Population-based case control study of seroprevalence of Mycobacterium paratuberculosis in patients with Crohn's disease and ulcerative colitis

There is renewed enthusiasm for exploring the possibility that Mycobacterium paratuberculosis may be causative in Crohn's disease (CD). We aimed to determine whether CD subjects are more likely to be M. paratuberculosis seropositive than controls. Using our population-based University of Manitoba Inflammatory Bowel Disease Research Registry, we recruited CD and ulcerative colitis (UC) subjects between 18 and 50 years of age for a study involving detailed questionnaires and venipuncture. We accessed the population-based databases of Manitoba Health (single provincial health insurer) to get age-, gender-, and geography-matched controls to our inflammatory bowel disease (IBD) population. We asked enrolling IBD subjects for potential nonaffected sibling controls. We used an enzyme-linked immunosorbent assay (ELISA) for serum antibodies to M. paratuberculosis initially developed for cattle but adapted for human use. The rate of positive ELISA results, based on previously published interpretation criteria, was significantly higher for all study groups. There was no difference in M. paratuberculosis seropositivity rate among CD patients (37.8%; n = 283), UC patients (34.7%; n = 144), healthy controls (33.6%; n = 402), and nonaffected siblings (34.1%; n = 138). For siblings, there was no correlation between M. paratuberculosis serological status and that of the corresponding IBD affected sibling. None of the demographic or questionnaire variables studied were predictive of M. paratuberculosis status. Subjects with CD and UC were less likely to have ingested unpasteurized milk and less likely to have had a non-tap water source as a primary water source. In conclusion, in this population-based case control study, the M. paratuberculosis seropositivity rate was approximately 35% for all groups and there was no difference in rates between CD patients, UC patients, healthy controls, or nonaffected siblings. The much higher rate of seropositivity for subjects from Manitoba, Canada, than for those from Denmark or Wisconsin cannot be obviously explained. While these data seem to refute any association of CD with M. paratuberculosis, the high seroprevalence in Manitobans raises the possibility that the high rates of CD in Manitoba could be related to high exposure rates for M. paratuberculosis. Hence, the possibility of an association between M. paratuberculosis and CD remains inconclusive.     

Susceptibility testing of multidrug-resistant Staphylococcus aureus with the Sceptor microdilution system.

The antimicrobial susceptibilities of Staphylococcus aureus isolates were concurrently determined by the Sceptor system (BBL Microbiology Systems, Cockeysville, Md.) and by the standard disk diffusion method. For the methicillin-resistant isolates, there was greater than 98% agreement between the two test results with penicillin G, erythromycin, clindamycin, tetracycline, gentamicin, and tobramycin. Major disagreements (susceptible by one method and resistant by the other) were 7% for methicillin, 13.5% for cephalothin, 3.5% for cefamandole, and 27% for amikacin. The major discrepancies for methicillin were eliminated by supplementing the inoculum broth with salt. For methicillin-susceptible isolates, agreement between the two methods was 96 to 100% for all antibiotics except amikacin.     

Evaluation of the Interaction Between TGF <Emphasis Type="Italic">β</Emphasis> and Nitric Oxide in the Mechanisms of Progression of Colon Carcinoma

It is recognised that stromal cells determine cancer progression. We have previously shown that active TGFβ produced by rat colon carcinoma cells modulated NO production in rat endothelial cells. To elucidate the role of TGFβ and NO in the mechanisms of interaction of colon carcinoma cells with stromal cells and in cancer progression, we transfected REGb cells, a regressive colon carcinoma clone secreting latent TGFβ, with a cDNA encoding for a constitutively-secreted active TGFβ. Out of 20 injected rats only one tumour progressed, which was resected and sub-cultured (ReBeta cells). ReBeta cells secreted high levels of active TGFβ. The adhesive properties of REGb and Rebeta cells to endothelial cells were similar, showing that the secretion of active TGFβ is not involved in tumour cell adhesion to endothelial cells. ReBeta, but not REGb, cell culture supernatants inhibited cytokine-dependent NO secretion by endothelial cells, but inhibition of NO production was similar in co-cultures of REGb or ReBeta cells with endothelial cells. Therefore, secretion of active TGFβ regulated endothelial NO synthase activity when tumour cells were distant from, but not in direct contact with, endothelial cells. However, only ReBeta cells inhibited cytokine-dependent secretion of NO in coculture with macrophages, indicating that the active-TGFβ–NO axis confers an advantage for tumour cells in their interaction with macrophages rather than endothelial cells in cancer progression.     

Currant allergy and the Rosaceae-grass pollen allergy syndrome: a case report.

