Tuberculosis specific responses following therapy for TB: Impact of HIV co-infection.

Characterizing perturbations in the immune response to tuberculosis in HIV can develop insights into the pathogenesis of coinfection. HIV+ TB+ and TB monoinfected (TB+) subjects recruited from clinics in Bamako prior to initiation of TB treatment were evaluated at time-points following initiation of therapy. Flow cytometry assessed CD4+/CD8+ T cell subsets and activation markers CD38/HLA-DR. Antigen specific responses to TB proteins were assessed by intracellular cytokine detection and proliferation. HIV+ TB+ subjects had significantly higher markers of immune activation in the CD4+ and CD8+ T cells compared to TB+ subjects. HIV+ TB+ had lower numbers of TB-specific CD4+ T cells at baseline. Plasma IFNγ levels were similar between HIV+ TB+ and TB+ subjects. No differences were observed in in-vitro proliferative capacity to TB antigens between HIV+ TB+ and TB+ subjects. Subjects with HIV+ TB+ coinfection demonstrate in vivo expansion of TB-specific CD4+ T cells. Immunodeficiency associated with CD4+ T cell depletion may be less significant compared to immunosuppression associated with HIV viremia or untreated TB infection.

A flow cytometry-based assay to assess RSV-specific neutralizing antibody is reproducible, efficient and accurate.

Respiratory syncytial virus (RSV) is an important cause of respiratory infection in people of all ages, and is the leading cause of hospitalization in infants. Although commercially available monoclonal antibody is available for passive prophylaxis of neonates at risk of severe disease, there is no available vaccine to prevent RSV. Measurement of neutralizing activity will be a key endpoint for vaccine evaluation. Assessment of neutralizing antibody against RSV has been limited to traditional plaque reduction, which is time-consuming and inherently operator dependent and highly variable. Here, we describe a flow cytometry-based RSV-specific neutralization assay which is more rapid than traditional methods, highly sensitive and highly reproducible.

Heavy metal fractionation during the co-composting of biosolids, deinking paper fibre and green waste.

Due to the introduction of the European Union Landfill Directive, composting has become a potentially viable disposal route for some organic wastes. As waste-derived compost is frequently added to soil to improve soil quality, it is important to quantify the environmental risk posed by potentially toxic elements contained within it. Here we used a sequential chemical extraction procedure to investigate the temporal dynamics of heavy metals (Cu, Zn, Pb and Ni) during the co-composting of biosolids, deinking paper fibre and green waste. Overall, composting over 26 weeks reduced the availability of Ni, had no effect on Pb and slightly increased the availability of Cu and Zn. We conclude that although the total Cu and Ni concentrations in the compost exceed legislative guidelines for land application, due to their recalcitrant nature within the compost, this compost posed very little threat to soil or plant quality if used in agriculture or land restoration.

Contaminated land clean-up using composted wastes and impacts of VOCs on land.

This paper describes experiments that demonstrate the effects and potential for remediation of a former steelworks site in Wales polluted with polycyclic aromatic hydrocarbons (PAHs) and volatile organic compounds (VOCs). Under field conditions, PAH-contaminated soil was composted in-vessel, with or without organic feedstocks, receiving forced aeration for 80 days followed by 4 months maturation. Treatments compared PAH removal in contaminated soil to contaminated soil mixed with three different organic waste mixes after composting and after composts were spread to land. After composting, PAH concentrations declined in all treatments, by up to 38%. Sixteen months after the composts were landspread and vegetation was established, only those containing contaminated soil with organic additions exhibited further PAH removal, by up to 29%. Composting resulted in a decline in the relative concentration of small PAHs, whereas the landspreading-vegetation phase saw a decline in the relative concentration of medium PAHs in two of the three composts exhibiting PAH removal. Under controlled glasshouse conditions, vegetated soil columns of differing depths were exposed to VOCs from beneath. VOC vapour affected both shoot and root growth and soil microbial activity; effects varied with distance from the VOC source. This work demonstrated that on-site remediation of aged PAH-contaminated land can be successfully initiated by in-vessel co-composting followed by land spreading and vegetation, within a practical timeframe.

Patterns of immune response to a vaccine or virus as measured by intracellular cytokine staining in flow cytometry: hypothesis generation and comparison of groups.

Candidate HIV vaccines must show an immune response in order to be considered for further testing and development. What constitutes a "response," however, is still not clear. While the hunt for a protective vaccine continues, hypotheses are being formed by studying the immune responses across cohorts of people with differing responses to the infection, as well as the immune responses formed by healthy people to other viruses, ones that are generally common and well controlled. Here we examine the functional profile of the immune responses of a group of HIV+ long-term non-progressors as measured by intracellular cytokine staining using polychromatic flow cytometry, and compare these responses to those of a larger group of other HIV+ people. We describe some of the types of patterns in immune response that are of interest to vaccine researchers, and compare several statistical tests appropriate for this type of data.

