Toll-like receptor 4 (TLR4) is an immune protein which binds lipopolysaccharide (LPS) present on the outer membrane of Gram-negative bacteria and activates the innate immune response. In mice, an mRNA splice variant composed of only the extracellular domain of TLR4 was shown to encode a soluble product (sTLR4) capable of inhibiting inflammatory response to LPS. sTLR4 has been recovered from human saliva and demonstrated to dampen the production of pro-inflammatory cytokines by macrophages. Prior work showed that TLR4 was also present in endometrial glands, uterine tube epithelia and endocervical glands. However, there are no published studies exploring the presence or role of sTLR4 in lower genital tract secretions. We tested primary female genital epithelial cells’ supernatants as well as human endocervical cytobrush and vaginal swab samples for the presence of sTLR4 by using a chemiluminescent immunoassay. We found sTLR4 in cervicovaginal secretions, with increased concentration of sTLR4 present in participants with endocervical ectopy and in those sampled during the proliferative phase of the menstrual cycle. Supernatants from endocervical cell lines possessed higher levels of sTLR4 than those derived from ectocervical or vaginal cells. sTLR4 concentration was not correlated with the presence of bacterial vaginosis, age, the concentration of common vaginal Gram-negative bacteria or with genetic variation in the TLR4 locus. By western blotting, we demonstrated that sTLR4 is composed of a ~100 kDa polypeptide, corresponding to the entire TLR4 ectodomain. In a reporter monocytic cell line, we showed dose-dependent inhibition of the LPS/Interferon-regulatory factor (IRF) pathway when LPS was preincubated with endocervical cells’ supernatants. These results point to an unappreciated form of innate immune regulation in the cervicovaginal niche, which could potentially open new avenues for understanding inflammatory disorders such as cervicitis and pelvic inflammatory disease.

PARP inhibitors are a revolutionary precision cancer therapy that inhibits PARP function and causes synthetic lethality in cells with homologous recombination deficiency (HRD) such as cells with BRCA1/2 loss. As such, BRCA1 presence plays a key role in PARP inhibitor sensitivity, providing a basis to predict treatment response based on BRCA1 expression in patient tumors. In addition to protein loss mutations, hypermethylation of BRCA1 gene may also result in the loss of BRCA1 expression, and subsequent HRD and potential PARP inhibitor sensitivity. The NRG-GY005 clinical trial focuses on the development of a clinically useful predictor of PARP inhibitor sensitivity/resistance to spare toxicities for patients unlikely to derive benefit. I will first characterize BRCA1 expression in randomized and blinded tumor samples as a biomarker for response to PARP inhibitors in patients. I will then relate BRCA1 protein expression to methylation status of BRCA1. Combining these two aims will describe BRCA1 function/presence in tumors, and better define overall homologous recombination deficiency to optimize patient-specific treatment between PARP inhibitors or other precision therapies. I conducted immunohistochemistry (IHC), a targeted staining with antibody MS110, towards BRCA1 to identify protein presence in tumor tissue and have compiled and analyzed droplet digital PCR (ddPCR), IHC, and clinical treatment reports. I am currently working through Aim 1 by performing IHC staining on tissues from the first phase of the NRG-GY005 clinical trial and will continue investigating whether a lack of BRCA1 expression is associated with cancer cells that are responsive to therapies. In combining IHC and ddPCR assays, we can compare BRCA1 presence with methylation to analyze tumor cases without BRCA1 expression and contribute toward identifying biomarkers to inform patient-specific treatment for individuals with recurrent ovarian cancer. I anticipate that cases lacking BRCA1 expression have an increased likelihood of hypermethylation in the tumor, causing loss of BRCA1.

Apolipoprotein E (APOE) is a polymorphic gene with 3 main alleles: APOE2, APOE3, APOE4.The APOE4 allele is the greatest genetic risk factor of late-onset Alzheimer’s disease (AD). Carriage of the APOE4 allele results in increased amyloid-β (Aβ) accumulation, aggregation, and deposition in the brain compared to other genotypes, thus contributing to AD pathogenesis. Preeclampsia (PE) is a pregnancy-specific disorder associated with maternal and perinatal mortality. PE has also been linked to protein misfolding pathology, characterized by the accumulation of misfolded proteins (including Aβ) in the placenta, urine, and blood. Due to the similarities with AD pathology, we investigated the relationship between APOE4 and the development of PE-symptomatology in a transgenic mouse model carrying human APOE. To assess this relationship, we induced a PE-like syndrome in APOE3 +/+ and APOE4 +/+ pregnant females on gestational day 13.5 using the Reduced Uteroplacental Perfusion Pressure (RUPP) procedure (n=5-6/group). Sham procedures were performed on the same number of females in each group as controls. On gestational day 17.5, differences in blood pressure, proteinuria, kidney integrity, placental efficiency, placental morphology, and fetal weights in response to RUPP were assessed and compared between APOE3 and APOE4 mice. Our results show that RUPP induced significant kidney damage in APOE4 mice (p=0.014), while APOE3 mice maintained normal kidney morphology. Moreover, RUPP was associated with a significant reduction in placental efficiency in both groups, but only APOE4 fetuses developed fetal growth restriction (FGR, p=0.045). APOE3 was also associated with mild changes in placental morphology, but APOE4 was not. Blood pressure or proteinuria did not vary in response to RUPP in either genotype. Our findings suggest that APOE4 confers an increased susceptibility to kidney damage and FGR in response to RUPP compared to APOE3. Further investigation is needed to determine the molecular basis of these genotype-dependent effects.

Pregnant individuals infected with influenza A viruses (IAV) have higher risks of mortality, hospitalization, preterm birth, and stillbirth. The objective was to determine how the transcriptional program induced by IAV infection in the lung differs between pregnant and non-pregnant states. I hypothesized that a cluster of genes linked to aggravation of influenza disease would be upregulated in the pregnant lung early in IAV infection versus the non-pregnant lung. We used a non-human primate model [NHP; pregnant (N=10), non-pregnant (N=10); Macaca nemestrina, pigtail macaque] to investigate the transcriptional response in the lung of pregnant versus non-pregnant NHPs infected with the IAV CA/04/2009 (H1N1) strain. Maternal lung tissues were collected from the animals at necropsy 5 days after infection. mRNA-Seq was performed by first extracting mRNA from tissues, preparing mRNA libraries, and aligning raw sequencing data, using Spliced Transcripts Alignment to a Reference (STAR), to the macaque genome. I performed normalization of the raw gene count matrix using EdgeR in R Studio and alignment to the macaque reference genome. Next, I performed a single gene analysis using Limma-voom to determine differentially expressed genes (DEG). A total of 115 genes were significantly differentially expressed (>2-fold change, p<0.05) with 77 upregulated and 38 downregulated. Remarkably, genes linked to aggravation of influenza A viral disease, tissue injury, or acidification were upregulated in the infected pregnant versus non-pregnant lung 5 days after infection (MMP8, ATP12A, LGR4, NUP58, KBTBD6; log2fold change 1.28 - 2.8, all p<0.05). Next steps include gene set enrichment analysis and ingenuity pathway analysis to further investigate the gene networks linked to these upregulated genes. In summary, pregnancy was associated with upregulation of genes in the lungs 5 days after IAV infection that may predispose to greater tissue injury versus the non-pregnant lung.