CD4+ T cells with latent HIV-1 have reduced proliferative responses to T cell receptor stimulation.

The latent reservoir for HIV-1 in resting CD4+ T cells persists despite antiretroviral therapy as a barrier to cure. The antigen-driven proliferation of infected cells is a major mechanism of reservoir persistence. However, activation through the T cell antigen receptor (TCR) can induce latent proviruses, leading to viral cytopathic effects and immune clearance. In single-cell studies, we show that, relative to uninfected cells or cells with a defective provirus, CD4+ T cells with an intact provirus have a profound proliferative defect in response to TCR stimulation. Virion production was observed in only 16.5% of cultures with an intact provirus, but proliferation was reduced even when no virion production was detected. Proliferation was inversely correlated with in vivo clone size. These results may reflect the effects of previous in vivo proliferation and do not support attempts to reduce the reservoir with antiproliferative agents, which may have greater effects on normal T cell responses.

Bispecific antibodies promote natural killer cell-mediated elimination of HIV-1 reservoir cells.

The persistence of CD4+ T cells carrying latent human immunodeficiency virus-1 (HIV-1) proviruses is the main barrier to a cure. New therapeutics to enhance HIV-1-specific immune responses and clear infected cells will probably be necessary to achieve reduction of the latent reservoir. In the present study, we report two single-chain diabodies (scDbs) that target the HIV-1 envelope protein (Env) and the human type III Fcγ receptor (CD16). We show that the scDbs promoted robust and HIV-1-specific natural killer (NK) cell activation and NK cell-mediated lysis of infected cells. Cocultures of CD4+ T cells from people with HIV-1 on antiretroviral therapy (ART) with autologous NK cells and the scDbs resulted in marked elimination of reservoir cells that was dependent on latency reversal. Treatment of human interleukin-15 transgenic NSG mice with one of the scDbs after ART initiation enhanced NK cell activity and reduced reservoir size. Thus, HIV-1-specific scDbs merit further evaluation as potential therapeutics for clearance of the latent reservoir.

Last in first out: SIV proviruses seeded later in infection are harbored in short-lived CD4+ T cells.

HIV can persist in a latent form as integrated DNA (provirus) in resting CD4+ T cells of infected individuals and as such is unaffected by antiretroviral therapy (ART). Despite being a major obstacle for eradication efforts, the genetic variation and timing of formation of this latent reservoir remains poorly understood. Previous studies on when virus is deposited in the latent reservoir have come to contradictory conclusions. To reexamine the genetic variation of HIV in CD4+ T cells during ART, we determined the divergence in envelope sequences collected from 10 SIV infected rhesus macaques. We found that the macaques displayed a biphasic decline of the viral divergence over time, where the first phase lasted for an average of 11.6 weeks (range 4-28 weeks). Motivated by recent observations that the HIV-infected CD4+ T cell population is composed of short- and long-lived subsets, we developed a model to study the divergence dynamics. We found that SIV in short-lived cells was on average more diverged, while long-lived cells harbored less diverged virus. This suggests that the long-lived cells harbor virus deposited starting earlier in infection and continuing throughout infection, while short-lived cells predominantly harbor more recent virus. As these cell populations decayed, the overall proviral divergence decline matched that observed in the empirical data. This model explains previous seemingly contradictory results on the timing of virus deposition into the latent reservoir, and should provide guidance for future eradication efforts.

Learning from Persistent Viremia: Mechanisms and Implications for Clinical Care and HIV-1 Cure.

