Coeliac disease: investigation of proposed causal variants in the CTLA4 gene region.

Coeliac disease (CD) is an immune-mediated enteropathy triggered by gluten in genetically predisposed individuals. Patients with CD have an increased prevalence of other autoimmune disorders, including type 1 diabetes (T1D) and Graves' disease (GD). CD shares with these conditions certain HLA susceptibility alleles. A number of studies have also shown association of autoimmune diseases, including CD, with the CD28-cytotoxic T lymphocyte antigen 4 (CTLA4)-inducible costimulator (ICOS) region of chromosome 2q33, but until recently the precise causal variant has remained unknown. Recently, it was shown that, in GD, CT60 (+6230G>A), a single nucleotide polymorphism (SNP) at the end of the CTLA4 transcript, is associated with an alteration in the ratio of splice forms of the CTLA4 gene and that this ratio affects disease susceptibility. A similar but weaker association was found with T1D. There is also an independent association of GD and T1D with the SNP MH30 (-23 327G>C), which possibly affects promoter region function. Hypothesizing that CT60 and MH30 may be causal variants in other autoimmune disorders, we investigated these SNPs in CD using 149 family trios and 100 unrelated/unaffected controls. No association was detected with either SNP using both the transmission disequilibrium test (TDT) and case-control methods. Our study appears to have good power to detect moderate genetic effects, but possibly these SNPs exert too weak an effect on risk of CD to have been detected in our sample. Alternatively, the previously noted association of CD with the CTLA4 gene region may be due to different causal variants. Unlike T1D and GD, CD is not a true autoimmune disease, and CD has different associations at the CTLA4 exon 1 SNP +49G>A from all other autoimmune disorders. MH30, CT60, and other SNPs in the region may still warrant further investigation in other CD samples.

Coeliac disease: in vivo toxicity of the putative immunodominant epitope.

BACKGROUND: Peptides from alpha-gliadins have been used to characterise the immunodominant coeliac toxic epitope. A peptide corresponding to amino acid residues 57-73 of A-gliadin causes peripheral blood mononuclear cells from coeliac patients to secrete interferon gamma (IFN-gamma); gluten specific small intestinal T cell clones proliferate in response to peptides corresponding to residues 57-68 and 62-75 of alpha-gliadins. We wished to investigate whether a peptide corresponding to residues 56-75 of alpha-gliadins exacerbates coeliac disease in vivo. METHODS: Four adults with coeliac disease, all of whom were on a gluten free diet, underwent three challenges. Peptic-tryptic gliadin (PTG 1 g) served as a positive control. The test peptide and a negative control peptide were studied on separate occasions. The peptides were instilled into the duodenum and biopsies were taken before the infusion, and two, four, and six hours after commencing the infusions, using a Quinton hydraulic multiple biopsy capsule. Biopsy specimens were assessed blindly for villus height to crypt depth ratio (VH:CD), enterocyte cell height (ECH), and intraepithelial lymphocyte (IEL) count. We used the Mann-Whitney U test, with 95% confidence intervals, for statistical analysis. RESULTS: VH:CD and ECH fell, and IEL increased significantly 4-6 hours after commencing infusions with both PTG and the test peptide in all subjects. The negative control peptide caused no significant changes to villus morphology, enterocyte height, or IEL count in any patient. CONCLUSION: We have confirmed that the putative immunodominant epitope, a peptide corresponding to residues 56-75 of alpha-gliadins, exacerbates coeliac disease in vivo.

Investigation of the putative immunodominant T cell epitopes in coeliac disease.

