Transglutaminase 2 expression is enhanced synergistically by interferon-γ and tumour necrosis factor-α in human small intestine.

Transglutaminase 2 (TG2) is expressed ubiquitously, has multiple physiological functions and has also been associated with inflammatory diseases, neurodegenerative disorders, autoimmunity and cancer. In particular, TG2 is expressed in small intestine mucosa where it is up-regulated in active coeliac disease (CD). The aim of this work was to investigate the induction of TG2 expression by proinflammatory cytokines [interleukin (IL)-1, IL-6, tumour necrosis factor (TNF)-α, interferon (IFN)-γ and IL-15] and the signalling pathways involved, in human epithelial and monocytic cells and in intestinal tissue from controls and untreated CD patients. Here we report that IFN-γ was the most potent inducer of TG2 expression in the small intestinal mucosa and in four [Caco-2, HT-29, Calu-6 and human acute monocytic leukaemia cell line (THP-1)] of five cell lines tested. The combination of TNF-α and IFN-γ produced a strong synergistic effect. The use of selective inhibitors of signalling pathways revealed that induction of TG2 by IFN-γ was mediated by phosphoinositide 3-kinase (PI3K), while c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK) were required for TNF-α activation. Quantitative polymerase chain reaction (PCR), flow cytometry and Western blot analysis showed that TG2 expression was blocked completely when stimulation by either TNF-α or IFN-γ was performed in the presence of nuclear factor (NF)-κB inhibitors (sulphasalazine and BAY-117082). TG2 was up-regulated substantially by TNF-α and IFN-γ in intestinal mucosa in untreated CD compared with controls. This study shows that IFN-γ, a dominant cytokine in intestinal mucosa in active CD, is the most potent inducer of TG2, and synergism with TNF-α may contribute to exacerbate the pathogenic mechanism of CD. Selective inhibition of signalling pathways may be of therapeutic benefit.

Analysis of immune cells draining from the abdominal cavity as a novel tool to study intestinal transplant immunobiology.

During intestinal transplant (ITx) operation, intestinal lymphatics are not reconstituted. Consequently, trafficking immune cells drain freely into the abdominal cavity. Our aim was to evaluate whether leucocytes migrating from a transplanted intestine could be recovered from the abdominal draining fluid collected by a peritoneal drainage system in the early post-ITx period, and to determine potential applications of the assessment of draining cellular populations. The cell composition of the abdominal draining fluid was analysed during the first 11 post-ITx days. Using flow cytometry, immune cells from blood and draining fluid samples obtained the same day showed an almost complete lymphopenia in peripheral blood, whereas CD3(+) CD4(+) CD8(-) , CD3(+) CD4(-) CD8(+) and human leucocyte antigen D-related (HLA-DR)(+) CD19(+) lymphocytes were the main populations in the draining fluid. Non-complicated recipients evolved from a mixed leucocyte pattern including granulocytes, monocytes and lymphocytes to an exclusively lymphocytic pattern along the first post-ITx week. At days 1-2 post-Itx, analysis by short tandem repeats fingerprinting of CD3(+) CD8(+) sorted T cells from draining fluid indicated that 50% of cells were from graft origin, whereas by day 11 post-ITx this proportion decreased to fewer than 1%. Our results show for the first time that the abdominal drainage fluid contains mainly immune cells trafficking from the implanted intestine, providing the opportunity to sample lymphocytes draining from the grafted organ along the post-ITx period. Therefore, this analysis may provide information useful for understanding ITx immunobiology and eventually could also be of interest for clinical management.

Higher constitutive IL15R alpha 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. IL15R alpha 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. IL15R alpha 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, IL15R alpha 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 IFN gamma (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.

An innovative sandwich ELISA system based on an antibody cocktail for gluten analysis.

