An active HIV reservoir during ART is associated with maintenance of HIV-specific CD8+ T cell magnitude and short-lived differentiation status.

Before initiation of antiretroviral therapy (ART), HIV-specific CD8+ T cells are dysfunctional and short lived. To better understand the relationship between the HIV reservoir in CD4+ T cells and the magnitude and differentiation status of HIV-specific CD8+ T cells, we investigated these cells from acute and chronic HIV-infected individuals after 2 years of ART. Although both the HIV reservoir and the CD8+ T cell responses declined significantly after 2 years of ART, sustained HIV-specific CD8+ T cell responses correlated with a greater reduction of integrated HIV provirus. However, the magnitude of CD8+ T cells specific for HIV Gag, Pol, Nef, and Vif proteins positively associated with the active reservoir size during ART, measured as cell-associated RNA. Importantly, high HIV DNA levels strongly associate with maintenance of short-lived HIV-specific CD8+ T cells, regardless of ART initiation time. Our data suggest that the active reservoir maintains HIV-specific CD8+ T cell magnitude but prevents their differentiation into functional cells.

Initial productive and latent HIV infections originate in vivo by infection of resting T cells.

Productively infected cells are generally thought to arise from HIV infection of activated CD4+ T cells, and these infected activated cells are thought to be a recurring source of latently infected cells when a portion of the population transitions to a resting state. We discovered and report here that productively and latently infected cells can instead originate from direct infection of resting CD4+ T cell populations in lymphoid tissues in Fiebig I, the earliest stage of detectable HIV infection. We found that direct infection of resting CD4+ T cells was correlated with the availability of susceptible target cells in lymphoid tissues largely restricted to resting CD4+ T cells in which expression of pTEFb enabled productive infection, and we documented persistence of HIV-producing resting T cells during antiretroviral therapy (ART). Thus, we provide evidence of a mechanism by which direct infection of resting T cells in lymphoid tissues to generate productively and latently infected cells creates a mechanism by which the productively infected cells can replenish both populations and maintain two sources of virus from which HIV infection can rebound, even if ART is instituted at the earliest stage of detectable infection.

HIV rapidly targets a diverse pool of CD4+ T cells to establish productive and latent infections.

Upon infection, HIV disseminates throughout the human body within 1-2 weeks. However, its early cellular targets remain poorly characterized. We used a single-cell approach to retrieve the phenotype and TCR sequence of infected cells in blood and lymphoid tissue from individuals at the earliest stages of HIV infection. HIV initially targeted a few proliferating memory CD4+ T cells displaying high surface expression of CCR5. The phenotype of productively infected cells differed by Fiebig stage and between blood and lymph nodes. The TCR repertoire of productively infected cells was heavily biased, with preferential infection of previously expanded and disseminated clones, but composed almost exclusively of unique clonotypes, indicating that they were the product of independent infection events. Latent genetically intact proviruses were already archived early in infection. Hence, productive infection is initially established in a pool of phenotypically and clonotypically distinct T cells, and latently infected cells are generated simultaneously.

Long-term antiretroviral therapy initiated in acute HIV infection prevents residual dysfunction of HIV-specific CD8+ T cells.

