Peptic-tryptic Digest Of Gliadin Research Articles

Abstract 154: Endothelial Dysfunction In Experimental Celiac Disease Is Mediated By Gut-Derived Vascular Inflammation And Oxidative Stress

Objective: Celiac disease (CeD) is an autoimmune enteropathy triggered by dietary gluten, with a prevalence around 1%. Since many patients show heterogeneous symptoms, it is vastly underdiagnosed and yet without any treatment option besides lifelong gluten restriction. Vascular function in CeD patients compared to healthy controls has been assessed and hypothesized CeD as a cardiovascular risk factor. Herewith we aimed to explore the cardiovascular effects of CeD in a murine model, with special emphasis on endothelial dysfunction, vascular inflammation, and oxidative stress, known triggers for cardiovascular disease. Methods: NOD.DQ8 mice were raised on a zein-based, gluten-free diet. During the experiment, mice received gavage with peptic-tryptic digest of gliadin and were switched to a gluten-containing diet (Gliadin group). A control group (Zein group) was continued on gluten-free diet and received accordingly digested zein. Results: Blood pressure measurements revealed arterial hypertension in the Gliadin group. Endothelium-dependent relaxation was impaired in the Gliadin group compared to Zein, whereas no change in endothelium-independent was observed, tested by isometric tension recordings. Pro-inflammatory genes, like Tnfa and Nox2 were upregulated in the Gliadin group measured by quantitative rtPCR in aortic and intestinal tissue. Infiltration by intraepithelial T-lymphocytes into the epithelium of the small intestine was shown by IHC. In cardiac tissue, gliadin-treatment increased the oxidative stress parameters 3-nitrotyrosine and 4-hydroxy-2-nonenal shown in dot blots. To investigate the inflammatory link between gut and the cardiovascular system plasma proteomics were performed and revealed elevated levels of IL-17A. Conclusion: CeD is a cardiovascular risk factor. However, the mechanism remains unresolved. In a mouse model, we demonstrate that gluten treatment elevates blood pressure and impairs vascular function. Intestinal inflammation conveys to the cardiovascular system, leads to oxidative stress, and thereby impairs endothelial function. A potential mediator might be IL-17A, whose role in other autoimmunity-based cardiovascular risk factors (e.g., psoriasis) has already been demonstrated.

Gliadin Peptide P31-43 Induces mTOR/NFkβ Activation and Reduces Autophagy: The Role of Lactobacillus paracasei CBA L74 Postbiotc.

Celiac disease (CD) is an autoimmune disease characterized by an altered immune response stimulated by gliadin peptides that are not digested and cause damage to the intestinal mucosa. The aim of this study was to investigate whether the postbiotic Lactobacillus paracasei (LP) could prevent the action of gliadin peptides on mTOR, autophagy, and the inflammatory response. Most of the experiments performed were conducted on intestinal epithelial cells Caco-2 treated with a peptic-tryptic digest of gliadin (PTG) and P31-43. Furthermore, we pretreated the Caco-2 with the postbiotic LP before treatment with the previously described stimuli. In both cases, we evaluated the levels of pmTOR, p70S6k, and p4EBP-1 for the mTOR pathway, pNFkβ, and pERK for inflammation and LC 3 and p62 for autophagy. For autophagy, we also used immunofluorescence analysis. Using intestinal organoids derivate from celiac (CD) patients, we analyzed the effect of gliadin after postbiotic pretreatment with LP on inflammation marker NFkβ. Through these experiments, we showed that gliadin peptides are able to induce the increase of the inflammatory response in a more complex model of intestinal epithelial cells. LP postbiotic was able to induce autophagy in Caco-2 cells and prevent gliadin effects. In conclusion, postbiotic pretreatment with LP could be considered for in vivo clinical trials.