Identification of the cystic fibrosis transmembrane conductance regulator domains that are important for interactions with ROMK2.

In addition to functioning as a cAMP-activated chloride channel, the cystic fibrosis transmembrane conductance regulator (CFTR) plays an important role in conferring regulatory properties on other ion channels. It is known, with respect to CFTR regulation of ROMK2 (renally derived K(ATP) channel), that the first transmembrane domain and the first nucleotide binding fold domain (NBF1) of CFTR are necessary for this interaction to occur. It has been shown that under conditions that promote phosphorylation, the ROMK2-CFTR interaction is attenuated. To elucidate the complex nature of this interaction, CFTR constructs were co-expressed with ROMK2 in Xenopus oocytes, and two microelectrode voltage clamp experiments were performed. Although the second half of CFTR can act as a functional chloride channel, our results suggest that it does not confer glibenclamide sensitivity on ROMK2, as does the first half of CFTR. The attenuation of the ROMK2-CFTR interaction under conditions that promote phosphorylation is dependent on at least the presence of the R domain of CFTR. We conclude that transmembrane domain 1, NBF1, and the R domain are the CFTR domains involved in the ROMK2-CFTR interaction and that NBF2 and transmembrane domain 2 are not essential. Lastly, the R domain of CFTR is necessary for the attenuation of the ROMK2-CFTR interaction under conditions that promote phosphorylation.

Fathers with severe mental illness: characteristics and comparisons.

Among patients with severe mental illness attending a large, urban, outpatient mental health clinic, fathers are described and compared with nonfathers and with mothers on demographic, clinical, and child-related characteristics, and on resources and service needs. While fathers and nonfathers with mental illness differed significantly on most variables, fathers and mothers with mental illness were remarkably similar except on child-related characteristics. Issues regarding fathers' experiences and service needs are discussed.

A functional CFTR-NBF1 is required for ROMK2-CFTR interaction.

In a previous study on inside-out patches of Xenopus oocytes, we demonstrated that the cystic fibrosis transmembrane conductance regulator (CFTR) enhances the glibenclamide sensitivity of a coexpressed inwardly rectifying K+ channel, ROMK2 (C. M. McNicholas, W. B. Guggino, E. M. Schwiebert, S. C. Hebert, G. Giebisch, and M. E. Egan. Proc. Natl. Acad. Sci. USA 93: 8083-8088, 1996). In the present study, we used the two-microelectrode voltage-clamp technique to measure whole cell K+ currents in Xenopus oocytes, and we further characterized the enhanced sensitivity of ROMK2 to glibenclamide by CFTR. Glibenclamide inhibited K+ currents by 56% in oocytes expressing both ROMK2 and CFTR but only 11% in oocytes expressing ROMK2 alone. To examine the role of the first nucleotide binding fold (NBF1) of CFTR in the ROMK2-CFTR interaction, we studied the glibenclamide sensitivity of ROMK2 when coexpressed with CFTR constructs containing mutations in or around the NBF1 domain. In oocytes coinjected with ROMK2 and a truncated construct of CFTR with an intact NBF1 (CFTR-K593X), glibenclamide inhibited K+ currents by 46%. However, in oocytes coinjected with ROMK2 and a CFTR mutant truncated immediately before NBF1 (CFTR-K370X), glibenclamide inhibited K+ currents by 12%. Also, oocytes expressing both ROMK2 and CFTR mutants with naturally occurring NBF1 point mutations, CFTR-G551D or CFTR-A455E, display glibenclamide-inhibitable K+ currents of only 14 and 25%, respectively. Because CFTR mutations that alter the NBF1 domain reduce the glibenclamide sensitivity of the coexpressed ROMK2 channel, we conclude that the NBF1 motif is necessary for the CFTR-ROMK2 interaction that confers sulfonylurea sensitivity.

Resolution of infection status of human immunodeficiency virus (HIV)-seroindeterminate donors and high-risk seronegative individuals with polymerase chain reaction and virus culture: absence of persistent silent HIV type 1 infection in a high-prevalence area.

To address concerns over the prevalence of silent (antibody-negative) infections among blood donors and high-risk populations, a combination of proviral amplification by polymerase chain reaction (PCR) and viral isolation by co-culture techniques was employed to resolve the human immunodeficiency virus type 1 (HIV-1) infection status of well-characterized groups of suspect blood donors and others identified in the blood bank setting. No silent infections were found in 65 follow-up samples from 26 persistently HIV-1-seroindeterminate blood donors, 16 persistently seronegative heterosexual partners of infected transfusion recipients, and 6 high-risk seronegative homosexual men identified through donor look-back investigations. In contrast, 21 seropositive controls tested positive. These results suggest a low prevalence of persistently silent infections in at-risk populations, even in high HIV prevalence regions. The PCR assay, with a co-detected internal positive control, and appropriate confirmatory algorithms, was found to be a useful direct assay to rule out infection, especially in concert with confirmatory virus isolation.