Expression and reactivation of HIV in a chemokine induced model of HIV latency in primary resting CD4+ T cells

<p>Abstract</p> <p>Background</p> <p>We recently described that HIV latent infection can be established <it>in vitro </it>following incubation of resting CD4+ T-cells with chemokines that bind to CCR7. The main aim of this study was to fully define the post-integration blocks to virus replication in this model of CCL19-induced HIV latency.</p> <p>Results</p> <p>High levels of integrated HIV DNA but low production of reverse transcriptase (RT) was found in CCL19-treated CD4+ T-cells infected with either wild type (WT) NL4.3 or single round envelope deleted NL4.3 pseudotyped virus (NL4.3- Δenv). Supernatants from CCL19-treated cells infected with either WT NL4.3 or NL4.3- Δenv did not induce luciferase expression in TZM-bl cells, and there was no expression of intracellular p24. Following infection of CCL19-treated CD4+ T-cells with NL4.3 with enhanced green fluorescent protein (EGFP) inserted into the <it>nef </it>open reading frame (NL4.3- Δnef-EGFP), there was no EGFP expression detected. These data are consistent with non-productive latent infection of CCL19-treated infected CD4+ T-cells. Treatment of cells with phytohemagluttinin (PHA)/IL-2 or CCL19, prior to infection with WT NL4.3, resulted in a mean fold change in unspliced (US) RNA at day 4 compared to day 0 of 21.2 and 1.1 respectively (p = 0.01; n = 5), and the mean expression of multiply spliced (MS) RNA was 56,000, and 5,000 copies/million cells respectively (p = 0.01; n = 5). In CCL19-treated infected CD4+ T-cells, MS-RNA was detected in the nucleus and not in the cytoplasm; in contrast to PHA/IL-2 activated infected cells where MS RNA was detected in both. Virus could be recovered from CCL19-treated infected CD4+ T-cells following mitogen stimulation (with PHA and phorbyl myristate acetate (PMA)) as well as TNFα, IL-7, prostratin and vorinostat.</p> <p>Conclusions</p> <p>In this model of CCL19-induced HIV latency, we demonstrate HIV integration without spontaneous production of infectious virus, detection of MS RNA in the nucleus only, and the induction of virus production with multiple activating stimuli. These data are consistent with <it>ex vivo </it>findings from latently infected CD4+ T-cells from patients on combination antiretroviral therapy, and therefore provide further support of this model as an excellent <it>in vitro </it>model of HIV latency.</p

Adding 6 months of androgen deprivation therapy to postoperative radiotherapy for prostate cancer: a comparison of short-course versus no androgen deprivation therapy in the RADICALS-HD randomised controlled trial

