A. Salvador Peña

TNF-α promoter polymorphisms, production and susceptibility to multiple sclerosis in different groups of patients

TNF-alpha production in whole blood cultures upon stimulation with LPS was determined in 179 individuals from 61 families in order to characterise the magnitude of inherited differences in TNF-alpha production. The three families characterised by highest TNF production showed 7.1 +/- 0.3 ng TNF/ml upon culture with 10 ng LPS and 10.2 +/- 0.2 ng TNF/ml upon culture with 1000 ng LPS. in contrast to the three families characterised by the lowest TNF production that showed a production of 1.6 +/- 0.1 ng TNF upon culture with 10 ng and 2.5 +/- 0.2 ng/ml upon culture with 1000 ng LPS/ml. This difference could not be attributed to the promoter polymorphisms -308 G to A. -238 G to A or -376 G to A, although the -238 GA donors produced 2.1 +/- 0.9 ng TNF upon culture with 10 ng endotoxin compared to 3.2 +/- 2.2 ng TNF for the -238 GG donors. In line with these results the frequency of the -238 GG genotype was increased in hospitalized MS patients in a nursing home (100% 238GG, n = 57) compared to MS patients in an outpatients clinic (94% 238GG, n = 98) or Dutch controls (90% 238GG, n = 180). These results suggest that the -238 GG genotype is differently distributed in hospitalized MS patients in a nursing home.

European Trial of Cyclosporine in Chronic Active Crohn's Disease: A 12-Month Study

BACKGROUND & AIMSnThe role of cyclosporine in Crohns disease is controversial. This study aimed to delineate the long-term effect of cyclosporine in chronic active Crohns disease.nnnMETHODSnOne hundred eighty-two patients from 33 European centers were included. The patient cohort was stratified at entry into a stratum with low Crohns Disease Activity Index (CDAI) ( < 200) and high CDAI ( > 200). The low-activity group continued to receive the pretrial steroid dose for 2 months, and the high-activity group received 1 mg.kg-1.day-1 prednisone initially. During months 3 and 4, the dose of steroids was reduced stepwise to 5 mg/day in all patients. Placebo and cyclosporine (5 mg.kg-1.day-1) were administered throughout the 12-month study period. The main parameter of efficacy was the CDAI, and the main end point was the number of patients in remission at month 12.nnnRESULTSnDuring cyclosporine therapy, 35% (95% confidence interval [95% Cl], 25%-46%) of the patients achieved a full remission (CDAI, < 150) after 4 months compared with 27% (95% Cl, 18%-38%) in the placebo group (P > 0.05). At month 12, only 20% (95% Cl, 12%-31%) vs. 20% (95% Cl, 12%-31%) of the patients had maintained a continuous remission. No major differences between treatment groups were found within each of the two strata.nnnCONCLUSIONSnThe long-term treatment of chronic active Crohns disease with cyclosporine plus low-dose steroids does not offer an advantage compared with low-dose steroids alone.