A cocktail sandwich ELISA based on the employ of two monoclonal antibodies (MAbs) as coating antibodies and a third MAb conjugated to horseradish peroxidase has been developed for the analysis of gluten in foods. Given that each MAb displays a wide specificity spectrum for wheat, barley, rye and oats prolamins, their combination for ELISA ensures a high crossreactivity with most of the potentially toxic gliadin, hordein, secalin and avenin protein family. One of the unprecedented features of the cocktail sandwich ELISA is that it permits for the first time analysis of barley hordeins in foods, which is unattainable using conventional or commercial ELISA kits. Besides, gliadins, hordeins and secalins are recognised to the same extent. The system provides a high detection sensitivity for gliadins, hordeins, secalins and avenins (1.5, 0.05, 0.15 and 12 ng/ml, respectively). The working linear range comprises 3-100 ng/ml with a gliadin detection limit of 1.5 ppm. This limit of detection is even better than that demanded in the latest Codex recommendation, 10 ppm. Cocktail ELISA data were contrasted with those of commercial ELISA kits and confirmed by mass spectrometry, a non-immunological technique which provides evidence for the occurrence of false positive results with the commercial kits.

Evaluation of coeliac disease serological markers in Down syndrome patients.

BACKGROUND: The increased incidence of coeliac disease in patients with Down syndrome makes screening of coeliac disease in this population advisable. AIM: Evaluation of efficiency of different serological markers to detect coeliac disease in Down syndrome patients. PATIENTS: A total of 56 Down syndrome patients (aged: 1-17 years) were included in study. METHODS: Patients were evaluated for both IgG and IgA anti-transglutaminase antibodies and for anti-gliadin IgA and IgG antibodies using either purified omega-gliadin, wheat ethanol extract or commercial gliadin. Patients who had at least one positive result were evaluated for antiendomysium antibodies using either monkey oesophagus or human umbilical cord by indirect immunofluorescence. Coeliac disease was diagnosed by typical histological changes on duodenal mucosa. RESULTS: Increased levels of at least one anti-gliadin IgA and IgG antibody marker were found in 27 out of 56 cases (26 for IgG and 9 for IgA). 11/56 were positive for IgG anti-transglutaminase antibodies and two of them were also positive for IgA anti-transglutaminase antibodies and anti-endomysium antibodies. These two patients were finally diagnosed as coeliacs. Gliadin antigenic fractions employed produced differences in the performance of the anti-gliadin IgA and IgG antibody test. The use of commercial gliadin or wheat ethanol extract showed low sensitivity in IgA anti-gliadin IgA and IgG antibody determination, whereas good sensitivity and specificity were observed with omega-gliadins. IgG anti-transglutaminase antibodies showed a high proportion of false positive results (9 out of 56), whereas anti-endomysium antibodies and IgA anti-transglutaminase antibodies presented an excellent correlation with presence of active coeliac disease. CONCLUSIONS: Two out of 56 Down syndrome patients were diagnosed as coeliacs, corresponding to an incidence of 3.6%. The use of omega-gliadin presented the best efficiency in anti-gliadin IgA and IgG antibody determination whereas IgA anti-transglutaminase antibodies and anti-endomysium antibody determination showed an absolute correlation with presence of active coeliac disease.

Analysis of the effects of heat treatment on gliadin immunochemical quantification using a panel of anti-prolamin antibodies.

Antigen-labeled capture enzyme-linked immunosorbent assay with four different anti-gliadin monoclonal antibodies and an anti-gliadin serum and two different sample systems (purified gliadin fractions heat-treated in soluble phase and a model of dough simulating a baking process) were employed to study the effects of heat treatment on gliadin quantification. The analysis of purified gliadins showed that there is no particularly heat stable fraction. Remarkably, omega-gliadin did not present a differential heat stability. Reactivity varied depending on the time-temperature conditions of the treatment, the antibody employed, and the fraction analyzed. Heated dough samples showed an impairment of protein extraction depending on the intensity of the treatment. Capillary electrophoresis analysis of extracts showed that each gliadin group is affected to a different extent; omega-gliadin is less modified. Immunochemical analysis of the heat-treated samples using either of the five antibodies showed a decrease in the quantified gliadin, in concordance to the loss in the extracted proteins. Among the different sources of error in gliadin immunochemical quantification, the impairment in extraction efficiency in heat-treated samples appears as a major drawback to be overcome.

