A cohort-based study of host gene expression: tumor suppressor and innate immune/inflammatory pathways associated with the HIV reservoir size.

The major barrier to an HIV cure is the HIV reservoir: latently-infected cells that persist despite effective antiretroviral therapy (ART). There have been few cohort-based studies evaluating host genomic or transcriptomic predictors of the HIV reservoir. We performed host RNA sequencing and HIV reservoir quantification (total DNA [tDNA], unspliced RNA [usRNA], intact DNA) from peripheral CD4+ T cells from 191 ART-suppressed people with HIV (PWH). After adjusting for nadir CD4+ count, timing of ART initiation, and genetic ancestry, we identified two host genes for which higher expression was significantly associated with smaller total DNA viral reservoir size, P3H3 and NBL1, both known tumor suppressor genes. We then identified 17 host genes for which lower expression was associated with higher residual transcription (HIV usRNA). These included novel associations with membrane channel (KCNJ2, GJB2), inflammasome (IL1A, CSF3, TNFAIP5, TNFAIP6, TNFAIP9, CXCL3, CXCL10), and innate immunity (TLR7) genes (FDR-adjusted q<0.05). Gene set enrichment analyses further identified significant associations of HIV usRNA with TLR4/microbial translocation (q = 0.006), IL-1/NRLP3 inflammasome (q = 0.008), and IL-10 (q = 0.037) signaling. Protein validation assays using ELISA and multiplex cytokine assays supported these observed inverse host gene correlations, with P3H3, IL-10, and TNF-α protein associations achieving statistical significance (p<0.05). Plasma IL-10 was also significantly inversely associated with HIV DNA (p = 0.016). HIV intact DNA was not associated with differential host gene expression, although this may have been due to a large number of undetectable values in our study. To our knowledge, this is the largest host transcriptomic study of the HIV reservoir. Our findings suggest that host gene expression may vary in response to the transcriptionally active reservoir and that changes in cellular proliferation genes may influence the size of the HIV reservoir. These findings add important data to the limited host genetic HIV reservoir studies to date.

Differences in expression of tumor suppressor, innate immune, inflammasome, and potassium/gap junction channel host genes significantly predict viral reservoir size during treated HIV infection.

The major barrier to an HIV cure is the persistence of infected cells that evade host immune surveillance despite effective antiretroviral therapy (ART). Most prior host genetic HIV studies have focused on identifying DNA polymorphisms (e.g., CCR5Δ32 , MHC class I alleles) associated with viral load among untreated "elite controllers" (~1% of HIV+ individuals who are able to control virus without ART). However, there have been few studies evaluating host genetic predictors of viral control for the majority of people living with HIV (PLWH) on ART. We performed host RNA sequencing and HIV reservoir quantification (total DNA, unspliced RNA, intact DNA) from peripheral CD4+ T cells from 191 HIV+ ART-suppressed non-controllers. Multivariate models included covariates for timing of ART initiation, nadir CD4+ count, age, sex, and ancestry. Lower HIV total DNA (an estimate of the total reservoir) was associated with upregulation of tumor suppressor genes NBL1 (q=0.012) and P3H3 (q=0.012). Higher HIV unspliced RNA (an estimate of residual HIV transcription) was associated with downregulation of several host genes involving inflammasome ( IL1A, CSF3, TNFAIP5, TNFAIP6, TNFAIP9 , CXCL3, CXCL10 ) and innate immune ( TLR7 ) signaling, as well as novel associations with potassium ( KCNJ2 ) and gap junction ( GJB2 ) channels, all q<0.05. Gene set enrichment analyses identified significant associations with TLR4/microbial translocation (q=0.006), IL-1ß/NRLP3 inflammasome (q=0.008), and IL-10 (q=0.037) signaling. HIV intact DNA (an estimate of the "replication-competent" reservoir) demonstrated trends with thrombin degradation ( PLGLB1 ) and glucose metabolism ( AGL ) genes, but data were (HIV intact DNA detected in only 42% of participants). Our findings demonstrate that among treated PLWH, that inflammation, innate immune responses, bacterial translocation, and tumor suppression/cell proliferation host signaling play a key role in the maintenance of the HIV reservoir during ART. Further data are needed to validate these findings, including functional genomic studies, and expanded epidemiologic studies in female, non-European cohorts. Author Summary: Although lifelong HIV antiretroviral therapy (ART) suppresses virus, the major barrier to an HIV cure is the persistence of infected cells that evade host immune surveillance despite effective ART, "the HIV reservoir." HIV eradication strategies have focused on eliminating residual virus to allow for HIV remission, but HIV cure trials to date have thus far failed to show a clinically meaningful reduction in the HIV reservoir. There is an urgent need for a better understanding of the host-viral dynamics during ART suppression to identify potential novel therapeutic targets for HIV cure. This is the first epidemiologic host gene expression study to demonstrate a significant link between HIV reservoir size and several well-known immunologic pathways (e.g., IL-1ß, TLR7, TNF-α signaling pathways), as well as novel associations with potassium and gap junction channels (Kir2.1, connexin 26). Further data are needed to validate these findings, including functional genomic studies and expanded epidemiologic studies in female, non-European cohorts.