Real-time polymerase chain reaction to diagnose lymphogranuloma venereum

To the Editor: An outbreak of rectal lymphogranuloma venereum (LGV) has been detected in the Netherlands among men who have sex with men (1–4). More cases of LGV in other European countries such as Belgium, France, and the United Kingdom have been reported, and the first cases have been detected in the United States as well. This infection is encountered not only by clinicians who treat sexually transmitted diseases but also by gastroenterologists. Both the European Surveillance of Sexually Transmitted Infections (http://www.essti.org) and the Centers for Disease Control and Prevention (http://www.cdc.gov) are working on outbreak warning and response systems to increase the awareness and the direct management of the LGV outbreak (5,6). n nDifferent approaches have been described to diagnose LGV infections (Figure). The first 3 approaches have serious disadvantages: cell culture is rarely available in routine diagnostic settings, polymerase chain reaction (PCR)-based restriction fragment length polymorphism (RFLP) analysis (usually nested PCR approaches are used) needs post-PCR restriction enzyme profiling, and sequencing requires additional analyses of sequence data to identify the Chlamydia trachomatis serovar responsible for infection. In addition, all 3 techniques are time consuming (at least 1–4 days to get a result), laborious, and require specially trained personnel in a sophisticated laboratory setting. Therefore, we developed a real-time PCR approach (TaqMan and Rotorgene) that can easily identify LGV strains in 2 hours with equipment that is available in almost all diagnostic settings. n n n nFigure n nDiagnosis of lymphogranuloma venereum. MIF, microimmunofluorescence; STI, sexually transmitted infection; PCR, polymerase chain reaction; RFLP, restriction fragment length polymorphism; PAA, poly acrylamide; BLAST, basic local alignment search tool. n n n nWe used the polymorphic membrane protein H gene (pmp gene) as a PCR target because it has a unique gap in LGV strains of C. trachomatis, compared to other serovars, which makes it highly specific. The following primers and probes were selected: LGV-F 5´ CTG TGC CAA CCT CAT CAT CAA 3´, LGV-R 5´ AGA CCC TTT CCG AGC ATC ACT 3´, and LGV MGB-probe 6-FAM-CCT GCT CCA ACA GT. Real-time PCR conditions (20-μL format) for TaqMan were as follows: 2× TaqMan Universal Mastermix (Applied Biosystems, Foster City, CA, USA), 18 pmol each primer, 0.2 μmol/L probe, and 2 μL (LGV L2) DNA or clinical sample; 2 min at 50°C, 10 min at 95°C, and 40 cycles of 15 sec at 95°C and 1 min at 60°C. Conditions for Rotorgene were as follows: 10× buffer (Hoffman-La Roche Ltd, Basel, Switzerland), 10 pmol each primer, 0.04 μmol/L probe, 2 μL (LGV L2) DNA or clinical sample; 2 min at 50°C, 10 min at 95°C, and 45 cycles of 15 sec at 95°C and 1 min at 60°C. By using a previously described serial dilution of LGV L2 (7), sensitivity was assessed as 0.01 inclusion-forming units for both real-time PCR assays. n nTo determine specificity, we tested different C. trachomatis serovars and serovariants A, B, Ba, C, D, Da, D-, E, F, G, Ga, H, I, Ia, I-, J, Jv, K, L1, L2, L2b, L3, C. muridarum (MoPn), C. pneumoniae, C. pecorum, C. psittaci, and 32 other microorganisms that normally reside in the human perianal and urogenital region and in the oropharynx. These organisms included gram-positive and gram-negative bacteria and yeast: Acinetobacter baumannii, Campylobacter jejuni, Candida albicans, other yeast, Enterobacter agglomerans, Enterococcus faecalis, Escherichia coli, Streptococcus spp., Haemophilus influenzae, Klebsiella pneumoniae, Mycoplasma spp., Neisseria meningitidis, Pasteurella spp., Pseudomonas aeruginosa, Salmonella enteritidis, Shigella sonnei, Staphylococcus aureus, and others. Only LGV strains L1, L2, L2b, and L3 tested positive in both the TaqMan and Rotorgene assays, which shows the analytical specificity of real-time PCR. n nSubsequently, we determined in a blinded setting the presence of LGV in a selected group of patients (clinical spectrum and epidemiology described elsewhere [8]) according to C. trachomatis–positive rectal swab (Chlamydia 2SP Collection & Transport Kit [Quelab] by commercially available PCR (COBAS AMPLICOR, Hoffman-La Roche Ltd). By using the 2 reference standard techniques to type C. trachomatis serovars (PCR-based RFLP of the omp1 gene or sequencing the variable segment 2 [VS-2] of the omp1 gene) (9,10) with DNA isolated from rectal swab specimens (standard isopropanol DNA isolation method), we identified 28 of 125 men as LGV-positive. These 28 samples were also positive in both the TaqMan and Rotorgene assays. We also identified 2 additional LGV infections, which were initially typed and then retested as single-strain infections with serovars E and D by both PCR-based RFLP analysis and VS-2 sequencing. This discrepancy is most likely due to a double infection, which will, in most cases, result in the preferential amplification of 1 strain in the omp1 PCR and PCR-based sequencing methods; in the TaqMan and Rotorgene assays, only LGV strains can be amplified. Whether this outbreak is partially technically driven must be assessed in the future by retrospectively investigating the presence of these LGV infections in men who have sex with men and the presence of the L2b strain in the past, since at present only LGV infections from 2003 to 2005 have been investigated.

Slow Epidemic of Lymphogranuloma Venereum L2b Strain

We traced the Chlamydia trachomatis L2b variant in Amsterdam and San Francisco. All recent lymphogranuloma venereum cases in Amsterdam were caused by the L2b variant. This variant was also present in the 1980s in San Francisco. Thus, the current outbreak is most likely a slowly evolving epidemic.