Background Previous evidence indicates that adjuvant, short-course androgen deprivation therapy (ADT) improves metastasis-free survival when given with primary radiotherapy for intermediate-risk and high-risk localised prostate cancer. However, the value of ADT with postoperative radiotherapy after radical prostatectomy is unclear. Methods RADICALS-HD was an international randomised controlled trial to test the efficacy of ADT used in combination with postoperative radiotherapy for prostate cancer. Key eligibility criteria were indication for radiotherapy after radical prostatectomy for prostate cancer, prostate-specific antigen less than 5 ng/mL, absence of metastatic disease, and written consent. Participants were randomly assigned (1:1) to radiotherapy alone (no ADT) or radiotherapy with 6 months of ADT (short-course ADT), using monthly subcutaneous gonadotropin-releasing hormone analogue injections, daily oral bicalutamide monotherapy 150 mg, or monthly subcutaneous degarelix. Randomisation was done centrally through minimisation with a random element, stratified by Gleason score, positive margins, radiotherapy timing, planned radiotherapy schedule, and planned type of ADT, in a computerised system. The allocated treatment was not masked. The primary outcome measure was metastasis-free survival, defined as distant metastasis arising from prostate cancer or death from any cause. Standard survival analysis methods were used, accounting for randomisation stratification factors. The trial had 80% power with two-sided α of 5% to detect an absolute increase in 10-year metastasis-free survival from 80% to 86% (hazard ratio [HR] 0·67). Analyses followed the intention-to-treat principle. The trial is registered with the ISRCTN registry, ISRCTN40814031, and ClinicalTrials.gov, NCT00541047. Findings Between Nov 22, 2007, and June 29, 2015, 1480 patients (median age 66 years [IQR 61–69]) were randomly assigned to receive no ADT (n=737) or short-course ADT (n=743) in addition to postoperative radiotherapy at 121 centres in Canada, Denmark, Ireland, and the UK. With a median follow-up of 9·0 years (IQR 7·1–10·1), metastasis-free survival events were reported for 268 participants (142 in the no ADT group and 126 in the short-course ADT group; HR 0·886 [95% CI 0·688–1·140], p=0·35). 10-year metastasis-free survival was 79·2% (95% CI 75·4–82·5) in the no ADT group and 80·4% (76·6–83·6) in the short-course ADT group. Toxicity of grade 3 or higher was reported for 121 (17%) of 737 participants in the no ADT group and 100 (14%) of 743 in the short-course ADT group (p=0·15), with no treatment-related deaths. Interpretation Metastatic disease is uncommon following postoperative bed radiotherapy after radical prostatectomy. Adding 6 months of ADT to this radiotherapy did not improve metastasis-free survival compared with no ADT. These findings do not support the use of short-course ADT with postoperative radiotherapy in this patient population

Duration of androgen deprivation therapy with postoperative radiotherapy for prostate cancer: a comparison of long-course versus short-course androgen deprivation therapy in the RADICALS-HD randomised trial

Background Previous evidence supports androgen deprivation therapy (ADT) with primary radiotherapy as initial treatment for intermediate-risk and high-risk localised prostate cancer. However, the use and optimal duration of ADT with postoperative radiotherapy after radical prostatectomy remains uncertain. Methods RADICALS-HD was a randomised controlled trial of ADT duration within the RADICALS protocol. Here, we report on the comparison of short-course versus long-course ADT. Key eligibility criteria were indication for radiotherapy after previous radical prostatectomy for prostate cancer, prostate-specific antigen less than 5 ng/mL, absence of metastatic disease, and written consent. Participants were randomly assigned (1:1) to add 6 months of ADT (short-course ADT) or 24 months of ADT (long-course ADT) to radiotherapy, using subcutaneous gonadotrophin-releasing hormone analogue (monthly in the short-course ADT group and 3-monthly in the long-course ADT group), daily oral bicalutamide monotherapy 150 mg, or monthly subcutaneous degarelix. Randomisation was done centrally through minimisation with a random element, stratified by Gleason score, positive margins, radiotherapy timing, planned radiotherapy schedule, and planned type of ADT, in a computerised system. The allocated treatment was not masked. The primary outcome measure was metastasis-free survival, defined as metastasis arising from prostate cancer or death from any cause. The comparison had more than 80% power with two-sided α of 5% to detect an absolute increase in 10-year metastasis-free survival from 75% to 81% (hazard ratio [HR] 0·72). Standard time-to-event analyses were used. Analyses followed intention-to-treat principle. The trial is registered with the ISRCTN registry, ISRCTN40814031, and ClinicalTrials.gov , NCT00541047 . Findings Between Jan 30, 2008, and July 7, 2015, 1523 patients (median age 65 years, IQR 60–69) were randomly assigned to receive short-course ADT (n=761) or long-course ADT (n=762) in addition to postoperative radiotherapy at 138 centres in Canada, Denmark, Ireland, and the UK. With a median follow-up of 8·9 years (7·0–10·0), 313 metastasis-free survival events were reported overall (174 in the short-course ADT group and 139 in the long-course ADT group; HR 0·773 [95% CI 0·612–0·975]; p=0·029). 10-year metastasis-free survival was 71·9% (95% CI 67·6–75·7) in the short-course ADT group and 78·1% (74·2–81·5) in the long-course ADT group. Toxicity of grade 3 or higher was reported for 105 (14%) of 753 participants in the short-course ADT group and 142 (19%) of 757 participants in the long-course ADT group (p=0·025), with no treatment-related deaths. Interpretation Compared with adding 6 months of ADT, adding 24 months of ADT improved metastasis-free survival in people receiving postoperative radiotherapy. For individuals who can accept the additional duration of adverse effects, long-course ADT should be offered with postoperative radiotherapy. Funding Cancer Research UK, UK Research and Innovation (formerly Medical Research Council), and Canadian Cancer Society

