Complex decay dynamics of HIV virions,intact and defective proviruses,and 2LTR circles following initiation of antiretroviral therapy

In persons living with HIV-1 (PLWH) who start antiretroviral therapy (ART), plasma virus decays in a biphasic fashion to below the detection limit. The first phase reflects the short half-life (<1 d) of cells that produce most of the plasma virus. The second phase represents the slower turnover (t_1/2 = 14 d) of another infected cell population, whose identity is unclear. Using the intact proviral DNA assay (IPDA) to distinguish intact and defective proviruses, we analyzed viral decay in 17 PLWH initiating ART. Circulating CD4⁺ T cells with intact proviruses include few of the rapidly decaying first-phase cells. Instead, this population initially decays more slowly (t_1/2 = 12.9 d) in a process that largely represents death or exit from the circulation rather than transition to latency. This more protracted decay potentially allows for immune selection. After ∼3 mo, the decay slope changes, and CD4⁺ T cells with intact proviruses decay with a half-life of 19 mo, which is still shorter than that of the latently infected cells that persist on long-term ART. Two-long-terminal repeat (2LTR) circles decay with fast and slow phases paralleling intact proviruses, a finding that precludes their use as a simple marker of ongoing viral replication. Proviruses with defects at the 5′ or 3′ end of the genome show equivalent monophasic decay at rates that vary among individuals. Understanding these complex early decay processes is important for correct use of reservoir assays and may provide insights into properties of surviving cells that can constitute the stable latent reservoir.

For persons living with HIV-1 (PLWH), lifelong adherence to antiretroviral therapy (ART) is critical for maintaining suppression of viral replication and forestalling the development of fatal immunodeficiency. Following initiation of ART, plasma virus levels decay rapidly to below the limit of detection of clinical assays (1–6). Because antiretroviral drugs block new infection of susceptible cells, but not virus production by cells that have an integrated viral genome, this decay must reflect the loss of productively infected cells, cells that were infected prior to the initiation of ART. Productively infected cells could die from viral cytopathic effects, cytolytic host effector mechanisms, or virus-independent T cell turnover. In principle, the decay of plasma virus could also be explained by transition to a nonproductive or latent state of infection. Importantly, the decay is biphasic, indicating the presence of two populations of productively infected cells with different half-lives. Most of the plasma virus is produced by cells that decay very rapidly, with a half-life of less than 1 d. Perelson et al. (4) showed that after most of these cells have decayed, the slope changes, reflecting the slower elimination of a second population of productively infected cells. This population decays with a variable half-life (mean ∼ 2 wk). Although this biphasic decay is a consistent feature of the response to ART, there is still uncertainty about the nature, anatomic location, and fate of the cells responsible for virus production during the first and second phases of decay (referred to here as first- and second-phase cells, respectively). The differences between these two populations have never been elucidated.The first and second phases of decay bring viremia down to below the limit of detection of clinical assays (typically 20 to 50 copies of HIV-1 RNA per mL of plasma) within months of ART initiation, initially raising hope for eradication. However, a latent form of the virus persists in resting memory CD4⁺ T cells (7–14). Initial studies used a quantitative viral outgrowth assay (QVOA) to demonstrate that latently infected resting CD4⁺ T cells purified from PLWH on long-term suppressive ART could be induced to produce replication-competent virus by global T cell activation (8, 9). Longitudinal studies using the QVOA demonstrated that the half-life of the latent reservoir in resting CD4⁺ T cells is 44 mo in PLWH who are adherent to ART. This half-life is long enough to guarantee lifetime persistence of HIV-1 despite ART (12–14). Strategies targeting the latent reservoir in resting CD4⁺ T cells are a major focus of HIV cure research (15–17). In addition to resting CD4⁺ T cells, other cell types may contribute to HIV-1 persistence (18–20).Prior to and immediately following initiation of ART, the frequency of latently infected cells detected by QVOA is substantially higher than frequencies observed in PLWH on long-term ART (21). In principle, several different types of decay processes occurring over the first 6 to 12 mo of treatment could reduce the frequency of latently infected cells to the more stable frequencies observed in PLWH on long-term ART. Early studies by Jerome Zack and Mario Stevenson demonstrated that infected resting CD4⁺ T cells could harbor linear, unintegrated HIV-1 DNA in a state of preintegration latency (22, 23). Following cellular activation, linear unintegrated HIV-1 DNA can be integrated and transcribed, allowing production of virus (22, 23). The half-life of linear, unintegrated forms of the viral genome is not clear, with some studies suggesting that these forms are labile (22, 24–26). Some reverse-transcribed viral genomes can undergo homology-dependent or end-to-end ligation, generating one-long-terminal repeat or two-long-terminal repeat (2LTR) circles, respectively (reviewed in ref. 27). The stability of these forms is also controversial, but they are clearly replication-defective (27–31). Following integration of linear viral cDNA, decay dynamics depend on dynamics of the infected host cells, which can be eliminated by viral cytopathic effects, immune cytolytic effector mechanisms, and normal contraction-phase death of previously activated CD4⁺ T cells (32, 33).While the QVOA provides a definitive minimal estimate of the frequency of latently infected cells, it underestimates reservoir size because not all proviruses in resting CD4⁺ T cells are induced upon one round of maximum T cell activation (34–36). Many replication-competent proviruses require multiple rounds of stimulation for induction. As an alternative to the QVOA, many studies use PCR-based assays to measure proviral DNA. However, the vast majority of HIV-1 proviruses are defective due to apolipoprotein B messenger RNA editing enzyme, catalytic polypeptide-like (APOBEC)-mediated hypermutation or large internal deletions (34, 37–39). PCR-based assays do not distinguish between defective and intact proviruses (40, 41). Although infected cell dynamics have been explored using PCR-based assays (42), the results likely reflect the dynamics of defective proviruses (41). The recently developed intact proviral DNA assay (IPDA) uses two carefully chosen amplicons to probe informative regions of individual proviruses to provide better discrimination between intact and defective proviruses (41, 43). This assay has proven useful in evaluating the long-term dynamics of cells with intact and defective proviruses, demonstrating differences in decay rates that may reflect some vulnerability of cells with intact proviruses to immune effector mechanisms (41, 44, 45).In this study, we use the IPDA to explore the decay of intact and defective proviruses at early time points following initiation of ART. We identify decay processes occurring over intermediate time scales, but with pronounced differences between intact and defective proviruses. Of particular importance is the second-phase decay because infected cells that survive second-phase decay may down-regulate HIV-1 gene expression and enter the stable latent reservoir. Our findings also provide insight into mechanisms for the elimination of the cells with intact viral genomes and into the proper use of assays for the latent reservoir.     

