Human Galectin-9 Is a Potent Mediator of HIV Transcription and Reactivation.

Identifying host immune determinants governing HIV transcription, latency and infectivity in vivo is critical to developing an HIV cure. Based on our recent finding that the host factor p21 regulates HIV transcription during antiretroviral therapy (ART), and published data demonstrating that the human carbohydrate-binding immunomodulatory protein galectin-9 regulates p21, we hypothesized that galectin-9 modulates HIV transcription. We report that the administration of a recombinant, stable form of galectin-9 (rGal-9) potently reverses HIV latency in vitro in the J-Lat HIV latency model. Furthermore, rGal-9 reverses HIV latency ex vivo in primary CD4+ T cells from HIV-infected, ART-suppressed individuals (p = 0.002), more potently than vorinostat (p = 0.02). rGal-9 co-administration with the latency reversal agent "JQ1", a bromodomain inhibitor, exhibits synergistic activity (p<0.05). rGal-9 signals through N-linked oligosaccharides and O-linked hexasaccharides on the T cell surface, modulating the gene expression levels of key transcription initiation, promoter proximal-pausing, and chromatin remodeling factors that regulate HIV latency. Beyond latent viral reactivation, rGal-9 induces robust expression of the host antiviral deaminase APOBEC3G in vitro and ex vivo (FDR<0.006) and significantly reduces infectivity of progeny virus, decreasing the probability that the HIV reservoir will be replenished when latency is reversed therapeutically. Lastly, endogenous levels of soluble galectin-9 in the plasma of 72 HIV-infected ART-suppressed individuals were associated with levels of HIV RNA in CD4+ T cells (p<0.02) and with the quantity and binding avidity of circulating anti-HIV antibodies (p<0.009), suggesting a role of galectin-9 in regulating HIV transcription and viral production in vivo during therapy. Our data suggest that galectin-9 and the host glycosylation machinery should be explored as foundations for novel HIV cure strategies

May Measurement Month 2018: a pragmatic global screening campaign to raise awareness of blood pressure by the International Society of Hypertension

Aims Raised blood pressure (BP) is the biggest contributor to mortality and disease burden worldwide and fewer than half of those with hypertension are aware of it. May Measurement Month (MMM) is a global campaign set up in 2017, to raise awareness of high BP and as a pragmatic solution to a lack of formal screening worldwide. The 2018 campaign was expanded, aiming to include more participants and countries. Methods and results Eighty-nine countries participated in MMM 2018. Volunteers (≥18 years) were recruited through opportunistic sampling at a variety of screening sites. Each participant had three BP measurements and completed a questionnaire on demographic, lifestyle, and environmental factors. Hypertension was defined as a systolic BP ≥140 mmHg or diastolic BP ≥90 mmHg, or taking antihypertensive medication. In total, 74.9% of screenees provided three BP readings. Multiple imputation using chained equations was used to impute missing readings. 1 504 963 individuals (mean age 45.3 years; 52.4% female) were screened. After multiple imputation, 502 079 (33.4%) individuals had hypertension, of whom 59.5% were aware of their diagnosis and 55.3% were taking antihypertensive medication. Of those on medication, 60.0% were controlled and of all hypertensives, 33.2% were controlled. We detected 224 285 individuals with untreated hypertension and 111 214 individuals with inadequately treated (systolic BP ≥ 140 mmHg or diastolic BP ≥ 90 mmHg) hypertension. Conclusion May Measurement Month expanded significantly compared with 2017, including more participants in more countries. The campaign identified over 335 000 adults with untreated or inadequately treated hypertension. In the absence of systematic screening programmes, MMM was effective at raising awareness at least among these individuals at risk

Independent and combined effects of improved water, sanitation, and hygiene, and improved complementary feeding, on child stunting and anaemia in rural Zimbabwe: a cluster-randomised trial.

