Isolation and Expression of a Gene Cluster Responsible for Biosynthesis of the Glycopeptidolipid Antigens of \u3cem\u3eMicobacterium avium\u3c/em\u3e

Bacteria within the Mycobacterium avium complex are prominent in the environment and are a source of serious disseminated infections in patients with AIDS. Serovars of the M. avium complex are distinguished from all other mycobacteria and from one another by the presence of highly antigenic glycolipids, the glycopeptidolipids, on their surfaces. A genomic library of DNA from serovar 2 of the M. avium complex was constructed in the Escherichia coli-Mycobacterium shuttle cosmid, pYUB18, and used to clone and express in Mycobacterium smegmatis the genes responsible for the biosynthesis of the oligosaccharide segment of the M. avium serovar 2-specific glycopeptidolipid. The responsible gene cluster was mapped to a 22- to 27-kb functional region of the M. avium genome. The recombinant glycolipid was also isolated by high-pressure liquid chromatography and chemically characterized, by gas chromatography-mass spectrometry and fast atom bombardment-mass spectrometry, to demonstrate that the lipopeptide core originated in M. smegmatis, whereas the oligosaccharide segment arose from the cloned M. avium genes. This first-time demonstration of the cloning and expression, in a nonpathogenic mycobacterium, of the genes encoding complex cell wall glycoconjugates from a pathogenic mycobacterium presents a new approach for studying the role of such products in disease processes

Context-dependent representation of within- and between-model uncertainty: Aggregating probabilistic predictions in infectious disease epidemiology

Probabilistic predictions support public health planning and decision making, especially in infectious disease emergencies. Aggregating outputs from multiple models yields more robust predictions of outcomes and associated uncertainty. While the selection of an aggregation method can be guided by retrospective performance evaluations, this is not always possible. For example, if predictions are conditional on assumptions about how the future will unfold (e.g. possible interventions), these assumptions may never materialize, precluding any direct comparison between predictions and observations. Here, we summarize literature on aggregating probabilistic predictions, illustrate various methods for infectious disease predictions via simulation, and present a strategy for choosing an aggregation method when empirical validation cannot be used. We focus on the linear opinion pool (LOP) and Vincent average, common methods that make different assumptions about between-prediction uncertainty. We contend that assumptions of the aggregation method should align with a hypothesis about how uncertainty is expressed within and between predictions from different sources. The LOP assumes that between-prediction uncertainty is meaningful and should be retained, while the Vincent average assumes that between-prediction uncertainty is akin to sampling error and should not be preserved. We provide an R package for implementation. Given the rising importance of multi-model infectious disease hubs, our work provides useful guidance on aggregation and a deeper understanding of the benefits and risks of different approaches

IL-21 acts directly on B cells to regulate Bcl-6 expression and germinal center responses

During T cell–dependent responses, B cells can either differentiate extrafollicularly into short-lived plasma cells or enter follicles to form germinal centers (GCs). Interactions with T follicular helper (Tfh) cells are required for GC formation and for selection of somatically mutated GC B cells. Interleukin (IL)-21 has been reported to play a role in Tfh cell formation and in B cell growth, survival, and isotype switching. To date, it is unclear whether the effect of IL-21 on GC formation is predominantly a consequence of this cytokine acting directly on the Tfh cells or if IL-21 directly influences GC B cells. We show that IL-21 acts in a B cell–intrinsic fashion to control GC B cell formation. Mixed bone marrow chimeras identified a significant B cell–autonomous effect of IL-21 receptor (R) signaling throughout all stages of the GC response. IL-21 deficiency profoundly impaired affinity maturation and reduced the proportion of IgG1+ GC B cells but did not affect formation of early memory B cells. IL-21R was required on GC B cells for maximal expression of Bcl-6. In contrast to the requirement for IL-21 in the follicular response to sheep red blood cells, a purely extrafollicular antibody response to Salmonella dominated by IgG2a was intact in the absence of IL-21

B cell priming for extrafollicular antibody responses requires Bcl-6 expression by T cells

Bcl-6 expression in CD4+ T cells is required to generate extrafollicular antibody responses

Presence of Mycoplasma fermentans in the bloodstream of Mexican patients with rheumatoid arthritis and IgM and IgG antibodies against whole microorganism

<p>Abstract</p> <p>Background</p> <p>Increasing evidence incriminates bacteria, especially <it>Mycoplasma fermentans</it>, as possible arthritogenic agents in humans. The purpose of this study was to investigate <it>M. fermentans </it>in the bloodstream of patients with rheumatoid arthritis.</p> <p>Methods</p> <p>Two hundred and nineteen blood samples from patients with rheumatoid arthritis, systemic lupus erythematosus, antiphospholipid syndrome, and healthy individuals were screened by bacterial culture and direct PCR in order to detect mycoplasmas; IgM and IgG against <it>M. fermentans </it>PG18 were also detected by ELISA and Immunoblotting assays in patients with rheumatoid arthritis and healthy individuals.</p> <p>Results</p> <p>Blood samples from patients with antiphospholipid syndrome and healthy individuals were negative for mycoplasma by culture or direct PCR. In blood samples from patients with systemic lupus erythematosus were detected by direct PCR <it>M. fermentans </it>in 2/50 (2%), <it>M. hominis </it>in 2/50 (2%) and <it>U. urealyticum </it>in 1/50 (0.5%). In patients with RA <it>M. fermentans </it>was detected by culture in 13/87 blood samples and in 13/87 by direct PCR, however, there was only concordance between culture and direct PCR in six samples, so <it>M. fermentans </it>was detected in 20/87(23%) of the blood samples from patients with RA by either culture or PCR. Antibody-specific ELISA assay to <it>M. fermentans </it>PG18 was done, IgM was detected in sera from 40/87 patients with RA and in sera of 7/67 control individuals, IgG was detected in sera from 48/87 RA patients and in sera from 7/67 healthy individuals. Antibody-specific immunoblotting to <it>M. fermentans </it>PG18 showed IgM in sera from 35/87 patients with RA and in sera from 4/67 healthy individuals, IgG was detected in sera from 34/87 patients and in sera from 5/67 healthy individuals.</p> <p>Conclusion</p> <p>Our findings show that only <it>M. fermentans </it>produce bacteremia in a high percentage of patients with RA. This finding is similar to those reported in the literature. IgM and IgG against <it>M. fermentans </it>PG18 were more frequent in patients with RA than healthy individuals.</p