Similar Responses of Intestinal T Cells From Untreated Children and Adults With Celiac Disease to Deamidated Gluten Epitopes

Celiac disease is a chronic small intestinal inflammatory disorder mediated by an immune response to gluten peptides in genetically susceptible individuals. Celiac disease is often diagnosed in early childhood, but some patients receive a diagnosis late in life. It is uncertain whether pediatric celiac disease is distinct from adult celiac disease. It has been proposed that gluten-reactive T cells in children recognize deamidated and native gluten epitopes, whereas T cells from adults only recognize deamidated gluten peptides. We studied the repertoire of gluten epitopes recognized by T cells from children and adults. We examined T-cell responses against gluten by generating T-cell lines and T-cell clones from intestinal biopsies of adults and children and tested proliferative response to various gluten peptides. We analyzed T cells from 14 children (2-5 years old) at high risk for celiac disease who were followed for celiac disease development. We also analyzed T cells from 6 adults (26-55 years old) with untreated celiac disease. All children and adults were positive for HLA-DQ2.5. Biopsies were incubated with gluten digested with chymotrypsin (modified or unmodified by the enzyme transglutaminase 2) or the peptic-tryptic digest of gliadin (in native and deamidated forms) before T-cell collection. Levels of T-cell responses were higher to deamidated gluten than to native gluten in children and adults. T cells from children and adults each reacted to multiple gluten epitopes. Several T-cell clones were cross-reactive, especially clones that recognized epitopes from γ-and ω-gliadin. About half of the generated T-cell clones from children and adults reacted to unknown epitopes. T-cell responses to different gluten peptides appear to be similar between adults and children at the time of diagnosis of celiac disease.

Increase in Neuroendocrine Cells in the Duodenal Mucosa of Patients with Refractory Celiac Disease

Several immune-mediated gastrointestinal disorders, including celiac disease (CD), are associated with neuroendocrine cell hyperplasia. However, neuroendocrine cells have never been explored in refractory CD (RCD). Serial duodenal sections from 17 patients with RCD (6 type 1 and 11 type 2), 16 uncomplicated CD patients before and after gluten-free diet, 14 patients with potential CD, 27 patients with non-CD villous atrophy, i.e., common variable immunodeficiency (n=12), Whipple's disease (n=10) and giardiasis (n=5), and 16 healthy subjects were processed for the immunohistochemical detection of chromogranin A (CgA), serotonin, and somatostatin. Mucosal tryptophan hydroxylase (TpH)-1 and serotonin-selective reuptake transporter (SERT) transcripts were measured by quantitative reverse transcription-PCR. Serum CgA and 24-h urine 5-hydroxyindoleacetic acid (5-HIAA) were assessed. Biopsies from treated CD patients were cultured with serotonin or peptic tryptic digest of gliadin (PT-gliadin), and interferon (IFN)-γ was detected by ELISA in culture supernatants. Epithelial cells positive for CgA and serotonin, but not somatostatin, were significantly increased in RCD. Raised mucosal transcripts of TpH-1, but not SERT, were found in RCD. On biopsies from treated CD patients, serotonin upregulated IFN-γ production at levels comparable to those induced by PT-gliadin. Serum CgA, but not urine 5-HIAA, was increased in RCD. No significant difference was found between RCD type 1 and type 2 in terms of neuroendocrine cells, mucosal TpH-1 transcripts, and serum CgA. Serotonin-producing neuroendocrine cells are increased in RCD mucosa. IFN-γ upregulation induced by serotonin suggests that this monoamine may have a role in sustaining the local inflammatory response in CD.