BACKGROUND: Child stunting reduces survival and impairs neurodevelopment. We tested the independent and combined effects of improved water, sanitation, and hygiene (WASH), and improved infant and young child feeding (IYCF) on stunting and anaemia in in Zimbabwe. METHODS: We did a cluster-randomised, community-based, 2 × 2 factorial trial in two rural districts in Zimbabwe. Clusters were defined as the catchment area of between one and four village health workers employed by the Zimbabwe Ministry of Health and Child Care. Women were eligible for inclusion if they permanently lived in clusters and were confirmed pregnant. Clusters were randomly assigned (1:1:1:1) to standard of care (52 clusters), IYCF (20 g of a small-quantity lipid-based nutrient supplement per day from age 6 to 18 months plus complementary feeding counselling; 53 clusters), WASH (construction of a ventilated improved pit latrine, provision of two handwashing stations, liquid soap, chlorine, and play space plus hygiene counselling; 53 clusters), or IYCF plus WASH (53 clusters). A constrained randomisation technique was used to achieve balance across the groups for 14 variables related to geography, demography, water access, and community-level sanitation coverage. Masking of participants and fieldworkers was not possible. The primary outcomes were infant length-for-age Z score and haemoglobin concentrations at 18 months of age among children born to mothers who were HIV negative during pregnancy. These outcomes were analysed in the intention-to-treat population. We estimated the effects of the interventions by comparing the two IYCF groups with the two non-IYCF groups and the two WASH groups with the two non-WASH groups, except for outcomes that had an important statistical interaction between the interventions. This trial is registered with ClinicalTrials.gov, number NCT01824940. FINDINGS: Between Nov 22, 2012, and March 27, 2015, 5280 pregnant women were enrolled from 211 clusters. 3686 children born to HIV-negative mothers were assessed at age 18 months (884 in the standard of care group from 52 clusters, 893 in the IYCF group from 53 clusters, 918 in the WASH group from 53 clusters, and 991 in the IYCF plus WASH group from 51 clusters). In the IYCF intervention groups, the mean length-for-age Z score was 0·16 (95% CI 0·08-0·23) higher and the mean haemoglobin concentration was 2·03 g/L (1·28-2·79) higher than those in the non-IYCF intervention groups. The IYCF intervention reduced the number of stunted children from 620 (35%) of 1792 to 514 (27%) of 1879, and the number of children with anaemia from 245 (13·9%) of 1759 to 193 (10·5%) of 1845. The WASH intervention had no effect on either primary outcome. Neither intervention reduced the prevalence of diarrhoea at 12 or 18 months. No trial-related serious adverse events, and only three trial-related adverse events, were reported. INTERPRETATION: Household-level elementary WASH interventions implemented in rural areas in low-income countries are unlikely to reduce stunting or anaemia and might not reduce diarrhoea. Implementation of these WASH interventions in combination with IYCF interventions is unlikely to reduce stunting or anaemia more than implementation of IYCF alone. FUNDING: Bill & Melinda Gates Foundation, UK Department for International Development, Wellcome Trust, Swiss Development Cooperation, UNICEF, and US National Institutes of Health.The SHINE trial is funded by the Bill & Melinda Gates Foundation (OPP1021542 and OPP113707); UK Department for International Development; Wellcome Trust, UK (093768/Z/10/Z, 108065/Z/15/Z and 203905/Z/16/Z); Swiss Agency for Development and Cooperation; US National Institutes of Health (2R01HD060338-06); and UNICEF (PCA-2017-0002)

The meningeal lymphatic system: a route for HIV brain migration?

Two innovative studies recently identified functional lymphatic structures in the meninges that may influence the development of HIV-associated neurological disorders (HAND). Until now, blood vessels were assumed to be the sole transport system by which HIV-infected monocytes entered the brain by bypassing a potentially hostile blood-brain barrier through inflammatory-mediated semi-permeability. A cascade of specific chemokine signals promote monocyte migration from blood vessels to surrounding brain tissues via a well-supported endothelium, where the cells differentiate into tissue macrophages capable of productive HIV infection. Lymphatic vessels on the other hand are more loosely organized than blood vessels. They absorb interstitial fluid from bodily tissues where HIV may persist and exchange a variety of immune cells (CD4(+) T cells, monocytes, macrophages, and dendritic cells) with surrounding tissues through discontinuous endothelial junctions. We propose that the newly discovered meningeal lymphatics are key to HIV migration among viral reservoirs and brain tissue during periods of undetectable plasma viral loads due to suppressive combinational antiretroviral therapy, thus redefining the migration process in terms of a blood-lymphatic transport system

Human Galectin-9 Is a Potent Mediator of HIV Transcription and Reactivation.

