Loss and Gain of Tolerance to Pancreatic Glycoprotein 2 in Celiac Disease

Background

Autoantibodies against pancreatic secretory-granule membrane glycoprotein 2 (GP2) have been demonstrated in patients with Crohn’s disease but recently also with celiac disease (CD). Both entities are characterized by intestinal barrier impairment with increased gut permeability. Pathophysiological hallmark of CD is a permanent loss of tolerance to alimentary gliadin and a transient loss of tolerance to the autoantigen human tissue transglutaminase (tTG). Therefore, we explored the behavior of loss of tolerance to GP2 reported in CD.

Methods

We assessed prevalences and levels of autoantibodies against GP2, CD-specific antibodies to endomysial antigens and tTG as well as Crohn’s disease-specific anti-Saccharomyces cerevisiae antibodies in sera of 174 patients with active CD, 84 patients under gluten-free diet (GFD) and 129 controls. Furthermore, we looked for an association between anti-GP2 antibody positivity and degree of mucosal damage in CD.

Results

We found significantly elevated anti-GP2 IgA positivity in active CD patients (19.5%) compared to CD patients under GFD (0.0%) and controls (5.4%, p < 0.001, respectively). Anti-GP2 IgA levels correlated significantly with CD-specific antibodies (p < 0.001). Anti-GP2 autoantibody positivity disappeared under GFD similarly to CD-specific autoantibodies against tTG and endomysial antigens. For the first time, IgA antibody levels to GP2 are demonstrated to be associated with degree of villous atrophy according to Marsh classification.

Conclusions

Anti-GP2 IgA seems to be associated with disease activity in a distinct subgroup of patients with CD. The observed loss of tolerance to GP2 in a subset of patients with CD is transient and disappears under GFD.     

Serological Assessment for Celiac Disease in IgA Deficient Adults

Purpose

Selective immunoglobulin A deficiency is the most common primary immunodeficiency disorder that is strongly overrepresented among patients with celiac disease (CD). IgG antibodies against tissue transglutaminase (tTG) and deamidated gliadin peptides (DGP) serve as serological markers for CD in IgA deficient individuals, although the diagnostic value remains uncertain. The aim of this study was to investigate the prevalence of these markers in a large cohort of IgA deficient adults with confirmed or suspected CD and relate the findings to gluten free diet.

Methods

Sera from 488,156 individuals were screened for CD in seven Swedish clinical immunology laboratories between 1998 and 2012. In total, 356 out of 1,414 identified IgA deficient adults agreed to participate in this study and were resampled. Forty-seven IgA deficient blood donors served as controls. Analyses of IgG antibodies against tTG and DGP as well as HLA typing were performed and a questionnaire was used to investigate adherence to gluten free diet. Available biopsy results were collected.

Results

Out of the 356 IgA deficient resampled adults, 67 (18.8%) were positive for IgG anti-tTG and 79 (22.2%) for IgG anti-DGP, 54 had biopsy confirmed CD. Among the 47 IgA deficient blood donors, 4 (9%) were positive for IgG anti-tTG and 8 (17%) for anti-DGP. Four were diagnosed with biopsy verified CD, however, 2 of the patients were negative for all markers. Sixty-eight of 69 individuals with positive IgG anti-tTG were HLA-DQ2/DQ8 positive whereas 7 (18.9%) of the 37 individuals positive for IgG anti-DGP alone were not.

Conclusions

IgG anti-tTG seems to be a more reliable marker for CD in IgA deficient adults whereas the diagnostic specificity of anti-DGP appears to be lower. High levels of IgG antibodies against tTG and DGP were frequently found in IgA deficient adults despite adhering to gluten free diet.     