Electrochemical Synthesis of Zinc Oxide Nanostructures on Flexible Substrate and Application as an Electrochemical Immunoglobulin-G Immunosensor

Immunoglobulin G (IgG), a type of antibody, represents approximately 75% of serum antibodies in humans, and is the most common type of antibody found in blood circulation. Consequently, the development of simple, fast and reliable systems for IgG detection, which can be achieved using electrochemical sandwich-type immunosensors, is of considerable interest. In this study we have developed an immunosensor for human (H)-IgG using an inexpensive and very simple fabrication method based on ZnO nanorods (NRs) obtained through the electrodeposition of ZnO. The ZnO NRs were treated by electrodepositing a layer of reduced graphene oxide (rGO) to ensure an easy immobilization of the antibodies. On Indium Tin Oxide supported on Polyethylene Terephthalate/ZnO NRs/rGO substrate, the sandwich configuration of the immunosensor was built through different incubation steps, which were all optimized. The immunosensor is electrochemically active thanks to the presence of gold nanoparticles tagging the secondary antibody. The immunosensor was used to measure the current density of the hydrogen development reaction which is indirectly linked to the concentration of H-IgG. In this way the calibration curve was constructed obtaining a logarithmic linear range of 10–1000 ng/mL with a detection limit of few ng/mL and good sensitivity.     

An infant with cystic fibrosis and a mother with an eating disorder: A difficult combination

The feeding of an infant with cystic fibrosis and associated malabsorption was made difficult by the mother's attitudes to body shape and food. She had a history of anorexia nervosa. Such parents may require special advice and support. © 1994 by John Wiley & Sons, Inc.     

Cerebral and tumoral blood flow in adult gliomas: a systematic review of results from magnetic resonance imaging

Objective:Blood flow is the rate of blood movement and relevant to numerous processes, though understudied in gliomas. The aim of this review was to pool blood flow metrics obtained from MRI modalities in adult supratentorial gliomas.Methods:MEDLINE, EMBASE and the Cochrane database were queried 01/01/2000–31/12/2019. Studies measuring blood flow in adult Grade II–IV supratentorial gliomas using dynamic susceptibility contrast (DSC) MRI, dynamic contrast enhanced MRI (DCE-MRI) or arterial spin labelling (ASL) were included. Absolute and relative cerebral blood flow (CBF), peritumoral blood flow and tumoral blood flow (TBF) were reported.Results:34 studies were included with 1415 patients and 1460 scans. The mean age was 52.4 ± 7.3 years. Most patients had glioblastoma (n = 880, 64.6%). The most common imaging modality was ASL (n = 765, 52.4%) followed by DSC (n = 538, 36.8%). Most studies were performed pre-operatively (n = 1268, 86.8%). With increasing glioma grade (II vs IV), TBF increased (70.8 vs 145.5 ml/100 g/min, p < 0.001) and CBF decreased (85.3 vs 49.6 ml/100 g/min, p < 0.001). In Grade IV gliomas, following treatment, CBF increased in ipsilateral (24.9 ± 1.2 vs 26.1 ± 0.0 ml/100 g/min, p < 0.001) and contralateral white matter (25.6 ± 0.2 vs 26.0± 0.0 ml/100 g/min, p < 0.001).Conclusion:Our findings demonstrate that increased mass effect from high-grade gliomas impairs blood flow within the surrounding brain that can improve with surgery.Advances in knowledge:This systematic review demonstrates how mass effect from brain tumours impairs blood flow in the surrounding brain parenchyma that can improve with treatment.     

