J. B. A. Crusius

Secretion of Tumour Necrosis Factor α and Lymphotoxin α in Relation to Polymorphisms in the TNF Genes and HLA-DR Alleles. Relevance for Inflammatory Bowel Disease

The genes for tumour necrosis factor alpha (TNFα) and lymphotoxin alpha (LTα; TNFβ) are tandemly arranged in the central region of the MHC. They may, therefore, be of importance for the aetiology of MHC‐associated diseases. The authors have prospectively studied the secretion of TNFα and LTα in relation to polymorphisms at positions ‐308 and ‐238 in the TNFα gene (TNFA), and two polymorphisms in the first intron of the LTα gene (LTA), as well as HLA‐DR in 30 patients with chronic inflammatory bowel diseases (IBD) and 12 healthy controls. In the Dutch population, the alleles of these four polymorphisms are present in only five combinations, called TNF‐haplotypes: TNF‐C, ‐E, ‐H, ‐I, and ‐P. Significant associations between TNF haplotypes and TNFα and LTα secretion were found when PBMC were cultured with T‐cell activators, irrespective of disease. Mean TNFα secretion of individuals carrying the HLA‐DR3 associated TNF‐E haplotype was significantly higher, as compared to individuals without this haplotype (26u2003441u2003pg/ml versus 19u2003629u2003pg/ml; Pu2003=u20030.014). Individuals carrying the TNF‐C haplotype produced the lowest amount of TNFα (17u2003408u2003pg/ml; Pu2003=u20030.022). The TNF‐C and TNF‐E haplotypes differ only at position ‐308 in the promoter of TNFA. Individuals carrying the HLA‐DR1 associated TNF‐I haplotype produced significantly less LTα when compared to those who lack this haplotype (1979u2003pg/ml versus 3462u2003pg/ml; Pu2003=u20030.006). As the TNF‐I haplotype is also associated with low TNFα secretion, this haplotype thus defines a ‘low secretor phenotype’. In conclusion, this is the first study to show associations between TNF haplotypes and TNFα and LTα secretion when T‐cell stimulators are used. These findings will contribute to define disease heterogeneity in IBD and may be of relevance for understanding the pathogenesis of autoimmune diseases.

Allelic polymorphism in IL‐1β and IL‐1 receptor antagonist (IL‐1Ra) genes in inflammatory bowel disease

Recent reports have shown that allele 2 of the IL‐1 receptor antagonist (IL‐1Ra) gene is over‐represented in ulcerative colitis (UC). Healthy individuals carrying allele 2 of this gene have increased production of IL‐1Ra protein. Since the final outcome of the biological effects of IL‐1β may depend on the relative proportion of these two cytokines, we have studied if a TaqI polymorphism in the IL‐1β gene, which is relevant to IL‐1β protein production, may be involved in the genetic susceptibility to UC and Crohns disease (CD), in association with the established IL‐1Ra gene polymorphism. Polymorphisms in the closely linked genes for IL‐1β and IL‐1Ra were typed in 100 unrelated Dutch patients with UC, 79 with CD, and 71 healthy controls. The polymorphic regions in exon 5 of the IL‐1β gene and in intron 2 of the IL‐1Ra gene, were studied by polymerase chain reaction (PCR)‐based methods. The IL‐1β allele frequencies in UC and CD patients did not differ from those in healthy controls. In order to study if the IL‐1β gene polymorphism might participate synergistically with the IL‐1Ra gene polymorphism in susceptibility to UC and CD, individuals were distributed into carriers and non‐carriers of allele 2 of the genes encoding IL‐1β and IL‐1Ra, in each of the patient groups and controls. Results indicated a significant association of this pair of genes, estimated by the odds ratio (OR) after performing Fishers exact test, in the UC group (P= 0·023, OR = 2·81), as well as in the CD group (P= 0·01, OR = 3·79). Thus, non‐carriers of IL‐1β allele 2 were more often present in the subgroup of patients carrying the IL‐1Ra allele 2. By contrast, no association of these alleles was detected in the group of healthy controls (P= 1·00, OR = 0·92). These results suggest that the IL‐1β/IL‐1Ra allelic cluster may participate in defining the biological basis of predisposition to chronic inflammatory bowel diseases.