Host variation in type I interferon signaling genes (MX1), C-C chemokine receptor type 5 gene, and major histocompatibility complex class I alleles in treated HIV+ noncontrollers predict viral reservoir size.

OBJECTIVE: Prior genomewide association studies have identified variation in major histocompatibility complex (MHC) class I alleles and C-C chemokine receptor type 5 gene (CCR5Δ32) as genetic predictors of viral control, especially in 'elite' controllers, individuals who remain virally suppressed in the absence of therapy. DESIGN: Cross-sectional genomewide association study. METHODS: We analyzed custom whole exome sequencing and direct human leukocyte antigen (HLA) typing from 202 antiretroviral therapy (ART)-suppressed HIV+ noncontrollers in relation to four measures of the peripheral CD4+ T-cell reservoir: HIV intact DNA, total (t)DNA, unspliced (us)RNA, and RNA/DNA. Linear mixed models were adjusted for potential covariates including age, sex, nadir CD4+ T-cell count, pre-ART HIV RNA, timing of ART initiation, and duration of ART suppression. RESULTS: Previously reported 'protective' host genetic mutations related to viral setpoint (e.g. among elite controllers) were found to predict smaller HIV reservoir size. The HLA 'protective' B∗57:01 was associated with significantly lower HIV usRNA (q = 3.3 × 10-3), and among the largest subgroup, European ancestry individuals, the CCR5Δ32 deletion was associated with smaller HIV tDNA (P = 4.3 × 10-3) and usRNA (P = 8.7 × 10-3). In addition, genomewide analysis identified several single nucleotide polymorphisms in MX1 (an interferon stimulated gene) that were significantly associated with HIV tDNA (q = 0.02), and the direction of these associations paralleled MX1 gene eQTL expression. CONCLUSIONS: We observed a significant association between previously reported 'protective' MHC class I alleles and CCR5Δ32 with the HIV reservoir size in noncontrollers. We also found a novel association between MX1 and HIV total DNA (in addition to other interferon signaling relevant genes, PPP1CB, DDX3X). These findings warrant further investigation in future validation studies.

The impact of low-dose aspirin in the Brain Injury Guidelines on outcomes in traumatic brain injury: A retrospective cohort study.

