José Antonio Garrote

High Levels of Proinflammatory Cytokines, but Not Markers of Tissue Injury, in Unaffected Intestinal Areas from Patients with IBD

Intestinal alterations in IBD are triggered and maintained by an overexpression of proinflammatory cytokines. Additionally, increased immune activation has been found in the adjacent intestinal areas without displaying any apparent histological alterations, however, the regulatory environment is not well established. Biopsy specimens from patients with ulcerative colitis (UC) and Crohns disease (CD), from both affected and unaffected areas, and also from a group of colonic biopsies from healthy controls, were included in our study. Cytokines and markers of mucosal damage were analyzed by real-time PCR, and some of the results confirmed by western-blot and ELISA. Levels of IFNγ, TNFα, IL-6, IL-15, IL-18, and IL-23 were increased (above healthy controls) in both affected and unaffected areas from IBD. IL-1β, IL-6, IL-12, and IL-27 were higher in affected areas compared to unaffected ones in UC but not CD. In general, a correlation was observed between mRNA levels of these cytokines and both iNOS and Granzyme B. SOCS-2 and SOCS-3 were also increased in the affected areas. In conclusion, the unaffected areas from IBD show increased levels of a restricted set of cytokines that may exert immune activating roles in these areas without being able to trigger tissue damage.

Influence of Environmental and Genetic Factors Linked to Celiac Disease Risk on Infant Gut Colonization by Bacteroides Species

ABSTRACT Celiac disease (CD) is an immune-mediated enteropathy involving genetic and environmental factors whose interaction might influence disease risk. The aim of this study was to determine the effects of milk-feeding practices and the HLA-DQ genotype on intestinal colonization of Bacteroides species in infants at risk of CD development. This study included 75 full-term newborns with at least one first-degree relative suffering from CD. Infants were classified according to milk-feeding practice (breast-feeding or formula feeding) and HLA-DQ genotype (high or low genetic risk). Stools were analyzed at 7 days, 1 month, and 4 months by PCR and denaturing gradient gel electrophoresis (DGGE). The Bacteroides species diversity index was higher in formula-fed infants than in breast-fed infants. Breast-fed infants showed a higher prevalence of Bacteroides uniformis at 1 and 4 months of age, while formula-fed infants had a higher prevalence of B. intestinalis at all sampling times, of B. caccae at 7 days and 4 months, and of B. plebeius at 4 months. Infants with high genetic risk showed a higher prevalence of B. vulgatus, while those with low genetic risk showed a higher prevalence of B. ovatus, B. plebeius, and B. uniformis. Among breast-fed infants, the prevalence of B. uniformis was higher in those with low genetic risk than in those with high genetic risk. Among formula-fed infants, the prevalence of B. ovatus and B. plebeius was increased in those with low genetic risk, while the prevalence of B. vulgatus was higher in those with high genetic risk. The results indicate that both the type of milk feeding and the HLA-DQ genotype influence the colonization process of Bacteroides species, and possibly the disease risk.

Celiac disease pathogenesis: the proinflammatory cytokine network.

In susceptible individuals, the adaptive response, mediated by the activation of antigen-specific T lymphocytes, drives a proinflammatory response, which ends in an immune-mediated enteropathy characterized by villous atrophy, crypt hyperplasia, and recruitment of intraepithelial lymphocytes. In addition, some gluten peptides are able to induce an innate immune response in intestinal mucosa. The molecular mechanisms and the cells involved in the initial stages of the gluten–intestinal mucosa interaction are poorly understood to date. There is evidence of a direct toxic effect of gluten peptides in several biological models. However, the failure to control the inflammatory response may be one of the factors underlying gluten intolerance in these individuals. The cytokine network involved in celiac disease is characterized by abundant interferon-γ in the intestinal mucosa. In addition, the production of interleukin (IL)-15, IL-18, and IL-21 is linked to gluten intake, which can drive the inflammatory response probably sustained by IL-18, IL-21, and perhaps IL-27 through STAT1 and STAT5 pathways, whereas neither IL-12 nor IL-23 plays a significant role in pathogenic mechanisms. Herein we describe the involvement of these activation pathways in the context of the pathogenesis of celiac disease.