Accuracy of Serologic Tests and HLA-DQ Typing for Diagnosing Celiac Disease

Context The value of adding HLA genetic typing to serologic testing for celiac disease is not well defined. Contribution In this prospective study of patients referred for evaluation of celiac disease, the test performance of combinations of genetic typing and serologic testing was similar to that of either strategy alone. Caution The small number of cases of celiac disease precluded meaningful comparisons of testing strategies. Implications The combination of genetic typing and serologic testing is about as accurate as either strategy alone. Neither is a substitute for small-bowel biopsy in the diagnosis of celiac disease. The Editors The high prevalence and clinical heterogeneity of celiac disease necessitate noninvasive tests for diagnosis. Specifically, tests are needed to select which patients should undergo small-bowel biopsy. Although celiac disease serologic tests, especially IgA tissue antitransglutaminase antibodies (TGA) and IgA antiendomysium antibodies (EMA), are often used for this purpose because of their reported high sensitivity (1), they may perform less well in the clinical setting (2). Most studies have not defined the usefulness of serologic tests prospectively (37), and in addition, some authors doubt the high sensitivity of these tests (8, 9). Susceptibility to celiac disease is related to the presence of distinct HLA-DQ heterodimersthe DQ2 heterodimer encoded by the alleles HLA-DQA1*05 and HLA-DQB1*02, and the DQ8 heterodimer encoded by the alleles HLA-DQA1*03 and HLA-DQB1*0302 (1014). One way to improve the selection of patients to undergo small-bowel biopsy may be to combine serologic tests with HLA-DQ typing (15, 16). We designed a prospective study to define the value of specific serologic tests, HLA-DQ typing, or both in diagnosing celiac disease. Methods The institutional review board of the VU University Medical Center, Amsterdam, the Netherlands, approved the study protocol. All participants received oral and written information according to the usual recommendations for medical research and the Declaration of Helsinki (17) and gave written informed consent. Patients The study was performed in an academic, mixed secondary and tertiary referral center that serves a population of about 200000 people. In the design phase of the study (19992000), the staff of departments of internal medicine and gastroenterology reviewed the literature and agreed that serologic tests could not substitute for small-bowel biopsy in the diagnostic work-up of celiac disease. Therefore, the policy was to perform small-bowel biopsy when celiac disease was suspected. Adults suspected of having celiac disease who were attending the endoscopy department for small-bowel biopsy were requested to give blood samples for serum antibody testing and HLA-DQ typing. We excluded patients younger than 18 years of age, those with known celiac disease, and patients who declined to undergo endoscopy. Endoscopy We performed upper gastrointestinal endoscopy with Olympus video endoscopes (GIF-NT140/160, Olympus Nederland, Zoeterwoude, the Netherlands) and obtained 4 oriented biopsy specimens from the distal duodenum (18). Serum Antibody Tests We performed serologic tests after obtaining small-bowel biopsy specimens in all patients to avoid referral bias. All serologic tests were determined anonymously without knowledge of the clinical status or histologic result. We determined IgA and IgG antigliadin antibodies (AGA-IgA and AGA-IgG, respectively) by using enzyme-linked immunosorbent assay (ELISA). We tested for EMA according to the method of Lerner and colleagues (19) by indirect immunofluorescence assay using monkey esophagus (16). Finally, TGA was determined by ELISA, essentially as described by Dieterich and colleagues (20), with guinea pig TGA (gp-TGA) (Sigma-Aldrich, Poole, United Kingdom; coating 10 g/mL in Tris hydrochloride [pH, 7.5] with 5-mmol/L of CaCl2) as the substrate (20). Sera were diluted and preincubated (30 minutes at room temperature) with 1% bovine serum albumin to avoid nonspecific binding (16, 21). The cutoff values for the titers of AGA and gp-TGA tests are based on measurements in control groups (blood donors, patients without celiac disease, and the general population age 2 to 4 years), and optimization was done by a receiver-operating characteristic curve analysis in well-defined patient groups. Because the recombinant human TGA (rh-TGA) assay became available when the study was already ongoing, we retrospectively reevaluated all samples from patients with an abnormal result on serologic testing or histologic examination by using rh-TGA as substrate (Roboscreen, Leipzig, Germany; coating 5 g/mL and same conditions as those for gp-TGA). When serologic test results did not match histologic findings, we measured total serum IgA and repeated the serologic tests. In cases of IgA deficiency, we evaluated TGA-IgG antibodies. We defined seropositivity as 1 or more