BACKGROUND: Harnessing CD8+ T cell responses is being explored to achieve HIV remission. Although HIV-specific CD8+ T cells become dysfunctional without treatment, antiretroviral therapy (ART) partially restores their function. However, the extent of this recovery under long-term ART is less understood. METHODS: We analyzed the differentiation status and function of HIV-specific CD8+ T cells after long-term ART initiated in acute or chronic HIV infection ex vivo and upon in vitro recall. FINDINGS: ART initiation in any stage of acute HIV infection promoted the persistence of long-lived HIV-specific CD8+ T cells with high expansion (P<0·0008) and cytotoxic capacity (P=0·02) after in vitro recall, albeit at low cell number (P=0·003). This superior expansion capacity correlated with stemness (r=0·90, P=0·006), measured by TCF-1 expression, similar to functional HIV-specific CD8+ T cells found in spontaneous controllers. Importanly, TCF-1 expression in these cells was associated with longer time to viral rebound ranging from 13 to 48 days after ART interruption (r =0·71, P=0·03). In contrast, ART initiation in chronic HIV infection led to more differentiated HIV-specific CD8+ T cells lacking stemness properties and exhibiting residual dysfunction upon recall, with reduced proliferation and cytolytic activity. INTERPRETATION: ART initiation in acute HIV infection preserves functional HIV-specific CD8+ T cells, albeit at numbers too low to control viral rebound post-ART. HIV remission strategies may need to boost HIV-specific CD8+ T cell numbers and induce stem cell-like properties to reverse the residual dysfunction persisting on ART in people treated after acute infection prior to ART release. FUNDING: U.S. National Institutes of Health and U.S. Department of Defense.

Anti-HIV antibody development up to 1 year after antiretroviral therapy initiation in acute HIV infection.

Early initiation of antiretroviral therapy (ART) in acute HIV infection (AHI) is effective at limiting seeding of the HIV viral reservoir, but little is known about how the resultant decreased antigen load affects long-term Ab development after ART. We report here that Env-specific plasma antibody (Ab) levels and Ab-dependent cellular cytotoxicity (ADCC) increased during the first 24 weeks of ART and correlated with Ab levels persisting after 48 weeks of ART. Participants treated in AHI stage 1 had lower Env-specific Ab levels and ADCC activity on ART than did those treated later. Importantly, participants who initiated ART after peak viremia in AHI developed elevated cross-clade ADCC responses that were detectable 1 year after ART initiation, even though clinically undetectable viremia was reached by 24 weeks. These data suggest that there is more germinal center (GC) activity in the later stages of AHI and that Ab development continues in the absence of detectable viremia during the first year of suppressive ART. The development of therapeutic interventions that can enhance earlier development of GCs in AHI and Abs after ART initiation could provide important protection against the viral reservoir that is seeded in individuals treated early in the disease.

Continuous Prophylactic Antiretrovirals/Antiretroviral Therapy Since Birth Reduces Seeding and Persistence of the Viral Reservoir in Children Vertically Infected With Human Immunodeficiency Virus.

BACKGROUND: Early antiretroviral therapy (ART) restricts the size of the human immunodeficiency virus (HIV) reservoir in infants. However, whether antiretroviral (ARV) prophylaxis given to exposed vertically infected children exerts similar effects remains unknown. METHODS: We measured total and integrated HIV DNA, as well as the frequency of CD4 T cells producing multiply spliced RNA (msRNA) after stimulation (inducible reservoir) in vertically infected Thai infants. Eighty-five infants were followed longitudinally for up to 3 years. We compared the size of the reservoir in children who received continuous ARV prophylaxis since birth vs those who never received or discontinued prophylaxis before initiating ART. We used samples from a cross-sectional cohort of 37 Thai children who had initiated ART within 6 months of life to validate our findings. RESULTS: Before ART, levels of HIV DNA and the frequencies of cells producing msRNA were significantly lower in infants who received continuous ARV prophylaxis since birth compared to those in whom ARV prophylaxis was discontinued or never initiated (P < .020 and P < .001, respectively). Upon ART initiation, total and integrated HIV DNA levels decayed significantly in both groups (P < .01 in all cases). Interestingly, the initial differences in the frequencies of infected cells persisted during 3 years on ART. The beneficial effect of prophylaxis on the size of the HIV reservoir was confirmed in the cross-sectional study. Importantly, no differences were observed between children who discontinued prophylactic ARVs before starting ART and those who delayed ART initiation without receiving prior prophylaxis. CONCLUSIONS: Neonatal ARV prophylaxis with direct transition to ART durably limits the size of the HIV reservoir.

A randomized trial of vorinostat with treatment interruption after initiating antiretroviral therapy during acute HIV-1 infection.