PURPOSE OF REVIEW: In this review, we discuss what persistent viremia has taught us about the biology of the HIV-1 reservoir during antiretroviral therapy (ART). We will also discuss the implications of this phenomenon for HIV-1 cure research and its clinical management. RECENT FINDINGS: While residual viremia (RV, 1-3 HIV-1 RNA copies/ml) can be detected in most of people on ART, some individuals experience non-suppressible viremia (NSV, > 20-50 copies/mL) despite optimal adherence. When issues of drug resistance and pharmacokinetics are ruled out, this persistent virus in plasma is the reflection of virus production from clonally expanded CD4+ T cells carrying proviruses. Recent work has shown that a fraction of the proviruses source of NSV are not infectious, due to defects in the 5'-Leader sequence. However, additional viruses and host determinants of NSV are not fully understood. The study of NSV is of prime importance because it represents a challenge for the clinical care of people on ART, and it sheds light on virus-host interactions that could advance HIV-1 remission research.

Antiretroviral therapy reveals triphasic decay of intact SIV genomes and persistence of ancestral variants.

The decay kinetics of HIV-1-infected cells are critical to understand virus persistence. We evaluated the frequency of simian immunodeficiency virus (SIV)-infected cells for 4 years of antiretroviral therapy (ART). The intact proviral DNA assay (IPDA) and an assay for hypermutated proviruses revealed short- and long-term infected cell dynamics in macaques starting ART ∼1 year after infection. Intact SIV genomes in circulating CD4+T cells showed triphasic decay with an initial phase slower than the decay of the plasma virus, a second phase faster than the second phase decay of intact HIV-1, and a stable third phase reached after 1.6-2.9 years. Hypermutated proviruses showed bi- or mono-phasic decay, reflecting different selective pressures. Viruses replicating at ART initiation had mutations conferring antibody escape. With time on ART, viruses with fewer mutations became more prominent, reflecting decay of variants replicating at ART initiation. Collectively, these findings confirm ART efficacy and indicate that cells enter the reservoir throughout untreated infection.

Clonally expanded HIV-1 proviruses with 5'-leader defects can give rise to nonsuppressible residual viremia.

BackgroundAntiretroviral therapy (ART) halts HIV-1 replication, decreasing viremia to below the detection limit of clinical assays. However, some individuals experience persistent nonsuppressible viremia (NSV) originating from CD4+ T cell clones carrying infectious proviruses. Defective proviruses represent over 90% of all proviruses persisting during ART and can express viral genes, but whether they can cause NSV and complicate ART management is unknown.MethodsWe undertook an in-depth characterization of proviruses causing NSV in 4 study participants with optimal adherence and no drug resistance. We investigated the impact of the observed defects on 5'-leader RNA properties, virus infectivity, and gene expression. Integration-site specific assays were used to track these proviruses over time and among cell subsets.ResultsClones carrying proviruses with 5'-leader defects can cause persistent NSV up to approximately 103 copies/mL. These proviruses had small, often identical deletions or point mutations involving the major splicing donor (MSD) site and showed partially reduced RNA dimerization and nucleocapsid binding. Nevertheless, they were inducible and produced noninfectious virions containing viral RNA, but lacking envelope.ConclusionThese findings show that proviruses with 5'-leader defects in CD4+ T cell clones can give rise to NSV, affecting clinical care. Sequencing of the 5'-leader can help in understanding failure to completely suppress viremia.FundingOffice of the NIH Director and National Institute of Dental and Craniofacial Research, NIH; Howard Hughes Medical Institute; Johns Hopkins University Center for AIDS Research; National Institute for Allergy and Infectious Diseases (NIAID), NIH, to the PAVE, BEAT-HIV, and DARE Martin Delaney collaboratories.

Genome-wide CRISPR screens identify combinations of candidate latency reversing agents for targeting the latent HIV-1 reservoir.