Are Veterans Getting Their Preferred Depression Treatment? A National Observational Study in the Veterans Health Administration

BackgroundPhysician responsiveness to patient preferences for depression treatment may improve treatment adherence and clinical outcomes.ObjectiveTo examine associations of patient treatment preferences with types of depression treatment received and treatment adherence among Veterans initiating depression treatment.DesignPatient self-report surveys at treatment initiation linked to medical records.SettingVeterans Health Administration (VA) clinics nationally, 2018–2020.ParticipantsA total of 2582 patients (76.7% male, mean age 48.7 years, 62.3% Non-Hispanic White)Main MeasuresPatient self-reported preferences for medication and psychotherapy on 0–10 self-anchoring visual analog scales (0=“completely unwilling”; 10=“completely willing”). Treatment receipt and adherence (refilling medications; attending 3+ psychotherapy sessions) over 3 months. Logistic regression models controlled for socio-demographics and geographic variables.Key ResultsMore patients reported strong preferences (10/10) for psychotherapy than medication (51.2% versus 36.7%, McNemar χ²₁=175.3, p<0.001). A total of 32.1% of patients who preferred (7–10/10) medication and 21.8% who preferred psychotherapy did not receive these treatments. Patients who strongly preferred medication were substantially more likely to receive medication than those who had strong negative preferences (odds ratios [OR]=17.5; 95% confidence interval [CI]=12.5–24.5). Compared with patients who had strong negative psychotherapy preferences, those with strong psychotherapy preferences were about twice as likely to receive psychotherapy (OR=1.9; 95% CI=1.0–3.5). Patients who strongly preferred psychotherapy were more likely to adhere to psychotherapy than those with strong negative preferences (OR=3.3; 95% CI=1.4–7.4). Treatment preferences were not associated with medication or combined treatment adherence. Patients in primary care settings had lower odds of receiving (but not adhering to) psychotherapy than patients in specialty mental health settings. Depression severity was not associated with treatment receipt or adherence.ConclusionsMismatches between treatment preferences and treatment type received were common and associated with worse treatment adherence for psychotherapy. Future research could examine ways to decrease mismatch between patient preferences and treatments received and potential effects on patient outcomes.Supplementary InformationThe online version contains supplementary material available at 10.1007/s11606-021-07136-2.KEY WORDS: major depression, treatment preferences, treatment adherence, Veterans     

Earning Patient Trust: More Than a Question of Signaling

Laura Specker Sullivan's article “Trust, Risk, and Race in American Medicine” is a philosophically grounded and highly practical call for medical professionals to take on the task of comprehending the sources of patients’ mistrust. This is not only a clinical competence but also a moral obligation, in particular, when mistrust is warranted—as with African American patients who rely on medical institutions that have breached and continue to breach the trust of their communities. While Specker Sullivan focuses on how clinicians can signal comprehension once it has already been attained, I wish to step back and examine the nature of the efforts to gain knowledge. The effort to comprehend requires, at a minimum, the following actions: educating oneself about medical racism, examining one's implicit biases, and engaging, with empathic curiosity, with the patient or family members in the clinical encounter at hand.     

GPER-induced signaling is essential for the survival of breast cancer stem cells

G protein-coupled estrogen receptor-1 (GPER), a member of the G protein-coupled receptor (GPCR) superfamily, mediates estrogen-induced proliferation of normal and malignant breast epithelial cells. However, its role in breast cancer stem cells (BCSCs) remains unclear. Here we showed greater expression of GPER in BCSCs than non-BCSCs of three patient-derived xenografts of ER⁻/PR⁺ breast cancers. GPER silencing reduced stemness features of BCSCs as reflected by reduced mammosphere forming capacity in vitro, and tumor growth in vivo with decreased BCSC populations. Comparative phosphoproteomics revealed greater GPER-mediated PKA/BAD signaling in BCSCs. Activation of GPER by its ligands, including tamoxifen (TMX), induced phosphorylation of PKA and BAD-Ser118 to sustain BCSC characteristics. Transfection with a dominant-negative mutant BAD (Ser118Ala) led to reduced cell survival. Taken together, GPER and its downstream signaling play a key role in maintaining the stemness of BCSCs, suggesting that GPER is a potential therapeutic target for eradicating BCSCs.