Computed tomography and granulocyte scintigraphy in active inflammatory bowel disease. Comparison with endoscopy and operative findings.

The accuracy of computed tomography (CT) and [99mTc]HMPAO granulocyte scintigraphy (GS) for detection of bowel localization, inflammatory activity, and complications in acute inflammatory bowel disease (IBD) was prospectively studied in 32 patients. Of each bowel segment, findings on CT and GS were scored by one blinded observer. Findings on operation or endoscopy served as the gold standard. In Crohns disease (CD, 17 patients), CT detected bowel pathology (sensitivity 71%, specificity 98%), abscesses (sensitivity and specificity 100%), and fistulas (sensitivity 80%, specificity 100%). In CD, GS had a sensitivity of 79% and a specificity of 98% for detection of inflammatory activity. The detection of complications with GS was poor. Segmental inflammatory activity correlated with endoscopy-operative findings for CT (r=0.86,P<0.0001) and GS (r=0.86,P<0.0001). In ulcerative colitis (UC, 15 patients), GS predicted proximal extension of bowel involvement better than CT. In CD, CT is superior to GS for localization of both active and fibrostenotic bowel disease, and in detection of abscesses and fistulas. In UC, GS showed proximal extension more accurately than CT.

CTLA-4 and CD28 gene polymorphisms in susceptibility, clinical course and progression of multiple sclerosis.

The balance between CD28 and CTLA-4 signalling is important for regulation of the immune response. We were interested whether a genetically mediated disturbance of this balance could be related to susceptibility or severity of multiple sclerosis (MS). We examined three polymorphisms in these genes, CTLA-4-318, CTLA-4+49 and CD28-I3+17, in 514 patients with MS and 181 controls. As the loci cannot be assumed independent of each other, we analysed the effects of each of the three polymorphisms corrected for the presence of the other two. We found no association between carriership of any of the alleles either with susceptibility to MS or with clinical features. For a subgroup of patients, longitudinal magnetic resonance imaging (MRI) data were available. We observed no effects of the polymorphisms on brain and lesion volumes. These data suggest that the polymorphisms under investigation do not affect the risk of developing MS and have no influence on the course of disease.

Microscopic enteritis: Bucharest consensus

Microscopic enteritis (ME) is an inflammatory condition of the small bowel that leads to gastrointestinal symptoms, nutrient and micronutrient deficiency. It is characterised by microscopic or sub-microscopic abnormalities such as microvillus changes and enterocytic alterations in the absence of definite macroscopic changes using standard modern endoscopy. This work recognises a need to characterize disorders with microscopic and submicroscopic features, currently regarded as functional or non-specific entities, to obtain further understanding of their clinical relevance. The consensus working party reviewed statements about the aetiology, diagnosis and symptoms associated with ME and proposes an algorithm for its investigation and treatment. Following the 5(th) International Course in Digestive Pathology in Bucharest in November 2012, an international group of 21 interested pathologists and gastroenterologists formed a working party with a view to formulating a consensus statement on ME. A five-step agreement scale (from strong agreement to strong disagreement) was used to score 21 statements, independently. There was strong agreement on all statements about ME histology (95%-100%). Statements concerning diagnosis achieved 85% to 100% agreement. A statement on the management of ME elicited agreement from the lowest rate (60%) up to 100%. The remaining two categories showed general agreement between experts on clinical presentation (75%-95%) and pathogenesis (80%-90%) of ME. There was strong agreement on the histological definition of ME. Weaker agreement on management indicates a need for further investigations, better definitions and clinical trials to produce quality guidelines for management. This ME consensus is a step toward greater recognition of a significant entity affecting symptomatic patients previously labelled as non-specific or functional enteropathy.