OBJECTIVE AND DESIGN: A randomized, open-label pilot study in individuals treated with antiretroviral therapy (ART) since acute HIV infection (AHI) with a regimen including a histone deacetylase inhibitor to induce HIV from latency and control HIV replication during subsequent treatment interruption (TI). METHODS: Fifteen participants who initiated ART at AHI were randomized to vorinostat/hydroxychloroquine/maraviroc (VHM) plus ART (n â€‹= â€‹10) or ART alone (n â€‹= â€‹5). The VHM arm received three 14-day vorinostat cycles within 10 weeks before TI. ART was resumed for plasma viral load (VL) â€‹> â€‹1,000 HIV RNA copies/mL. Primary outcome was proportion of participants on VHM â€‹+ â€‹ART versus ART only with VL â€‹< â€‹50 copies/mL for 24 weeks after TI. RESULTS: Fifteen participants on ART (median: 178 weeks: range 79-295) enrolled. Two on VHM â€‹+ â€‹ART experienced serious adverse events. Fourteen participants underwent TI; all experienced VL rebound with no difference in time between arms: VHM â€‹+ â€‹ART (n â€‹= â€‹9) median: 4 weeks and ART only (n â€‹= â€‹5) median: 5 weeks. VHM induced a 2.2-fold increase in VL (p â€‹= â€‹0.008) by single-copy HIV RNA assay after the first cycle. Neopterin levels increased significantly following the first two cycles. After VHM treatment, the frequencies of peripheral blood mononuclear cells harboring total HIV DNA and cell-associated RNA were unchanged. All participants achieved VL suppression following ART re-initiation. CONCLUSIONS: Administration of VHM increased HIV VL in plasma, but this was not sustained. VHM did not impact time to viral rebound following TI and had no impact on the size of the HIV reservoir, suggesting that HIV reservoir elimination will require alternative treatment strategies.

Plasmacytoid dendritic cells sense HIV replication before detectable viremia following treatment interruption.

Plasmacytoid dendritic cells (pDCs) are robust producers of IFNα and one of the first immune cells to respond to SIV infection. To elucidate responses to early HIV-1 replication, we studied blood pDCs in 29 HIV-infected participants who initiated antiretroviral therapy during acute infection and underwent analytic treatment interruption (ATI). We observed an increased frequency of partially activated pDCs in the blood before detection of HIV RNA. Concurrent with peak pDC frequency, we detected a transient decline in the ability of pDCs to produce IFNα in vitro, which correlated with decreased phosphorylation of IFN regulatory factory 7 (IRF7) and NF-κB. The levels of phosphorylated IRF7 and NF-κB inversely correlated with plasma IFNα2 levels, implying that pDCs were refractory to in vitro stimulation after IFNα production in vivo. After ATI, decreased expression of IFN genes in pDCs inversely correlated with the time to viral detection, suggesting that pDC IFN loss is part of an effective early immune response. These data from a limited cohort provide a critical first step in understanding the earliest immune response to HIV-1 and suggest that changes in blood pDC frequency and function can be used as an indicator of viral replication before detectable plasma viremia.

Screening for Opioid Misuse in the Nonhospitalized Seriously Ill Patient.

Background: Responding to an epidemic of opioid-related deaths, guidelines and laws have been implemented to promote safe opioid prescribing practices. Objective: This study evaluates differences in screening practices and knowledge of laws between oncologists and cardiologists who prescribe opiates. Design: Surveys regarding screening practices and knowledge of opioid prescribing laws were distributed in March 2017 to oncology and congestive heart failure (CHF) clinicians at the University of Virginia. Chi-square and Wilcoxon rank sum tests were used. Results: Forty-six of 129 (35.6%) oncology providers and 7 of 14 (50%) CHF providers reported prescribing opiates in their clinic with usable survey results. The majority of oncology (65.22%) and cardiology (85.71%) providers report screening for substance abuse "when indicated" (p = 0.053). Only 19.6% of oncologists reported always using the prescription monitoring program (PMP), while 71.43% of cardiologists reported using it always (p = 0.014). Of the oncology providers, 66.67% report never using the urine drug screen (UDS), while 86.7% of cardiologists reported using it "when indicated" (p = 0.0086). Up to 34.78% of the oncologists and 57.14% of the cardiologists reported of never screening the family members for misuse (p = 0.317). Knowledge of laws was similar between groups, with 14.29% of cardiology and 17.39% of oncology providers reporting no knowledge of opioid prescribing laws (p = 0.2869). Conclusions: Routine screening for substance misuse risk was uncommon for both groups, but cardiology providers were more likely to use the PMP or UDS. Knowledge gaps regarding Virginia laws were noted in both groups. Improved education regarding best practices and laws, as well as programs to promote screening, is needed for all providers.

