Codon bias and plasticity in immunoglobulins

Immunoglobulin genes experience Darwinian evolution twice. In addition to the germline evolution all genes experience, immunoglobulins are subjected, upon exposure to antigen, to somatic hypermutation. This is accompanied by selection for high affinity to the eliciting antigen and frequently results in a significant increase in the specificity of the responding population. The hypermutation mechanism displays a strong sequence specificity. Thus arises the opportunity to manipulate codon bias in a site-specific manner so as to direct hypermutation to those parts of the gene that encode the antigen-binding portions of the molecule and away from those that encode the structurally conserved regions. This segregation of mutability would clearly be advantageous; it would enhance the generation of potentially useful variants while keeping mutational loss to acceptably low levels. But it is not clear that the advantage gained would be large enough to produce a measurable effect within the background stochasticity of the evolutionary process. I have performed a pair of statistical tests to determine whether site- specific codon bias in human immunoglobulin genes is correlated with the sequence specificity of the somatic mutation mechanism. The sequence specificity of the mutator was determined by analysis of a database of published immunoglobulin intron sequences that had experienced somatic mutation but not selection. The site-specific codon bias was determined by analysis of published sequences of human germline immunoglobulin V genes. Both tests strongly suggest that evolution has acted to enhance the plasticity of immunoglobulin genes under somatic hypermutation.      

A novel receptor for calcitonin gene-related peptide (CGRP) mediates secretion in the rat colon: implications for secretory function in colitis.

The receptor responsible for CGRP-induced ion transport and permeability was examined in tissues from animals treated 7 days previously with trinitrobenzenesulfonic acid to induce colitis or in controls. CGRP caused a concentration-dependent increase in short circuit current (I(sc), EC(50) 21 nM), which was abolished in chloride-free buffer but was not blocked by CGRP(8-37) or tetrodotoxin (TTX). Amylin and adrenomedullin caused only a modest increase in I(sc). The responses to the linear CGRP(2) receptor agonists [Cys(Et)(2,7)] hCGRPalpha and [Cys(Acm)(2,7)] hCGRPalpha were considerably smaller than the response to CGRP. These responses were abolished in chloride-free buffer and were TTX sensitive. Atropine, doxantrazole, and indomethacin did not block the effects of CGRP or the CGRP(2) agonists. The response to [Cys(Et)(2,7)] hCGRPalpha was not affected by prior desensitization of the CGRP receptor and vice versa. Inflamed rats had a similar secretory response to CGRP (I(sc), EC(50) 15 nM) and [Cys(Et)(2,7)] hCGRPalpha as control tissues, while being hyporesponsive to carbachol. CGRP application increased electrical conductance of inflamed preparations. Taken together, these data suggest that CGRP may play an important role in the maintenance of host defense in colitis through an apparently novel CGRP receptor located on the colonic enterocyte.     

Major Challenges in Clinical Management of TB/HIV Coinfected Patients in Eastern Europe Compared with Western Europe and Latin America

Objectives

Rates of TB/HIV coinfection and multi-drug resistant (MDR)-TB are increasing in Eastern Europe (EE). We aimed to study clinical characteristics, factors associated with MDR-TB and predicted activity of empiric anti-TB treatment at time of TB diagnosis among TB/HIV coinfected patients in EE, Western Europe (WE) and Latin America (LA).

Design and Methods

Between January 1, 2011, and December 31, 2013, 1413 TB/HIV patients (62 clinics in 19 countries in EE, WE, Southern Europe (SE), and LA) were enrolled.

Results

Significant differences were observed between EE (N = 844), WE (N = 152), SE (N = 164), and LA (N = 253) in the proportion of patients with a definite TB diagnosis (47%, 71%, 72% and 40%, p<0.0001), MDR-TB (40%, 5%, 3% and 15%, p<0.0001), and use of combination antiretroviral therapy (cART) (17%, 40%, 44% and 35%, p<0.0001). Injecting drug use (adjusted OR (aOR) = 2.03 (95% CI 1.00–4.09), prior anti-TB treatment (3.42 (1.88–6.22)), and living in EE (7.19 (3.28–15.78)) were associated with MDR-TB. Among 585 patients with drug susceptibility test (DST) results, the empiric (i.e. without knowledge of the DST results) anti-TB treatment included ≥3 active drugs in 66% of participants in EE compared with 90–96% in other regions (p<0.0001).