The chronic effects of a combination of herbal extracts (Euphytose®) on psychological mood state and response to a laboratory stressor: A randomised, placebo-controlled, double blind study in healthy humans.

Background: Global lifetime prevalence of anxiety disorders has been estimated at approximately 16.6%, with subclinical prevalence likely much higher. Herbal approaches to reduce anxiety may be as effective as pharmacological treatments and are less likely to be associated with adverse side effects. The herbal species valerian, passionflower, hawthorn and ballota have a long history of use as anxiolytics in traditional medicine, further supported by recent pre-clinical and clinical trials. Aims: To assess the effects of chronic (14 days) supplementation with a multi-herb extract preparation (Euphytose®) on psychological state and psychological and physiological stress responses during a laboratory stressor. Methods: In this crossover study, 31 healthy participants (aged 19 – 58 years) received a multi-herb extract preparation and placebo for 14 days with a 28-day washout. Anxiety (State-Trait Anxiety Inventory), mood, and physiological measures of stress (heart rate, galvanic skin response, salivary α-amylase and cortisol levels) were measured before and after an Observed Multitasking Stressor. Cognitive performance was also assessed. Results: Multi-herb extract preparation was associated with reduced tension-anxiety (p = .038), with participants showing an attenuated response to the observed multitasking psychosocial stressor following multi-herb extract preparation, evidenced by lower salivary α-amylase (p = .041) and galvanic skin response (p = .004). Conclusions: The combination of herbal extracts contained within the multi-herb extract preparation reduced subjective anxiety in a healthy population and lowered electrodermal skin conductance and concentration of salivary α-amylase in response to a psychosocial stressor, compared to placebo. The study was registered on clinicaltrials.gov (identifier: NCT03909906). Declaration of interest/funding: This work was sponsored by Bayer Healthcare

Comparison of Sequence Analysis and a Novel Discriminatory Real-Time PCR Assay for Detection and Quantification of Lamivudine-Resistant Hepatitis B Virus Strains

We report a rapid and accurate real-time PCR-based method to quantify wild-type and lamivudine-resistant hepatitis B virus by using a common forward primer paired with different reverse primers. Excellent concordance was demonstrated between sequencing and the discriminatory real-time assay; however, a mixture of quasispecies was more frequently detected by discriminatory real-time PCR

Reduced Hepatitis B Virus (HBV)-Specific CD4(+) T-Cell Responses in Human Immunodeficiency Virus Type 1-HBV-Coinfected Individuals Receiving HBV-Active Antiretroviral Therapy

Functional hepatitis B virus (HBV)-specific T cells are significantly diminished in individuals chronically infected with HBV compared to individuals with self-limiting HBV infection or those on anti-HBV therapy. In individuals infected with human immunodeficiency virus type 1 (HIV-1), coinfection with HBV is associated with an increased risk of worsening liver function following antiviral therapy and of more rapid HBV disease progression. Total HBV-specific T-cell responses in subjects with diverse genetic backgrounds were characterized by using a library of 15-mer peptides overlapping by 11 amino acids and spanning all HBV proteins. The magnitude and breadth of CD4(+) and CD8(+) T-cell responses to HBV in peripheral blood were examined by flow cytometry to detect gamma interferon production following stimulation with HBV peptide pools. Chronic HBV carriers (n = 34) were studied, including individuals never treated for HBV infection (n = 7), HBV-infected individuals receiving anti-HBV therapy (n = 13), and HIV-1-HBV-coinfected individuals receiving anti-HBV therapy (n = 14). CD4(+) and CD8(+) HBV-specific T-cell responses were more frequently detected and the CD8(+) T-cell responses were of greater magnitude and breadth in subjects on anti-HBV treatment than in untreated chronic HBV carriers. There was a significant inverse correlation between detection of a HBV-specific T-cell response and HBV viral load. HBV-specific CD4(+) and CD8(+) T-cell responses were significantly (fivefold) reduced compared with HIV-specific responses. Although, the frequency and breadth of HBV-specific CD8(+) T-cell responses were comparable in the monoinfected and HIV-1-HBV-coinfected groups, HBV-specific CD4(+) T-cell responses were significantly reduced in HIV-1-HBV-coinfected individuals. Therefore, HIV-1 infection has a significant and specific effect on HBV-specific T-cell immunity