Expression and splicing of Ikaros family members in murine and human thymocytes.

The Ikaros family of transcription factors includes five highly homologous members that can homodimerize or heterodimerize in any combination. Dimerization is essential for their ability to bind DNA and function as transcription factors. Previous studies showed that eliminating the function of the entire family blocks lymphocyte development while deletion of individual family members has relatively minor defects. These data indicate that multiple family members function during T cell development, so we examined the changes in expression of each family member as thymocytes progressed from the CD4-CD8- double negative (DN) to the CD4+CD8+ double positive (DP) developmental stage. Further, we compared the expression of each family member in murine and human thymocytes. In both species, Ikaros and Aiolos mRNA levels increased as thymocytes progressed through the DN to DP transition, but the corresponding increases in protein levels were only observed in mice. Further, Ikaros and Aiolos underwent extensive alternative splicing in mice, whereas only Ikaros was extensively spliced in humans. Helios mRNA and protein levels decreased during murine T cell development, but increased during human T cell development. These differences in the expression and splicing of Ikaros family members between human and murine thymocytes strongly suggest that the Ikaros family of transcription factors regulates murine and human T cell development differently, although the similarities across Ikaros family members may allow different proteins to fulfill similar functions.

Variations in mRNA and protein levels of Ikaros family members in pediatric T cell acute lymphoblastic leukemia.

BACKGROUND: Pediatric T cell acute lymphoblastic leukemia (T-ALL) is a highly heterogeneous disease in which the cells share phenotypic characteristics with normal human thymocytes. The Ikaros family of transcription factors includes five members that are required for normal T cell development and are implicated in leukemogenesis. The goal of this work was to correlate the pattern of expression of Ikaros family members with the phenotype of the T-ALL cells. METHODS: We obtained twenty-four samples from pediatric T-ALL patients and used multi-parameter flow cytometry to characterize each sample, comparing the phenotype of the leukemic cells with normal human thymocytes. Then, we defined the expression levels of each Ikaros family member to determine whether the mRNA levels or splicing or protein levels were similar to the normal patterns seen during human T cell development. RESULTS: Multi-parameter analysis of the phenotype of T-ALL cells revealed that each patient's cells were unique and could not be readily correlated with stages of T cell development. Similarly, the pattern of Ikaros expression varied among patients. In most patients, Ikaros mRNA was the dominant family member expressed, but some patients' cells contained mostly Helios, Aiolos, or Eos mRNA. Despite that most patients had elevated mRNA levels of Ikaros family members and unique patterns of mRNA splicing, most patients had significantly reduced protein levels of Ikaros and Aiolos. CONCLUSIONS: Our analysis of the cell phenotype and Ikaros expression levels in T-ALL cells revealed the extent of heterogeneity among patients. While it is rarely possible to trace leukemic cells to their developmental origin, we found distinct patterns of Ikaros family mRNA levels in groups of patients. Further, mRNA and protein levels of Ikaros and Aiolos did not correlate, indicating that mRNA and protein levels are regulated via distinct mechanisms.

Expression patterns of Ikaros family members during positive selection and lineage commitment of human thymocytes.