Reversing HIV-1 latency promotes killing of infected cells and is essential for cure strategies; however, no single latency reversing agent (LRA) or LRA combination have been shown to reduce HIV-1 latent reservoir size in persons living with HIV-1 (PLWH). Here, we describe an approach to systematically identify LRA combinations to reactivate latent HIV-1 using genome-wide CRISPR screens. Screens on cells treated with suboptimal concentrations of an LRA can identify host genes whose knockout enhances viral gene expression. Therefore, inhibitors of these genes should synergize with the LRA. We tested this approach using AZD5582, an activator of the noncanonical nuclear factor κB (ncNF-κB) pathway, as an LRA and identified histone deacetylase 2 (HDAC2) and bromodomain-containing protein 2 (BRD2), part of the bromodomain and extra-terminal motif (BET) protein family targeted by BET inhibitors, as potential targets. Using CD4+ T cells from PLWH, we confirmed synergy between AZD5582 and several HDAC inhibitors and between AZD5582 and the BET inhibitor, JQ1. A reciprocal screen using suboptimal concentrations of an HDAC inhibitor as an LRA identified BRD2 and ncNF-κB regulators, especially BIRC2, as synergistic candidates for use in combination with HDAC inhibition. Moreover, we identified and validated additional synergistic drug candidates in latency cell line cells and primary lymphocytes isolated from PLWH. Specifically, the knockout of genes encoding CYLD or YPEL5 displayed synergy with existing LRAs in inducing HIV mRNAs. Our study provides insights into the roles of host factors in HIV-1 reactivation and validates a system for identifying drug combinations for HIV-1 latency reversal.

TCR-mimic bispecific antibodies to target the HIV-1 reservoir.

HIV-1 infection is incurable due to the persistence of the virus in a latent reservoir of resting memory CD4+ T cells. "Shock-and-kill" approaches that seek to induce HIV-1 gene expression, protein production, and subsequent targeting by the host immune system have been unsuccessful due to a lack of effective latency-reversing agents (LRAs) and kill strategies. In an effort to develop reagents that could be used to promote killing of infected cells, we constructed T cell receptor (TCR)-mimic antibodies to HIV-1 peptide-major histocompatibility complexes (pMHC). Using phage display, we panned for phages expressing antibody-like variable sequences that bound HIV-1 pMHC generated using the common HLA-A*02:01 allele. We targeted three epitopes in Gag and reverse transcriptase identified and quantified via Poisson detection mass spectrometry from cells infected in vitro with a pseudotyped HIV-1 reporter virus (NL4.3 dEnv). Sequences isolated from phages that bound these pMHC were cloned into a single-chain diabody backbone (scDb) sequence, such that one fragment is specific for an HIV-1 pMHC and the other fragment binds to CD3ε, an essential signal transduction subunit of the TCR. Thus, these antibodies utilize the sensitivity of T cell signaling as readouts for antigen processing and as agents to promote killing of infected cells. Notably, these scDbs are exquisitely sensitive and specific for the peptide portion of the pMHC. Most importantly, one scDb caused killing of infected cells presenting a naturally processed target pMHC. This work lays the foundation for a novel therapeutic killing strategy toward elimination of the HIV-1 reservoir.

A Study on Risk Factors Associated with Reflux Esophagitis in Patients Undergoing Esophageal Cancer Surgery.

Objective: To investigate the risk factors associated with reflux esophagitis in patients undergoing esophageal cancer surgery and to provide reference for the prevention and treatment of reflux esophagitis. Methods: In the manner retrospective study, the data of 300 patients with esophageal cancer who received the surgical treatment in our hospital (January 2018-December 2020) were retrospectively reviewed. The 300 patients were divided into the occurrence group (n = 45) and nonoccurrence group (n = 255) depending on whether they had reflux esophagitis after surgery. The social demographic data and clinical data of the patients in the two groups were collected. These data were classified into the personal factors and surgical factors. The single-factor analysis method was adopted to analyze the effects of the personal and surgical factors on reflux esophagitis. The factors with statistically significant differences in the single-factor analysis were analyzed by logistic regression to verify the factors were the risk factors associated with reflux esophagitis in patients undergoing esophageal cancer surgery. Results: The differences in the bodyweight, body mass index (BMI), length of the resected esophagus, surgical approach, intraoperative blood loss, gastrointestinal decompression volume, and surgery time between the two groups were of statistical significance (P < 0.05). After being tested by the logistics multivariate analysis, length of the resected esophagus, whole stomach reconstruction, intraoperative blood loss, and surgery time were identified as the risk factors associated with reflux esophagitis in patients undergoing esophageal cancer surgery. Conclusion: The length of the resected esophagus, whole stomach reconstruction, intraoperative blood loss, and surgery time were the risk factors associated with reflux esophagitis in patients undergoing esophageal cancer surgery. It is necessary to choose the appropriate surgical approach according to the patients' conditions in practice and to strengthen the prevention and treatment of reflux esophagitis.

