PlmCas12e (CasX2) cleavage of CCR5: impact of guide RNA spacer length and PAM sequence on cleavage activity.

Gene editing using CRISPR/Cas (clustered regularly interspaced palindromic repeats/CRISPR-associated) is under development as a therapeutic tool for the modification of genes in eukaryotic cells. While much effort has focused on CRISPR/Cas9 systems from and , alternative CRISPR systems have been identified from non-pathogenic microbes, including previously unknown class 2 systems, adding to a diverse toolbox of CRISPR/Cas enzymes. The Cas12e enzymes from non-pathogenic Deltaproteobacteria (CasX1, DpeCas12e) and Planctomycetes (CasX2, PlmCas12e) are smaller than Cas9, have a selective protospacer adjacent motif (PAM), and deliver a staggered cleavage cut with a 5-7 nucleotide overhang.

CAS12e (CASX2) CLEAVAGE OF CCR5: IMPACT OF GUIDE RNA LENGTH AND PAM SEQUENCE ON CLEAVAGE ACTIVITY.

CRISPR/Cas is under development as a therapeutic tool for the cleavage, excision, and/or modification of genes in eukaryotic cells. While much effort has focused on CRISPR/Cas from (SpCas9) and (SaCas9), alternative CRISPR systems have been identified using metagenomic datasets from non-pathogenic microbes, including previously unknown class 2 systems, adding to a diverse toolbox of gene editors. The Cas12e (CasX1, CasX2) endonucleases from non-pathogenic Deltaproteobacteria (DpeCas12e) and Planctomycetes (PlmCas12e) are more compact than SpCas9, have a more selective protospacer adjacent motif (PAM) requirement, and deliver a staggered cleavage cut with 5-7 base overhangs.

Analysis of CRISPR/Cas9 Guide RNA Efficiency and Specificity Against Genetically Diverse HIV-1 Isolates.

Gene editing approaches using CRISPR/Cas9 are being developed as a means for targeting the integrated HIV-1 provirus. Enthusiasm for the use of gene editing as an anti-HIV-1 therapeutic has been tempered by concerns about the specificity and efficacy of this approach. Guide RNAs (gRNAs) that target conserved sequences across a wide range of genetically diverse HIV-1 isolates will have greater clinical utility.

A mathematical model of HIV dynamics treated with a population of gene-edited haematopoietic progenitor cells exhibiting threshold phenomenon.

The use of gene-editing technology has the potential to excise the CCR5 gene from haematopoietic progenitor cells, rendering their differentiated CD4-positive (CD4+) T cell descendants HIV resistant. In this manuscript, we describe the development of a mathematical model to mimic the therapeutic potential of gene editing of haematopoietic progenitor cells to produce a class of HIV-resistant CD4+ T cells. We define the requirements for the permanent suppression of viral infection using gene editing as a novel therapeutic approach.

Toll-like receptor agonists are potent inhibitors of human immunodeficiency virus-type 1 replication in peripheral blood mononuclear cells.

Innate immune responses to microbial pathogens are initiated following the binding of ligand to specific pattern recognition receptors. Each pattern recognition receptor, which includes members of the Toll-like receptor (TLR) family, is specific for a particular type of pathogen associated molecular pattern ensuring that the organism can respond rapidly to a wide range of pathogens including bacteria, viruses, and fungi. We studied the extent to which agonists to endosomal TLR could induce anti-HIV-1 activity in peripheral blood mononuclear cells (PBMCs).

Gardiquimod: a Toll-like receptor-7 agonist that inhibits HIV type 1 infection of human macrophages and activated T cells.

Immune response modifiers are being studied as therapeutic agents for viral infections and cancer. These molecules include agonists for the Toll-like receptors (TLR), a family of innate immune receptors. TLR7 and 8, located in cellular endosomes, bind single-stranded RNA characteristic of viral genomes, and trigger intracellular signaling pathways that induce inflammatory cytokines and antiviral innate immune factors.

Breast milk from Tanzanian women has divergent effects on cell-free and cell-associated HIV-1 infection in vitro.

Transmission of HIV-1 during breastfeeding is a significant source of new pediatric infections in sub-Saharan Africa. Breast milk from HIV-positive mothers contains both cell-free and cell-associated virus; however, the impact of breast milk on HIV-1 infectivity remains poorly understood. In the present study, breast milk was collected from HIV-positive and HIV-negative Tanzanian women attending antenatal clinics in Dar es Salaam.

Nanoparticles containing siRNA to silence CD4 and CCR5 reduce expression of these receptors and inhibit HIV-1 infection in human female reproductive tract tissue explants.

Human Immunodeficiency Virus-type 1 (HIV-1) binds to CD4 and CCR5 receptors on target cells in the human female reproductive tract. We sought to determine whether reducing levels of messenger RNA (mRNA) transcripts that encode these receptors in female reproductive tract cells could protect mucosal tissue explants from HIV-1 infection. Explants prepared from the endometrium, endocervix, and ectocervix of hysterectomy tissues from HIV-1 sero-negative women were exposed to nanoparticles containing CD4- and CCR5-specific short-interfering RNA (siRNA) sequences.