A combined biochemical, biophysical and immunological approach towards the identification of celiac disease-specific wheat antigens

Celiac disease (CD) is an inflammatory affliction of the small bowel caused by an immunological hypersensitivity to ingested wheat antigens affecting almost 1 % of the population. The gliadin fraction of wheat has been shown to contain the pathogenic antigens which react with antibodies and T cells. However, there is only limited knowledge regarding the precise nature of the wheat antigens recognized by IgA antibodies from CD patients and diagnostic tests based on the gliadin fraction have been demonstrated to give frequently false positive results. The aim of this study was the characterization of wheat antigens specifically recognized by IgA antibodies of CD patients. We developed a combined biochemical, biophysical, and immunological approach for the identification of celiac disease-specific wheat antigens. It is based on sub-fractionation of the wheat gliadin fraction using two ion exchange chromatography steps, the localization of CD-specific antigens by immunoblotting with IgA antibodies from CD patients, subsequent digestion followed by electro spray ionization–liquid chromatography/mass spectrometry (LC–ESI–MS/MS) and N-terminal sequencing by Edman degradation. Through the sub-fractionation procedure it was possible to separate CD-specific IgA-reactive wheat antigens from other wheat antigens which were also recognized by IgA antibodies of individuals without CD or by CD patients on gluten-free diet. Analysis by LC–ESI–MS/MS and N-terminal sequencing of the sub-fractions and the proteins specifically recognized by CD patients identified certain γ-gliadins with molecular mass of 37,000 and 45,000 as CD-specific wheat antigens. The CD-specific γ-gliadins with the molecular mass of 37,000 and 45,000 should be useful to study pathomechanisms of the disease and to improve the specificity of diagnostic tests for CD.     

Application of Deamidated Gliadin Antibodies in the Follow-Up of Treated Celiac Disease

Introduction

The role of serological tests such as IgA anti-transglutaminase autoantibodies has become increasingly important in celiac disease (CD) diagnosis. However, the efficiency of these tests for patient follow-up is controversial. We investigated the correlation of 12 different serological tests, including recent deamidated gliadin and actin IgA tests, with villous atrophy (VA) in a retrospective cohort of treated celiac patients.

Materials and Methods

Serum samples were collected from 100 treated CD patients who had intestinal biopsy in the course of their follow-up. Antibodies against transglutaminase, deamidated gliadin peptides, and native gliadin were measured, along with IgA anti-actin. The biopsy slides were all blind-reviewed and scored according to Marsh classification.

Results

For all deamidated gliadin and transglutaminase tests, we found that a positive result was significantly associated with persistence of intestinal VA, with a diagnostic efficacy up to 80%. Furthermore, antibodies titers directly correlated with the degree of VA, indicating a strong link between disease activity and presence of antibodies in the serum. Interestingly, the tests with the highest association with persistent VA were those for deamidated gliadin IgG. Using a test positivity pattern analysis, we were also able to identify several groups of patients with distinct antibody profiles that showed significant differences in intestinal damage and diet compliance.

Conclusions

Altogether, these results show that deamidated gliadin antibodies are strongly correlated with VA and should be considered valuable tools in CD follow-up and that multiplex serologic analysis for treated CD represents a promising tool for personalized patient management.     

Antibodies in the Diagnosis of Coeliac Disease: A Biopsy-Controlled,International, Multicentre Study of 376 Children with Coeliac Disease and 695 Controls

Diagnosis of coeliac disease (CD) relies on a combination of clinical, genetic, serological and duodenal morphological findings. The ESPGHAN suggested that biopsy may not be necessary in all cases. New guidelines include omission of biopsy if the concentration of CD-specific antibodies exceeds 10 times the upper limit of normal (10 ULN) and other criteria are met. We analysed the 10 ULN criterion and investigated multiple antibody-assays. Serum was collected from 1071 children with duodenal biopsy (376 CD patients, 695 disease-controls). IgA-antibodies to tissue transglutaminase (IgA-aTTG), IgG-antibodies to deamidated gliadin peptides (IgG-aDGL) and IgA-endomysium antibodies (IgA-EMA) were measured centrally. We considered 3 outcomes for antibody test procedures utilizing IgA-aTTG and/or IgG-aDGL: positive (≥10 ULN, recommend gluten-free diet), negative (<1 ULN, no gluten-free diet) or unclear (perform biopsy). Positive (PPV) and negative (NPV) predictive values were based on clear test results. We required that they and their lower confidence bounds (LCB) be simultaneously very high (LCB >90% and PPV/NPV >95%). These stringent conditions were met for appropriate antibody-procedures over a prevalence range of 9–57%. By combining IgG-aDGL with IgA-aTTG, one could do without assaying total IgA. The PPV of IgG-aDGL was estimated to be extremely high, although more studies are necessary to narrow down the LCB. The proportion of patients requiring a biopsy was <11%. The procedures were either equivalent or even better in children <2 years compared to older children. All 310 of the IgA-aTTG positive children were also IgA-EMA positive. Antibody-assays could render biopsies unnecessary in most children, if experienced paediatric gastroenterologists evaluate the case. This suggestion only applies to the kits used here and should be verified for other available assays. Confirming IgA-aTTG positivity (≥10 ULN) by EMA-testing is unnecessary if performed on the same blood sample. Prospective studies are needed.     