Altered Expression of Type-1 and Type-2 Cannabinoid Receptors in Celiac Disease

Anandamide (AEA) is the prominent member of the endocannabinoid family and its biological action is mediated through the binding to both type-1 (CB1) and type-2 (CB2) cannabinoid receptors (CBR). The presence of AEA and CBR in the gastrointestinal tract highlighted their pathophysiological role in several gut diseases, including celiac disease. Here, we aimed to investigate the expression of CBR at transcriptional and translational levels in the duodenal mucosa of untreated celiac patients, celiac patients on a gluten-free diet for at least 12 months and control subjects. Also biopsies from treated celiac patients cultured ex vivo with peptic-tryptic digest of gliadin were investigated. Our data show higher levels of both CB1 and CB2 receptors during active disease and normal CBR levels in treated celiac patients. In conclusion, we demonstrate an up-regulation of CB1 and CB2 mRNA and protein expression, that points to the therapeutic potential of targeting CBR in patients with celiac disease.

Involvement of CD40–CD40 Ligand in Uncomplicated and Refractory Celiac Disease

Cognate interaction between CD40 on antigen-presenting cells and CD40 ligand (CD40L) on T cells is a crucial costimulatory signal in T-cell activation. In this study, we investigated CD40-CD40L expression in the duodenum of uncomplicated and refractory celiac disease patients, and explored the ex vivo effects of CD40L blockade on cytokine production and the T-helper cell type 1-specific transcription factor T-bet. CD40L and colocalization of CD40 with the dendritic cell markers CD11c and CD123 were investigated by confocal microscopy on tissue sections of duodenal biopsy samples obtained from 14 uncomplicated celiac patients before and after 12 months of gluten-free diet, 5 refractory celiac patients, and 12 controls. CD40 was also analyzed by flow cytometry on single cell suspension of mucosal biopsies. Treated celiac biopsies were stimulated with peptic-tryptic digest of gliadin (PT-gliadin) with or without an anti-CD40L-neutralizing antibody. Interferon (IFN)-γ and interleukin (IL)-17 were measured by ELISA (enzyme-linked immunosorbent assay). T-bet, CD40, and CD40L were determined by immunoblotting. CD40 and CD40L expression was higher in uncomplicated untreated and refractory celiac patients than in controls; the expression returned to normal after gluten-free diet in uncomplicated patients. Flow cytometric analysis confirmed that most CD40(+) cells were dendritic cells. The addition of the anti-CD40L antibody to treated celiac biopsies significantly inhibited the PT-gliadin-induced production of IFN-γ and IL-17, and mucosal T-bet. Our results indicate that the CD40-CD40L pathway has a key role in celiac disease. Disruption of CD40-CD40L interaction may offer a therapeutic alternative in refractory celiac disease.

In Vitro Differentiation of Human Monocytes into Dendritic Cells by Peptic–Tryptic Digest of Gliadin Is Independent of Genetic Predisposition and the Presence of Celiac Disease

This study was done to further reveal the role of the innate immune system in celiac disease. Dendritic cells were matured from venous blood of patients with active or treated celiac disease and DQ2-DQ8-positive or negative controls. Dendritic cells were treated with a peptic-tryptic digest of gliadin (500 microg/ml) and their activation was analyzed by fluorescent-activated cell sorting analysis, cytokine secretion, and their ability to elicit T cell proliferation. Gliadin upregulated interleukin (IL)-6, IL-8, and IL-12 (p40) secretion in dendritic cells and induced strong expression of the maturation markers human leukocyte antigen (HLA)-DR, CD25, CD83, and CD86 of all subjects irrespective of their genotype or the presence of disease, whereas the digest of bovine serum albumin showed no effect. However, gliadin-stimulated dendritic cells from active celiac showed enhanced stimulation of autologous T cells compared to the other groups. Further research should be aimed at identifying the mechanisms that control inflammation in healthy individuals.

Characterizing one of the DQ2 candidate epitopes in coeliac disease: A-gliadin 51-70 toxicity assessed using an organ culture system.

