Biology subjects

Levy, C. N.

Publications and source records attributed to Levy, C. N..

5 recordsLinked to original sources

bioRxiv2026

Menstruation is associated with cyclical granulysin peaks in vaginal secretions despite stable expression by cervicovaginal immune cells

ProblemThe anti-microbial protein granulysin is present in vaginal secretions during the follicular phase of the menstrual cycle but nearly disappears during the luteal phase. The reason for this change is unknown. Method of studyParticipants (n = 23) with regular menstrual cycles collected daily vaginal swabs for granulysin ELISAs. Endocervical cytobrushes, ectocervical biopsies, vaginal biopsies, and PBMC were collected across the cycle to enumerate granulysin-expressing cells by flow cytometry. Cycle phase was determined by daily urinary luteinizing hormone testing and confirmed by serum progesterone levels. ResultsGranulysin levels in secretions were up to 10,000 times higher during menstruation than during the luteal phase (menstruation, median 3,924 pg/mL [IQR 400-17,280]; luteal, median and IQR undetectable [<7.81 pg/mL]). In the endocervical canal, granulysin-expressing cells were much more abundant during menstruation than during the mid-follicular or mid-luteal phases. In contrast, the number of granulysin-expressing cells in the ectocervix and vagina remained stable during the cycle. The most abundant granulysin-expressing cell types in the mucosa were CD8 T cells and NK cells. In a minority of participants, granulysin was consistently detected in luteal-phase swabs; this phenomenon was associated with parity. ConclusionsGranulysin in vaginal secretions is associated with menstruation and concomitant with a spike in granulysin-expressing cells in the endocervical canal. This result explains the much higher granulysin levels in secretions during the follicular than the luteal phase. In contrast, immune cells from ectocervical and vaginal biopsies express granulysin independently of the menstrual cycle, indicating their continuous ability to respond to microbial infection.

immunology↗

bioRxiv2025

NF-κB dependent gene expression and plasma IL-1β, TNFα and GCSF drive transcriptomic diversity and CD4:CD8 ratio in people with HIV on ART.

Despite antiretroviral therapy (ART), people with HIV (PWH) on ART experience higher rates of morbidity and mortality vs. age-matched HIV negative controls, which may be driven by chronic inflammation due to persistent virus. We performed bulk RNA sequencing (RNA-seq) on peripheral CD4+ T cells, as well as quantified plasma immune marker levels from 154 PWH on ART to identify host immune signatures associated with immune recovery (CD4:CD8) and HIV persistence (cell-associated HIV DNA and RNA). Using a novel dimension reduction tool - Pairwise Controlled Manifold Approximation (PaCMAP), we defined three distinct participant transcriptomic clusters. We found that these three clusters were largely defined by differential expression of genes regulated by the transcription factor NF-{kappa}B. While clustering was not associated with HIV reservoir size, we observed an association with CD4:CD8 ratio, a marker of immune recovery and prognostic factor for mortality in PWH on ART. Furthermore, distinct patterns of plasma IL-1{beta}, TNF- and GCSF were also strongly associated with the clusters, suggesting that these immune markers play a key role in CD4+ T cell transcriptomic diversity and immune recovery in PWH on ART. These findings reveal novel subgroups of PWH on ART with distinct immunological characteristics, and define a transcriptional signature associated with clinically significant immune parameters for PWH. A deeper understanding of these subgroups could advance clinical strategies to treat HIV-associated immune dysfunction.

microbiology↗

bioRxiv2023

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., CCR5{Delta}32, 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-1{beta}/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 SummaryAlthough 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-1{beta}, TLR7, TNF- 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.

genetics↗

bioRxiv2019

Oral tenofovir disoproxil fumarate/emtricitabine for HIV pre-exposure prophylaxis increases expression of type I/III interferon-stimulated factors in the gastrointestinal tract but not in the blood

Tenofovir disoproxil fumarate (TDF) and emtricitabine (FTC) are used for HIV treatment and prevention. Previously, we found that topical rectal tenofovir gel caused immunological changes in the mucosa. Here we assessed the effect of oral TDF/FTC in three HIV pre-exposure prophylaxis trials, two with gastrointestinal and one with cervicovaginal biopsies. TDF/FTC induced type I/III interferon-related (IFN I/III) genes in the gastrointestinal tract, but not blood, with strong correlations between the two independent rectal biopsy groups (Spearman r=0.91) and between the rectum and duodenum (r=0.81). Gene set testing also indicated stimulation of type I/III pathways in the ectocervix, as well as of cellular proliferation in the duodenum. mRNA sequencing, digital droplet PCR, proteomics, and immunofluorescence staining confirmed IFN I/III pathway stimulation in the gastrointestinal tract. Thus, oral TDF/FTC stimulates an IFN-I/III signature throughout the gut, which could increase antiviral efficacy but also cause chronic immune activation in HIV prevention and treatment settings.

immunology↗

bioRxiv2018

Cryopreservation of human mucosal tissues

BACKGROUNDCryopreservation of leukocytes isolated from the cervicovaginal and colorectal mucosa is useful for the study of cellular immunity (see Hughes SM et al. PLOS ONE 2016). However, some questions about mucosal biology and sexually transmitted infections are better addressed with intact mucosal tissue, for which there is no standard cryopreservation protocol.\n\nMETHODS AND FINDINGSTo find an optimal preservation protocol for mucosal tissues, we tested slow cooling (1%C/min) with 10% dimethylsulfoxide (designated \"cryopreservation\") and fast cooling (plunge in liquid nitrogen) with 20% dimethylsulfoxide and 20% ethylene glycol (\"vitrification\"). We compared fresh and preserved human cervicovaginal and colorectal tissues in a range of assays, including metabolic activity, human immunodeficiency virus infection, cell phenotype, tissue structure by hematoxylin-and-eosin staining, cell number and viability, production of cytokines, and microbicide drug concentrations. Metabolic activity, HIV infectability, and tissue structure were similar in cryopreserved and vitrified vaginal tissues. However, vitrification led to poor cell recovery from the colorectal mucosa, with 90% fewer cells recovered after isolation from vitrified colorectal tissues than from cryopreserved. HIV infection rates were similar for fresh and cryopreserved ectocervical tissues, whereas cryopreserved colorectal tissues were less easily infected than fresh tissues (hazard ratio 0.7 [95% confidence interval 0.4, 1.2]). Finally, we compared isolation of cells before and after cryopreservation. Cell recoveries were higher when cells were isolated after freezing and thawing (71% [59-84%]) than before (50% [38-62%]). Cellular function was similar to fresh tissue in both cases. Microbicide drug concentrations were lower in cryopreserved explants compared to fresh ones.\n\nCONCLUSIONSCryopreservation of intact cervicovaginal and colorectal tissues with dimethylsulfoxide works well in a range of assays, while the utility of vitrification is more limited. Cell yields are higher from cryopreserved intact tissue pieces than from thawed cryopreserved single cell suspensions isolated before freezing, but T cell functions are similar.

immunology↗