The transcription factor ZIP-1 promotes resistance to intracellular infection in Caenorhabditis elegans.

Defense against intracellular infection has been extensively studied in vertebrate hosts, but less is known about invertebrate hosts; specifically, the transcription factors that induce defense against intracellular intestinal infection in the model nematode Caenorhabditis elegans remain understudied. Two different types of intracellular pathogens that naturally infect the C. elegans intestine are the Orsay virus, which is an RNA virus, and microsporidia, which comprise a phylum of fungal pathogens. Despite their molecular differences, these pathogens induce a common host transcriptional response called the intracellular pathogen response (IPR). Here we show that zip-1 is an IPR regulator that functions downstream of all known IPR-activating and regulatory pathways. zip-1 encodes a putative bZIP transcription factor, and we show that zip-1 controls induction of a subset of genes upon IPR activation. ZIP-1 protein is expressed in the nuclei of intestinal cells, and is at least partially required in the intestine to upregulate IPR gene expression. Importantly, zip-1 promotes resistance to infection by the Orsay virus and by microsporidia in intestinal cells. Altogether, our results indicate that zip-1 represents a central hub for triggers of the IPR, and that this transcription factor has a protective function against intracellular pathogen infection in C. elegans.

Engaging innate immunity in HIV-1 cure strategies.

Combination antiretroviral therapy (ART) can block multiple stages of the HIV-1 life cycle to prevent progression to AIDS in people living with HIV-1. However, owing to the persistence of a reservoir of latently infected CD4+ T cells, life-long ART is necessary to prevent viral rebound. One strategy currently under consideration for curing HIV-1 infection is known as 'shock and kill'. This strategy uses latency-reversing agents to induce expression of HIV-1 genes, allowing for infected cells to be cleared by cytolytic immune cells. The role of innate immunity in HIV-1 pathogenesis is best understood in the context of acute infection. Here, we suggest that innate immunity can also be used to improve the efficacy of HIV-1 cure strategies, with a particular focus on dendritic cells (DCs) and natural killer cells. We discuss novel latency-reversing agents targeting DCs as well as DC-based strategies to enhance the clearance of infected cells by CD8+ T cells and strategies to improve the killing activity of natural killer cells.

HSF1 inhibition attenuates HIV-1 latency reversal mediated by several candidate LRAs In Vitro and Ex Vivo.

HIV-1 latency is a major barrier to cure. Identification of small molecules that destabilize latency and allow immune clearance of infected cells could lead to treatment-free remission. In vitro models of HIV-1 latency involving cell lines or primary cells have been developed for characterization of HIV-1 latency and high-throughput screening for latency-reversing agents (LRAs). We have shown that the majority of LRAs identified to date are relatively ineffective in cells from infected individuals despite activity in model systems. We show here that, for diverse LRAs, latency reversal observed in model systems involves a heat shock factor 1 (HSF1)-mediated stress pathway. Small-molecule inhibition of HSF1 attenuated HIV-1 latency reversal by histone deactylase inhibitors, protein kinase C agonists, and proteasome inhibitors without interfering with the known mechanism of action of these LRAs. However, latency reversal by second mitochondria-derived activator of caspase (SMAC) mimetics was not affected by inhibition of HSF1. In cells from infected individuals, inhibition of HSF1 attenuated latency reversal by phorbol ester+ionomycin but not by anti-CD3+anti-CD28. HSF1 promotes elongation of HIV-1 RNA by recruiting P-TEFb to the HIV-1 long terminal repeat (LTR), and we show that inhibition of HSF1 attenuates the formation of elongated HIV-1 transcripts. We demonstrate that in vitro models of latency have higher levels of the P-TEFb subunit cyclin T1 than primary cells, which may explain why many LRAs are functional in model systems but relatively ineffective in primary cells. Together, these studies provide insights into why particular LRA combinations are effective in reversing latency in cells from infected individuals.

