Jacques Schmitz

Selective expansion of intraepithelial lymphocytes expressing the HLA-E–specific natural killer receptor CD94 in celiac disease

Abstract Background & Aims: Celiac disease is a gluten-induced enteropathy characterized by the presence of gliadin-specific CD4+ T cells in the lamina propria and by a prominent intraepithelial T-cell infiltration of unknown mechanism. The aim of this study was to characterize the subset(s) of intraepithelial lymphocytes (IELs) expanding during active celiac disease to provide insights into the mechanisms involved in their expansion. Methods: Flow-cytometric analysis of isolated IELs and/or immunohistochemical staining of frozen sections were performed in 51 celiac patients and 50 controls with a panel of monoclonal antibodies against T-cell and natural killer (NK) receptors. In addition, in vitro studies were performed to identify candidate stimuli for NK receptor expression. Results: In normal intestine, different proportions of IELs, which were mainly T cells, expressed the NK receptors CD94/NKG2, NKR-P1A, KIR2D/3D, NKp46, Pen5, or CD56. During the active phase of celiac disease, the frequency of CD94+ IELs, which were mostly αβ T cells, was conspicuously increased over controls. In contrast, the expression of other NK markers was not modified. Furthermore, expression of CD94 could be selectively induced in vitro by T-cell receptor activation and/or interleukin 15, a cytokine produced by intestinal epithelial cells. Conclusions: The gut epithelium favors the development of T cells that express NK receptors. In active celiac disease, there is a specific and selective increase of IELs expressing CD94, the HLA-E–specific NK receptor that may be related to T-cell receptor activation and/or interleukin 15 secretion. GASTROENTEROLOGY 2000;118:867-879

CTLA-4 gene polymorphism is associated with predisposition to coeliac disease

Background—Susceptibility to coeliac disease is strongly associated with particular HLA class II alleles. However, non-HLA genetic factors are likely to be required for the development of the disease. Among candidate genes is the CTLA-4 (cytotoxic T lymphocyte associated) gene located on chromosome 2q33 in humans, which encodes a cell surface molecule providing a negative signal for T cell activation. Aims—To investigate CTLA-4 exon 1 polymorphism (position 49 A/G) in patients with coeliac disease. Patients—101 patients with coeliac disease and 130 healthy controls. Methods—Allele specific hybridisation and restriction enzyme digestion of polymerase chain reaction amplified genomic DNA. Results—The A allele of the CTLA-4 position 49 polymorphism was found on 82.2% of chromosomes in patients with coeliac disease compared with 65.8% in controls (p<0.0001), mostly in the homozygous form (68.3% in patients versus 47.7% in controls; odds ratio (OR) 2.36, 95% confidence interval (CI) 1.37 to 4.06, p=0.002). Four patients only had the G/G genotype compared with 21 controls (OR 0.21, CI 10.07 to 0.64, p=0.002). These differences were maintained when subjects were stratified according to the HLA class II phenotype, in particular when patients and controls were matched for the presence of the predisposing HLA DQB1*02 (DQ2) allele or HLA-DQA1*0501/DQB1*02 heterodimer. Conclusion—The CTLA-4 gene polymorphism is a non-HLA determinant that predisposes to coeliac disease. Whether it directly contributes to disease susceptibility or represents a marker for a locus in linkage disequilibrium with CTLA-4 needs further investigation.

A prospective study of the efficacy and tolerance of a chimeric antibody to tumor necrosis factors (remicade) in severe pediatric crohn disease.

ObjectivesTo evaluate the efficacy and toxicity of infliximab in children with severe Crohn disease (CD), the authors prospectively monitored 21 children aged 15 ± 2 years with severe CD who they treated with infliximab (5 mg/kg) on days 0, 15, and 45. One patient received only one injection. Eighteen patients were corticosteroid dependent, and 6 were receiving parenteral nutrition. Three patients were corticoid resistant (1 mg/kg/d >15 days). Sixteen had perianal disease. ResultsThe Harvey-Bradshaw index (HB) decreased from 8 ± 3 on day 0 to 1 ± 2 on day 45 (P = 0.001). The inflammation factors decreased (P = 0.001), and albumin increased (P = 0.002). Nineteen children were in complete remission (HB < 4) on day 45, and 2 had improved (HB = −6 points). Tumor necrosis factor-&agr; (TNF&agr;) in the stools (n = 16) decreased (P = 0.04). All perianal fistulas (n = 12) were closed by day 90. Fourteen of 21 patients had stopped taking steroids at 3 months, and all had stopped parenteral nutrition. Growth velocity was significantly greater after infliximab administration (Z score, +0.5) than before (−0.45;P = 0.004). Nineteen of 21 patients had relapsed (90%) at 1 year despite continued immunosuppressors. Seven had surgery because of an uncontrolled relapse (5), stenosis (1), or fistula (1). Six patients developed antinuclear antibodies (1/40–1/640e), and two had anti-DNA antibodies. Epstein-Barr virus (EBV) polymerase chain reaction (PCR) values increased (>100-fold) in eight patients. One child developed an anaphylactic reaction to the medication, and one had a catheter-related sepsis. ConclusionInfliximab produces spectacular results for children with severe CD and is well tolerated. However, its effect is transitory for many (90%), with frequent relapses despite continued immunosuppressors. Long-term management with infliximab should be tested despite its worrying side effects.