BACKGROUND: Current Brain Injury Guidelines (BIG) characterize patients with intracranial hemorrhage taking antiplatelet or anticoagulant agents as BIG 3 (the most severe category) regardless of trauma severity. This study assessed the risk of in-hospital mortality or need for neurosurgery in patients taking low-dose aspirin who otherwise would be classified as BIG 1. METHODS: This was a retrospective study at an academic level 1 trauma center. Patients were included if they were admitted with traumatic intracerebral hemorrhage and were evaluated by the BIG criteria. Exclusion criteria included indeterminate BIG status or patients with missing primary outcomes documentation. Patients were categorized as BIG 1, BIG 2, BIG 3, or BIG 1 on aspirin (patients with BIG 1 features taking low-dose aspirin). The primary endpoint was a composite of neurosurgical intervention and all-cause in-hospital mortality. Key secondary endpoints include rate of intracranial hemorrhage progression, and intensive care unit- and hospital-free days. RESULTS: A total of 1,520 patients met the inclusion criteria. Median initial Glasgow Coma Scale was 14 (interquartile range [IQR], 12-15), Injury Severity Scale score was 17 (IQR, 10-25), and Abbreviated Injury Scale subscore head and neck (AIS Head ) was 3 (IQR, 3-4). The rate of the primary outcome for BIG 1, BIG 1 on aspirin, BIG 2, and BIG 3 was 1%, 2.2%, 1%, and 27%, respectively; the difference between BIG 1 on aspirin and BIG 3 was significant ( p < 0.001). CONCLUSION: Patients taking low-dose aspirin with otherwise BIG 1-grade injuries experienced mortality and required neurosurgery significantly less often than other patients categorized as BIG 3. Inclusion of low-dose aspirin in the BIG criteria should be reevaluated. LEVEL OF EVIDENCE: Therapeutic/Care Management; Level IV.

Everolimus, an mTORC1/2 inhibitor, in ART-suppressed individuals who received solid organ transplantation: A prospective study.

Pharmacologic inhibition of the mammalian target of rapamycin (mTOR) in the setting of renal transplantation has previously been associated with lower human immunodeficiency virus 1 (HIV-1) DNA burden, and in vitro studies suggest that mTOR inhibition may lead to HIV transcriptional silencing. Because prospective clinical trials are lacking, we conducted an open-label, single-arm study to determine the impact of the broad mTOR inhibitor, everolimus, on residual HIV burden, transcriptional gene expression profiles, and immune responses in HIV-infected adult solid organ transplant (SOT) recipients on antiretroviral therapy. Whereas everolimus therapy did not have an overall effect on cell-associated HIV-1 DNA and RNA levels in the entire cohort, participants who maintained everolimus time-averaged trough levels >5 ng/mL during the first 2 months of therapy had significantly lower RNA levels up to 6 months after the cessation of study drug. Time-averaged everolimus trough levels significantly correlated with greater inhibition of mTOR gene pathway transcriptional activity. Everolimus treatment also led to decreased PD-1 expression on certain T cell subsets. These data support the rationale for further study of the effects of mTOR inhibition on HIV transcriptional silencing in non-SOT populations, either alone or in combination with other strategies. Trial Registration: ClinicalTrials.gov NCT02429869.

Correction to: Rapid development of HIV elite control in a patient with acute infection.

After publication of the original article [1], we were notified in Table 1 a column should be removed.

Rapid development of HIV elite control in a patient with acute infection.

BACKGROUND: Elite controllers (EC), a small subset of the HIV-positive population (< 1%), suppress HIV viremia below the limit of quantification of clinical viral load assays in the absence of antiretroviral therapy (ART). However, there is a paucity of longitudinal data detailing the viral and immune dynamics or HIV reservoir seeding during acute infection in individuals that go on to become Elite Controllers. CASE PRESENTATION: In this report, we describe a case of a 42 year old woman diagnosed during acute infection who rapidly and permanently suppressed her viremia in the absence of antiretroviral therapy (ART). Rapid antibody/antigen testing was either negative or equivocal during acute infection, despite subsequent viral load testing at that time point with 71,550 plasma HIV RNA copies/mL, making initial diagnosis challenging. The patient subsequently developed detectable anti-HIV antibodies and an increase in HIV-specific CD8+ T cell responses to overlapping subtype C HIV gag peptide; very low-level plasma viremia (0.84 RNA copies/mL) was detected by an ultrasensitive assay 2 years following infection. Subsequently, she was started on ART for multifocal furunculosis despite continued suppression of virus and stable CD4+ T cell counts. Following ART initiation, HIV specific antibody levels and CD8+ T cell responses increased, but no HIV DNA or RNA was able to be isolated from large numbers of peripheral blood CD4+ T cells. CONCLUSION: This case provides important information regarding the establishment of elite HIV control during acute infection and also demonstrates an increase in HIV-specific immune responses following ART despite undetectable peripheral blood cellular measures of HIV persistence. This case also highlights the challenges in diagnosing acute HIV infection without the use of viral load testing in this rare elite controller phenotype.