BACKGROUND: Despite the increasing use of currants in culinary recipes, currant allergy has rarely been reported. OBJECTIVES: To study a case of currant allergy and to explore cross-reactivity between grass pollen and Rosaceae family fruit allergens. METHODS: Skin prick tests to pollen and skin prick-to-prick tests with currants and peach were performed. Specific IgE levels were determined using the CAP method. We prepared a protein extract of 0.1 mg/mL in phosphate-buffered saline using red currant in the presence of protease inhibitors. Immunoblot inhibition studies were performed to explore cross-reactivity between grass pollen and currant allergens. RESULTS: Skin prick test results were positive to Dactylis, arizonic, and olive pollens. Results of skin prick-to-prick tests with fresh red and black currants were negative and positive, respectively, to peach. The specific IgE level was 5.7 KU/L to red currant and 2.92 KU/L to peach (CAP). Western blot analysis with red currant extract revealed specific IgE protein bands of 37 and 26 kDa. Preincubation of sera with extracts from red currant and peach inhibited both IgE bands, and preincubation with Dactylis pollen inhibited the 37-kDa band only. CONCLUSIONS: We report a case of allergy to grass pollen with an oral allergy syndrome involving several fruits from 2 different families of the Rosidae subclass confirmed by in vitro tests. Inhibition studies demonstrated cross-reactivity between different fruits (currant and raspberry) from the Rosidae subclass and were incomplete with grass pollen allergens.     

Sensitive and Specific Identification of Neospora caninum Infection of Cattle Based on Detection of Serum Antibodies to Recombinant Ncp29

Neosporosis is an economically important disease of dairy cattle caused by the protozoan Neospora caninum. Diagnostic tests for neosporosis are complicated by the potential for cross-reaction of antibodies to antigens that are similar between N. caninum and closely related parasites Toxoplasma gondii and Sarcocystis cruzi. To provide a sensitive and specific assay for detecting antibodies to N. caninum in the serum of infected animals, we have investigated a recombinant form of the antigen known as Ncp29 (rNcp29), which is a major surface protein of the parasite. Ncp29 is encoded by a gene that is homologous to the SAG1 gene previously characterized from T. gondii. An enzyme-linked immunosorbent assay (ELISA) was used to screen animals for the presence of serum antibodies specific to rNcp29. The rNcp29 ELISA readily distinguished between cattle known to be infected with N. caninum (optical density [OD] > 1.2 at 1:500 or greater dilution) and negative controls (OD < 0.5 at 1:500). Additionally, sera from animals that were infected with T. gondii or S. cruzi were negative. The rNcp29 ELISA developed here provides a specific and sensitive assay for detecting neosporosis in cattle.     

Polyclonal antibody responses against Listeria monocytogenes 4b surface proteins in asymptomatic human carriers

Listeria monocytogenes 4b surface protein extract was used in an immunoblot assay to analyze the antibody profile in sera from 40 healthy urban workers (U group), 40 healthy slaughterhouse workers (W group) and four healthy carriers with positive L. monocytogenes 4b feces culture (positive controls). In addition, pooled rabbit sera, before and after immunization with L. monocytogenes 4b whole‐cell suspension, were analyzed against L. monocytogenes 4b surface protein extract in order to determine the specific L. monocytogenes 4b antibody pattern. The degree of similarity (S) between such a pattern and each of those obtained with serum samples from the three subject groups was assessed. For U and W group sera, mean S values were 24.3 ± 13.5 and 32.8 ± 14.3, respectively. An S value greater than 65, corresponding to mean S_U value ± 3 standard deviation, was considered as an indicator of a healthy carrier. Thus, the estimated healthy carrier percentages found in U and W groups were 2.5 and 5%, respectively. The proposed immunoblot assay may prove a useful tool for epidemiological surveys to determine whether a healthy person is a L. monocytogenes 4b carrier.     

Video analysis of communication in paediatric consultations in primary care.

BACKGROUND: There is a paucity of research evidence concerning communication in paediatric consultations between GPs, adults, and child patients. AIM: This study was carried out to identify features of the interaction between a doctor, a child patient aged 6-12 years, and their carer in the consultation associated with the child's participation. DESIGN OF STUDY: A qualitative analysis of video recordings of 31 primary care paediatric consultations was undertaken, using strategies from the methodology of conversation analysis. SETTING: Primary care, Suffolk, UK. METHOD: NHS GPs from three primary care trusts (PCTs), were invited to participate in this study. Sixteen volunteers from this sample took part. RESULTS: Analysis of the interaction in the consultations revealed that the children had little involvement. Children participated when invited to do so, and took more time than adults to answer a doctor's question. An adult carer was less likely to answer on behalf of a child, when they were in a position to see that the doctor's gaze was directed at the child, and the doctor addressed the child by name. Adult carers, who had not voiced their own concerns first, were seen to interrupt doctor-child talk. In consultations where the participants sat in a triangular arrangement, all parties being an equal distance apart, triadic talk was noted. CONCLUSION: Child involvement in the primary care consultation is associated with adult carers being able to voice their own concerns early in the consultation, and children being invited to speak with the appropriate recipient design.     