Human uterine natural killer cells but not blood natural killer cells inhibit human immunodeficiency virus type 1 infection by secretion of CXCL12.

Natural killer (NK) cells derived from the human female reproductive tract (FRT) are phenotypically and functionally distinct from those obtained from peripheral blood. Because the FRT is a primary site of human immunodeficiency virus type 1 (HIV-1) infection in women, we determined whether soluble factors secreted by uterine-derived NK (uNK) cells inhibit HIV-1 infection. Clonal populations of uNK cells were activated with interleukin-12 (IL-12) and IL-15, and conditioned media (CM) from these cultures evaluated for their ability to inhibit infection of cells by HIV-1(IIIB), HIV-1(NL4.

HIV type 1 infection in women: increased transcription of HIV type 1 in ectocervical tissue explants.

Background: Mucosal surfaces of the female reproductive tract are the main routes of heterosexual transmission of human immunodeficiency virus type 1 (HIV-1), but the contribution of each of the reproductive sites to mucosal transmission is unknown.

Methods: We compared levels of HIV-1 transcription between ectocervical and endometrial tissue explants infected ex vivo with HIV-1.

Results: We detected higher levels of HIV-1 transcription in the ectocervix.

Innate factors in human breast milk inhibit cell-free HIV-1 but not cell-associated HIV-1 infection of CD4+ cells.

Background: Transmission of HIV from mother to child through breast-feeding remains a global health challenge, particularly in developing countries. Breast milk from an HIV-infected women may contain both cell-free HIV-1 and cell-associated virus; however, the impact of human breast milk on HIV infection and replication in CD4 cells remain poorly understood.

Objectives: In the present study, we evaluated the effects of breast milk in vitro on infection of CD4 cells with cell-free HIV-1, including effects on HIV-1 receptor expression, reverse transcription, integration, and viral transcription.

Estradiol and progesterone regulate HIV type 1 replication in peripheral blood cells.

Endogenous levels of estradiol and progesterone fluctuate in the peripheral blood of premenopausal women during the reproductive cycle. We studied the effects of these sex hormones on HIV-1 replication in peripheral blood mononuclear cells (PBMCs). We compared HIV-1 replication in PBMCs infected in the presence of mid-secretory (high concentrations) and mid-proliferative (low concentrations) or in the absence of sex hormones.

Toward a VA Women's Health Research Agenda: setting evidence-based priorities to improve the health and health care of women veterans.

The expansion of women in the military is reshaping the veteran population, with women now constituting the fastest growing segment of eligible VA health care users. In recognition of the changing demographics and special health care needs of women, the VA Office of Research & Development recently sponsored the first national VA Women's Health Research Agenda-setting conference to map research priorities to the needs of women veterans and position VA as a national leader in Women's Health Research. This paper summarizes the process and outcomes of this effort, outlining VA's research priorities for biomedical, clinical, rehabilitation, and health services research.

HIV-1 infection of the female reproductive tract.

The human immunodeficiency virus type 1 (HIV-1) infects cells within mucosal tissues, including those of the female reproductive tract (FRT). The mechanism of viral transmission within the FRT and the mode of viral spread to the periphery are not completely understood. We performed phenotypical analyses and infectivity studies of primary FRT cells to identify potential targets of infection within the FRT.

Chemokine receptor expression in the human ectocervix: implications for infection by the human immunodeficiency virus-type I.

Human immunodeficiency virus-type 1 (HIV-1) is a sexually transmitted pathogen that can infect cells in the female reproductive tract (FRT). The mechanism of viral transmission within the FRT and the mode of viral spread to the periphery are not well understood. To characterize the frequency of potential targets of HIV infection within the FRT, we performed a systematic study of the expression of HIV receptors (CD4, galactosyl ceramide (GalCer)) and coreceptors (CXCR4 and CCR5) on epithelial cells and leucocytes from the ectocervix.

Transmission of HIV-1 by primary human uterine epithelial cells and stromal fibroblasts.

Women can become infected with human immunodeficiency virus type 1 (HIV-1) after the heterosexual transmission of virus from an infected male partner. To understand the events that result in transmission of HIV-1 across the female reproductive tract, we characterized the life-cycle events of HIV-1 in primary cultures of human uterine epithelial cells and stromal fibroblasts. Epithelial cells and stromal fibroblasts released virus particles after exposure to either X4- or R5-tropic strains of HIV-1.

Intravenous fentanyl increases natural killer cell cytotoxicity and circulating CD16(+) lymphocytes in humans.

Unlabelled: Opioids, including fentanyl, are often administered to patients who may be at risk for the consequences of impaired immune function. We performed a clinical study to test the effects of the synthetic opioid fentanyl on human immune function. Participants received an IV fentanyl initial dose of 3 microg/kg followed by a 2-h IV infusion of 1.