positive measured antibody test results and seronegativity as negative results on all 4 tests. HLA-DQ Typing Whole blood was obtained for HLA-DQA1 and HLA-DQB1 genotyping. Polymerase chain reactionamplified exon 2 amplicons were generated for low- to medium-resolution typing in a combined, single-stranded conformation polymorphismheteroduplex assay by a semiautomated electrophoresis and gel-staining method on the PhastSystem (Amersham Pharmacia Biotech, Uppsala, Sweden). Alleles DQA1*05 and DQB1*02 (encoding the HLA-DQ2 heterodimer) and alleles DQA1*03 and DQB1*0302 (encoding the HLA-DQ8 heterodimer) could be reliably characterized in homozygous and heterozygous states. This method has been validated by using a panel of reference DNA against the Dynall Allset sequence-specific primers high-resolution typing kits (Dynal A.S., Oslo, Norway) (16, 22). Histologic Studies A gastrointestinal pathologist who was masked to clinical data evaluated the biopsy material, and an independent pathologist reviewed the samples when histologic examination was abnormal. Consensus was reached on the final diagnosis. Villous (crypt) anatomy and density of intraepithelial lymphocytes were assessed uniformly by using hematoxylineosin and immunohistologic anti-CD3 staining, respectively. Appendix Figures 1 and 2 show the histologic grading of abnormalities, based on the most severe change found according to the modified Marsh classification (23, 24). Appendix Figure 1. Small-bowel histologic findings (Marsh 0 to II). A. B. C. Appendix Figure 2. Small-bowel histologic findings (Marsh IIIa to IIIc). A. B. C. Diagnosis and Follow-up of Celiac Disease The diagnosis of celiac disease was based on the European Society for Paediatric Gastroenterology, Hepatology and Nutrition criteria, revised in 1989 and published in 1990, by identifying characteristic histologic findings (Marsh III) on small-bowel biopsy and unequivocal clinical resolution after a gluten-free diet was initiated (25). Thus, by this definition and for this study, the diagnosis of celiac disease did not require follow-up biopsy. However, we assessed histologic response in most patients and serologic response in all patients who were found to have celiac disease. We defined a serologic response as the disappearance of initially positive celiac disease antibody test results and histologic response as the regression of villi to Marsh 0 to II on a repeated biopsy at least 12 months after a gluten-free diet was initiated (24). Statistical Analysis We compared results of serologic tests and HLA-DQ typing with the diagnosis of celiac disease as previously defined. We performed statistical analysis by using SPSS software, version 11.0 (SPSS, Chicago, Illinois). To calculate exact binomial CIs, we used StatXact software, version 7.0.0 (Cytel Software, Cambridge, Massachusetts). We used 2 2 tables (Bayes theorem) to calculate sensitivities and specificities, predictive values, and likelihood ratios. We used the t test and Fisher exact test to compare continuous data and categorical data, respectively. We calculated posttest probabilities (and CIs) of celiac disease for different diagnostic tests or a combination thereof by using the method recommended by Altman (26). Role of the Funding Source The study received no funding. Results Patients Between January 2001 and January 2004, 502 consecutive patients (originally from community practices in the Amsterdam area) were referred from outpatient internal medicine and gastroenterology clinics for endoscopy and small-bowel biopsy for the diagnosis of celiac disease. Another 16 inpatients were referred from the internal medicine and gastroenterology inpatient wards. No referred patient was under the care of the investigators. We excluded 55 (10.6%) patients because they declined to participate in the study after the small-bowel biopsy. Age, sex, body mass index, ethnicity, and indications for referral did not statistically significantly differ between those included in and those excluded from the study (data not shown). The available serologic data (n = 20), HLA-DQ data (n = 4), and histologic examinations (n = 55) suggested the absence of celiac disease in excluded patients. Therefore, 463 patients were included in the study: 346 (75%) were unrelated Dutch Caucasian persons and 117 (25%) were not Caucasian (Indian, Chinese, North African [Arab], and Central African [black], in descending order of frequency). All patients were on a normal diet at the time of inclusion. Table 1 summarizes the general characteristics and indications for small-bowel biopsy of patients without and with celiac disease. Table 1. Characteristics of and Indications for Referral in Patients without and with Celiac Disease Patients with Celiac Disease Of the 463 patients enrolled, 16 (3.46% [95% CI, 1.99% to 5.55%]) fulfilled the diagnostic criteria for celiac disease (Figure) (25) within a median follow-up interval of 22 months (range, 11 to 44 months). Biopsy readings of the 2 pathologists were concor