Understanding factors that modulate the establishment of HIV latency in resting CD4+ T-Cells in <i>vitro</i>

Developing robust in vitro models of HIV latency is needed to better understand how latency is established, maintained and reversed. In this study, we examined the effects of donor variability, HIV titre and co-receptor usage on establishing HIV latency in vitro using two models of HIV latency. Using the CCL19 model of HIV latency, we found that in up to 50% of donors, CCL19 enhanced latent infection of resting CD4+ T-cells by CXCR4-tropic HIV in the presence of low dose IL-2. Increasing the infectious titre of CXCR4-tropic HIV increased both productive and latent infection of resting CD4+ T-cells. In a different model where myeloid dendritic cells (mDC) were co-cultured with resting CD4+ T-cells, we observed a higher frequency of latently infected cells in vitro than CCL19-treated or unstimulated CD4+ T-cells in the presence of low dose IL-2. In the DC-T-cell model, latency was established with both CCR5- and CXCR4-tropic virus but higher titres of CCR5-tropic virus was required in most donors. The establishment of latency in vitro through direct infection of resting CD4+ T-cells is significantly enhanced by CCL19 and mDC, but the efficiency is dependent on virus titre, co-receptor usage and there is significant donor variability

No Increase in Hepatitis B Virus (HBV)-Specific CD8+ T Cells in Patients with HIV-1-HBV Coinfections following HBV-Active Highly Active Antiretroviral Therapy ▿

Following treatment of hepatitis B virus (HBV) monoinfection, HBV-specific T-cell responses increase significantly; however, little is known about the recovery of HBV-specific T-cell responses following HBV-active highly active antiretroviral therapy (HAART) in HIV-HBV coinfected patients. HIV-HBV coinfected patients who were treatment naïve and initiating HBV-active HAART were recruited as part of a prospective cohort study in Thailand and followed for 48 weeks (n = 24). Production of gamma interferon (IFN-γ) and tumor necrosis factor α (TNF-α) in both HBV- and HIV-specific CD8+ T cells was quantified using intracellular cytokine staining on whole blood. Following HBV-active HAART, the median (interquartile range) log decline from week 0 to week 48 for HBV DNA was 5.8 log (range, 3.4 to 6.7) IU/ml, and for HIV RNA it was 3.1 (range, 2.9 to 3.5) log copies/ml (P < 0.001 for both). The frequency of HIV Gag-specific CD8+ T-cell responses significantly decreased (IFN-γ, P < 0.001; TNF-α, P = 0.05). In contrast, there was no significant change in the frequency (IFN-γ, P = 0.21; TNF-α, P = 0.61; and IFN-γ and TNF-α, P = 0.11) or magnitude (IFN-γ, P = 0.13; TNF-α, P = 0.13; and IFN-γ and TNF-α, P = 0.13) of HBV-specific CD8+ T-cell responses over 48 weeks of HBV-active HAART. Of the 14 individuals who were HBV e antigen (HBeAg) positive, 5/14 (36%) lost HBeAg during the 48 weeks of follow-up. HBV-specific CD8+ T cells were detected in 4/5 (80%) of patients prior to HBeAg loss. Results from this study show no sustained change in the HBV-specific CD8+ T-cell response following HBV-active HAART. These findings may have implications for the duration of treatment of HBV in HIV-HBV coinfected patients, particularly in HBeAg-positive disease