BACKGROUND: Coeliac disease (CD) is an enteropathy mediated by gluten specific T cells which secrete interferon gamma (IFN-gamma) when stimulated by gluten peptides presented by HLA-DQ2 or DQ8 molecules. Residues 62-75 of alpha(2) gliadin have been proposed as the immunodominant epitope in the majority of CD patients. Deamidation by tissue transglutaminase (tTG) of the glutamine (Q) at position 65 to glutamic acid (E) is essential for T cell stimulation. AIMS: To investigate the antigenicity of this peptide and to establish whether its T cell activating properties can be downregulated by the formation of altered peptide ligands. PATIENTS: Individuals with known CD. METHODS: Peptide G4 corresponding to alpha(2) gliadin residues 62-75, Q-E65 and analogues, substituting each amino acid, except E65, in turn for alanine residues, were synthesised. Small intestinal biopsies were obtained from patients. Biopsies were cultured overnight with a peptic/tryptic digest of gliadin (PTG). Lymphocytes were cultured and restimulated with tTG treated PTG. A T cell line was cloned and clones tested for stimulation and IFN-gamma production in response to G4 and its analogues. RESULTS: Some high activity clones were isolated with, for example, a stimulation index (SI) of 15 to G4 and secreting 327 pg/ml of IFN-gamma. Substitution of amino acids at several positions abolished or downregulated stimulation and IFN-gamma production. CONCLUSIONS: Peptide G4 is highly immunogenic. Certain amino acid substitutions in peptide G4 abolish T cell reactivity while others are partial agonists which may have potential in immunomodulation in this condition.

Analysis of candidate genes on chromosome 19 in coeliac disease: an association study of the KIR and LILR gene clusters.

Coeliac disease is strongly heritable, with more than half of the genetic susceptibility estimated to come from genes outside the HLA region. Several candidate regions have been suggested from genome-wide linkage studies including chromosome 19q13.4 where linkage has been replicated between populations. The natural killer (NK) cell immunoglobulin-like receptors (KIRs) and leukocyte immunoglobulin-like receptor (LILR, also known as ILT and LIR) gene clusters lie within this region in the leukocyte receptor cluster (LRC). KIR molecules are involved in cytotoxic lymphocyte function and expressed by intraepithelial T and NK cells in the duodenum. We studied 132 unrelated UK Caucasian coeliac patients and their parents together with a control group of 171 UK Caucasians. PCR-SSP for KIR2DL1, KIR2DL2, KIR2DL3, KIR2DL5, LILRA3 (ILT6), LILRA3 deletion and an LILRA3 exon 3 single nucleotide polymorphism (SNP) allowed classification of KIR genotypes into five categories and determination of homozygosity or heterozygosity for the common A and B type KIR haplotypes (as defined in the text) and for the LILRA3 deletion. Case-control analysis found no association of the five KIR genotype categories, the A or B KIR haplotypes, the LILRA3 gene deletion or the LILRA3 exon 3 SNP with coeliac disease. A transmission disequilibrium test also found no association of the A and B KIR haplotypes or the LILRA3 gene deletion with coeliac disease.

Coeliac disease: follow-up linkage study provides further support for existence of a susceptibility locus on chromosome 11p11.

Susceptibility to coeliac disease has a strong genetic component. The HLA associations have been well described but it is clear that other genes outside this region must also be involved in disease development. Two previous genome-wide linkage studies using the affected sib pair method produced conflicting results. Our own family based linkage study of 16 highly informative pedigrees identified 17 possibly linked regions, each of which produced a result significant at p & 0.05 or less. We have now investigated these 17 regions in a larger set of pedigrees using more finely spaced markers. Fifty multiply affected families were studied, comprising the 16 pedigrees from the original genome screen plus 34 new highly informative pedigrees. A total of 128 microsatellite markers were genotyped with an average spacing between markers of 5 cM. Two-point and three-point linkage analysis using classical and model free methods identified five potential susceptibility loci with heterogeneity lod scores > 2.0, at 6p12, 11p11, 17q12, 18q23 and 22q13.3. The most significant was a heterogeneity lod of 2.6 at D11S914 on chromosome 11p11. This marker maps to a position implicated in one of the two previous genome scans and taken together these results provide strong support for the existence of a susceptibility locus in this region.

Comparison of 2 hemostatic agents for the prevention of postextraction hemorrhage in patients on anticoagulants.