Distribution of four polymorphisms in the tumour necrosis factor (TNF) genes in patients with inflammatory bowel disease (IBD)

In 153 patients with IBD, 64 with Crohns disease (CD), and 89 with ulcerative colitis (UC), as well as in 54 healthy controls (HC), the frequencies of four known di‐allelic polymorphisms in the genes for TNF‐α and lymphotoxin alpha (LTα) were investigated. In the Dutch population, the alleles of these four polymorphisms are present in only five combinations, called TNF haplotypes: TNF‐C, ‐E, ‐H, ‐I, ‐P. Furthermore, the relation with the presence of perinuclear anti‐neutrophil cytoplasmic autoantibodies (P‐ANCA) was studied. A small, but statistically significant, association between the polymorphism at position ‐308 in the promoter region of the TNF‐α gene and UC was found. The frequency of the uncommon TNF‐α ‐308 allele 2 was found to be decreased in patients with UC compared with HC (allele frequency of allele 2 in UC patients 0±15 versus 0±25 in HC, P= 0±044). No significant differences in distribution of the TNF haplotypes were found between IBD patients and HC, although there was a tendency towards a higher frequency of the TNF‐C haplotype in UC patients compared with controls (haplotype frequency 22%versus 13%; P= 0±19). No statistically significant differences in distribution of the TNF haplotypes were observed between P‐ANCA‐positive and P‐ANCA‐negative UC patients. The strength of the associations indicates that TNF genes are not markers for the predisposition to suffer from IBD. They may, however, be markers of subsets of patients with UC and CD.

Genetic markers in clinically well defined patients with ulcerative colitis (UC)

Results of genetic association studies in UC are conflicting. We propose that the power of candidate gene studies will increase when disease heterogeneity is taken into account. Phenotype frequencies of molecularly defined HLA‐DR alleles, polymorphisms in the tumour necrosis factor‐alpha (TNF‐α), lymphotoxin‐alpha (LT‐α), IL‐1 receptor antagonist (IL‐1Ra) and IL‐1β genes were determined in 98 clinically well characterized UC patients with a mean period of follow up of 10 years, and ethnically matched healthy controls (HC). The alleles HLA‐DRB1*0103 (phenotype frequency 6% versus 0.2%; Pu2003=u20030.0002; odds ratio (OR) 27.6) and DRB1*15 (41% versus 26%; Pu2003=u20030.001; ORu2003=u20032.0, compared with HC) were associated with overall disease susceptibility. Subgroup analysis revealed that DRB1*15 was only increased in females (53% versus 24%; Pu2003<u20030.0001; ORu2003=u20033.5), but not in males. With regard to disease localization, all DRB1*0103+ patients had extensive disease (Pu2003<u20030.002; ORu2003=u200333.5), and DRB1*15 was found in 59% of females with extensive colitis (Pu2003<u20030.0001; ORu2003=u20034.4). DRB1*0103 was significantly increased in patients undergoing colectomy (Pu2003<u20030.0002; ORu2003=u200384). No association between overall disease susceptibility and the cytokine gene polymorphisms were found. Subgroup analysis revealed several significant associations, but most did not retain significance when corrected for multiple comparisons. However, a noticeable finding was that haplotype TNF‐C was significantly associated with progression in extent of disease (Pu2003=u20030.003, ORu2003=u200320.4). This study provides additional evidence for the role of DRB1 alleles in the susceptibility to UC, and supports the hypothesis that these alleles may determine the severity of the disease. The cytokine gene polymorphisms evaluated in this study do not seem to be strong risk factors for the overall disease susceptibility in UC, but may be involved in determining the severity of the disease.