Serum miR-101-3p combined with pepsinogen contributes to the early diagnosis of gastric cancer.

BACKGROUND: This study aimed to explore the diagnostic value of serum miR-101-3p combined with pepsinogen (PG) on early diagnosis of gastric cancer (GC). METHODS: A total of 61 atrophic gastritis (AG) and 86 GC patients, and 50 healthy volunteers were enrolled. The serum expression of miR-101-3p was measured by qRT-PCR. The serum content of carcinoembryonic antigen (CEA) was measured by Electrochemiluminescence immunoassay. The serum contents of PGI and PGII were measured by Enzyme linked immunosorbent assay. The diagnostic value of serum markers on AG and GC was analyzed by receiver operating characteristic (ROC) analysis. RESULTS: The expression of miR-101-3p, the content of PGI and the ratio of PGI/II were significantly decreased, and the content of PGII was significantly increased in AG patients compared with those in normal controls. The changes of the above serum indicators were more obvious in GC patients than those in AG patients. The content of CEA was significantly higher in GC patients than that in AG patients. In addition, the expression of miR-101-3p was negatively associated with the submucosal infiltration in GC patients. MiR-101-3p exhibited high diagnostic value on AG (AUC 0.8493, sensitivity 80.33%, specificity 80%) and GC (AUC 0.8749, sensitivity 72.09%, specificity 86.49%). MiR-101-3p + PGI + PGI/II (AUC 0.856, sensitivity 80.23%, specificity 77.05%) exhibited a high diagnostic value in distinguishing between AG and GC. CONCLUSIONS: MiR-101-3p was a potential diagnostic marker for AG and GC. MiR-101-3p + PGI + PGI/II was effective in distinguishing between AG and GC.

Comparison of the safety of the application of painless gastroscopy and ordinary gastroscopy in chronic hypertension patients combined with early gastric cancer.

The aim of the present study was to compare the safety of the application of painless gastroscopy and ordinary gastroscopy for chronic hypertension patients combined with early gastric cancer. A total of 123 patients with early gastric cancer were selected at the Dongying People's Hospital from June, 2014 to August, 2016. The patients were randomly divided into the painless (n=63) and ordinary (n=60) gastroscopy groups. Proper pretreatment was performed according to whether anesthesia was performed or not. Arterial pressure, heart rate, and blood oxygen saturation were detected and compared before anesthesia, when gastroscope passed through the esophageal entrance plane, and after recovery from anesthesia. The incidence of nausea and vomiting, cough, dysphoria, throat discomfort and other adverse reactions during and after surgery were recorded and compared. Compared with the levels before anesthesia, the mean arterial pressure, heart rate and blood oxygen saturation were significantly reduced in painless gastroscopy when the gastroscope passed through the esophageal entrance plane (P<0.05). In the ordinary gastroscopy group, the mean arterial pressure, heart rate and blood oxygen saturation were significantly increased when the gastroscope passed through the esophageal entrance plane compared with the levels before anesthesia (P<0.05). Blood pressure decreased in the painless gastroscopy group whereas it increased in the ordinary gastroscopy group after anesthesia. The decrease in the painless gastroscopy group was lower than in the ordinary group. The incidence of intraoperative and postoperative adverse reactions including nausea, vomiting, cough, dysphoria, pharyngeal discomfort and other adverse reactions was significantly decreased in the painless gastroscopy group than in the ordinary gastroscopy group (P<0.05). The results suggest that the application of painless gastroscopy in chronic hypertension patients can significantly reduce the incidence of intraoperative and postoperative adverse reactions compared with that of the Gastric cancer ordinary gastroscopy. Thus, painless gastroscopy is safer than ordinary gastroscopy.