Determination of B-Cell Epitopes in Patients with Celiac Disease: Peptide Microarrays

Background

Most antibodies recognize conformational or discontinuous epitopes that have a specific 3-dimensional shape; however, determination of discontinuous B-cell epitopes is a major challenge in bioscience. Moreover, the current methods for identifying peptide epitopes often involve laborious, high-cost peptide screening programs. Here, we present a novel microarray method for identifying discontinuous B-cell epitopes in celiac disease (CD) by using a silicon-based peptide array and computational methods.

Methods

Using a novel silicon-based microarray platform with a multi-pillar chip, overlapping 12-mer peptide sequences of all native and deamidated gliadins, which are known to trigger CD, were synthesized in situ and used to identify peptide epitopes.

Results

Using a computational algorithm that considered disease specificity of peptide sequences, 2 distinct epitope sets were identified. Further, by combining the most discriminative 3-mer gliadin sequences with randomly interpolated3- or 6-mer peptide sequences, novel discontinuous epitopes were identified and further optimized to maximize disease discrimination. The final discontinuous epitope sets were tested in a confirmatory cohort of CD patients and controls, yielding 99% sensitivity and 100% specificity.

Conclusions

These novel sets of epitopes derived from gliadin have a high degree of accuracy in differentiating CD from controls, compared with standard serologic tests. The method of ultra-high-density peptide microarray described here would be broadly useful to develop high-fidelity diagnostic tests and explore pathogenesis.     

Lack of Serologic Evidence to Link IgA Nephropathy with Celiac Disease or Immune Reactivity to Gluten

IgA nephropathy is the most common form of primary glomerulonephritis worldwide. Mucosal infections and food antigens, including wheat gluten, have been proposed as potential contributing environmental factors. Increased immune reactivity to gluten and/or association with celiac disease, an autoimmune disorder triggered by ingestion of gluten, have been reported in IgA nephropathy. However, studies are inconsistent about this association. We aimed to evaluate the proposed link between IgA nephropathy and celiac disease or immune reactivity to gluten by conducting a comprehensive analysis of associated serologic markers in cohorts of well-characterized patients and controls. Study participants included patients with biopsy-proven IgA nephropathy (n = 99), unaffected controls of similar age, gender, and race (n = 96), and patients with biopsy-proven celiac disease (n = 30). All serum specimens were tested for IgG and IgA antibodies to native gliadin and deamidated gliadin, as well as IgA antibody to transglutaminase 2 (TG2). Anti-TG2 antibody-positive nephropathy patients and unaffected controls were subsequently tested for IgA anti-endomysial antibody and genotyped for celiac disease-associated HLA-DQ2 and -DQ8 alleles. In comparison to unaffected controls, there was not a statistically significant increase in IgA or IgG antibody reactivity to gliadin in individuals with IgA nephropathy. In addition, the levels of celiac disease-specific serologic markers, i.e., antibodies to deamidated gliadin and TG2, did not differ between IgA nephropathy patients and unaffected controls. Results of the additional anti-endomysial antibody testing and HLA genotyping were corroborative. The data from this case-control study do not reveal any evidence to suggest a significant role for celiac disease or immune reactivity to gluten in IgA nephropathy.     