The polymorphic IL-1B and IL-1RN genes in the aetiopathogenesis of peptic ulcer

Besides environmental factors, the genetic background of an individual may contribute to the development and final outcome of peptic ulcer disease. Interleukin‐1β (IL‐1β) and the interleukin‐1 receptor antagonist (IL‐1ra) are cytokines that play a key role in modulating the inflammatory response in the gastrointestinal mucosa. This study aimed to investigate whether polymorphisms in the IL‐1B and IL‐RN genes are involved in the susceptibility to and final outcome of peptic ulcer disease. DNA from 179 unrelated Spanish Caucasian patients with peptic ulcer diseases and 99 ethnically matched healthy controls was typed for the TaqI polymorphism at position +u200a3954 in the IL‐1B gene and the variable number of tandem repeats polymorphism in intron 2 of the IL‐1RN gene. The determination of Helicobacter pylori status and non‐steroidal anti‐inflammatory drug (NSAIDs) use was studied in all patients and in controls. H. pylori infection and NSAID use were more frequent in ulcer patients than in controls. There were no significant differences in carriage rate, genotype and allele frequencies of the IL‐1RN and the IL‐1B+3954 gene polymorphisms between peptic ulcer patients and controls. However, a strong allelic association between IL‐1B and IL‐1RN genes was found in duodenal ulcer patients (Pu2003<u20030·0006). Logistic regression identified H. pylori infection and NSAIDs use as independent risk factors for peptic ulcer diseases whereas the simultaneous carriage of IL‐1B+3954 allele 2 and IL‐1RN allele 2 was associated with reduced risk for duodenal ulcer disease (OR: 0·37, 95% CIu2003=u20030·14–0·9). Our data suggest that IL‐1B and IL‐1RN genes in addition to bacterial and environmental factors play a key role in determining the final outcome of peptic ulcer disease.

Evidence for genetic heterogeneity in inflammatory bowel disease (IBD); HLA genes in the predisposition to suffer from ulcerative colitis (UC) and Crohn's disease (CD)

Family and epidemiological studies support a genetic susceptibility to UC and CD. Conflicting reports regarding associations between UC and HLA‐DR2 and between CD and various HLA alleles have been published. The aim of this study was to determine whether molecularly defined HLA‐DR genes are associated with these diseases in a Dutch group of patients. Fifty‐nine unrelated Dutch UC patients and 89 CD patients were typed using DNA‐based methods. A total of 2400 healthy local blood donors served as controls. The phenotype frequency of the HLA‐DRB1*15 allele was increased in UC patients compared with controls (42% versus 26% in controls; Pu2003=u20030.006; odds ratio (OR)u2003=u20032.1), and was predominantly found in female patients (53% versus 24%; Pu2003=u20030.001; ORu2003=u20033.5). The DRB1*15 allele was increased in UC patients having a positive family history (Pu2003=u20030.01; ORu2003=u20035.8). Among the 16 patients who showed an increase in extent of disease during follow up, 10 were DRB1*15+ (Pu2003=u20030.002; ORu2003=u20034.8). The frequency of the DRB1*13 allele was decreased in patients with UC (15% versus 28% in controls; Pu2003=u20030.04; ORu2003=u20030.5). In CD, no association was observed between disease or particular clinical subgroups and any allele tested. The present study provides additional evidence for the genetic association between UC and HLA‐DRB1*15, and supports recent findings that the susceptibility gene(s) for CD is not located in the HLA class II region.