The Ikaros family of transcription factors is essential for normal T-cell development, but their expression pattern in human thymocytes remains poorly defined. Our goal is to determine how protein levels of Ikaros, Helios and Aiolos change as human thymocytes progress through the positive selection and lineage commitment stages. To accomplish this goal, we used multi-parameter flow cytometry to define the populations in which positive selection and lineage commitment are most likely to occur. After human thymocytes express CD3 and receive positive selection signals, the cells down-regulate expression of CD4 to become transitional single-positive (TSP) CD8+ thymocytes. At this stage, there was a transient increase in the Ikaros, Helios and Aiolos protein levels. After the TSP CD8+ developmental stage, some thymocytes re-express CD4 and become CD3hi double-positive thymocytes before down-regulating CD8 to become mature single-positive CD4+ thymocytes. Except for regulatory T cells, Helios protein levels declined and Aiolos protein levels transiently increased during CD4+ T-cell maturation. For thymocytes progressing toward the CD8+ T-cell lineage, TSP CD8+ thymocytes increase their expression of CD3 and maintain high levels of Aiolos protein as the cells complete their maturation. In summary, we defined the TSP CD8+ developmental stage in human T-cell development and propose that this stage is where CD4/CD8 lineage commitment occurs. Ikaros, Helios and Aiolos each undergo a transient increase in protein levels at the TSP stage before diverging in their expression patterns at later stages.

Breast Cancer Screening and Prevention.

This issue provides a clinical overview of breast cancer screening and prevention, focusing on risk assessment, screening, prevention, and practice improvement. The content of In the Clinic is drawn from the clinical information and education resources of the American College of Physicians (ACP), including MKSAP (Medical Knowledge and Self-Assessment Program). Annals of Internal Medicine editors develop In the Clinic in collaboration with the ACP's Medical Education and Publishing divisions and with the assistance of additional science writers and physician writers.

Ikaros, Helios, and Aiolos protein levels increase in human thymocytes after ß selection.

In human T cell development, the mechanisms that regulate cell fate decisions after TCRß expression remain unclear. We defined the stages of T cell development that flank TCRß expression and found distinct patterns of human T cell development. In half the subjects, T cell development progressed from the CD4(-)CD8(-) double-negative stage to the CD4(+)CD8(+) double-positive (DP) stage through an immature single-positive (ISP) CD4(+) intermediate. However, in some patients, CD4 and CD8 were expressed simultaneously and the ISP population was small. In each group of patients, CD3(-) ISP and DP thymocytes were subdivided into ISP1, ISP2, DP1, DP2, DP3, DP4, and DP5 developmental stages according to their expression of CD28, CD44, CD1a, CD7, CD45RO, and CD38. The ISP2, DP2, and DP3 thymocyte populations proliferated more robustly than ISP1 and DP1 and expressed markers consistent with TCRß expression. After the DP3 stage, proliferation returned to baseline levels. We then analyzed protein levels of Ikaros, Helios, and Aiolos, the three Ikaros family members most abundantly expressed in human thymocytes. Ikaros and Helios expression increased transiently at the ISP2, DP2, and DP3 populations. Aiolos expression also increased at the ISP2, DP2, and DP3 stages, but its expression remained elevated throughout the DP4 and DP5 stages. In summary, we propose a model of human T cell development that reflects the asynchronous nature of TCRß expression and we define the subpopulations of thymocytes that are highly proliferative and express Ikaros family members.

Women's Awareness of Their Contraceptive Benefits Under the Patient Protection and Affordable Care Act.

The Patient Protection and Affordable Care Act mandates that there be no out-of-pocket cost for Food and Drug Administration-approved contraceptive methods. Among 987 privately insured reproductive aged Pennsylvania women, fewer than 5% were aware that their insurance covered tubal sterilization, and only 11% were aware that they had full coverage for an intrauterine device. For the Affordable Care Act contraceptive coverage mandate to affect effective contraception use and reduce unintended pregnancies, public awareness of the expanded benefits is essential.

Web-based curriculum. A practical and effective strategy for teaching women's health.