Prevalence of depression and anxiety in patients with chronic digestive system diseases: A multicenter epidemiological study.

AIM: To investigate the prevalence of depression and anxiety in patients with chronic digestive system diseases. METHODS: A total of 1736 patients with chronic digestive system diseases were included in this cross-sectional study, including 871 outpatients and 865 in-patients. A self-designed General Information for Patients of the Department of Gastroenterology of General Hospitals questionnaire was used to collect each patient's general information, which included demographic data (including age, sex, marital status, and education) and disease characteristics (including major diseases, disease duration, principal symptoms, chronic pain, sleep disorder, and limited daily activities). RESULTS: The overall detection rate was 31.11% (540/1736) for depression symptoms alone, 27.02% (469/1736) for anxiety symptoms alone, 20.68% (359/1736) for both depression and anxiety symptoms, and 37.44% (650/1736) for either depression or anxiety symptoms. Subjects aged 70 years or above had the highest detection rate of depression (44.06%) and anxiety symptoms (33.33%). χ2 trend test showed: the higher the body mass index (BMI), the lower the detection rate of depression and anxiety symptoms (χ2trend = 13.697, P < 0.001; χ2trend = 9.082, P = 0.003); the more severe the limited daily activities, the higher the detection rate of depression and anxiety symptoms (χ2trend = 130.455, P < 0.001, χ2trend = 108.528, P < 0.001); and the poorer the sleep quality, the higher the detection rate of depression and anxiety symptoms (χ2trend = 85.759, P < 0.001; χ2trend = 51.969, P < 0.001). Patients with digestive system tumors had the highest detection rate of depression (57.55%) and anxiety (55.19%), followed by patients with liver cirrhosis (41.35% and 48.08%). Depression and anxiety symptoms were also high in subjects with comorbid hypertension and coronary heart disease. CONCLUSION: Depression and anxiety occur in patients with tumors, liver cirrhosis, functional dyspepsia, and chronic viral hepatitis. Elderly, divorced/widowed, poor sleep quality, and lower BMI are associated with higher risk of depression and anxiety.

Microsporidia Intracellular Development Relies on Myc Interaction Network Transcription Factors in the Host.

Microsporidia are ubiquitous parasites that infect a wide range of animal hosts, and these fungal-related microbes undergo their entire replicative lifecycle inside of host cells. Despite being widespread in the environment and causing medical and agricultural harm, virtually nothing is known about the host factors important to facilitate their growth and development inside of host cells. Here, we perform a genetic screen to identify host transcription factors important for development of the microsporidian pathogen Nematocida parisii inside intestinal cells of its natural host, the nematode Caenorhabditis elegans Through this screen, we identified the C. elegans Myc family of transcription factors as key host regulators of microsporidia growth and development. The Mad-like transcription factor MDL-1, and the Max-like transcription factors MXL-1 and MXL-2 promote pathogen levels, while the Myc-Mondo-like transcription factor MML-1 inhibits pathogen levels. We used epistasis analysis to show that MDL-1 and MXL-1, which are thought to function as a heterodimer, appear to be acting canonically. In contrast, MXL-2 and MML-1, which are also thought to function as a heterodimer, appear to be acting in separate pathways (noncanonically) in the context of pathogen infection. We also found that both MDL-1::GFP and MML-1::GFP are expressed in intestinal cells during infection. These findings provide novel insight into the host transcription factors that regulate microsporidia development.