Humoral Immunity Links Candida albicans Infection and Celiac Disease

Objective

The protein Hwp1, expressed on the pathogenic phase of Candida albicans, presents sequence analogy with the gluten protein gliadin and is also a substrate for transglutaminase. This had led to the suggestion that C. albicans infection (CI) may be a triggering factor for Celiac disease (CeD) onset. We investigated cross-immune reactivity between CeD and CI.

Methods

Serum IgG levels against recombinant Hwp1 and serological markers of CeD were measured in 87 CeD patients, 41 CI patients, and 98 healthy controls (HC). IgA and IgG were also measured in 20 individuals from each of these groups using microchips sensitized with 38 peptides designed from the N-terminal of Hwp1.

Results

CI and CeD patients had higher levels of anti-Hwp1 (p=0.0005 and p=0.004) and anti-gliadin (p=0.002 and p=0.0009) antibodies than HC but there was no significant difference between CeD and CI patients. CeD and CI patients had higher levels of anti-transglutaminase IgA than HC (p=0.0001 and p=0.0039). During CI, the increase in anti-Hwp1 paralleled the increase in anti-gliadin antibodies. Microchip analysis showed that CeD patients were more reactive against some Hwp1 peptides than CI patients, and that some deamidated peptides were more reactive than their native analogs. Binding of IgG from CeD patients to Hwp1 peptides was inhibited by γIII gliadin peptides.

Conclusions

Humoral cross-reactivity between Hwp1 and gliadin was observed during CeD and CI. Increased reactivity to Hwp1 deamidated peptide suggests that transglutaminase is involved in this interplay. These results support the hypothesis that CI may trigger CeD onset in genetically-susceptible individuals.     

Markers of Celiac Disease and Gluten Sensitivity in Children with Autism

Objective

Gastrointestinal symptoms are a common feature in children with autism, drawing attention to a potential association with celiac disease or gluten sensitivity. However, studies to date regarding the immune response to gluten in autism and its association with celiac disease have been inconsistent. The aim of this study was to assess immune reactivity to gluten in pediatric patients diagnosed with autism according to strict criteria and to evaluate the potential link between autism and celiac disease.

Methods

Study participants included children (with or without gastrointestinal symptoms) diagnosed with autism according to both the Autism Diagnostic Observation Schedule (ADOS) and the Autism Diagnostic Interview, Revised (ADI-R) (n = 37), their unaffected siblings (n = 27), and age-matched healthy controls (n = 76). Serum specimens were tested for antibodies to native gliadin, deamidated gliadin, and transglutaminase 2 (TG2). Affected children were genotyped for celiac disease associated HLA-DQ2 and -DQ8 alleles.

Results

Children with autism had significantly higher levels of IgG antibody to gliadin compared with unrelated healthy controls (p<0.01). The IgG levels were also higher compared to the unaffected siblings, but did not reach statistical significance. The IgG anti-gliadin antibody response was significantly greater in the autistic children with gastrointestinal symptoms in comparison to those without them (p<0.01). There was no difference in IgA response to gliadin across groups. The levels of celiac disease-specific serologic markers, i.e., antibodies to deamidated gliadin and TG2, did not differ between patients and controls. An association between increased anti-gliadin antibody and presence of HLA-DQ2 and/or -DQ8 was not observed.

Conclusions

A subset of children with autism displays increased immune reactivity to gluten, the mechanism of which appears to be distinct from that in celiac disease. The increased anti-gliadin antibody response and its association with GI symptoms points to a potential mechanism involving immunologic and/or intestinal permeability abnormalities in affected children.     

Design of a peptide for immunodetection of IgA antigliadin antibody for the purpose of screening of celiac disease

Celiac disease (CD) is gluten induced enteropathy which requires jejunal biopsy for diagnosis. To select the patients for endoscopoic procedure some serologic tests are popular in clinical practice for screening of CD. Although gliadin is one of the key immuno activator of the disease; serological screening by immuno-detection of gliadin is not recommended. In this context we have designed a peptide using tools of computational biology keeping molecular pathogenesis of the disease into consideration such that antigliadin antibody detection based sensitive and specific cost effective tool for screening of celiac disease can be developed. The designed peptide QPFPEP interacts in a stable manner with dimeric immunoglobin A1 molecule and its parent peptide QPFPQP are sequentially present in maximum number of gliadin epitopes. This hexapeptide is predicted to interact with dimeric IgA1, which increases in the biofluids of the CD patients. ABBREVIATIONS: CD - Celiac disease, TT - Tissue transglutamase, IgA - Immunoglobulin A, AGA - antigliadin antibody, Immunoglobulin G - IgG.     