Long‐term follow‐up of 61 coeliac patients diagnosed in childhood: evolution toward latency is possible on a normal diet

Background/Aims: Whether a life-long gluten-free diet (GFD) is necessary in all children with diagnosed coeliac disease (CD) remains debated. To address this question, a retrospective analysis of the clinical and biological status of adult coeliac patients diagnosed in childhood, who remained on a normal diet after gluten challenge and were clinically silent, was carried out. Methods: Patients aged 18–65 years with CD diagnosed in childhood were included. Clinical status, gluten intake, biological parameters of malabsorption, bone mineral density, human leucocyte antigen (HLA) genotype, serological markers of CD, and histological and immunohistochemical parameters in duodenal biopsies were recorded. Results: Sixty-one patients had resumed a normal diet and were asymptomatic. Forty-eight showed different degrees of villous atrophy (silent CD), while 13 had no detectable atrophy (latent CD) on duodenal biopsies. Latent CD patients had significantly less osteopenia/osteoporosis (1/9 (11%) vs 23/33 (70%), p<0.001)), and lower T cell receptor (TCR) αβ+ intraepithelial T cell counts (38±20 vs 55±15, p<0.01) than silent CD patients. The mean age at diagnosis and first GFD was lower in latent than in silent patients (14.4±5 vs 40.1±47 months, p<0.05). Latent patients did not differ significantly from the seven control patients on a long-term GFD, except for a higher frequency of CD-specific serum antibodies. However, two latent patients relapsed clinically and histologically during subsequent follow-up. Conclusions: Long-term latency developed in about 20% of CD patients who remained symptom free after gluten reintroduction. This latency can be transient and thus a regular follow-up is mandatory. In silent patients, the increased risk of osteoporosis substantiates the need for a GFD.

Severe osteopenia in symptom-free adults with a childhood diagnosis of coeliac disease

The frequency of osteopenia in symptom-free adults diagnosed with coeliac disease during childhood and who resumed a normal diet during adolescence is unknown. Severe osteopenia (a bone mineral density below two standard deviations of the mean) was found in up to a third of symptom-free young adults on a normal diet. These patients should not be thought to be disease-free but should receive long-term follow-up and most of them should be advised to resume a gluten-free diet.

Characteristics of inflammatory bowel disease with onset during the first year of life

Background: Inflammatory bowel disease (IBD) is recognized in young children, however, only rare data on onset and evolution are available in children younger than 1 year. In the present clinical study, we aimed to analyze characteristics and clinical course of children with very early onset IBD. We were particularly interested in the relationship between bacterial infections and the use of antibiotics before the onset of IBD. Patients and Methods: The IBD database of Necker-Enfants-Malades-Hospital was screened for patients with IBD with disease onset during the first year of life and a follow-up of at least 2.5 years. Ten patients were identified during the period 1996–2002. Results: All patients presented with rectal bleeding and had colonic involvement. Four patients had definitive diagnosis of Crohn disease; ulcerative or indeterminate colitis was seen in 2 and 4 children, respectively. Five of the patients had a positive history of neonatal or early-onset bacterial infection with use of antibiotics before onset of IBD, 4 patients were still breastfed and 3 just weaned when GI symptoms started. Seven patients had a severe onset of disease requiring bowel rest, parenteral nutrition and steroid medication, followed by azathioprine or cyclosporine medication. Surgery was necessary in 3 of 10 patients. Disease relapses were frequent and observed in 8 of 10 children. Discussion: Very early onset IBD may reflect a subgroup of patients characterized by a particular sensitivity to modifications of the intestinal flora. Neonatal IBD was most often severe in presentation and evolution.

Syndrome of Intractable Diarrhoea with Persistent Villous Atrophy in Early Childhood: A Clinicopathological Survey of 47 Cases