Identifying host immune determinants governing HIV transcription, latency and infectivity in vivo is critical to developing an HIV cure. Based on our recent finding that the host factor p21 regulates HIV transcription during antiretroviral therapy (ART), and published data demonstrating that the human carbohydrate-binding immunomodulatory protein galectin-9 regulates p21, we hypothesized that galectin-9 modulates HIV transcription. We report that the administration of a recombinant, stable form of galectin-9 (rGal-9) potently reverses HIV latency in vitro in the J-Lat HIV latency model. Furthermore, rGal-9 reverses HIV latency ex vivo in primary CD4+ T cells from HIV-infected, ART-suppressed individuals (p = 0.002), more potently than vorinostat (p = 0.02). rGal-9 co-administration with the latency reversal agent "JQ1", a bromodomain inhibitor, exhibits synergistic activity (p<0.05). rGal-9 signals through N-linked oligosaccharides and O-linked hexasaccharides on the T cell surface, modulating the gene expression levels of key transcription initiation, promoter proximal-pausing, and chromatin remodeling factors that regulate HIV latency. Beyond latent viral reactivation, rGal-9 induces robust expression of the host antiviral deaminase APOBEC3G in vitro and ex vivo (FDR<0.006) and significantly reduces infectivity of progeny virus, decreasing the probability that the HIV reservoir will be replenished when latency is reversed therapeutically. Lastly, endogenous levels of soluble galectin-9 in the plasma of 72 HIV-infected ART-suppressed individuals were associated with levels of HIV RNA in CD4+ T cells (p<0.02) and with the quantity and binding avidity of circulating anti-HIV antibodies (p<0.009), suggesting a role of galectin-9 in regulating HIV transcription and viral production in vivo during therapy. Our data suggest that galectin-9 and the host glycosylation machinery should be explored as foundations for novel HIV cure strategies

rGal-9 induces HIV transcription and reactivation in a glycan-dependent manner.

<p>(<b>A</b>) Effects of anti-Tim-3 antibody, anti-CD44 antibody, or anti-PDI antibody administration on rGal-9-mediated reactivation of HIV in J-Lat 5A8 cells. Antibodies were added 30 minutes prior to administration of 200 nM rGal-9. α-lactose (30 mM) was used as a positive control. (<b>B</b>, <b>C</b>) Treatment of J-Lat 5A8 cells with either 1 μg/ml tunicamycin, or with an enzymatic deglycosylation mix for 24 hours prior to rGal-9 stimulation. J-Lat cells were analyzed by flow cytometry to assess HIV-encoded GFP expression. Statistical comparisons were performed using two-tailed Mann-Whitney tests. (<b>D</b>) Effects of deglycosylation enzyme combinations on rGal-9-mediated HIV latency reversal in J-Lat 5A8 cells. N = PNGase F (Elizabethkingia miricola); O = O-Glycosidase (recombinant from Streptococcus pneumonia); S = α-(2→3,6,8,9)-Neuraminidase (recombinant from Arthrobacter ureafaciens); B = β(1→4)-Galactosidase (recombinant from Streptococcus pneumonia) + β-N-Acetylglucosaminidase (recombinant from Streptococcus pneumonia). Mean ± SEM is displayed, and statistical comparisons were performed using two-tailed unpaired t tests. * = p<0.05; ** = p<0.01, *** = p<0.001, and **** = p<0.0001.</p

rGal-9 partially activates primary CD4+ T cells and induces proliferation primarily in naïve CD4+ T cells.