Down-Regulation of T-Cell Proliferation in Response to Soluble Anti-CD3 Antibodies through Development of Redirected Cytolytic Activity Eliminating Costimulatory Cells

CD4⁺ T-depleted spleen cells (CD8⁺ T cells) activated by anti-CD3 antibodies (aCD3) suppressed proliferation of CD8⁺ T-depleted spleen cells (CD4⁺ T cells) and fresh normal T cells in response to aCD3. Antigen-nonspecific cytolytic activity was induced in splenic CD8⁺ T cells by stimulation with aCD3 and showed the peak level on day 3, whereas cytolytic activity induced in CD4⁺ T cells was weak. Intact Ig but not F(ab')₂ of aCD3 induced and mediated cytolytic activity. Correspondingly, the cytolytic activity induced by aCD3 was directed against target cells bearing Ig-binding Fc-receptor activity and cytolysis was inhibited by the addition of free Ig into the assay system. We showed that aCD3-activated T cells carried a high level of aCD3 on their surface at the time after the peak proliferation when they attained high cytolytic activity. This raised the possibility that the anti-CD3-induced aCD3-redirected cytolytic activity eliminated Fc-receptor-bearing costimulatory cells in the culture for down-regulation of the T-cell proliferation. This view was supported by partial restoration of anti-CD3-induced low responsiveness of CD8⁺ T cells by the addition of fresh costimulatory cells. These results suggested a new pathway of down-regulation of T-cell proliferation by aCD3-activated cytolytic CD8⁺ T cells.     

Equilibrium and kinetic analysis of the unusual binding behavior of a highly immunogenic gluten peptide to HLA-DQ2

HLA-DQ2 predisposes an individual to celiac sprue by presenting peptides from dietary gluten to intestinal CD4(+) T cells. A selectively deamidated multivalent peptide from gluten (LQLQPFPQPELPYPQPELPYPQPELPYPQPQPF; underlined residues correspond to posttranslational Q --> E alterations) is a potent trigger of DQ2 restricted T cell proliferation. Here we report equilibrium and kinetic measurements of interactions between DQ2 and (i) this highly immunogenic multivalent peptide, (ii) its individual constituent epitopes, (iii) its nondeamidated precursor, and (iv) a reference high-affinity ligand of HLA-DQ2 that is not recognized by gluten-responsive T cells from celiac sprue patients. The deamidated 33-mer peptide efficiently exchanges with a preloaded peptide in the DQ2 ligand-binding groove at pH 5.5 as well as pH 7.3, suggesting that the peptide can be presented to T cells comparably well through the endocytic pathway or via direct loading onto extracellular HLA-DQ2. In contrast, the monovalent peptides, and the nondeamidated precursor, as well as the tight-binding reference peptide show a much poorer ability to exchange with a preloaded peptide in the DQ2 binding pocket, especially at pH 7.3, suggesting that endocytosis of these peptides is a prerequisite for T cell presentation. At pH 5.5 and 7.3, dissociation of the deamidated 33-mer peptide from DQ2 is much slower than dissociation of its constituent monovalent epitopes or the nondeamidated precursor but faster than dissociation of the reference high-affinity peptide. Oligomeric states involving multiple copies of the DQ2 heterodimer bound to a single copy of the multivalent 33-mer peptide are not observed. Together, these results suggest that the remarkable antigenicity of the 33-mer gluten peptide is primarily due to its unusually efficient ability to displace existing ligands in the HLA-DQ2 binding pocket, rather than an extremely low rate of dissociation.     