To investigate, using an organ culture system, in-vitro toxicity of region 51-70 of A-gliadin (SQQPYLQLQPFPQPQLPYSQ), a peptide overlapping some of the sequences recently characterized as DQ2-restricted T-cell epitopes in coeliac disease. Jejunal biopsies obtained from each of ten coeliac patients (eight treated, two untreated) and two non-coeliac patients were cultured in vitro for 18 h in the presence of A-gliadin amino acids 51-70 (200 microg/ml), organ culture medium only, peptic-tryptic digest of gliadin (1 mg/ml) or ovalbumin (1 mg/ml), the last two acting as positive and negative controls, respectively. Morphometric analysis involved measuring the cell height of 30 enterocytes, selected at random from the middle third of different villi for each section. Mean enterocyte cell heights (ECH) were compared with values for specimens cultured in medium alone. Levels of tissue transglutaminase antibody in biopsy supernatants were assessed by enzyme-linked immunosorbent assay (ELISA). In eight of ten coeliac patients, A-gliadin 51-70 was significantly toxic, causing a 30% decrease in ECH when compared with medium alone. In two of ten subjects, the peptide did not show any toxic effect. In all ten cases, we found that both positive and negative controls worked as expected. The peptide was non-toxic in the non-coeliac individuals. Tissue transglutaminase antibody titre in the supernatant was not found to be related to mucosal damage. We showed that the peptide corresponding to amino acids 51-70 of A-gliadin is characterized by in-vitro toxicity to the jejunal coeliac mucosa, correlating with recent findings of an immunological role of similar peptides. The lack of response in two of ten subjects suggests that this epitope may not be relevant in all cases of coeliac disease.

Gliadin increases iNOS gene expression in interferon-gamma-stimulated RAW 264.7 cells through a mechanism involving NF-kappa B.

Nitric oxide (NO) plays an important role in the pathogenesis of the histological changes seen in coeliac disease. We have investigated the effect of peptic-tryptic digest of gliadin (Pt-G) and gliadin (G) on inducible nitric oxide synthase (iNOS) protein expression in RAW 264.7 macrophages stimulated with interferon-gamma (IFN-gamma). Pt-G and G enhanced in a concentration and time-dependent manner NO production by IFN-gamma-stimulated RAW 264.7 cells. The increase of iNOS protein expression was correlated with NF-kappa B/DNA binding activity and occurred at transcriptional level. Pyrrolidine dithiocarbamate and N-alpha-para-tosyl-L-lysine chloromethyl ketone, two known inhibitors of NF-kappa B activation, decreased significantly NO production and iNOS protein expression as well as NF-kappa B/DNA binding activity. Our results show that the effect of Pt-G and G on enhancement of iNOS protein expression in IFN-gamma-treated RAW 264.7 cells is mainly mediated through NF-kappa B and suggest that blockage of NF-kappa B activation reduces enhancing effect of gluten on NO production in inflamed mucosa of coeliac patients.

Gluten sensitivity in a subset of children with insulin dependent diabetes mellitus.

The association between celiac disease and insulin dependent diabetes mellitus (IDDM) is well established. Rectal gluten challenge has been used in patients with celiac disease and in first degree relatives as a tool to assess the mucosal immune response to gluten. The aim of this study was to assess the mucosal immune response to gluten in IDDM children by rectal gluten challenge. Rectal biopsy specimens were obtained from 19 children with IDDM before and 6 h after rectal challenge with 2 g of a peptic tryptic digest of gliadin. A total of 16 treated celiac patients and 10 control subjects were also investigated. Epithelium and lamina propria CD3(+) and gamma delta(+) lymphocytes were counted with reference to a standard reference area of muscularis mucosae (10(4) microm(2)). After a local instillation of gliadin, a significant (>mean + 1 SD) percentage increment of lamina propria and epithelium CD3(+) and of lamina propria and epithelium gamma delta(+) lymphocytes was observed in five IDDM children, as compared to 11 and 13 celiac patients and one and two controls, respectively. A discriminant analysis allowed correct classification of 100% of patients with celiac disease and controls. The same analysis classified four of 19 IDDM children in the group of celiac patients. The positivity was associated with normal serology (antigliadin antibody, antiendomysial antibody, and antitissue transglutaminase antibodies) and a morphologically normal jejunal mucosa. All four patients had HLA-DQ alleles associated with celiac disease. Approximately 20% of IDDM children react to rectal instillation of gliadin. Long term follow-up is necessary to establish whether these subjects are at increased risk for developing celiac disease.