Higher constitutive IL15Rα expression and lower IL-15 response threshold in coeliac disease patients

The IL‐15 triggering effect of gliadin is not exclusive to coeliac disease (CD) patients, whereas the secondary response is CD specific. We have studied the expression of the IL‐15 receptor, and the IL‐15 response upon stimulation, in non‐CD and CD patients, and the possible existence of a lower immunological threshold in the latter. Forty‐two CD patients (20 on a gluten‐containing diet, GCD, and 22 on gluten‐free diet, GFD) and 24 non‐CD healthy individuals were studied. IL15Rα mRNA expression, and tissue characterization, were assayed in the duodenum. Biopsies from six CD patients on GFD and 10 non‐CD individuals were studied in vitro using organ culture in basal conditions, as well as after IL‐15 stimulation discarding basal IL‐15 production. Secretion of immune mediators was measured in the culture supernatants. IL15Rα mRNA expression was increased in CD patients, as compared with non‐CD controls (on GFD P = 0·0334, on GCD P = 0·0062, respectively), and confirmed also by immunofluorescence. No differences were found between CD patients on GFD and on GCD. After in vitro IL‐15 stimulation, IL15Rα expression was only triggered in non‐CD controls (P = 0·0313), though it remained increased in CD patients. Moreover, IL‐15 induced a more intense immunological response in CD patients after triggering the production of both nitrites and IFNγ (P = 0·0313, P = 0·0313, respectively). Gliadin‐induced IL15 has a lower response threshold in CD patients, leading to the production of other immune mediators and the development of the intestinal lesion, and thus magnifying its effects within the CD intestine.

One-step immunochromatographic assay for screening of coeliac disease

Tissue transglutaminase is the autoantigen that elicits endomysial antibodies, which are the serological hallmarks of coeliac disease. We describe a simple, rapid immunochromatographic assay for IgA and IgG antibodies to transglutaminase, which is highly accurate for diagnosis of this disease. Results were positive for all samples from 50 untreated coeliac patients, and negative for 40 non-coeliac patients with gastrointestinal disorders. The assay seems to be a useful alternative to biopsy for mass screening for coeliac disease.

Is gliadin really safe for non-coeliac individuals? Production of interleukin 15 in biopsy culture from non-coeliac individuals challenged with gliadin peptides

Nowadays it is assumed that an innate immunity to gluten plays a key role in the development of coeliac disease (CD).1 This innate response, mediated by interleukin (IL) 15 and elicited by “toxic peptides”, like the 19-mer, through a DQ2-independent mechanism, induces epithelial stress and reprogrammes intraepithelial lymphocytes into natural killer (NK)-like cells2 leading to enterocyte apoptosis and an increase in epithelium permeability. Thus, immunodominant peptides, like the 33-mer, can reach the lamina propria to trigger adaptive immunity. However, although an innate specific response in CD has been reported,3 no differential factors between patients with and without CD have been described controlling the innate immune response. Thus, since the toxic 19-mer elicits its harmful effect through a DQ2-independent mechanism, we hypothesise that the innate response is common in patients with and without CD, whereas the adaptive response is exclusive of susceptible patients with CD. To test the hypothesis, biopsy cultures were taken from at least three patients with CD who are on a gluten-free diet (GFD) and three patients without …

Interleukin 18 maintains a long-standing inflammation in coeliac disease patients

Dietary gluten induces an early response in the intestine of coeliac disease patients (CD), within a few hours, and this is driven by high levels of proinflammatory cytokines, including IFNγ and IL‐15, as has been thoroughly shown by gluten stimulation of biopsy explants. Our aim was to identify the immune mediators involved in the long‐standing inflammation in untreated CD patients at diagnosis. mRNA and protein levels of TNFα, IL‐12(p35), IL‐12(p40), IL‐15, IL‐18 and IL‐23(p19) were quantified in biopsies from active CD patients, CD patients on a gluten‐free diet (GFD), healthy controls, and patients with non‐CD inflammation and mild histological changes in the intestine. Biopsies from CD patients on a GFD were also stimulated in vitro with gliadin, and protein expression of IL‐15 and IL‐18 was analysed. Levels of IL‐12 and IL‐23 mRNA are nearly absent, and TNFα levels remain unchanged among different groups. Both the active and inactive forms of IL‐18 protein have been found in all samples from active CD, and protein expression was only localized within the crypts. Levels of IL‐15 mRNA remain unchanged, and protein expression, localized within the lamina propria, is found in a small number of samples. In vitro stimulation with gluten induces the expression of IL‐15 and IL‐18. In active CD, the early response following gluten intake characterized by high IFNγ levels is driven by IL‐18, and probably IL‐15, and this alternates with periods of long‐standing inflammation with moderate IFNγ levels, maintained by IL‐18 alone.