In vivo antigen challenge in celiac disease identifies a single transglutaminase-modified peptide as the dominant A-gliadin T-cell epitope

Celiac disease (CD) is an increasingly diagnosed enteropathy (prevalence, 1:200-1:300) that is induced by dietary exposure to wheat gliadins (as well as related proteins in rye and barley) and is strongly associated with HLA-DQ2 (alpha1*0501, beta1*0201), which is present in over 90% of CD patients. Because a variety of gliadin peptides have been identified as epitopes for gliadin-specific T-cell clones and as bioactive sequences in feeding studies and in ex vivo CD intestinal biopsy challenge, it has been unclear whether a 'dominant' T-cell epitope is associated with CD. Here, we used fresh peripheral blood lymphocytes from individual subjects undergoing short-term antigen challenge and tissue transglutaminase-treated, overlapping synthetic peptides spanning A-gliadin to demonstrate a transient, disease-specific, DQ2-restricted, CD4 T-cell response to a single dominant epitope. Optimal gamma interferon release in an ELISPOT assay was elicited by a 17-amino-acid peptide corresponding to the partially deamidated peptide of A-gliadin amino acids 57-73 (Q65E). Consistent with earlier reports indicating that host tissue transglutaminase modification of gliadin enhances gliadin-specific CD T-cell responses, tissue transglutaminase specifically deamidated Q65 in the peptide of A-gliadin amino acids 56-75. Discovery of this dominant epitope may allow development of antigen-specific immunotherapy for CD.     

Identification of a peptide from alpha-gliadin resistant to digestive enzymes: implications for celiac disease

Current knowledge indicates that both innate and adaptive immune responses are involved in Celiac disease (CD) driven by different gliadin peptides. By studying a representative recombinant alpha-gliadin form, a further 25-mer peptide resistant to gastric, pancreatic, and human intestinal brush-border membrane enzymes was detected. This peptide latter encompasses the sequence 31-43 known to elicit the innate immune response in CD. The resistance of 25-mer, as well as that of the already described 33-mer related to the CD adaptive immune response, was confirmed on a standard flour wheat sample representative of the most widespread European varieties.     

Human genome search in celiac disease: mutated gliadin T-cell-like epitope in two human proteins promotes T-cell activation

Discovery of a number of novel and known human genes whose protein products bear striking similarity to two or more wheat gliadin domains raised the possibility that human intestinal non-HLA peptides homologous to celiac T-cell epitopes could play a role in non-HLA gene specification in celiac disease. Database searching of the entire human genome identified only 11 gut-expressed proteins with high T-cell epitope homology, particularly to the DQ2-gamma-I-gliadin epitope (i.e. TFIIA, FOXJ2 and IgD; mean BestFit quality score=40 versus random value of 24). Others were similar to DQ2-alpha-I-gliadin (i.e. PAX9; BestFit quality 46 versus 20 for random), or DQ2-alpha-II-gliadin (PHLDA1, known in mice as the T-cell death-associated gene; BestFit quality 43 versus 30 for random) epitopes. Among proteins previously screened for gliadin homology, noteworthy was achaete scute homologous protein (DQ2-alpha-I-gliadin; BestFit quality 41 versus 22 for random). With the exception of IgD, all are nuclear factors. Paying particular attention to the position of potential major histocompatibility complex (MHC) anchor residues, several were selected for testing in a DQ2-gamma-I-gliadin-restricted T-cell system. All native 10-mer peptides were inactive, even when deamidated, but V96F substitution of deamidated TFIIA amino acid residues 91-100 stimulated IL-2 release at levels exceeding the wheat gliadin positive control. Also active, but only slightly, was L1009F substitution of AIB3 amino acid residues 1004-1013. PlotSimilarity alignment of TFIIAs from eight species revealed subthreshold similarity score in the peptide region, in contrast to the highly conserved amino and carboxy termini. Molecular modeling of TFIIA[V96F] peptide points to an important juxtaposition of an upwardly projecting phenylalanine residue at peptide position 6 that likely contacts a receptor complementarity-determining region, and a downwardly projecting glutamic acid residue that fits into the shallow MHC P7 pocket. These observations tentatively point to a new multi-gene hypothesis for the initiation of celiac disease in which deamidated free human peptides with T-cell epitope homology (particularly those made more homologous by mutation) escape negative selection, as per deamidation of the HEL(48-62) peptide in the hen egg lysozyme model of autoimmunity. Deamidation following peptide release due to injury triggers inflammation, thereafter repeatedly provoked by dietary gliadin immunodominant peptides concentrated in the proximal small intestine.     