Conclusions

In EE, TB/HIV patients were less likely to receive a definite TB diagnosis, more likely to house MDR-TB and commonly received empiric anti-TB treatment with reduced activity. Improved management of TB/HIV patients in EE requires better access to TB diagnostics including DSTs, empiric anti-TB therapy directed at both susceptible and MDR-TB, and more widespread use of cART.     

Contractile activity of lymphatic vessels is altered in the TNBS model of guinea pig ileitis

The ability of the lymphatic system to actively remove fluid from the interstitium is critical to the resolution of edema. The response of the lymphatics to inflammatory situations is poorly studied, so we examined mesenteric lymphatic contractile activity in the 2,4,6-trinitrobenzenesulfonic acid (TNBS) model of guinea pig ileitis, a well-accepted animal model of intestinal inflammation, by videomicroscopy in vivo and in vitro 1, 3, and 6 days after induction of ileitis. Lymphatic function (diameter, constriction frequency, amplitude of constrictions, and calculated stroke volume and lymph flow rate) of isolated vessels from TNBS-treated guinea pigs were impaired compared with sham-treated controls. The dysfunction was well correlated with the degree of inflammation, with differences reaching significance (P < 0.05) at the highest inflammation-induced damage observed at day 3. In vivo, significantly fewer lymphatics exhibited spontaneous constrictions in TNBS-treated than sham-treated animals. Cyclooxygenase (COX) metabolites were suggested to be involved in this lymphatic dysfunction, since application of nonselective COX inhibitor (10 microM indomethacin) or a combination of COX-1 and COX-2 inhibitors (1 microM SC-560 and 10 microM celecoxib) markedly increased constriction frequency or induced them in lymphatics from TNBS-treated animals in vivo and in vitro. The present results demonstrate that lymphatic contractile function is altered in TNBS-induced ileitis and suggest a role for prostanoids in the lymphatic dysfunction.     

Antigenicity and immunogenicity of novel chimeric hepatitis B surface antigen particles with exposed hepatitis C virus epitopes

The small envelope protein of hepatitis B virus (HBsAg-S) can self-assemble into highly organized virus like particles (VLPs) and induce an effective immune response. In this study, a restriction enzyme site was engineered into the cDNA of HBsAg-S at a position corresponding to the exposed site within the hydrophilic a determinant region (amino acid [aa] 127-128) to create a novel HBsAg vaccine vector allowing surface orientation of the inserted sequence. We inserted sequences of various lengths from hypervariable region 1 (HVR1) of the hepatitis C virus (HCV) E2 protein containing immunodominant epitopes and demonstrated secretion of the recombinant HBsAg VLPs from transfected mammalian cells. A number of different recombinant proteins were synthesized, and HBsAg VLPs containing inserts up to 36 aa were secreted with an efficiency similar to that of wild-type HBsAg. The HVR1 region exposed on the particles retained an antigenic structure similar to that recognized immunologically during natural infection. VLPs containing epitopes from either HCV-1a or -1b strains were produced that induced strain-specific antibody responses in immunized mice. Injection of a combination of these VLPs induced antibodies against both HVR1 epitopes that resulted in higher titers than were achieved by vaccination with the individual VLPs, suggesting a synergistic effect. This may lead to the development of recombinant particles which are able to induce a broad anti-HCV immune response against the HCV quasispecies or other quasispecies-like infectious agents.     

