Natural variation in immune responses to neonatal mycobacterium bovis bacillus calmette-guerin (BCG) vaccination in a cohort of Gambian infants

Background There is a need for new vaccines for tuberculosis (TB) that protect against adult pulmonary disease in regions where BCG is not effective. However, BCG could remain integral to TB control programmes because neonatal BCG protects against disseminated forms of childhood TB and many new vaccines rely on BCG to prime immunity or are recombinant strains of BCG. Interferon-gamma (IFN-) is required for immunity to mycobacteria and used as a marker of immunity when new vaccines are tested. Although BCG is widely given to neonates IFN- responses to BCG in this age group are poorly described. Characterisation of IFN- responses to BCG is required for interpretation of vaccine immunogenicity study data where BCG is part of the vaccination strategy. Methodology/Principal Findings 236 healthy Gambian babies were vaccinated with M. bovis BCG at birth. IFN-, interleukin (IL)-5 and IL-13 responses to purified protein derivative (PPD), killed Mycobacterium tuberculosis (KMTB), M. tuberculosis short term culture filtrate (STCF) and M. bovis BCG antigen 85 complex (Ag85) were measured in a whole blood assay two months after vaccination. Cytokine responses varied up to 10 log-fold within this population. The majority of infants (89-98% depending on the antigen) made IFN- responses and there was significant correlation between IFN- responses to the different mycobacterial antigens (Spearman’s coefficient ranged from 0.340 to 0.675, p=10-6-10-22). IL-13 and IL-5 responses were generally low and there were more non-responders (33-75%) for these cytokines. Nonetheless, significant correlations were observed for IL-13 and IL-5 responses to different mycobacterial antigens Conclusions/Significance Cytokine responses to mycobacterial antigens in BCG-vaccinated infants are heterogeneous and there is significant inter-individual variation. Further studies in large populations of infants are required to identify the factors that determine variation in IFN- responses

Presentation and Outcome of Tuberculous Meningitis in a High HIV Prevalence Setting

Mycobacterium tuberculosis is a common, devastating cause of meningitis in HIV-infected persons. Due to international rollout programs, access to antiretroviral therapy (ART) is increasing globally. Starting patients with HIV-associated tuberculous meningitis (TBM) on ART during tuberculosis (TB) treatment may increase survival in these patients. We undertook this study to describe causes of meningitis at a secondary-level hospital in a high HIV/TB co-infection setting and to determine predictors of mortality in patients with TBM.A retrospective review of cerebrospinal fluid findings and clinical records over a six-month period (March 2009-August 2009). Definite, probable and possible TBM were diagnosed according to published case definitions.TBM was diagnosed in 120/211 patients (57%) with meningitis. In 106 HIV-infected patients with TBM, six-month all-cause mortality was lower in those who received antiretroviral therapy (ART) during TB treatment; hazard ratio = 0.30 (95% CI = 0.08-0.82). Factors associated with inpatient mortality in HIV-infected patients were 1) low CD4(+) count at presentation; adjusted odds ratio (AOR) = 1.4 (95% confidence interval [CI] = 1.03-1.96) per 50 cells/µL drop in CD4(+) count and, 2) higher British Medical Research Council TBM disease grade (2 or 3 versus 1); AOR = 4.8 (95% CI = 1.45-15.87).Starting ART prior to or during TB treatment may be associated with lower mortality in patients with HIV-associated TBM. Advanced HIV and worse stage of TBM disease predict in-hospital mortality in patients presenting with TBM

A Strong Deletion Bias in Nonallelic Gene Conversion

Gene conversion is the unidirectional transfer of genetic information between orthologous (allelic) or paralogous (nonallelic) genomic segments. Though a number of studies have examined nucleotide replacements, little is known about length difference mutations produced by gene conversion. Here, we investigate insertions and deletions produced by nonallelic gene conversion in 338 Drosophila and 10,149 primate paralogs. Using a direct phylogenetic approach, we identify 179 insertions and 614 deletions in Drosophila paralogs, and 132 insertions and 455 deletions in primate paralogs. Thus, nonallelic gene conversion is strongly deletion-biased in both lineages, with almost 3.5 times as many conversion-induced deletions as insertions. In primates, the deletion bias is considerably stronger for long indels and, in both lineages, the per-site rate of gene conversion is orders of magnitudes higher than that of ordinary mutation. Due to this high rate, deletion-biased nonallelic gene conversion plays a key role in genome size evolution, leading to the cooperative shrinkage and eventual disappearance of selectively neutral paralogs

Large introns in relation to alternative splicing and gene evolution: a case study of Drosophila bruno-3