Preparative fractionation of gliadins by electrophoresis at pH 3.1 (A-PAGE).

Purification of gliadin subclasses has been difficult since they share many biochemical and physicochemical properties. In this report, the optimization of a preparative electrophoretic method to fractionate gliadins is described. Separation was performed in preparative 7% polyacrylamide gels at pH 3.1. The separation performance was tested using analytical electrophoresis at pH 3.1 and capillary electrophoresis. Preparative gels of different lengths were employed. Using 5-cm preparative gels, several fractions of alpha-, beta-, and gamma-gliadins and fast-mobility and slow-mobility omega-gliadins were collected in 40 h of separation. Resolution was maintained at a protein load of up to 30 mg in each run. The highest efficiency of recovery was achieved using aluminum lactate as the collecting buffer. Fractionation with 10 cm in length gels improved resolution but increased operation times. Gels of 2 cm in length did not separate alpha/beta- and gamma-gliadins efficiently but were useful to separate the two main fractions of omega-gliadins in shorter times. In conclusion, preparative electrophoresis at low pH allowed the separation of alpha-, beta-, gamma-, and omega-gliadin fractions from crude material under nondenaturing conditions.

Evaluation of calprotectin level in intestinal content as an early marker for graft rejection.

INTRODUCTION: The diagnosis of rejection after intestinal transplantation is still performed by endoscopic biopsy monitoring. Less invasive diagnostic procedures are desirable, although they are not available so far. Calprotectin, a stable cytosolic granulocyte protein, which previously was used as a marker of inflammatory processes, has been proposed to be a biochemical marker for rejection. The aim of the present work was to analyze the concordance between calprotectin levels in intestinal content and the presence of graft rejection after small bowel transplantation. METHODS: Calprotectin level was measured using a commercial ELISA kit on 137 samples of intestinal content randomly collected during endoscopies performed on 11 intestinal transplantation patients during 2 years' follow-up. Calprotectin determinations were correlated with histological and clinical findings. The cut-off level was determined retrospectively by receiver-operator curve analysis. RESULTS: Based on histological findings and clinical records, samples were discerned as rejection positive (37 of 137), versus negative (35 of 137) samples or those with no clinical, endoscopic, or histological findings (65 of 137 samples). A cut-off value of 65 microg of calprotectin/g of intestinal content provided the best assay parameter according to the clinical findings: a 76% sensitivity and a 47% specificity. False positive results corresponded to patients with gastrointestinal infections (13%), systemic infections (13%), ulcers (10%), or nonspecific histological alterations of the mucosa (15%). The other false positive cases corresponded to postsurgical samples (4%), or patients with concomitant gastrointestinal symptoms (2%). Most false negative results (78%) were observed during recovery from severe acute rejection episodes, among successfully treated patients. In these cases, epithelial reconstitution and no mucosal infiltration was observed. If the latter group were discarded, sensitivity rose to 93%, and specificity, to 50% with a 96% negative predictive value. Furthermore, a weak correlation was observed between calprotectin levels and the severity of rejection. CONCLUSIONS: This study confirmed the results obtained by other groups: fecal calprotectin dosage showed a good sensitivity but low specificity for the diagnosis of intestinal rejection because high calprotectin levels can also be observed in other clinical conditions. Nevertheless, it might be used as a first line for continuous evaluation of intestinal transplantation status, like other biochemical parameters that are used in kidney or liver transplantation, before considering the need for a biopsy.

Quantitation of adenylate cyclase of Bordetella pertussis by enzyme linked immunosorbent assay.