Measuring soil microbial community diversity using polar lipid fatty acid and denaturing gradient gel electrophoresis data

The possibility of calculating useful microbial community diversity indices from environmental polar lipid fatty acid and 16S rDNA PCR-DGGE data was investigated. First, the behavior of the species richness, Shannon's, and Simpson's diversity indices were determined on polar lipid fatty acid profiles of 115 pure cultures, communities constructed from those profiles with different numbers of species, and constructed communities with different distributions of species. Differences in the species richness of these artificial communities was detected by all three diversity indices, but they were insensitive to the evenness of the distribution of species. Second, data from a field experiment with substrate addition to soil was used to compare the methods developed for lipid- and DNA-based diversity indices. Very good agreement was found between indices calculated from environmental polar lipid fatty acid profiles and denaturing gradient gel electrophoresis profiles from matched samples (Pearson's correlation coefficient r=0.95-0.96). A method for data pre-treatment for diversity calculations is described.     

Identification of iron-reducing<Emphasis Type="Italic"> Thermus</Emphasis> strains as<Emphasis Type="Italic"> Thermus scotoductus</Emphasis>

Thermus strain SA-01, previously isolated from a deep (3.2 km) South African gold mine, is closely related to Thermus strains NMX2 A.1 and VI-7 (previously isolated from thermal springs in New Mexico, USA, and Portugal, respectively). Thermus strains SA-01 and NMX2 A.1 have also been shown previously to grow using nitrate, Fe(III), Mn(IV) or S^O as terminal electron acceptors and to be capable of reducing Cr(VI), U(VI), Co(III), and the quinone-containing compound anthraquinone-2,6-disulfonate. The objectives of this study were to determine the phylogenetic positions of the three known metal-reducing Thermus strains and to determine the phylogenetic significance of metal reduction within the genus Thermus. Phylogenetic analyses of 16S rDNA sequences, BOX PCR genomic fingerprinting, and DNA–DNA reassociation analyses indicated that these strains belong to the previously described genospecies T. scotoductus. The morphologies and lipid fatty acid profiles of these metal-reducing strains are consistent with their identification as T. scotoductus; however, the T. scotoductus strains tested in this study evinced a wide intraspecies variability in some other phenotypic traits, e.g., carbon substrate utilization and pigmentation. Iron reduction occurred in all strains of T. scotoductus tested except the mixotrophic, sulfur-oxidizing strain IT-7254. Thermus strains belonging to other species did not reduce Fe(III) to Fe(II) or reduced it only poorly.Communicated by J. Wiegel     

Rhinovirus multistranded RNA: dependence of the replicative form on the presence of actinomycin D.

The multistranded and double-stranded RNAs synthesized in HeLa cells infected with rhinovirus in the presence and in the absence of actinomycin D have been characterized by polyacrylamide gel electrophoresis and hybridization studies. The replicative form is only found in infected cells treated with actinomycin D, whereas the replicative intermediate is found in both the presence and the absence of the drug. The significance of these results is discussed.     

Modelling habitat distributions for multiple species using phylogenetics

In this paper, we describe an empirical approach to model community structure using phylogenetic signals. That approach combines information about the species (i.e. traits and phylogeny) with information about the habitat (i.e. environmental conditions and spatial distribution of sampling sites) and their interactions to predict the species responses (e.g. the local densities). As an application, we use the approach to model fish densities in rivers. In the model, the different species and size classes were described using a functional trait, body length, and phylogenetic eigenvectors maps whereas the sites were described using water velocity, depth, substrate composition, macrophyte cover, degree‐days, total phosphorus, and spatial eigenvector maps. The model (estimated using a regularised Poisson‐family generalised linear modelling approach) fitted the data well (likelihood‐based R²_adj = 0.512) and showed fair predictive power (likelihood‐based cross‐validation R² = 0.283) to predict the density of fish pertaining to 48 species totalling 143 combinations of species and size classes in 15 unregulated Canadian rivers. Using the model as a baseline to estimate the effect of flow regulation on community composition, we found that, with few exceptions, the densities of most fish species were lower in regulated than in unregulated rivers. Phylogenetics have been proposed to study community structure, but this is, to our knowledge, the first time phylogenetic information is used explicitly for numerical habitat modelling. We expect that models of that type will be in increasing demand now that development projects are routinely assessed through impact studies.