Modulatory Effect of Gliadin Peptide 10-mer on Epithelial Intestinal CACO-2 Cell Inflammatory Response

Celiac Disease (CD) is a chronic inflammatory enteropathy, triggered in genetically susceptible individuals by dietary gluten. Gluten is able to elicit proliferation of specific T cells and secretion of inflammatory cytokines in the small intestine. In this study we investigated the possibility that p10-mer, a decapeptide from durum wheat (QQPQDAVQPF), which was previously shown to prevent the activation of celiac peripheral lymphocytes, may exert an inhibitory effect on peptic-tryptic digested gliadin (PT-Gly)-stimulated intestinal carcinoma CACO-2 cells. In these cells, incubated with PT-Gly or p31-43 α-gliadin derived peptide in the presence or in the absence of p10-mer, IRAK1 activation and NF-kB, ERK1/2 and p38 MAPK phosphorylation were measured by immunoblotting, Cyclooxigenase 2 (COX-2) activity by PGE-2 release assay, and production of cytokines in the cell supernatants by ELISA. Our results showed that pre-treatment of CACO-2 cells with p10-mer significantly inhibited IRAK1 activation and NF-kB, ERK1/2 and p38 MAPK phosphorylation, as well as COX-2 activity (i.e. PGE-2 release) and production of the IL-6 and IL-8 pro-inflammatory cytokines, induced by gliadin peptides. These findings demonstrate the inhibitory effect of the p10-mer peptide on inflammatory response in CACO-2 cells. The results of the present study show that this p10-mer peptide can modulate "in vitro" the inflammatory response induced by gliadin peptides, allowing to move towards new therapeutic strategies. Turning off the inflammatory response, may in fact represent a key target in the immunotherapy of celiac disease.     

Transglutaminase 2 in celiac disease: Minireview article

Summary. Celiac disease (CD) is an autoimmune pathology of the small intestine triggered, in genetically predisposed patients, by the exposition to gliadin, a flour protein, thus evoking local immune reactions and mucosal atrophy. The discovery that type 2 transglutaminase (TG2) is the main, if not the sole, target of the endomysium CD-specific autoantibodies assigned to this enzyme a master regulator role of CD. Two separated events, both based on the finding that gliadin is able to act as a TG2 substrate, have been described to indicate that TG2 is involved in both the humoral and cellular immune responses. In this paper we review the novel insights on the localization and enzymatic activity of TG2 in the small intestinal mucosa. Moreover, we report on the capability of gliadin and its peptides to act as TG2 substrates.     

Gamma-gliadin specific celiac disease antibodies recognize p31-43 and p57-68 alpha gliadin peptides in deamidation related manner as a result of cross-reaction

Celiac disease (CeD) is a T-cell-dependent enteropathy with autoimmune features where tissue transglutaminase (TG2)-mediated posttranslational modification of gliadin peptides has a decisive role in the pathomechanism. The humoral immune response is reported to target mainly TG2-deamidated γ-gliadin peptides. However, α-gliadin peptides, like p57-68, playing a crucial role in the T-cell response, and p31-43, a major trigger of innate responses, also contain B-cell gliadin epitopes and γ-gliadin like motifs. We aimed to identify if there are anti-gliadin-specific antibodies in CeD patients targeting the p31-43 and p57-68 peptides and to examine whether deamidation of these peptides could increase their antigenicity. We explored TG2-mediated deamidation of the p31-43 and p57-68 peptides, and investigated serum antibody reactivity toward the native and deamidated α and γ-gliadin peptides in children with confirmed CeD and in prospectively followed infants at increased risk for developing CeD. We affinity-purified antibody populations utilizing different single peptide gliadin antigens and tested their binding preferences for cross-reactivity in real-time interaction assays based on bio-layer interferometry. Our results demonstrate that there is serum reactivity toward p31-43 and p57-68 peptides, which is due to cross-reactive γ-gliadin specific antibodies. These γ-gliadin specific antibodies represent the first appearing antibody population in infancy and they dominate the serum reactivity of CeD patients even later on and without preference for deamidation. However, for the homologous epitope sequences in α-gliadins shorter than the core QPEQPFP heptapeptide, deamidation facilitates antibody recognition. These findings reveal the presence of cross-reactive antibodies in CeD patients recognizing the disease-relevant α-gliadins.