Gluten sensitivity in a subset of children with insulin dependent diabetes mellitus

Objectives The association between celiac disease and insulin dependent diabetes mellitus (IDDM) is well established. Rectal gluten challenge has been used in patients with celiac disease and in first degree relatives as a tool to assess the mucosal immune response to gluten. The aim of this study was to assess the mucosal immune response to gluten in IDDM children by rectal gluten challenge. Methods Rectal biopsy specimens were obtained from 19 children with IDDM before and 6 h after rectal challenge with 2 g of a peptic tryptic digest of gliadin. A total of 16 treated celiac patients and 10 control subjects were also investigated. Epithelium and lamina propria CD3 + and γδ + lymphocytes were counted with reference to a standard reference area of muscolaris mucosae (10 4 μm 2). Results After a local instillation of gliadin, a significant (>mean + 1 SD) percentage increment of lamina propria and epithelium CD3 + and of lamina propria and epithelium γδ + lymphocytes was observed in five IDDM children, as compared to 11 and 13 celiac patients and one and two controls, respectively. A discriminant analysis allowed correct classification of 100% of patients with celiac disease and controls. The same analysis classified four of 19 IDDM children in the group of celiac patients. The positivity was associated with normal serology (antigliadin antibody, antiendomysial antibody, and antitissue transglutaminase antibodies) and a morphologically normal jejunal mucosa. All four patients had HLA-DQ alleles associated with celiac disease. Conclusions Approximately 20% of IDDM children react to rectal instillation of gliadin. Long term follow-up is necessary to establish whether these subjects are at increased risk for developing celiac disease.

Enhanced peptide-binding capacities of small intestinal brush border membranes in celiac disease.

In pathogenesis of celiac disease, the significance of prolamin peptide interactions with enterocytes is controversial. Changes in cellular metabolism induced by gliadin peptides, as well as uptake and presentation by enterocytes, are discussed. We analyzed peptide binding to enterocytic membranes as a potential key event. Binding capacities of brush border membranes isolated from small intestinal biopsies of untreated (n = 49) and treated celiac patients on a gluten-free diet (n = 30), as well as control subjects (n = 43), were measured with a dot blot chemiluminescence assay. Synthetic gliadin peptides comprising amino acid position 8-19 (G XIV) and 30-41 (G XI) of alpha-gliadins, a peptic-tryptic digest of gliadin (PT-GLI), and a synthetic zein peptide were used. Comparing treated celiac patients with controls, we observed significantly enhanced membrane-binding of PT-GLI [mean 122.4 densitometric units/microg (95% confidence interval 116.0-128.9) vs 108.9 (102.1-115.7)] and of zein peptide [50.2 (38.4-61.9) vs 28.8 (13.4-44.2)], but only slightly increased binding of the synthetic gliadin peptides G XIV [65.5 (60.6-70.5) vs 62.4 (56.3-68.5) and G XI [75.2 (69.8-80.6) vs 65.9 (55.2-76.5)]. Independent of patient group, membrane-binding capacities for celiac-active gliadin peptides exceeded those of the zein peptide. Thus, interaction of gliadin peptides with the apical enterocytic membrane was not found exclusively in celiac disease. Furthermore, increased binding capacities in treated celiac disease were not confined to celiac-active peptides. Quantitative differences in gliadin peptide binding as a primary characteristic in celiac disease might contribute to pathogenetic effects exerted on small intestinal epithelial cells.