Bordetella pertussis produces extracytoplasmic adenylate cyclase toxin (AC) which has received considerable attention as a potential vaccine candidate. Great interest from laboratories involved in production, purification and quality control of acellular pertussis vaccine is focused on finding an appropriate technique for rapid and accurate quantitation of AC antigen. In this paper a competitive ELISA is proposed. A polystyrene microplate coated with purified AC was incubated with the sample to be tested plus anti-AC serum. The bound anti-AC antibodies were measured by sequential reaction with alkaline phosphatase-labelled anti-mouse IgG and p-nitrophenylphosphate. This method showed high specificity, with the 50% inhibition corresponding to 4 micrograms/ml of AC. It also proved to be useful to assess the presence of AC in culture supernatants, with high reproducibility.

Identification of casein as the major allergenic and antigenic protein of cow's milk.

The objective of this study was to analyze both the allergenicity and immunogenicity of cow's milk proteins. To this end, 80 milk-atopic patients were selected on the basis of the presence of cow's milk-specific IgE antibodies in serum and compatible clinical history. Fifteen patients allergic to other allergens and 10 nonatopic subjects were studied as controls. The specificity of serum IgG and IgE antibodies was determined by immunoblotting, employing both cow's milk and milk components, i.e., alpha- and beta-casein, beta-lactoglobulin, and alpha-lactalbumin separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The experiments showed that casein-specific IgE antibodies were present in all (80/80) sera examined; 10/80 showed reactivity to beta-lactoglobulin, and 5/80 showed reactivity to alpha-lactalbumin. None of the 25 negative control sera analyzed showed the presence of specific IgE antibodies against milk proteins. These results were similar to those corresponding to the detection, by the radioallergosorbent test, of IgE antibodies against the milk components coupled to paper disks. All sera from milk-atopic patients also showed IgE reactivity against a high-molecular-mass fraction that hardly enters the gel. This fraction, after separation by gel filtration and treatment with beta-mercaptoethanol and urea, was shown by SDS-PAGE analysis to be formed by casein monomers. All sera analyzed by immunoblotting reacted against the components corresponding to casein monomers. Inhibition of immunoblotting by adsorption with different milk components confirmed that those high-molecular-mass aggregates are formed by casein components. The results presented here strongly suggest that casein is the major allergenic component of cow's milk.

Detection and characterization of antibodies specific to food antigens (gliadin, ovalbumin and beta-lactoglobulin) in human serum, saliva, colostrum and milk.

Antibodies against food antigens are usually produced in healthy people. This humoral response can be detected both in serum and secretions. The characterization of this response can be useful for a better understanding of food-related immunological alterations. In this study, IgA and IgG antibodies specific to ovalbumin, beta-lactoglobulin or gliadin were measured in serum, saliva, colostrum and milk from 40 healthy breast-feeding women. Specific IgA and IgG to the three antigens were measured by indirect ELISA. Specific IgG levels were highest in serum and very low in the other biological fluids. No correlation between the IgG specific to the different antigens was found. Specific IgA reactivity was found in all the samples analysed. Levels observed were higher in colostrum and milk than in serum and saliva. In spite of being three different unrelated food antigens, a correlation between the levels of specific IgA was found in saliva, colostrum and milk samples of all subjects studied. The specificity of IgA anti-gliadin antibodies from serum, saliva and colostrum was analysed by immunoblotting of SDS-PAGE-separated wheat proteins. Each sample presented a unique pattern of recognition. No common pattern of recognition was found either among the same biological fluids of the different subjects tested, or among the different samples--either serum, colostrum or saliva--of the same individual. Different degrees of specificity to wheat proteins among IgA from colostrum, saliva or serum were observed, suggesting that the local IgA-producing populations are functionally different in the different tissues of the organism.

Presence of high levels of non-degraded gliadin in breast milk from healthy mothers.