BACKGROUND The syndrome of intractable diarrhoea of infancy is heterogeneous and includes several diseases with diverse aetiologies. This study determines whether diagnostic categories can be defined on the basis of clinicopathological analysis. METHODS European Society of Paediatric Gastroenterology, Hepatology and Nutrition members were surveyed to identify cases of intractable diarrhoea with persisting small intestinal enteropathy. A retrospective clinicopathological analysis was performed on cases showing life-threatening diarrhoea within the first 24 mo of life and requiring total parenteral nutrition, which were characterized by persistent villous atrophy, and resistance to therapy. RESULTS Forty-seven infants were identified with intractable diarrhoea. Villous atrophy was of varying degrees with (group I, n = 24) or without (group II, n = 18) lamina propria mononuclear cell infiltration. Group I presented later, had gut autoantibodies, and a higher prevalence of protein-losing enteropathy; a subset (group Ia, n = 12) also had extraintestinal symptoms of autoimmunity associated with a later onset of larger volume diarrhoea. Group II presented early; 8 cases (group IIa) had phenotypic abnormalities and a low birth weight; the remaining 10 (group IIb) showed mild-to-moderate villous atrophy, epithelial tufting, and abnormal crypts. Group III included five patients in whom no specific features were recognised. Twenty-one (45%) died at a median age of 24 months, 20 (43%) remained dependent on parenteral (n = 16) or enteral tube (n = 4) feeding, 4 (9%) received elimination diets plus other therapies, and 2 (4%) were lost to follow-up. CONCLUSIONS Clinicopathological analysis allowed distinct disease groups to be identified, allowing a provisional classification to be made. This straightforward approach forms a basis for future research in this exceptionally difficult paediatric condition.

Induction of T lymphocyte apoptosis by sulphasalazine in patients with Crohn’s disease

Background: Lamina propria T lymphocytes (LPL) of the intestinal mucosa are chronically activated in Crohn’s disease (CD). Defective apoptosis of activated LPL was proposed as a key pathogenic mechanism. In fact, increased expression of antiapoptotic molecules was observed in CD LPL. In the present work, we aimed to analyse the effects and underlying molecular mechanisms of 5-amino salicylic acid (5-ASA) and derivatives on apoptosis of LPL and peripheral blood lymphocytes (PBL) in patients with CD compared with ulcerative colitis (UC) and in non-inflammatory controls. Methods: PBL and LPL were isolated by Ficoll-Hypopaque gradient centrifugation and the EGTA-collagenase method, respectively. PBL/LPL were stimulated with FasL, 5-ASA, sulphapyridine, and sulphasalazine for 24/48 hours and apoptosis was quantified by flow cytometry (annexin V- propidium iodide method) and immunofluorescence. The molecular mechanisms of drug induced apoptosis were analysed in wild-type and FADD−/− Jurkat T cells using western blots and caspase assays. Results: While PBL displayed a normal apoptosis pattern after Fas stimulation in patients with active CD, LPL from inflammatory areas were highly resistant. Comparable resistance to apoptosis was observed in LPL of UC patients. In contrast with 5-ASA, which did not induce apoptosis in lymphocytes, sulphasalazine proved to be a potent proapoptotic agent. Sulphasalazine induced T lymphocyte apoptosis was independent of the Fas pathway but associated with marked downregulation of antiapoptotic bcl-xl and bcl2, activation of the mitochondrial apoptosis signalling pathway, and subsequent activation of caspase-9 and caspase-3. Conclusion: The beneficial effect of sulphasalazine in treating inflammatory bowel disease is at least in part attributable to its proapoptotic effects on LPL which allows potent downregulation of lymphocyte activation.

Microvillous inclusion disease (microvillous atrophy)

Microvillous inclusion disease (MVID) or microvillous atrophy is a congenital disorder of the intestinal epithelial cells that presents with persistent life-threatening watery diarrhea and is characterized by morphological enterocyte abnormalities. MVID manifests either in the first days of life (early-onset form) or in the first two months (late-onset form) of life. MVID is a very rare disorder of unknown origin, probably transmitted as an autosomal recessive trait. To date, no prevalence data are available. Ultrastructural analyses reveal: 1) a partial to total atrophy of microvilli on mature enterocytes with apical accumulation of numerous secretory granules in immature enterocytes; 2) the highly characteristic inclusion bodies containing rudimentary or fully differentiated microvilli in mature enterocytes. Light microscopy shows accumulation of PAS-positive granules at the apical pole of immature enterocytes, together with atrophic band indicating microvillus atrophy and, in parallel, an intracellular PAS or CD10 positive line (marking the microvillous inclusion bodies seen on electron microscopy). Intestinal failure secondary to diarrhea is definitive. To date, no curative therapy exists and children with MVID are totally dependent on parenteral nutrition. Long-term outcome is generally poor, due to metabolic decompensation, repeated states of dehydration, infectious and liver complications related to the parenteral nutrition. As MVID is a very rare disorder, which is extremely difficult to diagnose and manage, children with MVID should be transferred to specialized pediatric gastro-intestinal centers, if possible, a center equipped to perform small bowel transplantation. Early small bowel transplantation resulting in intestinal autonomy gives new hope for disease management and outcome.

The efficacy of exclusive nutritional therapy in paediatric Crohn’s disease, comparing fractionated oral vs. continuous enteral feeding

Background  Nutritional therapy has an established role as induction therapy in paediatric Crohn’s disease. However, compliance is the main difficulty and may be greatly influenced by the administration route.