Characterization of translational frame exception patients in Duchenne/Becker muscular dystrophy

The clinical progression of Duchenne muscular dystrophy (DMD)patients with deletions can be predicted in 93% of cases bywhether the deletion maintains or disrupts the translationalreading frame (frameshift hypothesis). We have identified andstudied a number of patients who have deletions that do notconform to the translational frame hypothesis. The most commonexception to the frameshift hypothesis is the deletion of exons3 to 7 which disrupts the translational reading frame. We identifieda Becker muscular dystrophy (BMD) patient, an intermediate,and a DMD patient with this deletion. In all three cases, dystrophinwas detected and localized to the membrane. One DMD patientwith an inframe deletion of exons 4–18 produced no dystrophin.One patient with a mild intermediate phenotype and a deletionof exon 45, which shifts the reading frame, produced no dystrophin.Two patients with large inframe deletions had discordant phenotypes(exons 3–41, DMD; exons 13–48, BMD), but both produceddystrophin that localized to the sarcolemma. The DMD patient,113, indicates that dystrophin with an intact carboxy terminuscan be produced in Duchenne patients at levels equivalent tosome Beckers. The dystrophin analysis from these patients, togetherwith patients reported in the literature, indicate that morethan one domain can localize dystrophin to the sarcolemma. Lastely,the data shows that although most patients show correlationof clinical severity to molecular data, there are rare patientswhich do not conform.     

Partial gonadal dysgenesis in a patient with a marker Y chromosome

We evaluated a patient with partial gonadal dysgenesis including a right dysgenetic testis and a left streak gonad with rudimentary fallopian tube and uterus. She had ambiguous external genitalia and was raised female. Although her height is normal (25th centile at age 12 years), she has some findings of Ullrich–Turner syndrome. Her karyotype was reported to be 46, X, + marker; subsequent molecular investigations showed the marker to be the short arm of the Y chromosome. Genomic DNA, isolated from leukocytes of the patient and her father, was digested with a variety of restriction endonucleases and subjected to Southern blot analysis. A positive hybridization signal was obtained with probes for the short arm of the Y chromosome (pRsY0.55, SRY, ZFY, 47Z, pY-190, and YC-2) in DNA from the patient, indicating the presence of most if not all of the short arm, while long arm probes (HinfA and pY3.4) indicated that at least 75% of the long arm of the Y chromosome was missing. The gene responsible for testicular determination (TDF) is on the distal portion of the short arm of the Y chromosome; Yq has no known influence on sex determination. Hence, the deletion of the long arm of the Y chromosome cannot explain the gonadal dysgenesis in this patient. One explanation for the gonadal dysgenesis and Ullrich–Turner phenotype in the patient could be undetected 45, X/46,X, + marY mosaicism but no such mosaicism was observed in peripheral lymphocytes. Several investigators have suggested the presence of an “anti-Turner” gene near TDF. Hence it is possible that the clinical phenotype in our patient results from a Y chromosomal defect in sequences flanking TDF, which reduces the function of both TDF and the “anti-Turner” genes.     

Transfer of Tumor Necrosis Factor-α to Rat Lung Induces Severe Pulmonary Inflammation and Patchy Interstitial Fibrogenesis with Induction of Transforming Growth Factor-β1 and Myofibroblasts

Tumor necrosis factor-α is up-regulated in a variety of different human immune-inflammatory and fibrotic pulmonary pathologies. However, its precise role in these pathologies and, in particular, the mechanism(s) by which it may induce fibrogenesis are not yet elucidated. Using a replication-deficient adenovirus to transfer the cDNA of tumor necrosis factor-α to rat lung, we have been able to study the effect of transient but prolonged (7 to 10 days) overexpression of tumor necrosis factor-α in normal adult pulmonary tissue. We have demonstrated that local overexpression resulted in severe pulmonary inflammation with significant increases in neutrophils, macrophages, and lymphocytes and, to a lesser extent, eosinophils, with a peak at day 7. By day 14, the inflammatory cell accumulation had declined, and fibrogenesis became evident, with fibroblast accumulation and deposition of extracellular matrix proteins. Fibrotic changes were patchy but persisted to beyond day 64. To elucidate the mechanism underlying this fibrogenesis, we examined bronchoalveolar fluids for the presence of the fibrogenic cytokine transforming growth factor-β1 and tissues for induction of α-smooth muscle actin-rich myofibroblasts. Transforming growth factor-β1 was transiently elevated from day 7 (peak at day 14) immediately preceding the onset of fibrogenesis. Furthermore, there was a striking accumulation of myofibroblasts from day 7, with the most extensive and intense immunostaining at day 14, ie, coincident with the up-regulation of transforming growth factor-β1 and onset of fibrogenesis. Thus, we have provided a model of tumor necrosis factor-α-mediated pulmonary inflammation and fibrosis in normal adult lung, and we suggest that the fibrogenesis may be mediated by the secondary up-regulation of transforming growth factor-β1 and induction of pulmonary myofibroblasts.