Increased HIV-1 transcriptional activity and infectious burden in peripheral blood and gut-associated CD4+ T cells expressing CD30.

HIV-1-infected cells persist indefinitely despite the use of combination antiretroviral therapy (ART), and novel therapeutic strategies to target and purge residual infected cells in individuals on ART are urgently needed. Here, we demonstrate that CD4+ T cell-associated HIV-1 RNA is often highly enriched in cells expressing CD30, and that cells expressing this marker considerably contribute to the total pool of transcriptionally active CD4+ lymphocytes in individuals on suppressive ART. Using in situ RNA hybridization studies, we show co-localization of CD30 with HIV-1 transcriptional activity in gut-associated lymphoid tissues. We also demonstrate that ex vivo treatment with brentuximab vedotin, an antibody-drug conjugate (ADC) that targets CD30, significantly reduces the total amount of HIV-1 DNA in peripheral blood mononuclear cells obtained from infected, ART-suppressed individuals. Finally, we observed that an HIV-1-infected individual, who received repeated brentuximab vedotin infusions for lymphoma, had no detectable virus in peripheral blood mononuclear cells. Overall, CD30 may be a marker of residual, transcriptionally active HIV-1 infected cells in the setting of suppressive ART. Given that CD30 is only expressed on a small number of total mononuclear cells, it is a potential therapeutic target of persistent HIV-1 infection.

Elucidating the Burden of HIV in Tissues Using Multiplexed Immunofluorescence and In Situ Hybridization: Methods for the Single-Cell Phenotypic Characterization of Cells Harboring HIV In Situ.

Persistent tissue reservoirs of HIV present a major barrier to cure. Defining subsets of infected cells in tissues is a major focus of HIV cure research. Herein, we describe a novel multiplexed in situ hybridization (ISH) (RNAscope) protocol to detect HIV-DNA (vDNA) and HIV-RNA (vRNA) in formalin-fixed paraffin-embedded (FFPE) human tissues in combination with immunofluorescence (IF) phenotyping of the infected cells. We show that multiplexed IF and ISH (mIFISH) is suitable for quantitative assessment of HIV vRNA and vDNA and that multiparameter IF phenotyping allows precise identification of the cellular source of the ISH signal. We also provide semi-quantitative data on the impact of various tissue fixatives on the detectability of vDNA and vRNA with RNAscope technology. Finally, we describe methods to quantitate the ISH signal on whole-slide digital images and validation of the quantitative ISH data with quantitative real-time PCR for vRNA. It is our hope that this approach will provide insight into the biology of HIV tissue reservoirs and to inform strategies aimed at curing HIV.

HIV-1 persistence following extremely early initiation of antiretroviral therapy (ART) during acute HIV-1 infection: An observational study.

