal in the three groups, whereas 51Cr-EDTA permeability was increased in inflamed ileum. Ovalbumin passed both transcellularly and paracellularly, but dextran followed a strictly paracellular route. Both markers were subsequently endocytosed by cells of the lamina propria.. Noninflamed ileal mucosa from patients with CD shows increased epithelial permeability to ovalbumin, probably by augmented transcytosis. This increase in antigen load to the lamina propria could be an initiating pathogenic event in CD.

Topics: Adult; Aged; Colonic Neoplasms; Crohn Disease; Dextrans; Edetic Acid; Electrophysiology; Female; Humans; Ileum; Intestinal Mucosa; Macromolecular Substances; Male; Microscopy, Confocal; Middle Aged; Ovalbumin; Permeability

Ovalbumin loading enzyme immunoassay was made in 44 patients with ulcerative colitis (UC) and 8 patients with Crohn's disease (CD). Enhanced intestinal permeability for macromolecules was found in 87.5 and 65.9% of patients with CD and UC, respectively. Blood serum of UC patients suffering from combination of food intolerance with dysbacteriosis contained ovalbumin in amounts exceeding those in patients without the above disorders 3.4 times (p < 0.05). No significant relationship existed between UC patients' high intestinal permeability and such indices as age, duration of the disease, intestinal lesion extension, administration of corticosteroids. It was found desirable to include ovalbumin intestinal permeability test in examination of UC and CD patients to differentiate treatment policy.

Topics: Adolescent; Adult; Aged; Colitis, Ulcerative; Crohn Disease; Female; Humans; Intestinal Absorption; Intestinal Mucosa; Male; Middle Aged; Ovalbumin; Permeability

Regulation of immune cell activation in lymphocyte-bearing human tissues is a pivotal host function, and metabolites of arachidonic acid (prostaglandin E2 in particular) have been reported to serve this function at non-mucosal sites. However, it is unknown whether prostaglandin E2 is immunoregulatory for the large lymphocyte population in the lamina propria of intestine; whether low (nM) concentrations of prostaglandin E2 modulate immune responses occurring there; and whether adjacent inflammation per se abrogates prostaglandin E2's regulatory effects. To address these issues, intestine-derived lymphocytes and T hybridoma cells were assessed, T cell activation was monitored by release of independently quantitated lymphokines, and dose-response studies were performed over an 8-log prostaglandin E2 concentration range. IL-3 release by normal intestinal lamina propria mononuclear cells was reduced (up to 78%) in a dose-dependent manner by prostaglandin E2, when present in as low a concentration as 10(-10) M. PGE2 also inhibited (by > or = 60%) mucosal T lymphocytes' ability to destabilize the barrier function of human epithelial monolayers. Further, with an intestine-derived T lymphocyte hybridoma cell line, a prostaglandin E2 dose-dependent reduction in IL-3 and IL-2 (90 and 95%, respectively) was found; this was true for both mitogen- and antigen-driven T cell lymphokine release. Concomitant [3H] thymidine uptake studies suggested this was not due to a prostaglandin E2-induced reduction in T cell proliferation or viability. In contrast, cells from chronically inflamed intestinal mucosa were substantially less sensitive to prostaglandin E2, e.g., high concentrations (10(-6) M) of prostaglandin E2 inhibited IL-3 release by only 41%. We conclude that prostaglandin E2 in nM concentrations is an important modulator of cytokine release from T lymphocytes derived from the gastrointestinal tract, and it may play a central role in regulation of lamina propria immunocyte populations residing there.

Topics: Animals; Cell Line; Colon; Crohn Disease; Dinoprostone; Epithelial Cells; Epithelium; Humans; Hybridomas; Interleukin-2; Interleukin-3; Intestinal Mucosa; Mice; Ovalbumin; T-Lymphocytes; Thymidine; Tritium