Mycobacterium tuberculosis reactivates latent HIV-1 in T cells in vitro

Following proviral integration into the host cell genome and establishment of a latent state, the human immunodeficiency virus type 1 (HIV-1) can reenter a productive life cycle in response to various stimuli. HIV-1 reactivation occurs when transcription factors, such as nuclear factor-κB (NF-κB), nuclear factor of activated T cells (NFAT), and activator protein -1 (AP-1), bind cognate sites within the long terminal repeat (LTR) region of the HIV-1 provirus to promote transcription. Interestingly, pattern recognition receptors (PRRs) that recognize pathogen-associated molecular patterns (PAMPs) can reactivate latent HIV-1 through activation of the transcription factor NF-κB. Some PRRs are expressed on central memory CD4+ T cells (TCM), which in HIV-1 patients constitute the main reservoir of latent HIV-1. Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), interacts with PRRs through membrane components. However, the ability of Mtb to reactivate latent HIV-1 has not been extensively studied. Here we show that phosphatidylinositol mannoside 6 (PIM6), a component of the Mtb membrane, in addition to whole bacteria in co-culture, can reactivate HIV-1 in a primary TCM cell model of latency. Using a JLAT model of HIV-1 latency, we found this interaction to be mediated through Toll-like receptor-2 (TLR-2). Thus, we describe a mechanism by which Mtb can exacerbate HIV-1 infection. We hypothesize that chronic Mtb infection can drive HIV-1 reactivation. The phenomenon described here could explain, in part, the poor prognosis that characterizes HIV-1/Mtb co-infection

Characterization of the Molecular Determinants of Primary HIV-1 Vpr Proteins: Impact of the Q65R and R77Q Substitutions on Vpr Functions

Although HIV-1 Vpr displays several functions in vitro, limited information exists concerning their relevance during infection. Here, we characterized Vpr variants isolated from a rapid and a long-term non-progressor (LTNP). Interestingly, vpr alleles isolated from longitudinal samples of the LTNP revealed a dominant sequence that subsequently led to diversity similar to that observed in the progressor patient. Most of primary Vpr proteins accumulated at the nuclear envelope and interacted with host-cell partners of Vpr. They displayed cytostatic and proapoptotic activities, although a LTNP allele, harboring the Q65R substitution, failed to bind the DCAF1 subunit of the Cul4a/DDB1 E3 ligase and was inactive. This Q65R substitution correlated with impairment of Vpr docking at the nuclear envelope, raising the possibility of a functional link between this property and the Vpr cytostatic activity. In contradiction with published results, the R77Q substitution, found in LTNP alleles, did not influence Vpr proapoptotic activity

The IDENTIFY study: the investigation and detection of urological neoplasia in patients referred with suspected urinary tract cancer - a multicentre observational study

Objective To evaluate the contemporary prevalence of urinary tract cancer (bladder cancer, upper tract urothelial cancer [UTUC] and renal cancer) in patients referred to secondary care with haematuria, adjusted for established patient risk markers and geographical variation. Patients and Methods This was an international multicentre prospective observational study. We included patients aged ≥16 years, referred to secondary care with suspected urinary tract cancer. Patients with a known or previous urological malignancy were excluded. We estimated the prevalence of bladder cancer, UTUC, renal cancer and prostate cancer; stratified by age, type of haematuria, sex, and smoking. We used a multivariable mixed-effects logistic regression to adjust cancer prevalence for age, type of haematuria, sex, smoking, hospitals, and countries. Results Of the 11 059 patients assessed for eligibility, 10 896 were included from 110 hospitals across 26 countries. The overall adjusted cancer prevalence (n = 2257) was 28.2% (95% confidence interval [CI] 22.3–34.1), bladder cancer (n = 1951) 24.7% (95% CI 19.1–30.2), UTUC (n = 128) 1.14% (95% CI 0.77–1.52), renal cancer (n = 107) 1.05% (95% CI 0.80–1.29), and prostate cancer (n = 124) 1.75% (95% CI 1.32–2.18). The odds ratios for patient risk markers in the model for all cancers were: age 1.04 (95% CI 1.03–1.05; P < 0.001), visible haematuria 3.47 (95% CI 2.90–4.15; P < 0.001), male sex 1.30 (95% CI 1.14–1.50; P < 0.001), and smoking 2.70 (95% CI 2.30–3.18; P < 0.001). Conclusions A better understanding of cancer prevalence across an international population is required to inform clinical guidelines. We are the first to report urinary tract cancer prevalence across an international population in patients referred to secondary care, adjusted for patient risk markers and geographical variation. Bladder cancer was the most prevalent disease. Visible haematuria was the strongest predictor for urinary tract cancer

Immunogenicity and Safety of Three Doses of a Bivalent (B:4:P1.19,15 and B:4:P1.7-2,4) Meningococcal Outer Membrane Vesicle Vaccine in Healthy Adolescents