BACKGROUND: It is unknown if extremely early initiation of antiretroviral therapy (ART) may lead to long-term ART-free HIV remission or cure. As a result, we studied 2 individuals recruited from a pre-exposure prophylaxis (PrEP) program who started prophylactic ART an estimated 10 days (Participant A; 54-year-old male) and 12 days (Participant B; 31-year-old male) after infection with peak plasma HIV RNA of 220 copies/mL and 3,343 copies/mL, respectively. Extensive testing of blood and tissue for HIV persistence was performed, and PrEP Participant A underwent analytical treatment interruption (ATI) following 32 weeks of continuous ART. METHODS AND FINDINGS: Colorectal and lymph node tissues, bone marrow, cerebral spinal fluid (CSF), plasma, and very large numbers of peripheral blood mononuclear cells (PBMCs) were obtained longitudinally from both participants and were studied for HIV persistence in several laboratories using molecular and culture-based detection methods, including a murine viral outgrowth assay (mVOA). Both participants initiated PrEP with tenofovir/emtricitabine during very early Fiebig stage I (detectable plasma HIV-1 RNA, antibody negative) followed by 4-drug ART intensification. Following peak viral loads, both participants experienced full suppression of HIV-1 plasma viremia. Over the following 2 years, no further HIV could be detected in blood or tissue from PrEP Participant A despite extensive sampling from ileum, rectum, lymph nodes, bone marrow, CSF, circulating CD4+ T cell subsets, and plasma. No HIV was detected from tissues obtained from PrEP Participant B, but low-level HIV RNA or DNA was intermittently detected from various CD4+ T cell subsets. Over 500 million CD4+ T cells were assayed from both participants in a humanized mouse outgrowth assay. Three of 8 mice infused with CD4+ T cells from PrEP Participant B developed viremia (50 million input cells/surviving mouse), but only 1 of 10 mice infused with CD4+ T cells from PrEP Participant A (53 million input cells/mouse) experienced very low level viremia (201 copies/mL); sequence confirmation was unsuccessful. PrEP Participant A stopped ART and remained aviremic for 7.4 months, rebounding with HIV RNA of 36 copies/mL that rose to 59,805 copies/mL 6 days later. ART was restarted promptly. Rebound plasma HIV sequences were identical to those obtained during acute infection by single-genome sequencing. Mathematical modeling predicted that the latent reservoir size was approximately 200 cells prior to ATI and that only around 1% of individuals with a similar HIV burden may achieve lifelong ART-free remission. Furthermore, we observed that lymphocytes expressing the tumor marker CD30 increased in frequency weeks to months prior to detectable HIV-1 RNA in plasma. This study was limited by the small sample size, which was a result of the rarity of individuals presenting during hyperacute infection. CONCLUSIONS: We report HIV relapse despite initiation of ART at one of the earliest stages of acute HIV infection possible. Near complete or complete loss of detectable HIV in blood and tissues did not lead to indefinite ART-free HIV remission. However, the small numbers of latently infected cells in individuals treated during hyperacute infection may be associated with prolonged ART-free remission.

High-throughput Characterization of HIV-1 Reservoir Reactivation Using a Single-Cell-in-Droplet PCR Assay.

Reactivation of latent viral reservoirs is on the forefront of HIV-1 eradication research. However, it is unknown if latency reversing agents (LRAs) increase the level of viral transcription from cells producing HIV RNA or harboring transcriptionally-inactive (latent) infection. We therefore developed a microfluidic single-cell-in-droplet (scd)PCR assay to directly measure the number of CD4+ T cells that produce unspliced (us)RNA and multiply spliced (ms)RNA following ex vivo latency reversal with either an histone deacetylase inhibitor (romidepsin) or T cell receptor (TCR) stimulation. Detection of HIV-1 transcriptional activity can also be performed on hundreds of thousands of CD4+ T-cells in a single experiment. The scdPCR method was then applied to CD4+ T cells obtained from HIV-1-infected individuals on antiretroviral therapy. Overall, our results suggest that effects of LRAs on HIV-1 reactivation may be heterogeneous-increasing transcription from active cells in some cases and increasing the number of transcriptionally active cells in others. Genomic DNA and human mRNA isolated from HIV-1 reactivated cells could also be detected and quantified from individual cells. As a result, our assay has the potential to provide needed insight into various reservoir eradication strategies.

Reproductive health impact of a school health center.

PURPOSE: Although school health centers (SHCs) may improve access to reproductive health care services and contraception, published data on SHC service use and reproductive health impact are limited. METHODS: Reproductive health indicators among students at four urban high schools in a single building with an SHC in 2009 were compared with students in a school without an SHC, using a quasi-experimental research design (N = 2,076 students, 1,365 from SHC and 711 from comparison school). The SHC provided comprehensive reproductive health education and services, including on-site provision of hormonal contraception. RESULTS: Students in the SHC were more likely to report receipt of health care provider counseling and classroom education about reproductive health and a willingness to use an SHC for reproductive health services. Use of hormonal contraception measured at various time points (first sex, last sex, and ever used) was greater among students in the SHC. Most 10th-12th graders using contraception in the SHC reported receiving contraception through the SHC. Comparing students in the nonintervention school to SHC nonusers to SHC users, we found stepwise increases in receipt of education and provider counseling, willingness to use the SHC, and contraceptive use. CONCLUSIONS: Students with access to comprehensive reproductive health services via an SHC reported greater exposure to reproductive health education and counseling and greater use of hormonal contraception. SHCs can be an important access point to reproductive health care and a key strategy for preventing teen pregnancy.