     

Deamidation and cross-linking of gliadin peptides by transglutaminases and the relation to celiac disease

Activation of small intestinal gluten-reactive CD4+ T cells is a critical event in celiac disease. Such cells predominantly recognise gluten peptides in which specific glutamines are deamidated. Deamidation may be catalysed by intestinal tissue transglutaminase (TG2), a protein which is also the main autoantigen in celiac disease. Our aim was to study how the two main catalytic activities of transglutaminase--deamidation and transamidation (cross-linking) of an immunodominant gliadin epitope--are influenced by the presence of acceptor amines in the intestinal mucosa, and thereby contribute to further elucidation of the pathogenetic mechanisms in celiac disease. We prepared monoclonal antibodies, reacting specifically with the non-deamidated epitope QPFPQPQLPYPQPQ-amide and/or the deamidated epitope QPFPQPELPYPQPQ-amide. A solid phase immunoassay combined with gel filtration chromatography was used to analyse deamidation and cross-linking of these peptides to proteins. Our results show that QPFPQPQLPYPQPQ-amide was deamidated when incubated with purified TG2, with fresh mucosal sheets and with mucosal homogenates. Of other transglutaminases tested, only Streptoverticillium transglutaminase was able to generate the deamidated epitope. A fraction of the non-deamidated epitope was cross-linked to proteins, including TG2. The results suggest that intestinal TG2 is responsible for generation of the active deamidated epitope. As the epitope often occurs in a repeat structure, the result may be cross-linking of a deamidated, i.e., activated cell epitope. Alternatively, the deamidation may occur by reversal of the cross-linking reaction. The results provide a basis for the suggestion that binding of a peptide to a protein, in connection to its modification to a T cell epitope, might be a general explanation for the role of TG2 in celiac disease and a possible mechanism for the generation of autoantigens.     

Evaluation of IgG, IgM, and IgA antibodies to mycoplasma penetrans detected by ELISA and immunoblot in HIV-1-infected and STD patients, in São Paulo, Prazil

The aim of the present study was to determine the frequency of IgG, IgA, and IgM antibodies to Mycoplasma penetrans in HIV-1-infected patients and in patients with sexually transmitted diseases. We tested serum samples from 106 HIV-1-positive patients and 110 individuals with clinical symptoms of urethritis. ELISA and the immunoblot test were performed using M. penetrans lipid associated membrane proteins as antigen. By ELISA, we found a higher frequency (P < 0.05) of IgG against M. penetrans in HIV-1-infected and STD patients (25.5 and 17.3%) than in controls (1.2%), as well as a higher frequency of IgA (P < 0.05) (15.1 and 17.3% compared to 1.2%). For IgM, no differences were observed (P >/= 0.05) (3.8, 9.1, and 5. 8%, respectively). When the frequencies of IgG, IgM, and IgA antibodies of the HIV-1-infected patients were compared taking into account the CD4/CD8 cell ratios < 0.3 and >/= 0.3, no significant differences were observed between the two groups (13.3, 10, and 20%, compared to 20, 0, and 5%, respectively) (P > 0.05), possibly due to the low number of samples on which we could perform T-cell counts (53/106). The M. penetrans peptide of 38 kDa, considered immunodominant, was recognized in immunoblot by 51.8% of positive sera by ELISA for IgG, 50.0% for IgM, and 75% for IgA in the AIDS patients group, and by 47.4, 60.0, and 75.0%, respectively, in the sexually transmitted disease group. Cross-reactions in immunoblot for IgG were observed in sera from individuals infected with Mycoplasma pneumoniae and Mycoplasma hominis, and cross-reactions in immunoblot for IgA were observed in sera from individuals infected with M. hominis; all of them were ELISA negative to M. penetrans.