Pediatric staff and their perceptions of music therapy services.

PURPOSE: To reveal the benefits, needs, and limitations of music therapy observed by clinical pediatric staff at a children's hospital in the United States of America. DESIGN AND METHODS: Researchers developed an electronic 13-question survey and distributed the survey in the fall of 2022. Questions included demographics, Likert-type scale, and open-ended queries. Data was collected via Qualtrics and analyzed using descriptive statistics and content analysis. RESULTS: A total of 83 pediatric staff completed the survey. Staff observed positive benefits, where the highest reported areas were opportunities for dealing with anxiety/stress (94.7%), opportunities for social interaction (93.3%), and quality of life (89.3%). Analysis of free-response questions suggest that staff expect expertise and a nuanced understanding of the needs of each of their clinical units. CONCLUSION: Results suggest that staff possess an overall positive attitude toward music therapy in all settings served. Music therapists may be valuable for psychosocial and rehabilitative support to hospitalized children and their families. PRACTICE IMPLICATIONS: Nursing staff may utilize music therapists to improve patient outcomes and reduce the negative effects of hospitalization.

Combined noncanonical NF-κB agonism and targeted BET bromodomain inhibition reverse HIV latency ex vivo.

Latency reversal strategies for HIV cure using inhibitor of apoptosis protein (IAP) antagonists (IAPi) induce unprecedented levels of latent reservoir expression without immunotoxicity during suppressive antiretroviral therapy (ART). However, full targeting of the reservoir may require combinatorial approaches. A Jurkat latency model screen for IAPi combination partners demonstrated synergistic latency reversal with bromodomain (BD) and extraterminal domain protein inhibitors (BETi). Mechanistic investigations using CRISPR-CAS9 and single-cell RNA-Seq informed comprehensive ex vivo evaluations of IAPi plus pan-BET, bD-selective BET, or selective BET isoform targeting in CD4+ T cells from ART-suppressed donors. IAPi+BETi treatment resulted in striking induction of cell-associated HIV gag RNA, but lesser induction of fully elongated and tat-rev RNA compared with T cell activation-positive controls. IAPi+BETi resulted in HIV protein induction in bulk cultures of CD4+ T cells using an ultrasensitive p24 assay, but did not result in enhanced viral outgrowth frequency using a standard quantitative viral outgrowth assay. This study defines HIV transcriptional elongation and splicing as important barriers to latent HIV protein expression following latency reversal, delineates the roles of BET proteins and their BDs in HIV latency, and provides a rationale for exploration of IAPi+BETi in animal models of HIV latency.

Sequence Evaluation and Comparative Analysis of Novel Assays for Intact Proviral HIV-1 DNA.

The HIV proviral reservoir is the major barrier to cure. The predominantly replication-defective proviral landscape makes the measurement of virus that is likely to cause rebound upon antiretroviral therapy (ART)-cessation challenging. To address this issue, novel assays to measure intact HIV proviruses have been developed. The intact proviral DNA assay (IPDA) is a high-throughput assay that uses two probes to exclude the majority of defective proviruses and determine the frequency of intact proviruses, albeit without sequence confirmation. Quadruplex PCR with four probes (Q4PCR) is a lower-throughput assay that uses limiting dilution long-distance PCR amplification followed by quantitative PCR (qPCR) and near-full-length genome sequencing (nFGS) to estimate the frequency of sequence-confirmed intact proviruses and provide insight into their clonal composition. To explore the advantages and limitations of these assays, we compared IPDA and Q4PCR measurements from 39 ART-suppressed people living with HIV. We found that IPDA and Q4PCR measurements correlated with one another, but frequencies of intact proviral DNA differed by approximately 19-fold. This difference may be in part due to inefficiencies in long-distance PCR amplification of proviruses in Q4PCR, leading to underestimates of intact proviral frequencies. In addition, nFGS analysis within Q4PCR explained that some of this difference is explained by proviruses that are classified as intact by IPDA but carry defects elsewhere in the genome. Taken together, this head-to-head comparison of novel intact proviral DNA assays provides important context for their interpretation in studies to deplete the HIV reservoir and shows that together the assays bracket true reservoir size.IMPORTANCE The intact proviral DNA assay (IPDA) and quadruplex PCR (Q4PCR) represent major advances in accurately quantifying and characterizing the replication-competent HIV reservoir. This study compares the two novel approaches for measuring intact HIV proviral DNA in samples from 39 antiretroviral therapy (ART)-suppressed people living with HIV, thereby informing ongoing efforts to deplete the HIV reservoir in cure-related trials.

Longitudinal Dynamics of Intact HIV Proviral DNA and Outgrowth Virus Frequencies in a Cohort of Individuals Receiving Antiretroviral Therapy.