BACKGROUND: Secretion of dietary antigens into breast milk has been extensively documented. The presence of these antigens is of relevance because they could be involved in the modulation of the immune response in neonates. The objective of this study is to determine the gliadin concentration in milk, colostrum, and serum samples from healthy lactating mothers on a normal diet. Gliadin levels in milk samples from a group of six mothers after a brief period of gluten restriction were also determined. The molecular weight of secreted gliadins was also analysed. METHODS: Gliadin concentration was determined with a highly sensitive competitive enzyme-linked immunosorbent assay, modified so as to eliminate anti-gliadin antibody interference. The level of gliadin/IgA anti-gliadin immune complexes in milk, colostrum, and serum samples was determined. RESULTS: Gliadin was detected in all 49 milk samples. Its concentration varied between 5 and 1200 ng/ml (mean, 178 ng/ml). In colostrum (n = 14) gliadin levels were higher (range, 28-9000 ng/ml; mean, 883 ng/ml), not being detectable in one case. Gliadin was detectable in 14 of 31 serum samples, in which levels were lower than in milk and colostrum samples (mean, 41 ng/ml). Neither a correlation between gliadin levels in milk, colostrum, and serum samples from the same subject nor a relation between gluten intake and gliadin concentration in milk samples from six subjects under a 3-day gluten-free diet could be found. Higher levels of immune complexes were observed in colostrum samples than in milk and serum samples. No correlation was detected between gliadin concentration and the level of immune complexes. The analysis of milk and colostrum samples by immunoblotting showed bands of immunoreactive gliadin presenting Mr similar to those of native proteins from wheat extracts. CONCLUSIONS: Very high levels of gliadin were detected in milk samples from healthy mothers on an unrestricted diet. Gliadin levels were higher than those reported for dietary antigens in other studies. Breast milk contained non-degraded gliadins and gliadin/anti-gliadin IgA immune complexes.

Determination of anti-omega-gliadin antibodies in serologic tests for coeliac disease.

BACKGROUND: Serologic detection of coeliac disease in the general population or in subjects belonging to risk groups implies the use of a test with high efficiency, large-scale use, and low cost. The enzyme-linked immunosorbent assay (ELISA) technique is the most appropriate assay for performing this kind of studies. Even though anti-gliadin determination has been the test most frequently used as the first step in screening procedures, many false-positive results produced a low-specificity test. In a previous work a selective recognition of omega-gliadins, mainly by IgA antibodies, was observed. Results also indicated that omega-gliadins can be useful as antigens in serologic detection of coeliac disease. We therefore wanted to analyse the anti-gliadin antibody reactivity by using purified gliadins and to evaluate the actual performance of the anti-omega-gliadin antibody test. METHODS: A population consisting of 105 coeliac patients, 81 healthy controls, and 73 subjects in a disease control group was analysed. Anti-endomysium (EMA), both IgG and IgA anti-omega-gliadins, and anti-tissue transglutaminase (tTG) antibodies were determined. RESULTS: Concordant results, positive and negative, in the EMA and IgG and IgA anti-gliadin determinations were observed in 220 of 259 samples from the total population analysed. The three assays showed high efficiency, being 96.9%, 90.7%, and 91.1% for EMA and anti-omega-gliadins IgG and IgA, respectively. Anti-tTG determination was performed on 103 samples (69 controls and 34 coeliac patients), finding 4 false results (2 false positive and 2 false negative), whereas anti-omega-gliadins showed 10 false results (5 false negative and 5 false positive), 3 of which were coincident with anti-tTG determination. To compare the reactivity of anti-gliadin antibodies, alpha-, beta-, gamma- and omega-gliadins were isolated under non-denaturing conditions by acid preparative electrophoresis and cation-exchange fast protein liquid chromatography (FPLC) and used in an indirect ELISA test. The composition of these fractions was analysed by means of capillary electrophoresis, showing no cross-contamination among them. CONCLUSIONS: The comparison of results using purified gliadins shows that omega-gliadins present a differential reactivity that has not previously been documented. Results using omega-gliadins isolated by either preparative electrophoresis or FPLC were similar. Tests using the purified omega-gliadin fraction present the best performance when anti-gliadin antibodies are evaluated.

Analysis of anti-gliadin antibodies by immunoblot analysis and enzyme-linked immunosorbent assay using gliadin fractions as antigens.

