Objective Evaluate upper genital tract (UGT) presence of vaginal bacterial species using sensitive molecular methods capable of detecting fastidious bacterial vaginosis (BV)-associated bacteria. Study Design Vaginal swabs were collected prior to hysterectomy. The excised uterus was sterilely opened and swabs collected from endometrium and upper endocervix. DNA was tested in 11 quantitative PCR (qPCR) assays for 12 bacterial species: Lactobacillus iners, L. crispatus, L. jensenii, Gardnerella vaginalis, Atopobium vaginae, Megasphaera spp., Prevotella spp., Leptotrichia/Sneathia, BVAB1, BVAB2, BVAB3 and a broad-range16S rRNA gene assay. Endometrial fluid was tested with Luminex and ELISA for cytokines and defensins, and tissue for gene expression of defensins and cathelicidin. Results We enrolled 58 women: mean age 43 + 7 years, mostly white (n = 46; 79%) and BV-negative (n = 43; 74%). By species-specific qPCR, 55 (95%) had UGT colonization with at least one species (n = 52), or were positive by 16S PCR (n = 3). The most common species were L. iners (45% UGT, 61% vagina), Prevotella spp. (33% UGT, 76% vagina) and L. crispatus (33% UGT, 56% vagina). Median quantities of bacteria in the UGT were lower than vaginal levels by 2–4 log10 rRNA gene copies/swab. There were no differences in endometrial inflammatory markers between women with no bacteria, Lactobacillus only or any BV-associated species in the UGT. Conclusion Our data suggest that the endometrial cavity is not sterile in most women undergoing hysterectomy, and that the presence of low levels of bacteria in the uterus is not associated with significant inflammation.
We evaluate gene editing of HSV in a well-established mouse model, using adeno-associated virus (AAV)-delivered meganucleases, as a potentially curative approach to treat latent HSV infection. Here we show that AAV-delivered meganucleases, but not CRISPR/Cas9, mediate highly efficient gene editing of HSV, eliminating over 90% of latent virus from superior cervical ganglia. Single-cell RNA sequencing demonstrates that both HSV and individual AAV serotypes are non-randomly distributed among neuronal subsets in ganglia, implying that improved delivery to all neuronal subsets may lead to even more complete elimination of HSV. As predicted, delivery of meganucleases using a triple AAV serotype combination results in the greatest decrease in ganglionic HSV loads. The levels of HSV elimination observed in these studies, if translated to humans, would likely significantly reduce HSV reactivation, shedding, and lesions. Further optimization of meganuclease delivery and activity is likely possible, and may offer a pathway to a cure for HSV infection.
Polymorphonuclear neutrophils (PMN) infiltrate the respiratory tract early after viral infection and can contribute to both host defence and pathology. Coronaviruses are important causes of respiratory tract infections, ranging from mild to severe depending on the viral strain. This study evaluated the role of PMN during a non-fatal pulmonary coronavirus infection in the natural host. Rat coronavirus (RCoV) causes respiratory disease in adult rats, characterized by an early PMN response, viral replication and inflammatory lesions in the lungs, mild weight loss and effective resolution of infection. To determine their role during RCoV infection, PMN were depleted and the effects on disease progression, viral replication, inflammatory response and lung pathology were analysed. Compared with RCoV infection in control animals, PMN-depleted rats had worsened disease with weight loss, clinical signs, mortality and prolonged pulmonary viral replication. PMNdepleted animals had fewer macrophages and lymphocytes in the respiratory tract, corresponding to lower chemokine levels. Combined with in vitro experiments showing that PMN express cytokines and chemokines in response to RCoV-infected alveolar epithelial cells, these findings support a role for PMN in eliciting an inflammatory response to RCoV infection. Despite their critical role in the protection from severe disease, the presence of PMN was correlated with haemorrhagic lesions, epithelial barrier permeability and cellular inflammation in the lungs. This study demonstrated that while PMN are required for an effective antiviral response, they also contribute to lung pathology during RCoV infection.
Our previous studies indicate that hMRE11 plays a role in MMR, and this function of hMRE11 is most likely mediated by the hMLH1-hMRE11 interaction. Here, we explored the functional implications of the hMLH1-hMRE11 interaction in MMR and the effects of hMLH1 mutations on their interaction. Our in vitro MMR assay demonstrated that the dominant-negative hMRE11 452-634 mutant peptide (i.e., harboring only the hMLH1-interacting domain) imparted a significant reduction in both 3' excision and 3'-directed MMR activities. Furthermore, the expression of hMRE11 , and to a lesser extent hMRE11 1-634 (ATLD1), impaired G2/M checkpoint control in response to MNU and cisplatin treatments, rendering cells resistant to killings by these two anticancer drugs. Analysis of 38 hMLH1 missense mutations showed that the majority of mutations caused significant (> 50%) reductions in their interaction with hMRE11, suggesting a potential link between aberrant protein interaction and the pathogenic effects of hMLH1 variants.
Severe respiratory viral infections are associated with spread to the alveoli of the lungs. There are multiple murine models of severe respiratory viral infections that have been used to identify viral and host factors that contribute to disease severity. Primary cultures of murine alveolar epithelial cells provide a robust in vitro model to perform mechanistic studies that can be correlated with in vivo studies to identify cell type-specific factors that contribute to pathology within the alveoli of the lung during viral infection. In this study, we established an in vitro model to compare the responses of type I (ATI) and type II (ATII) alveolar epithelial cells to infection by respiratory viruses used in murine models: mouse-adapted severe acute respiratory syndrome-associated coronavirus (SARS-CoV, v2163), murine coronavirus MHV-1, and influenza A (H1N1) virus, strain PR8. Murine alveolar cells cultured to maintain an ATII cell phenotype, determined by expression of LBP180, were susceptible to infection by all three viruses. In contrast, ATII cells that were cultured to trans-differentiate into an ATI-like cell phenotype were susceptible to MHV-1 and PR8, but not mouse-adapted SARS-CoV. Epithelial cells produce cytokines in response to viral infections, thereby activating immune responses. Thus, virus-induced cytokine expression was quantified in ATI and ATII cells. Both cell types had increased expression of IL-1β mRNA upon viral infection, though at different levels. While MHV-1 and PR8 induced expression of a number of shared cytokines in ATI cells, there were several cytokines whose expression was induced uniquely by MHV-1 infection. In summary, ATI and ATII cells exhibited differential susceptibilities and cytokine responses to infection by respiratory viruses. This in vitro model will be critical for future studies to determine the roles of these specialized cell types in the pathogenesis of respiratory viral infection.
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