Association of interleukin 1 gene family polymorphisms with duodenal ulcer disease.

Cytokine genes taking part in the immunological response to Helicobacter pylori infection are good candidates to study for genetic predisposition to duodenal ulcer disease (DU). Among cytokines, interleukin (IL)‐1β and its natural specific inhibitor, the interleukin‐1 receptor antagonist, are cytokines that play a key role in regulating gastric acid secretion and modulating the immune response in the gastrointestinal mucosa. We aimed to investigate whether polymorphisms in the IL‐1B and IL‐1RN genes are involved in the susceptibility to duodenal ulcer. DNA from 131 unrelated Spanish Caucasian patients with DU and 105 ethnically matched healthy controls was typed for the IL‐1B‐511, IL‐1B‐31, and IL‐1Bu2003+u20033954 gene polymorphisms, and the VNTR polymorphism in intron 2 of the IL‐1RN gene by polymerase chain reaction (PCR)‐based methods and TaqMan assays. H. pylori status and non‐steroidal anti‐inflammatory drugs (NSAIDs) use was determined in all patients and controls. Logistic regression analysis identified H. pylori infection (OR: 9·74; 95%CIu2003=u20033·53–26·89) and NSAIDs use (OR: 8·82; 95%CIu2003=u20033·51–22·17) as independent risk factors for DU. In addition, the simultaneous carriage of IL‐1RN*2, IL‐1B‐511*C, IL‐1B‐31*T and IL‐1Bu2003+u20033954*C alleles was a genetic risk factor for DU in patients with H. pylori infection (OR: 3·22; 95%CIu2003=u20031·09–9·47). No significant differences in IL‐1RN and IL‐1B genotypes were found when patients were categorized according to gender, age of onset, smoking habit, NSAIDs use, type of complication and positive family history. Our results provide further evidence that host genetic factors play a key role in the pathogenesis of duodenal ulcer.

Coeliac disease: emerging in China?

We read with interest the leading article by Hunt and van Heel on recent advances in coeliac disease (CD) genetics.1 They suggested that further investigation of the coeliac-associated single nucleotide polymorphisms (SNPs) in other populations was needed. Our recent work may help to push this research work in the Chinese population. Here we report on a serological screening for CD in China. CD has been historically considered to be absent in the Far East (China, Japan, Korea, Malaysia, etc.).2 However, since the major known risk factors for CD are common in China, we used serological tests for immunoglobulin G (IgG) antigliadin antibodies (AGAs) and IgA antitissue transglutaminase antibodies (tTGs) to screen for CD in high risk patients,3 …

In vitro production of TNFα, IL-6 and sil-2R in Chinese patients with ulcerative colitis

AIM To determine the tumor necrosis factor alpha (TNFα), interleukin 6 (IL-6) and soluble interleukin 2 receptor (sIL-2r) from peripheral blood mononuclear cells (PBMC) in 25 Chinese patients with ulcerative colitis and 20 healthy controls. METHODS PBMC were isolated by density gradient centrifugation of heparinized blood and cultures for 24 or 48 hours by stimulation with LPS or PHA. TNFα and sIL-2r were measured by ELISA method and IL-6 measured by biossay. RESULTS TNFα production stimulated by LPS and sIL-2r production by PHA in ulcerative colitis were significantly lower than in healthy controls (TNFα 509(46-7244)ng/L vs 1995(117-18 950)ng/ L, P 0.05; sIL-2r 264U/ ml±115U/ml vs 236U/ml±139U/ml, P>0.05). IL-6 production by spontaneous release from PBMC in ulcerative colitis group was 109U/ml±94U/ml vs 44U/ml±39U/ml for those in healthy controls, P <0.01. IL-6 stimulated by LPS in ulcerative colitis group was (261U/ml±80U/ml) higher than in healthy controls (102U/ml±54U/ml, P<0.01). No correlation of TNFα , IL-6, sIL-2r production was found to disease activity, disease location and medication. CONCLUSION Cytokine production from PBMC was also disturbed in Chinese patients with ulcerative colitis.