IBD1 and IBD3 determine location of Crohn's Disease in the Spanish population

Background:Crohn’s disease is a heterogeneous disease from both genetic and clinical points of view. Aims:To look for associations between distinct genetic polymorphisms and clinical subgroups of the disease. Subjects:A total of 210 patients and 343 healthy control subjects, all adult, unrelated, white, Spanish individuals. Methods:DNA was purified from peripheral blood samples and was typed by sequence-specific oligonucleotide polymerase chain reaction (PCR) method for human leukocyte antigen (HLA)-DRB1 alleles (IBD3) and by allele-specific PCR for NOD2/CARD15 (IBD1) polymorphisms. Results:NOD2/CARD15 mutations and HLA-DRB1*07 confer susceptibility only to the ileal location of the disease, whereas HLA-DRB1*0103 is associated only with the colonic location of the disease. The IBD3 effect was overshadowed by IBD1 mutations when present. Conclusion:The studied genetic polymorphisms of Crohn’s disease basically determine the location of the disease and, only secondarily, the clinical form of the disease. This appears to be true for both inflammatory bowel diseases as HLA-DRB1*0103 is associated both with colonic Crohn’s disease and ulcerative colitis.

HLA-DRB1*1501 and Spinal Cord Magnetic Resonance Imaging Lesions in Multiple Sclerosis

BACKGROUNDnMultiple sclerosis (MS) is a heterogeneous neurologic disease with extensive variation with respect to the most affected central nervous system region (brain vs spinal cord).nnnOBJECTIVEnTo test the hypothesis that this variation in lesion location (brain vs spinal cord) might be (partially) genetically determined.nnnDESIGNnCandidate gene study.nnnSETTINGnAcademic research.nnnPATIENTSnPatients were selected for the availability of DNA material, clinical variables, and brain and spinal cord magnetic resonance images (evaluating T2-weighted lesion load in the brain and the number of spinal cord lesions).nnnMAIN OUTCOME MEASURESnFor genotyping, we used a DNA chip containing a set of genes mentioned in previous publications noting their relation to different phenotypes of MS. We assessed the association between brain and spinal cord abnormalities and the genotypes of the patients.nnnRESULTSnOne hundred fifty patients were included in the analysis. Five single-nucleotide polymorphisms within the major histocompatibility complex region were associated with the number of focal abnormalities in the spinal cord. The most significant was rs3135388 (surrogate marker for the HLA-DRB1*1501 allele). Carriers of HLA-DRB1*1501 had a median of 4 spinal cord lesions compared with 2 lesions for noncarriers (P < .001). No significant association was noted between the single-nucleotide polymorphisms and T2-weighted lesion load in the brain.nnnCONCLUSIONSnCarriership of HLA-DRB1*1501 (via rs3135388) was associated with the extent of focal abnormalities in the spinal cord. Spinal cord lesions might be an explanation for increased MS disease severity in patients carrying HLA-DRB1*1501.

The CD14 functional gene polymorphism -260 C>T is not involved in either the susceptibility to Chlamydia trachomatis infection or the development of tubal pathology

BackgroundThe functional polymorphism -260 C>T in the LPS sensing TLR4 co-receptor CD14 gene enhances the transcriptional activity and results in a higher CD14 receptor density. Individuals carrying the T/T genotype also have significantly higher serum levels of soluble CD14. The T allele of this polymorphism has recently been linked to Chlamydia pneumoniae infection. We investigated the role of the CD14 -260 C>T polymorphism in the susceptibility to and severity (defined as subfertility and/or tubal pathology) of C. trachomatis infection in Dutch Caucasian women.MethodsThe different CD14 -260 C>T genotypes were assessed by PCR-based RFLP analysis in three cohorts: 1) A cohort (n = 576) of women attending a STD clinic, 2) a cohort (n = 253) of women with subfertility, and 3) an ethnically matched control cohort (n = 170). The following variables were used in the analysis: In cohort 1 the CT-DNA status, CT IgG serology status, self-reported symptoms and in cohort 2, the CT IgG serology status and the tubal status at laparoscopy.ResultsIn the control cohort the CC, CT and TT genotype distribution was: 28.2%, 48.2%, and 23.5% respectively. No differences were found in the overall prevalence of CD14 -260 genotypes (28.1%, 50.7%, and 21.2%) in cohort 1 when compared to the control cohort. Also no differences were observed in women with or without CT-DNA, with or without serological CT responses, with or without symptoms, or in combinations of these three variables. In subfertile women with tubal pathology (cohort 2, n = 50) the genotype distribution was 28.0%, 48.0%, and 24.0% and in subfertile women without tubal pathology (n = 203), 27.6%, 49.3% and 23.2%. The genotype distribution was unchanged when CT IgG status was introduced in the analyses.ConclusionThe CD14 -260 C>T genotype distributions were identical in all three cohorts, showing that this polymorphism is not involved in the susceptibility to or severity of sequelae of C. trachomatis infection.