OBJECTIVE: To address the need for women's health education by designing, implementing, and evaluating a self-study, web-based women's health curriculum. DESIGN: Cohort of students enrolled in the ambulatory portion of the medicine clerkship with comparison group of students who had not yet completed this rotation. PARTICIPANTS/SETTING: Third- and fourth-year medical students on the required medicine clerkship (115 students completed the curriculum; 158 completed patient-related logs). INTERVENTION: Following an extensive needs assessment and formulation of competencies and objectives, we developed a web-based women's health curriculum completed during the ambulatory portion of the medicine clerkship. The modules were case based and included web links, references, and immediate feedback on posttesting. We discuss technical issues with implementation and maintenance. MEASUREMENTS AND MAIN RESULTS: We evaluated this curriculum using anonymous questionnaires, open-ended narrative comments, online multiple-choice tests, and personal digital assistant (PDA) logs of patient-related discussions of women's health. Students completing the curriculum valued learning women's health, preferred this self-directed learning over lecture, scored highly on knowledge tests, and were involved in more and higher-level discussions of women's health with faculty (P<.001). CONCLUSIONS: We present a model for the systematic design of a web-based women's health curriculum as part of a medicine clerkship. The web-based instruction resolved barriers associated with limited curriculum time and faculty availability, provided an accessible and standard curriculum, and met the needs of adult learners (with their motivation to learn topics they value and apply this knowledge in their daily work). We hypothesize that our web-based curriculum spurred students to later discuss these topics with faculty. Web-based learning may be particularly suited for women's health because of its multidisciplinary nature and need for vertical integration throughout medical school curricula.

Postmenopausal hormone therapy and its association with cognitive impairment.

BACKGROUND: Cognitive impairment is a common and potentially debilitating medical problem in older women. Postmenopausal hormone therapy (HT) has been associated with better cognitive function, but the literature is conflicting. Results of recent trials suggest that HT is inappropriate for prevention of heart disease, and we sought to determine the role of HT in the risk of cognitive impairment. METHODS: We measured HT use and cognitive function in a population-based cohort of 1462 postmenopausal women participating in the 5-year follow-up examination for the Epidemiology of Hearing Loss Study in 1998-2000. The cohort was defined in 1987-1988 by residency in Beaver Dam, Wis, and an age of 43 to 84 years. Women had also participated in the Beaver Dam Eye Study baseline examination in 1988-1990. Use of HT was assessed at the Beaver Dam Eye Study baseline (1988-1990), 5-year follow-up (1993-1995), and 10-year follow-up (1998-2000) visits. Cognitive impairment was defined as a low Mini-Mental State Examination score or a reported diagnosis of Alzheimer disease. RESULTS: Six percent of participants (n = 94) were impaired; these women were significantly older and less educated than those who were unimpaired. In age- and education-adjusted analysis, current HT use was not significantly associated with cognitive impairment (odds ratio, 0.6; 95% confidence interval, 0.2-1.3). Similarly, cognitive impairment was not associated with past HT use or duration of HT use. CONCLUSION: In this large population-based study, postmenopausal hormone therapy was not significantly associated with better cognitive function.

Postmenopausal hormone therapy: a concise guide to therapeutic uses, formulations, risks, and alternatives.

Postmenopausal hormone replacement therapy is helpful in relieving menopausal vasomotor symptoms and vaginal atrophy and can prevent osteoporosis; however, attendant risks include breast cancer, thromboembolism, gallbladder disease, stroke, CHD, dementia, and hypertriglyceridemia. Decision making must weigh these risks and benefits and also include potential benefits on mood, colorectal cancer prevention, and hip fracture reduction. Some areas, such as ovarian cancer risk and the impact of combination estrogen-progestin versus unopposed estrogen on risk, remain unclear. The physician and patient need to carefully assess, discuss, and monitor the individual's symptoms and risks when considering HT use. For those with contraindications or concerns about HT, there are alternative therapies of variable efficacy for vasomotor symptoms and vaginal atrophy.