Background: Alternative splicing (AS) of maturing mRNA can generate structurally and functionally distinct transcripts from the same gene. Recent bioinformatic analyses of available genome databases inferred a positive correlation between intron length and AS. To study the interplay between intron length and AS empirically and in more detail, we analyzed the diversity of alternatively spliced transcripts (ASTs) in the Drosophila RNA-binding Bruno-3 (Bru-3) gene. This gene was known to encode thirteen exons separated by introns of diverse sizes, ranging from 71 to 41,973 nucleotides in D. melanogaster. Although Bru-3's structure is expected to be conducive to AS, only two ASTs of this gene were previously described. Results: Cloning of RT-PCR products of the entire ORF from four species representing three diverged Drosophila lineages provided an evolutionary perspective, high sensitivity, and long-range contiguity of splice choices currently unattainable by high-throughput methods. Consequently, we identified three new exons, a new exon fragment and thirty-three previously unknown ASTs of Bru-3. All exon-skipping events in the gene were mapped to the exons surrounded by introns of at least 800 nucleotides, whereas exons split by introns of less than 250 nucleotides were always spliced contiguously in mRNA. Cases of exon loss and creation during Bru-3 evolution in Drosophila were also localized within large introns. Notably, we identified a true de novo exon gain: exon 8 was created along the lineage of the obscura group from intronic sequence between cryptic splice sites conserved among all Drosophila species surveyed. Exon 8 was included in mature mRNA by the species representing all the major branches of the obscura group. To our knowledge, the origin of exon 8 is the first documented case of exonization of intronic sequence outside vertebrates. Conclusion: We found that large introns can promote AS via exon-skipping and exon turnover during evolution likely due to frequent errors in their removal from maturing mRNA. Large introns could be a reservoir of genetic diversity, because they have a greater number of mutable sites than short introns. Taken together, gene structure can constrain and/or promote gene evolution

Changes in Mycobacterium tuberculosis Genotype Families Over 20 Years in a Population-Based Study in Northern Malawi

BACKGROUND: Despite increasing interest in possible differences in virulence and transmissibility between different genotypes of M. tuberculosis, very little is known about how genotypes within a population change over decades, or about relationships to HIV infection. METHODS AND PRINCIPAL FINDINGS: In a population-based study in rural Malawi we have examined smears and cultures from tuberculosis patients over a 20-year period using spoligotyping. Isolates were grouped into spoligotype families and lineages following previously published criteria. Time trends, HIV status, drug resistance and outcome were examined by spoligotype family and lineage. In addition, transmissibility was examined among pairs of cases with known epidemiological contact by assessing the proportion of transmissions confirmed for each lineage, on the basis of IS6110 RFLP similarity of the M tuberculosis strains. 760 spoligotypes were obtained from smears from 518 patients from 1986-2002, and 377 spoligotypes from cultures from 347 patients from 2005-2008. There was good consistency in patients with multiple specimens. Among 781 patients with first episode tuberculosis, the majority (76%) had Lineage 4 ("European/American") strains; 9% had Lineage 3 ("East-African/Indian"); 8% Lineage 1 ("Indo-Oceanic"); and 2% Lineage 2 ("East-Asian"); others unclassifiable. Over time the proportion of Lineage 4 decreased from >90% to 60%, with an increase in the other 3 lineages (p<0.001). Lineage 1 strains were more common in those with HIV infection, even after adjusting for age, sex and year. There were no associations with drug resistance or outcome, and no differences by lineage in the proportion of pairs in which transmission was confirmed. CONCLUSIONS: This is the first study to describe long term trends in the four M. tuberculosis lineages in a population. Lineage 4 has probably been longstanding in this population, with relatively recent introductions and spread of Lineages1-3, perhaps influenced by the HIV epidemic

Gain and Loss of Phototrophic Genes Revealed by Comparison of Two Citromicrobium Bacterial Genomes

Proteobacteria are thought to have diverged from a phototrophic ancestor, according to the scattered distribution of phototrophy throughout the proteobacterial clade, and so the occurrence of numerous closely related phototrophic and chemotrophic microorganisms may be the result of the loss of genes for phototrophy. A widespread form of bacterial phototrophy is based on the photochemical reaction center, encoded by puf and puh operons that typically are in a ‘photosynthesis gene cluster’ (abbreviated as the PGC) with pigment biosynthesis genes. Comparison of two closely related Citromicrobial genomes (98.1% sequence identity of complete 16S rRNA genes), Citromicrobium sp. JL354, which contains two copies of reaction center genes, and Citromicrobium strain JLT1363, which is chemotrophic, revealed evidence for the loss of phototrophic genes. However, evidence of horizontal gene transfer was found in these two bacterial genomes. An incomplete PGC (pufLMC-puhCBA) in strain JL354 was located within an integrating conjugative element, which indicates a potential mechanism for the horizontal transfer of genes for phototrophy

Temporal Dynamics of Interferon Gamma Responses in Children Evaluated for Tuberculosis

