Association of the eukaryotic vaginal virome with prophylactic antibiotic exposure and reproductive outcomes in a subfertile population undergoing in vitro fertilisation: a prospective exploratory study.

OBJECTIVE: The objective of this study was to use high-throughput sequencing to describe the vaginal eukaryotic DNA virome in patients undergoing in vitro fertilisation (IVF) to examine associations between the vaginal virome, antibiotic exposure and IVF outcomes. DESIGN: Prospective exploratory study. SETTING: Single academic fertility centre. POPULATION: Subfertile women age 18-43 years undergoing their first IVF cycle with a fresh embryo transfer. METHODS: The primary exposure was prophylactic azithromycin or no azithromycin before IVF. A mid-vaginal swab was obtained at the time of embryo transfer for virome analysis. MAIN OUTCOME MEASURES: The primary outcomes compared between exposure groups were characteristics of vaginal virome and clinical pregnancy rates. Secondary outcomes were virome associations with number of oocytes retrieved, number of blastocysts and implantation rate. RESULTS: Twenty-six women contributed a vaginal swab before embryo transfer. There were no significant differences in IVF outcomes between azithromycin groups. There was no association between viral diversity and clinical pregnancy overall. A higher diversity of herpesviruses and α-papillomaviruses was observed in samples from the azithromycin-treated group compared with the no azithromycin group (P = 0.04). In women that received azithromycin, viral diversity was higher in the group that did not achieve clinical pregnancy compared with those who did (P = 0.06). CONCLUSIONS: We demonstrate that the vaginal eukaryotic virome in women undergoing IVF is associated with antibiotic exposure. Additionally, we demonstrate an inverse trend between viral diversity and pregnancy, with a higher number of viruses detected associated with failure to achieve clinical pregnancy in the azithromycin group. TWEETABLE ABSTRACT: Higher viral diversity is associated with prophylactic antibiotic exposure in subfertile women undergoing IVF.

CD20: a gene in search of a function.

CD20 is a signature B-cell differentiation antigen. The function of CD20 is unknown, although it is thought to be involved in B-cell activation, regulation of B-cell growth, and transmembrane calcium flux. This review covers several topics important for understanding CD20 biology. These topics include the expression pattern and transcriptional regulation of the CD20 gene, the structure of the protein and its interaction with other cell surface molecules, as well as CD20 phosphorylation and putative functions. An understanding of CD20 function from signal transduction to biological implications may prove important for the mechanistic understanding of the treatment of certain types of cancer.

Interleukin-10 receptor signaling through the JAK-STAT pathway. Requirement for two distinct receptor-derived signals for anti-inflammatory action.

Interleukin-10 (IL-10) is a cytokine that has pleiotropic effects on a variety of different cell types. Although many of the biologic responses induced by IL-10 are also induced by other cytokines, such as IL-6, IL-10 is relatively unique in its ability to potently inhibit production of pro-inflammatory cytokines in macrophages. In this study, we have used gain-of-function and loss-of-function genetic approaches to define the intracellular components involved in the different biologic actions of IL-10. Herein, we demonstrate that the ability of IL-10 to inhibit tumor necrosis factor alpha (TNFalpha) production in lipopolysaccharide-stimulated macrophages requires the presence of Stat3, Jak1, and two distinct regions of the IL-10 receptor intracellular domain. Macrophages deficient in Stat3 or Jak1 were unable to inhibit lipopolysaccharide-induced TNFalpha production following treatment with murine IL-10. Structure-function analysis of the intracellular domain of the IL-10 receptor alpha chain showed that whereas two redundant Stat3 recruitment sites (427YQKQ430 and 477YLKQ480) were required for all IL-10-dependent effects on either B cells or macrophages, expression of IL-10-dependent anti-inflammatory function required the presence on the intracellular domain of the IL-10 receptor of a carboxyl-terminal sequence containing at least one functionally critical serine. These results thus demonstrate that IL-10-induced inhibition of TNFalpha production requires two distinct regions of the IL-10 receptor intracellular domain and thereby establish a distinctive molecular basis for the developmental versus the anti-inflammatory actions of IL-10.

Stat3 recruitment by two distinct ligand-induced, tyrosine-phosphorylated docking sites in the interleukin-10 receptor intracellular domain.

Recent work has shown that IL-10 induces activation of the JAK-STAT signaling pathway. To define the mechanism underlying signal transducer and activator of transcription (STAT) protein recruitment to the interleukin 10 (IL-10) receptor, the STAT proteins activated by IL-10 in different cell populations were first defined using electrophoretic mobility shift assays. In all cells tested, IL-10 activated Stat1 and Stat3 and induced the formation of three distinct DNA binding complexes that contained different combinations of these two transcription factors. IL-10 also activated Stat5 in Ba/F3 cells that stably expressed the murine IL-10 receptor. Using a structure-function mutagenesis approach, two tyrosine residues (Tyr427 and Tyr477) in the intracellular domain of the murine IL-10 receptor were found to be redundantly required for receptor function and for activation of Stat3 but not for Stat1 or Stat5. Twelve amino acid peptides encompassing either of these two tyrosine residues in phosphorylated form coprecipitated Stat3 but not Stat1 and blocked IL-10-induced Stat3 phosphorylation in a cell-free system. In contrast, tyrosine-phosphorylated peptides containing Tyr374 or Tyr396 did not interact with Stat3 or block Stat3 activation. These data demonstrate that Stat3 but not Stat1 or Stat5 is directly recruited to the ligand-activated IL-10 receptor by binding to specific but redundant receptor intracellular domain sequences containing phosphotyrosine. This study thus supports the concept that utilization of distinct STAT proteins by different cytokine receptors is dependent on the expression of particular ligand-activatable, tyrosine-containing STAT docking sites in receptor intracellular domains.

Disruption of the Jak1 gene demonstrates obligatory and nonredundant roles of the Jaks in cytokine-induced biologic responses.

Herein we report the generation of mice lacking the ubiquitously expressed Janus kinase, Jak1. Jak1-/- mice are runted at birth, fail to nurse, and die perinatally. Although Jak1-/- cells are responsive to many cytokines, they fail to manifest biologic responses to cytokines that bind to three distinct families of cytokine receptors. These include all class II cytokine receptors, cytokine receptors that utilize the gamma(c) subunit for signaling, and the family of cytokine receptors that depend on the gp130 subunit for signaling. Our results thus demonstrate that Jak1 plays an essential and nonredundant role in promoting biologic responses induced by a select subset of cytokine receptors, including those in which Jak utilization was thought to be nonspecific.

Targeted disruption of the Stat1 gene in mice reveals unexpected physiologic specificity in the JAK-STAT signaling pathway.

The JAK-STAT signaling pathway has been implicated in mediating biological responses induced by many cytokines. However, cytokines that promote distinct cellular responses often activate identical STAT proteins, thereby raising the question of how specificity is manifest within this signaling pathway. Here we report the generation and characterization of mice deficient in STAT1. STAT1-deficient mice show no overt developmental abnormalities, but display a complete lack of responsiveness to either IFN alpha or IFN gamma and are highly sensitive to infection by microbial pathogens and viruses. In contrast, these mice respond normally to several other cytokines that activate STAT1 in vitro. These observations document that STAT1 plays an obligate and dedicated role in mediating IFN-dependent biologic responses and reveal an unexpected level of physiologic specificity for STAT1 action.