Chemopreventive potential of β-Sitosterol in experimental colon cancer model - an In vitro and In vivo study

<p>Abstract</p> <p>Background</p> <p><it>Asclepias curassavica </it>Linn. is a traditional medicinal plant used by tribal people in the western ghats, India, to treat piles, gonorrhoea, roundworm infestation and abdominal tumours. We have determined the protective effect of β-sitosterol isolated from <it>A. curassavica </it>in colon cancer, using <it>in vitro </it>and <it>in vivo </it>models.</p> <p>Methods</p> <p>The active molecule was isolated, based upon bioassay guided fractionation, and identified as β-sitosterol on spectral evidence. The ability to induce apoptosis was determined by its <it>in vitro </it>antiradical activity, cytotoxic studies using human colon adenocarcinoma and normal monkey kidney cell lines, and the expression of β-catenin and proliferating cell nuclear antigen (PCNA) in human colon cancer cell lines (COLO 320 DM). The chemopreventive potential of β-sitosterol in colon carcinogenesis was assessed by injecting 1,2-dimethylhydrazine (DMH, 20 mg/kg b.w.) into male Wistar rats and supplementing this with β-sitosterol throughout the experimental period of 16 weeks at 5, 10, and 20 mg/kg b.w.</p> <p>Results</p> <p>β-sitosterol induced significant dose-dependent growth inhibition of COLO 320 DM cells (IC₅₀266.2 μM), induced apoptosis by scavenging reactive oxygen species, and suppressed the expression of β-catenin and PCNA antigens in human colon cancer cells. β-sitosterol supplementation reduced the number of aberrant crypt and crypt multiplicity in DMH-initiated rats in a dose-dependent manner with no toxic effects.</p> <p>Conclusion</p> <p>We found doses of 10-20 mg/kg b.w. β-sitosterol to be effective for future <it>in vivo </it>studies. β-sitosterol had chemopreventive potential by virtue of its radical quenching ability <it>in vitro</it>, with minimal toxicity to normal cells. It also attenuated β-catenin and PCNA expression, making it a potential anticancer drug for colon carcinogenesis.</p

Distinct Roles of ComK1 and ComK2 in Gene Regulation in Bacillus cereus

The B. subtilis transcriptional factor ComK regulates a set of genes coding for DNA uptake from the environment and for its integration into the genome. In previous work we showed that Bacillus cereus expressing the B. subtilis ComK protein is able to take up DNA and integrate it into its own genome. To extend our knowledge on the effect of B. subtilis ComK overexpression in B. cereus we first determined which genes are significantly altered. Transcriptome analysis showed that only part of the competence gene cluster is significantly upregulated. Two ComK homologues can be identified in B. cereus that differ in their respective homologies to other ComK proteins. ComK1 is most similar, while ComK2 lacks the C-terminal region previously shown to be important for transcription activation by B. subtilis ComK. comK1 and comK2 overexpression and deletion studies using transcriptomics techniques showed that ComK1 enhances and ComK2 decreases expression of the comG operon, when B. subtilis ComK was overexpressed simultaneously

Strong Evidence of a Combination Polymorphism of the Tyrosine Kinase 2 Gene and the Signal Transducer and Activator of Transcription 3 Gene as a DNA-Based Biomarker for Susceptibility to Crohn’s Disease in the Japanese Population

OBJECTIVE: An association between susceptibility to inflammatory bowel disease (IBD) and polymorphisms of both the tyrosine kinase 2 gene (TYK2) and the signal transducer and activator of transcription 3 gene (STAT3) was examined in a Japanese population in order to identify the genetic determinants of IBD. METHODS: The study subjects comprised 112 patients with ulcerative colitis, 83 patients with Crohn's disease (CD), and 200 healthy control subjects. Seven tag single-nucleotide polymorphisms (SNPs) in TYK2 and STAT3 were detected by PCR-restriction fragment length polymorphism. RESULTS: The frequencies of a C allele and its homozygous C/C genotype at rs2293152 SNP in STAT3 in CD patients were significantly higher than those in control subjects (P = 0.007 and P = 0.001, respectively). Furthermore, out of four haplotypes composed of the two tag SNPs (rs280519 and rs2304256) in TYK2, the frequencies of a Hap 1 haplotype and its homozygous Hap 1/Hap1 diplotype were significantly higher in CD patients in comparison to those in control subjects (P = 0.023 and P = 0.024, respectively). In addition, the presence of both the C/C genotype at rs2293152 SNP in STAT3 and the Hap 1/Hap 1 diplotype of TYK2 independently contributes to the pathogenesis of CD and significantly increases the odds ratio to 7.486 for CD (P = 0.0008). CONCLUSION: TYK2 and STAT3 are genetic determinants of CD in the Japanese population. This combination polymorphism may be useful as a new genetic biomarker for the identification of high-risk individuals susceptible to CD