BACKGROUND: The replication-competent human immunodeficiency virus (HIV) reservoir is the major barrier to cure. The quantitative viral outgrowth assay (QVOA), the gold-standard method to quantify replication-competent HIV, is resource intensive, which limits its application in large clinical trials. The intact proviral DNA assay (IPDA) requires minimal cell input relative to QVOA and quantifies both defective and intact proviral HIV DNA, the latter potentially serving as a surrogate marker for replication-competent provirus. However, there are limited cross-sectional and longitudinal data on the relationship between IPDA and QVOA measurements. METHODS: QVOA and IPDA measurements were performed on 156 resting CD4 T-cell (rCD4) samples from 83 antiretroviral therapy-suppressed HIV-positive participants. Longitudinal QVOA and IPDA measurements were performed on rCD4 from 29 of these participants. RESULTS: Frequencies of intact, defective, and total proviruses were positively associated with frequencies of replication-competent HIV. Longitudinally, decreases in intact proviral frequencies were strikingly similar to that of replication-competent virus in most participants. In contrast, defective proviral DNA frequencies appeared relatively stable over time in most individuals. CONCLUSIONS: Changes in frequencies of IPDA-derived intact proviral DNA and replication-competent HIV measured by QVOA are similar. IPDA is a promising high-throughput approach to estimate changes in the frequency of the replication-competent reservoir.

Impact of Biological Sex on Immune Activation and Frequency of the Latent HIV Reservoir During Suppressive Antiretroviral Therapy.

BACKGROUND: Persistent HIV infection of long-lived resting CD4 T cells, despite antiretroviral therapy (ART), remains a barrier to HIV cure. Women have a more robust type 1 interferon response during HIV infection relative to men, contributing to lower initial plasma viremia. As lower viremia during acute infection is associated with reduced frequency of latent HIV infection, we hypothesized that women on ART would have a lower frequency of latent HIV compared to men. METHODS: ART-suppressed, HIV seropositive women (n = 22) were matched 1:1 to 22 of 39 ART-suppressed men. We also compared the 22 women to all 39 men, adjusting for age and race as covariates. We measured the frequency of latent HIV using the quantitative viral outgrowth assay, the intact proviral DNA assay, and total HIV gag DNA. We also performed activation/exhaustion immunophenotyping on peripheral blood mononuclear cells and quantified interferon-stimulated gene (ISG) expression in CD4 T cells. RESULTS: We did not observe evident sex differences in the frequency of persistent HIV in resting CD4 T cells. Immunophenotyping and CD4 T-cell ISG expression analysis revealed marginal differences across the sexes. CONCLUSIONS: Differences in HIV reservoir frequency and immune activation appear to be small across sexes during long-term suppressive therapy.

CTLA-4 and PD-1 dual blockade induces SIV reactivation without control of rebound after antiretroviral therapy interruption.

The primary human immunodeficiency virus (HIV) reservoir is composed of resting memory CD4+ T cells, which often express the immune checkpoint receptors programmed cell death protein 1 (PD-1) and cytotoxic T lymphocyte-associated protein 4 (CTLA-4), which limit T cell activation via synergistic mechanisms. Using simian immunodeficiency virus (SIV)-infected, long-term antiretroviral therapy (ART)-treated rhesus macaques, we demonstrate that PD-1, CTLA-4 and dual CTLA-4/PD-1 immune checkpoint blockade using monoclonal antibodies is well tolerated, with evidence of bioactivity in blood and lymph nodes. Dual blockade was remarkably more effective than PD-1 blockade alone in enhancing T cell cycling and differentiation, expanding effector-memory T cells and inducing robust viral reactivation in plasma and peripheral blood mononuclear cells. In lymph nodes, dual CTLA-4/PD-1 blockade, but not PD-1 alone, decreased the total and intact SIV-DNA in CD4+ T cells, and SIV-DNA and SIV-RNA in B cell follicles, a major site of viral persistence during ART. None of the tested interventions enhanced SIV-specific CD8+ T cell responses during ART or viral control after ART interruption. Thus, despite CTLA-4/PD-1 blockade inducing robust latency reversal and reducing total levels of integrated virus, the degree of reservoir clearance was still insufficient to achieve viral control. These results suggest that immune checkpoint blockade regimens targeting PD-1 and/or CTLA-4, if performed in people living with HIV with sustained aviremia, are unlikely to induce HIV remission in the absence of additional interventions.

The 17th Rocky Mountain Virology Association Meeting.

Since 2000, scientists and students from the greater Rocky Mountain region, along with invited speakers, both national and international, have gathered at the Mountain Campus of Colorado State University to discuss their area of study, present recent findings, establish or strengthen collaborations, and mentor the next generation of virologists and prionologists through formal presentations and informal discussions concerning science, grantsmanship and network development. This year, approximately 100 people attended the 17th annual Rocky Mountain Virology Association meeting, that began with a keynote presentation, and featured 29 oral and 35 poster presentations covering RNA and DNA viruses, prions, virus-host interactions and guides to successful mentorship. Since the keynote address focused on the structure and function of Zika and related flaviviruses, a special session was held to discuss RNA control. The secluded meeting at the foot of the Colorado Rocky Mountains gave ample time for in-depth discussions amid the peak of fall colors in the aspen groves while the random bear provided excitement. On behalf of the Rocky Mountain Virology Association, this report summarizes the >50 reports.