THE MAJORITY OF RELATIVES OF CELIAC PATIENTS WITH POSITIVE RECTAL GLUTEN CHALLENGE HAVE A LOW GRADE SMALL INTESTINAL ENTEROPATHY

111 A significant infiltration of CD3 and γδ+ lymphocytes is induced in the rectal mucosa of coeliac patients by a gliadin enema. Interestingly, a subset of siblings of coeliac children also react to rectal instillation of gliadin. Aim of this study has been to correlate in coeliac relatives the results of the rectal gluten challenge with the immunohistochemical features of the small intestinal mucosa, particularly with density of cells infiltrating the epithelium and signs of activated T cell mediated immunity. In 16 treated coeliac children, 10 disease-controls and 30 siblings of coeliacs, rectal biopsies were obtained before and 6 hours after rectal challenge with a peptic-tryptic digest of gliadin (Gastroenterology 1996; 111: 318-24 ). CD3+ and γδ+ lymphocytes were counted and a discriminant score was calculated. Jejunal biopsies were obtained 13 siblings and cryostat sections were incubated with antiCD3, antiTCRγδ, antiCD25 (IL2 receptor), antiCD54 (ICAM1) antibodies, and processed by immunohistochemistry (PAP or APAAP techniques). After local instillation of gliadin, in coeliac patients, but not in controls, immunohistochemistry showed a significant increase in CD3+ and γδ+ cells. The discriminant analysis allowed correct classification of 100% coeliacs and controls, while 11/30 (37%) siblings were classified as coeliacs. All of them had a negative serology (AGA, EMA) and a normal architecture of the jejunal mucosa. Nevertheless, the immunohistochemical analysis of the jejunal biopsies showed in 5/6 siblings with a positive response to rectal gluten challenge, but only in 1/7 of those with a negative response, increased density of CD3+ and/or γδ+ lymphocytes. In no case increased number of CD25+ cells or enhanced expression of ICAM 1 was observed. Our results demonstrate that in siblings of coeliac patients the positivity to rectal gluten challenge is often accompanied in the small intestine by a low-grade enteropathy characterized by a lymphocyte infiltration of the epithelium. The rectal gluten challenge is proposed as a useful tool to detect an abnormal mucosal immune response to gluten.

Gliadin activates mucosal cell mediated immunity in cultured rectal mucosa from coeliac patients and a subset of their siblings

Background—CD3 and γδ cells in the rectal mucosa increase after local instillation of gluten in children with coeliac disease and in half of their siblings.Aim—To establish an in vitro system...

In siblings of celiac children, rectal gluten challenge reveals gluten sensitization not restricted to celiac HLA

BACKGROUND & AIMS: Inflammatory changes in the rectum of patients with celiac disease after local instillation of gluten have been reported. The aim of this study was to examine rectal mucosa after local gluten challenge in children with celiac disease and their siblings. METHODS: Rectal biopsy specimens were obtained before and 6 hours after rectal challenge with a peptictryptic digest of gliadin in 33 children with treated celiac disease, 12 controls, and 19 siblings of children with celiac disease. Epithelium and lamina propria volumes were determined, and CD3+ and gamma delta + lymphocytes were counted. RESULTS: After local instillation of gliadin, a significant increment in the absolute number of intraepithelial lymphocytes was noted in patients with celiac disease but not in controls. Immunohistochemical analysis showed a significant increase in CD3+ and gamma delta + cells, with the gamma delta/CD3 ratio remaining unchanged after challenge. A discriminant analysis allowed correct classification of 100% of patients with celiac disease and controls. The same analysis was used to classify 6 of 13 siblings as having celiac disease. The positivity was not associated with the presence of the heterodimer encoded by the DQA*0501 DQB1*0201 alleles in any of the siblings. CONCLUSIONS: All patients with celiac disease were identified by rectal gluten challenge. Approximately half of the siblings reacted to rectal instillation of gluten. The genetic background of such sensitization to gluten remains to be elucidated. (Gastroenterology 1996 Aug;111(2):318-24)

Gliadin induced changes in the expression of MHC-class II antigens by human small intestinal epithelium. Organ culture studies with coeliac disease mucosa.