TNFα and LTα gene polymorphisms as additional markers of celiac disease susceptibility in a DQ2-positive population

Abstract. TNFα and TNFβ, or linfotoxin (LTα), are two molecules playing an important role in inflammation. Their genes map on Chromosome 6, between the HLA class II and class I loci. Polymorphisms in, or near, TNF genes have been associated with susceptibility to several autoimmune diseases. Studies of TNF genes in celiac disease (CD) have presented contradictory results. We have assessed the role of TNFα and linfotoxin α (TNFβ) in CD and their relative value as CD markers in addition to the presence of DQ2. The TNFA –308 polymorphism and the polymorphism at the first intron of the LTA gene were typed in CD patients and healthy controls and the results were correlated with the presence of DQ2. Significant differences were found in genotype and allele frequencies for the TNFA and LTA genes between CD patients and controls, with an increase in the presence of the TNFA*2 and LTA*1 alleles in CD patients. These differences increase when DQ2-positive CD patients and DQ2-positive controls are compared. In DQ2-positive individuals, allele 2 (A) in position –308 of the promoter of TNFA and allele 1 (G) of the NcoI RFLP in the first intron of LTA are additional risk markers for CD.

Altered human gut dendritic cell properties in ulcerative colitis are reversed by Lactobacillus plantarum extracellular encrypted peptide STp

SCOPE The human/microbiota cross-talk is partially mediated by bacteria-derived peptides like Serine-Threonine peptide (STp), which is resistant to gut proteolysis, is found in the human healthy colon and induces regulatory properties on gut dendritic cells (DCs); here we characterized human gut DC in ulcerative colitis (UC) patients and studied the effect of STp on their properties. METHODS AND RESULTS Human colonic DC from healthy controls and UC patients were isolated, conditioned for 24 h +/- STp and characterized by flow cytometry, immunohistochemistry, and electron microscopy. Expression of immature DC markers DC-SIGN and ILT3, and Toll-like receptors were increased on gut UC-DC. Langerin (involved in phagocytosis), lymph node homing marker CCR7, and activation markers CD40/CD80/CD86 were decreased in UC. Gut DC had restricted stimulatory capacity for T-cells in UC. Conditioning of DC with STp in vitro reduced Toll-like receptor expression, increased CD40 and CD80 expression, and restored their stimulatory capacity. CONCLUSION Colonic DCs display an abnormal immature phenotype in UC, which was partially restored following STp treatment. Bacteria-derived metabolites, like STp, seem to have a role in gut homeostasis that is missing in UC so they might lead a new era of probiotic products setting the basis for nondrug dietary therapy in inflammatory bowel disease.

Advances in the Immunogenetics of Coeliac Disease. Clues for Understanding the Pathogenesis and Disease Heterogeneity

Recent studies using the technique of the human genome screening in families with multiple siblings suffering from coeliac disease have suggested the presence of at least four different chromosomes in the predisposition to suffer from coeliac disease. Two loci in chromosome 6 appear to be important in disease susceptibility. Other studies based on cytokine gene polymorphisms have found a strong association with a particular haplotype in the TNF locus. This haplotype carries a gene for a high secretor phenotype of TNFalpha. The finding may be important in understanding the heterogeneity of inflammatory response. Evidence has been presented in favour of a predominantly Th1 pattern of cytokine production by the coeliac disease associated HLA-DQ restricted T cell clones. HLA-DQ2 and -DQ8 restricted gliadin-specific T cells have been shown to produce IFN-gamma, which appears to be an indispensable cytokine in the damage to enterocytes encountered in the small intestine, since the histological changes can be blocked by anti-IFN-gamma antibodies in vitro. TNF-alpha, also produced by several T cell clones, may in conjunction with IFN-gamma have a toxic effect or enhance the IFN-gamma-induced increase of HLA-class II expression on surface enterocytes. In the lamina propria this leads to an increased expression of adhesion molecules such as ICAM-1 on T lymphocytes and macrophages. Th1 cells also activate cytotoxic CD8+ T cells that migrate in the epithelial layer, and stimulate further LPL macrophages to produce IFN-gamma and TNF-alpha enhancing the inflammatory response. During this process autoreactive T cells proliferate, creating a situation which is very similar to the process that takes place in autoimmune diseases. Occasionally, this inflammatory destruction of the small intestinal integrity initiated by gluten peptides goes further and develops into a proper autoimmune disease which requires the use of immunosuppressive drugs in addition to a gluten-free diet.