<p>(<b>A</b>, <b>B</b>) Effects of rGal-9 stimulation on the cell surface expression of CD69 and CD25 activation markers on CD4+ T cells isolated from six ART-suppressed individuals. Mean ± SEM is displayed. Asterisks represent statistically significant differences as compared to DMSO control (p < 0.05, two-tailed Wilcoxon signed-rank test). (<b>C</b>) Effects of rGal-9 stimulation on the proliferation of CD4+ T cells isolated from three ART-suppressed individuals. Primary CD4+ T cells were stained with CFSE and cultured for 5 days, stained with CD4 and CD45RA monoclonal antibodies, and proliferation was quantified as the percentage of CFSE_low cells on CD4+ CD45RA+ (Naïve, Tn) or CD4+ CD45RA- (Memory, Tm) T cells. Mean ± SEM is displayed. (<b>D</b>) Example of the flow cytometry gating strategy. (<b>E</b>, <b>F</b>) Effects of rGal-9 on proliferation of (<b>E)</b>, memory CD4+ T cells, and (<b>F)</b>, naïve CD4+ T cells.</p

rGal-9 treatment reduces viral infectivity.

<p>(<b>A</b>) Illustrative schematic of the viral infectivity experiment. (<b>B-D</b>) Effects of rGal-9 treatment of producer cells on HIV infectivity. The MOLT4-CCR5 cell line was infected for 6 hours, cells were washed and treated with either PBS, rGal-9 200nM, or interferon-α (5000 U/ml) for 24 hours, and cultures were incubated for 3 days. (<b>B</b>) HIV p24 levels produced by MOLT4-CCR5 cells were quantified after concentrating the culture supernatants. Concentrated culture supernatants were used to infect Jurkat cells by spinoculation. (<b>C</b>) Levels of integrated HIV DNA measured at days 3, 6, 9, and 12 post-infection of Jurkat cells. (<b>D</b>) Levels of integrated HIV DNA at days 3, 6, 9, and 12 post-infection of Jurkat cells, normalized to producer cell p24 supernatant levels. Mean ± SEM is displayed, and statistical comparisons were performed using two-tailed unpaired t test. * = p<0.05; ** = p<0.01, *** = p<0.001, and **** = p<0.0001.</p

rGal-9 induces the expression of several anti-HIV host restriction factors including APOBEC3G <i>ex vivo</i>.

<p>(<b>A</b>) Heat map representing expression levels of host restriction factors in CD4+ T cells isolated from ART-suppressed individuals, after treatment with either 0.5% DMSO as negative control, 500 nM rGal-9, 1000 nM rGal-9, 1μM vorinostat, or a combination of PMA (2 nM) and Ionomycin (0.5 μM). Heat colors indicate fold modulation compared to the DMSO control. Red indicates induction of expression, and blue indicates reduction of expression. Statistical comparisons were performed using t tests, and p values were adjusted for multiple comparisons using false discovery rate. Asterisks indicate >3-fold, statistically significant modulation of gene expression as compared to DMSO control, as follows: * = FDR<0.05; ** = FDR<0.01, and *** = FDR<0.001. (<b>B</b>) APOBEC3G expression in isolated CD4+ T cells from HIV-infected ART-suppressed individuals, treated as described in panel <b>A</b>. Statistical comparisons were performed using two-tailed Wilcoxon signed-rank tests compared to the DMSO-treated control. <b>(C-D)</b> APOBEC3G protein expression in CD4+ T cells treated with either 500 nM rGal-9 or interferon-α (5000 units/ml), as determined by western blot. Immunoblotting bands were quantified with ImageJ software. The quantified APOBEC3G protein expression levels were normalized to corresponding Tubulin protein levels to account for variation in loading.</p

sGal-9 levels correlate with measures of HIV transcription and viral production <i>in vivo</i>.

<p>Correlations between levels of soluble Gal-9 and (<b>A</b>) levels of HIV cell-associated RNA, and (<b>B</b>) anti-HIV-1 antibodies in the plasma of 72 HIV-infected ART-suppressed individuals. Correlations were evaluated using Spearman's rank correlation coefficient tests.</p