Jejunal biopsies from 16 treated coeliac disease patients and from nine controls were cultured with and without a peptic-tryptic digest of gliadin. Cultures with a peptic-tryptic digest of maize prolamins were also undertaken. Frozen sections of baseline and cultured mucosa were stained by immunofluorescence with an anti-HLA-DR monoclonal antibody. Before culture the villous epithelium from both controls and treated coeliac disease expressed DR molecules while the crypt epithelium did not. When biopsies from treated coeliac disease were cultured with gliadin the expression of DR was enhanced in the crypt epithelium in eight of 14 cultures and in 11 of 14 was reduced or absent on the villous epithelium. No change was observed in control cultures. We conclude that gliadin is capable of inducing HLA-DR on the crypt epithelium of in vitro cultured coeliac disease mucosa, providing indirect evidence that gliadin may activate cell mediated immune mechanisms within the small bowel mucosa. This model could prove useful in identifying the immunogenic sequence(s) of gliadins and related prolamins.

Coeliac activity of the gliadin peptides CT-1 and CT-2.

The coeliac active peptide B 3142, which has been isolated from a peptic-tryptic digest of gliadin and which consists of 53 amino-acid sequences, was partially hydrolyzed with alpha-chymotrypsin. The two fragment peptides CT-1 (positions 1-22 of B 3142) and CT-2 (positions 23-53) were separated by high-performance liquid chromatography on octadecyl silica gel and purified by gel filtration on Biogel P2. The examination in the organ-culture test including 18 coeliac patients on normal diet and 7 control persons have shown that the toxicity is preserved after the chymotryptic treatment and that the peptides B 3142, CT-1 and CT-2 do not significantly differ from one another according to their coeliac-specific effect.

Re-evaluation of the techique of organ culture for studying gluten toxicity in coeliac disease.

In vitro cytotoxicity of four different gluten fractions was tested in organ culture for up to 48 hours using flat intestinal biopsies from children with coeliac disease. The fractions were (1) a peptic-tryptic digest of gliadin containing a moderate amount of alpha-gliadin, (2) a peptic-tryptic digest of gluten (Frazer fraction III) froma strain of wheat with a high content of alpha-gliadin, (3) alpha-gliadin, and (4) alpha-GT-18,000, a tryptic fragment of alpha-gliadin. The latter three fractions were toxic to coeliac patients in vivo. In vitro, however, none of these fractions proved to be cytotoxic. When added to the culture medium they were not capable of inhibiting the regeneration of the surface epithelium as visualised by histology and electron microscopy. The only difference between cultures with and without gluten fractions was that the former produced slightly more mucus when maintained in vitro as observed in the dissecting microscope. Furthermore, for Frazer fraction III the absence of apparent toxicity was confirmed by the behaviour of brush border enzyme activities during culture. Our results are not in accordance with those reported in the literature. We believe that the criteria used at the present time for the assessment of gluten toxicity in vitro should be extended to include the process of enterocyte desquamation.

Circulating antibodies to wheat gliadin fractions in coeliac disease.

Titres of circulating antibodies to wheat gliadin fractions have been determined in children with active coeliac disease using a passive haemagglutination technique. The highest titres against fractions from S.E. Sephadex chromatography of a peptic-tryptic-pancreatic digest of gliadin were obtained against Fraction 9. Titres of serum antibodies in children with active coeliac disease against a peptictryptic digest of gliadin and its ultrafiltrate were significantly higher than those in children free from gastrointestinal disorders. Titres of sera from some asymptomatic sibs of the probands were abnormally high against the peptic-tryptic gliadin digest, suggesting a minor gut abnormality in these children, despite normal duodenal mucosal histology. Haemagglutination titres of sera from patients with active coeliac disease against the protein digests and fractions evaluated seemed to be related to the toxicity of these substances in this disease.