Common sexually transmitted infections in adolescents.

Adolescents are often at higher risk for acquiring sexually transmitted infections (STIs). Medical providers should be alert for both asymptomatic and symptomatic STIs, and follow appropriate screening guidelines. Moreover, providers need to know how to best administer adolescent-friendly confidential care, treatment, and health education in the primary care setting. This article addresses the most common adolescent STIs and pertinent recommendations for screening, diagnosis, and management of infections, in addition to a brief focused discussion on human immunodeficiency virus and adolescents.

Women's expectations and experiences with labour pain in medical and midwifery models of birth in the United States.

BACKGROUND: This research focuses on how women understand and experience labour as related to two competing views of childbirth pain. The biomedical view is that labour pain is abnormal and anaesthesia/analgesia use is encouraged to relieve the pain. The midwifery view is that pain is a normal part of labour that should be worked with instead of against. AIMS: To determine differences in the preparation for and experiences with labour pain by women choosing midwives versus obstetricians. METHODS: Prenatal and postpartum in-depth semi-structured interviews were conducted with a convenience sample of 80 women in Florida (United States): 40 who had chosen an obstetrician and 40 who had chosen a licensed midwife as their birth practitioner. FINDINGS: Women in both groups were concerned with the pain of childbirth before and after their labour experiences. Women choosing midwives discussed preparing for pain through various non-pharmaceutical coping methods, while women choosing physicians discussed pharmaceutical and non-pharmaceutical pain relief. CONCLUSIONS: Equal numbers of women expressed concerns with childbirth pain during the prenatal interviews, while more women choosing doctors spoke about pain after their births. Women had negative experiences when their planned pain relief method, either natural or medical, did not occur. The quandary facing women when it comes to labour pain relief is not choosing what they desire, but rather preparing themselves for the possibility that they may have to accept alternatives to their original preferences.

Women, birth practitioners, and models of pregnancy and birth-does consensus exist?

Women have differing beliefs about pregnancy and birth, and will be more suited to one type of practitioner versus another, depending on whether they believe that birth is a natural or a medical event. I hypothesize that if women and their practitioners have similar explanatory models, then the women may experience a better relationship with their practitioners, resulting in greater understanding of birth expectations, leading to improvements in experience and outcomes. In this article I explore how differing beliefs constitute identifiable models that can be distinguished as aligning with the midwifery model versus the medical model of birth.

Measuring school health center impact on access to and quality of primary care.

PURPOSE: School health centers (SHC) that provide comprehensive health care may improve access and quality of care for students; however, published impact data are limited. METHODS: We evaluated access and quality of health services at an urban high school with a SHC compared with a school without a SHC, using a quasiexperimental research design. Data were collected at the beginning of the school year, using a paper and pencil classroom questionnaire (n = 2,076 students). We measured SHC impact in several ways including grade by school interaction terms. RESULTS: Students at the SHC school were more likely to report having a regular healthcare provider, awareness of confidential services, support for health services in their school, and willingness to utilize those services. Students in the SHC school reported higher quality of care as measured by: respect for their health concerns, adequate time with the healthcare provider, understandable provider communications, and greater provider discussion at their last visit on topics such as sexual activity, birth control, emotions, future plans, diet, and exercise. Users of the SHC were also more likely to report higher quality of care, compared with either nonusers or students in the comparison school. CONCLUSIONS: Access to comprehensive health services via a SHC led to improved access to health care and improved quality of care. Impact was measureable on a school-wide basis but was greater among SHC users.