BACKGROUND: Anti-gliadin antibody (AGA) determination has been widely used in the screening test to detect celiac patients in the general population and in risk groups. Serological assays present variable efficiency, probably caused by differences in the antigenic mixtures employed as antigen. The objective of this work is to evaluate the use of purified gliadin fractions in an enzyme-linked immunosorbent assay (ELISA) test. METHODS: Anti-gliadin antibody reactivity was characterized in the sera of patients with celiac disease, and AGA levels were determined by immunoblot analysis using purified gliadin fractions after separation of wheat proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and acid-PAGE and after indirect ELISA. Seven antigenic mixtures were tested: commercial gliadin, ethanolic wheat extract, and five fast protein liquid chromatography-purified fractions (omega-gliadins, two mixtures of alpha-/beta- and beta-/gamma-gliadins). Immunoblot analysis after A-PAGE separation showed that immunoglobulin (Ig)A reactivity was frequently more restricted than that of IgG. Serum IgA in 15 of 23 patients showed intense reactivity against omega-gliadins. RESULTS: In seven cases, only omega-gliadins were detected. To compare the efficiency of ELISA tests, serum samples of 28 patients with celiac disease and 31 control subjects were tested against the seven gliadin fractions. Immunoglobulin G AGAs demonstrated similar levels against the different gliadin fractions, whereas IgA AGAs showed a heterogeneous reactivity that depended on the fraction tested. The lowest number of false-positive and false-negative results was obtained when the omega-gliadin fraction was used. Parameters for ELISA showed that the omega-gliadin fraction elicited the highest assay efficiency for determinations of both IgA and IgG AGAs. A good correlation was found between IgG and IgA anti-omega-gliadin and antiendomysial antibody determinations. Of the 28 biopsy-confirmed patients with celiac disease, 26 samples (23 positive and 3 negative) were found to have concordant results among the three determinations. CONCLUSIONS: In this study, an intense and, in many cases, selective recognition of omega-gliadins was observed. Results suggest that a higher performance in AGA determination could be achieved using omega-gliadin as an antigen in indirect ELISA.

Enfermedad celiaca. Nuevas perspectivas terapeuticas basadas en un mejor conocimiento de su patogenia molecular. / Celiac disease. New therapeutic alternatives based on a better knowledge of molecular pathogenesis

La enfermedad celiaca (EC) es una patologia gastrointestinalcronica de tipo autoinmune desencadenadapor un antigeno exogeno conocido (el gluten). Estafuertemente asociada al sistema HLA, aunque otrosfactores geneticos, ambientales e inmunologicos, aun nocompletamente identificados, determinarian el momentoy forma de presentacion. La respuesta inmuneen la mucosa intestinal frente a ciertos peptidos derivados de gliadinas es caracterizada por una fuerte respuestade tipo TH1 (con predominio de IFN secretadopor linfocitos T específicos), la que es probablementeprecedida por una respuesta inmune innata, mediadafundamentalmente por IL-15. La dieta estricta librede gluten es la forma mas eficaz de revertir las alteracionesde la mucosa intestinal y la sintomatologia. Sin embargo, las transgresiones y el bajo cumplimientode la dieta han conducido a formular nuevas estrategias terapeuticas que se discuten en esta revision.

Coeliac disease is a chronic autoimmune-like gastrointestinal disorder triggered by a known exogenous antigen (gluten). The disease is strongly linked to the HLA system, though other genetic, environmental and immunologic factors, may determine the type and timing of presentation. The immune response within the intestinal mucosa is characterized by a well defined TH1 response, where IFNgamma secreted by specific T cells is the predominant cytokine, as well as an innate immune response to certain gluten-derived peptides, mediated by IL-15. The strict gluten-exclusion diet is the best way of reversing both the symptoms and the histological changes in the intestinal mucosa. However, the frequency of transgressions and a low dietary compliance had led to the description of new therapeutic alternatives discussed in this review.