BACKGROUND: Development of T-cells based-Interferon gamma (IFNgamma) assays has offered new possibilities for the diagnosis of latent tuberculosis infection (LTBI) and active disease in adults. Few studies have been performed in children, none in France. With reference to the published data on childhood TB epidemiology in the Paris and Ile de France Region, we considered it important to evaluate the performance of IGRA (QuantiFERON TB Gold In Tube(R), QF-TB-IT) in the diagnosis and the follow-up through treatment of LTBI and active TB in a cohort of French children. METHODOLOGY/PRINCIPAL FINDINGS: 131 children were recruited during a prospective and multicentre study (October 2005 and May 2007; Ethical Committee St Louis Hospital, Paris, study number 2005/32). Children were sampled at day 0, 10, 30, 60 (except Healthy Contacts, HC) and 90 for LTBI and HC, and a further day 120, and day 180 for active TB children. Median age was 7.4 years, with 91% of the children BCG vaccinated. LTBI and active TB children undergoing therapy produced significant higher IFNgamma values after 10 days of treatment (p = 0.035). In addition, IFNgamma values were significantly lower at the end of treatment compared to IFNgamma values at day 0, although the number of positive patients was not significantly different between day 0 and end of treatment. CONCLUSIONS/ SIGNIFICANCE: By following quantitative IFNgamma values in each enrolled child with LTBI or active TB and receiving treatment, we were able to detect an increase in the IFNgamma response at day 10 of treatment which might allow the confirmation of a diagnosis. In addition, a decline in IFNgamma values during treatment makes it possible for clinicians to monitor the effect of preventive or curative therapy

Genome Analysis of Multi- and Extensively-Drug-Resistant Tuberculosis from KwaZulu-Natal, South Africa

The KZN strain family of Mycobacterium tuberculosis is a highly virulent strain endemic to the KwaZulu-Natal region of South Africa, which has recently experienced an outbreak of extensively-drug resistant tuberculosis. To investigate the causes and evolution of drug-resistance, we determined the DNA sequences of several clinical isolates - one drug-susceptible, one multi-drug resistant, and nine extensively drug-resistant - using whole-genome sequencing. Analysis of polymorphisms among the strains is consistent with the drug-susceptibility profiles, in that well-known mutations are observed that are correlated with resistance to isoniazid, rifampicin, kanamycin, ofloxacin, ethambutol, and pyrazinamide. However, the mutations responsible for rifampicin resistance in rpoB and pyrazinamide in pncA are in different nucleotide positions in the multi-drug-resistant and extensively drug-resistant strains, clearly showing that they acquired these mutations independently, and that the XDR strain could not have evolved directly from the MDR strain (though it could have arisen from another similar MDR strain). Sequencing of eight additional XDR strains from other areas of KwaZulu-Natal shows that they have identical drug resistant mutations to the first one sequenced, including the same polymorphisms at sites associated with drug resistance, supporting the theory that this represents a case of clonal expansion

Topological Analysis of MAPK Cascade for Kinetic ErbB Signaling

Ligand-induced homo- and hetero-dimer formation of ErbB receptors results in different biological outcomes irrespective of recruitment and activation of similar effector proteins. Earlier experimental research indicated that cells expressing both EGFR (epidermal growth factor receptor) and the ErbB4 receptor (E1/4 cells) induced E1/4 cell-specific B-Raf activation and higher extracellular signal-regulated kinase (ERK) activation, followed by cellular transformation, than cells solely expressing EGFR (E1 cells) in Chinese hamster ovary (CHO) cells. Since our experimental data revealed the presence of positive feedback by ERK on upstream pathways, it was estimated that the cross-talk/feedback pathway structure of the Raf-MEK-ERK cascade might affect ERK activation dynamics in our cell system. To uncover the regulatory mechanism concerning the ERK dynamics, we used topological models and performed parameter estimation for all candidate structures that possessed ERK-mediated positive feedback regulation of Raf. The structure that reliably reproduced a series of experimental data regarding signal amplitude and duration of the signaling molecules was selected as a solution. We found that the pathway structure is characterized by ERK-mediated positive feedback regulation of B-Raf and B-Raf-mediated negative regulation of Raf-1. Steady-state analysis of the estimated structure indicated that the amplitude of Ras activity might critically affect ERK activity through ERK-B-Raf positive feedback coordination with sustained B-Raf activation in E1/4 cells. However, Rap1 that positively regulates B-Raf activity might be less effective concerning ERK and B-Raf activity. Furthermore, we investigated how such Ras activity in E1/4 cells can be regulated by EGFR/ErbB4 heterodimer-mediated signaling. From a sensitivity analysis of the detailed upstream model for Ras activation, we concluded that Ras activation dynamics is dominated by heterodimer-mediated signaling coordination with a large initial speed of dimerization when the concentration of the ErbB4 receptor is considerably high. Such characteristics of the signaling cause the preferential binding of the Grb2-SOS complex to heterodimer-mediated signaling molecules