OBJECTIVE: The objective of this study was to compare the use of a resorbable oxycellulose dressing with a fibrin adhesive for the prevention of postextraction hemorrhage in patients taking anticoagulants. STUDY DESIGN: A control group of 26 patients with a preoperative international normalized ratio (INR) in the range of 2.0 to 4.2 had extractions performed with the use of local anesthesia and the socket(s) dressed with a resorbable oxycellulose dressing and sutured with a resorbable suture. The study group with a comparable INR range of 2.1 to 4.1 was treated in a similar manner, except the sockets were dressed with a fibrin adhesive. RESULTS: No discernible difference in the postoperative outcome with regard to hemorrhage was noted. Postoperative pain was reported more frequently in the group that used a resorbable oxycellulose dressing. Only 1 patient had significant postoperative bleeding. CONCLUSIONS: This study shows that in patients receiving warfarin whose INR is within the therapeutic range, the fibrin adhesive is as effective as the resorbable oxycellulose dressing in preventing postextraction hemorrhage.

Renal tubular injury is present in acute inflammatory bowel disease prior to the introduction of drug therapy.

BACKGROUND: 5-aminosalicylic acid (5-ASA) has been associated with renal complications in inflammatory bowel disease. Renal function is typically monitored using serum creatinine; however, significant disease may predate increases in creatinine. AIMS: To identify whether markers of early renal disease (urinary albumin, alpha-1-microglobulin [alpha-1-M] and N-acetyl-beta-D-glucosaminidase [NAG], and serum cystatin C) are useful in the assessment of renal function in inflammatory bowel disease patients receiving 5-ASA. METHODS: Twenty-one patients with a new diagnosis of inflammatory bowel disease were investigated. Samples were taken at diagnosis, and at 3-monthly intervals after the commencement of 5-ASA, for 1 year. RESULTS: Mean creatinine clearance was 100 mL/min and did not change following treatment. Inflammatory bowel disease was not associated with albuminuria. Urinary N-acetyl-beta-D-glucosaminidase and alpha-1-microglobulin at diagnosis were increased in 10 (48%) and 11 (52%) patients, respectively: treatment was not associated with consistent changes in urinary protein excretion. There was a significant correlation between cystatin C and creatinine clearance both at diagnosis (r=-0.533, P=0.0275) and combining the initial and follow-up data (r=-0.601, P < 0.01), but not between creatinine and creatinine clearance (P > 0.05). CONCLUSIONS: Tubular proteinuria is an extra-intestinal manifestation of inflammatory bowel disease irrespective of 5-ASA treatment. Tubular proteins are not useful predictors of an adverse renal response to 5-ASA. Serum cystatin C may be an improved marker of glomerular filtration rate in this setting.

A comparison of the attitudes shown by general practitioners, hospital doctors and medical students towards alternative medicine.

The aim of this study was to compare and contrast the views of general practitioners (GPs), hospital doctors and medical students to alternative medicine. A questionnaire was sent to a random sample of 100 GPs and 100 hospital doctors in the South West Thames Regional Health Authority (SWTRHA). A convenience sample of 237 pre-clinical medical students at St George's Hospital Medical School was also given a questionnaire. Eighty-seven GPs and 81 hospital doctors replied. Five therapies were investigated: acupuncture; chiropractice; homeopathy; naturopathy; and osteopathy. All respondents were asked about their attitude towards and knowledge of these therapies. Doctors were asked how often they referred patients for such treatment and whether they practised it themselves. GPs and hospital doctors had similar levels of knowledge of the therapies. Medical students were the least informed but the most enthusiastic respondents. Seventy per cent of hospital doctors and 93% of GPs had, on at least one occasion, suggested a referral for alternative treatment. GPs were making these referrals more frequently and earlier. Twelve per cent of hospital doctors and 20% of GPs were practising alternative medicine. The majority of the respondents felt that alternative medicine should be available on the National Health Service (NHS) and that medical students should receive some tuition about alternative therapies. A considerable proportion of those doctors referring patients to alternative practitioners were ignorant of their official qualifications.