An experimental bivalent meningococcal outer membrane vesicle (OMV) vaccine (B:4:P1.19,15 and B:4:P1.7-2,4) has been developed to provide wide vaccine coverage particularly of the circulating strains in Europe. A randomized, controlled phase II study (study identification number, 710158/002; ClinicalTrials.gov identifier number, NCT00137917) to evaluate the immunogenicity and safety of three doses of the OMV vaccine when given to healthy 12- to 18-year-olds on a 0-2-4 month (n = 162) or 0-1-6 month schedule (n = 159). A control group received two doses of hepatitis A and one of conjugated meningococcal serogroup C vaccine on a 0-1-6 month schedule (n = 157). Immune response, defined as a fourfold increase in serum bactericidal titer using a range of vaccine-homologous or PorA-related and heterologous strains, was determined for samples taken before and 1 month after vaccination; assays were performed at two laboratories. As measured at the GlaxoSmithKline (GSK) laboratory, the OMV vaccine induced an immune response against homologous or PorA-related strains (in at least 51% of subjects against strains of serosubtype P1.19,15 and at least 66% against strains of serosubtype P1.7-2,4) and against a set of three heterologous strains (in 28% to 46% of subjects). Both laboratories showed consistent results for immune response rates. The OMV vaccine had a similar reactogenicity profile for each schedule. Pain preventing normal activities occurred in approximately one-fifth of the subjects; this was significantly higher than in the control group. The immune responses induced by the bivalent OMV vaccine demonstrated the induction of bactericidal antibodies against the vaccine-homologous/PorA-related strains but also against heterologous strains, indicating the presence of protective antigens in OMVs and confirming the potential of clinical cross-protection

G2-arrest and pro-apoptotic activities of Vpr proteins from the LTNP and PR patients.

<p>HPB-ALL T lymphoid cells were co-transfected with vectors for expression of HA tagged Vpr<i>Lai</i> or the indicated primary Vpr proteins from the LTNP and PR patients, in combination with the GFP expression vector. A) Cellular expression of HA-tagged Vpr proteins. Lysates from HPB-ALL transfected cells were analyzed by western-blotting using anti-GFP and anti-HA antibodies. B) G2-arrest activity of primary Vpr proteins. 48 h after transfection, cells were fixed and the DNA content of GFP-positive cells was analyzed by flow cytometry after DNA staining with propidium iodide. Results are expressed as the G2M/G1 ratios relative to that of the VprLai. C) Pro-apoptotic activity of Vpr proteins. Cells co-expressing the GFP and HA-tagged Vpr proteins were assayed by flow cytometry 72 h following transfection for cell surface phosphatidylserine exposure using AnnexinV coupled to phycoerythrin. Values are means of three independent experiments; error bars represent one standard deviation from the mean.</p

Interaction of the Vpr proteins from the LTNP and PR patients with hCG1, UNG2 and DCAF1 in the yeast two-hybrid system.

<p>The L40 reporter yeast strain expressing Vpr<i>Lai</i> or the indicated primary Vpr proteins from the LTNP and PR patients fused to LexA (LexA hybrid), in combination with either hCG1, UNG2 or DCAF1 fused to the Gal4 activation domain (Gal4AD hybrid), was analyzed for histidine auxotrophy. Growth in the absence of histidine indicates interaction between hybrid proteins.</p

Description of patients and <i>vpr</i> alleles analyzed in the study.

<p>A) Immunological and viral profiles of the patients analyzed. These patients were selected from the HIV-1-infected patient cohort of the St-Antoine Hospital (Paris), and the samples were collected with written informed consent <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007514#pone.0007514-Lefrere1" target="_blank">[45]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007514#pone.0007514-Lefrere2" target="_blank">[46]</a>. The graphs show the time-course evolution of the blood CD4+ T cell counts (green curves) and plasmatic virus load (viral RNA, red curves); the PBMC samples analyzed in the present study are indicated by the blue arrows. B) Alignment of the amino acid sequences derived from DNA sequencing of the <i>vpr</i> alleles cloned from PBMC DNA samples. Excepted for LTNP-05 and LTNP-07 sequences that were obtained by direct sequencing of the bulk PCR fragments amplified from these samples, at least 40 independent clones were sequenced from each other samples. The sequences of the primary Vpr proteins are aligned with respect of the prototypic HIV-1<i>Lai</i> sequence (upper sequence). The <i>vpr</i> alleles from the LNTP (patient 5071) that were selected for subsequent functional analysis are in the box; the R77Q substitution identified in some <i>vpr</i> alleles is indicated in blue, while the Q65R substitution identified in the Vpr LTNP-04-2 is indicated in red.</p

Impact of the residue 77 on pro-apoptotic and G2-arrest activities of Vpr LTNP variants.

<p>The Arg77 residue from VprLai and Vpr LTNP-96 was replaced by a Gln, whereas the Gln residue from LTNP-86 and LTNP-04-1 was replaced by an Arg. The wild-type (grey bars) and mutated (hatched bars) Vpr proteins were then analyzed for apoptosis (A) and G2-arrest (B) activities as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007514#pone-0007514-g004" target="_blank">Figure 4</a>.</p

Relative frequency of primary Vpr alleles^a.

a<p><i>vpr</i> genes were amplified by PCR from each PBMC sample (indicated by blue arrows in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007514#pone-0007514-g001" target="_blank">Fig. 1A</a>), cloned into a shuttle plasmid, and DNA sequences from at least 40 independent clones were determined from each sample.</p>b<p>samples 1986, 1996 and 2004 are from the LTNP patient (5071), and sample PR is from the progressor patient (1200).</p>c<p>Vpr alleles correspond to the primary amino acid sequences reported in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007514#pone-0007514-g001" target="_blank">Fig. 1B</a>.</p