Characterization of High-Risk-Other Human Papillomavirus Genotypes in Papanicolaou Tests, High-Grade Squamous Intraepithelial Lesions, and Cervical Cancer.

Background: The objective of this study was to determine the human papillomavirus (HPV) genotypes of high-risk-other HPV Papanicolaou (Pap) tests and of biopsy tissues from patients with high-grade squamous intraepithelial lesion (HGSIL) or cervical cancer. High-risk-other HPV status was determined with the cobas HPV Test (Roche Diagnostics, North America) that identifies 12 high-risk, non-16/18 HPV genotypes. We hypothesized that we would find genotypes of HPV in our population that are not covered by the 9-valent HPV vaccine. Methods: For this retrospective cohort study, we randomly selected 50 high-risk-other HPV Pap test samples from 2018 from our pathology department registries for HPV genotype determination by Roche Linear Array (Roche Diagnostics, North America). Then we randomly selected 76 cervical biopsy samples of HGSIL or cervical cancer with high-risk-other HPV or HPV unknown status from 2016 to 2022 for HPV genotype determination by next-generation sequencing. Results are reported as counts and frequencies. Results: In the 50 high-risk-other HPV Pap test samples, 21 genotypes of HPV were noted; the most common were 53 (n=6), 51 (n=6), and 59 (n=5). In the samples with HGSIL or cervical cancer, 16 HPV genotypes were detected; the most common were 16 (n=26), 58 (n=12), and 33 (n=8). Among the patients with HGSIL or cervical cancer, the 9-valent HPV vaccine provided coverage for all the HPV variants found in 88% of patients, partial coverage in 8% of patients, and no coverage in 4% of patients. Conclusion: The 3 most common HPV genotypes seen in our high-risk-other HPV Pap test samples are not covered by the 9-valent HPV vaccine. For the HGSIL and cancer samples, 88% of the samples had full HPV genotype coverage with the 9-valent HPV vaccine. This study highlights a presence of HPV that will not be protected by vaccination in a high-risk population.

Contraceptive use in patients with gender dysphoria who were assigned female at birth receiving care at a specialty gender-affirming clinic.

BACKGROUND: The Centers for Disease Control and Prevention (CDC) recommend that all patients, regardless of gender identity or sexual orientation, should be offered family planning and contraceptive options without assumptions of sexual behaviors and pregnancy risk. Current research on family planning services for lesbian, gay, bisexual, transgender, and queer or questioning patients is limited, but patients who are sexual or gender minorities are at increased risk for unintended pregnancy. OBJECTIVES: The objective of this study was to describe contraceptive use in patients assigned female at birth with gender dysphoria at a gender-affirming primary care clinic. METHODS: A retrospective descriptive study was conducted. Patients were included if they were 18 to 44 years old, received care at University of New Mexico Truman Health Services in 2019, were diagnosed with gender dysphoria, and were assigned female at birth. Patients were excluded if they had never developed female reproductive organs. Data were collected from the electronic medical records. Potential differences in contraceptive use based on demographic characteristics, having a family planning discussion, and having a contraceptive use discussion were analyzed using chi-square analyses. Potential predictors of contraceptive use were identified using exploratory forward conditional logistic regression and univariate logistic regression analyses. RESULTS: A total of 163 patients were included; average age was 26.6 years; 71% identified as male, 5% identified as masculine, and 25% identified as nonbinary. Most patients (92%) were prescribed masculinizing therapy (testosterone). Forty-five (28%) patients had documented contraception use; the most common form was permanent contraception (76%). Most patients (68%) did not have any documented contraindications to contraception based on CDC US Medical Eligibility Criteria for contraceptive use. Of 113 patients with a documented sexual orientation, 45 patients (40%) reported having sex with persons who have a penis; only 13 (29%) of those patients had a documented form of contraception. Family planning discussions were documented for 82% of patients. Family planning discussions that specifically addressed contraception were documented in only 49% of patients. However, the odds of a patient having a documented use of contraception was 9.26 times higher when family planning discussions specifically addressed contraception. CONCLUSION: Documented contraception use was low in people assigned female at birth of childbearing age receiving care at a gender-affirming clinic. Due to increased risks of unintended pregnancy in this population and the teratogenic nature of testosterone, family planning discussion should also include discussions related to contraception, as this was associated with increased contraception use. Additional research is needed to address potential barriers to contraception use in this population.

Antiviral Functions of Type I and Type III Interferons in the Olfactory Epithelium.

The olfactory neuroepithelium (OE) is one of the few neuronal tissues where environmental pathogens can gain direct access. Despite this vulnerable arrangement, little is known about the protective mechanisms in the OE to prevent viral infection and its antiviral responses. We systematically investigated acute responses in the olfactory mucosa upon exposure to vesicular stomatitis virus (VSV) via RNA-seq. VSVs were nasally inoculated into C57BL/6 mice. Olfactory mucosae were dissected for gene expression analysis at different time points after viral inoculation. Interferon functions were determined by comparing the viral load in interferon receptor knockout (Ifnar1-/- and Ifnlr1-/-) with wildtype OE. Antiviral responses were observed as early as 24 h after viral exposure in the olfactory mucosa. The rapidly upregulated transcripts observed included specific type I as well as type III interferons (Ifn) and interferon-stimulated genes. Genetic analyses demonstrated that both type I and type III IFN signaling are required for the suppression of viral replication in the olfactory mucosa. Exogenous IFN application effectively blocks viral replication in the OE. These findings reveal that the OE possesses an innate ability to suppress viral infection. Type I and type III IFNs have prominent roles in OE antiviral functions.

MicroRNA expression associated with low-grade cervical intraepithelial neoplasia outcomes.

PURPOSE: Only a fraction of low-grade cervical intraepithelial neoplasia (CIN) progresses to high-grade CIN; however, the biological processes that differentiate progressive CIN from CIN that resolves naturally are poorly understood. MicroRNAs (miRNAs) are important epigenetic regulators of gene expression and thus, miRNA expression profiling can reveal the dysregulated biology underlying disease processes. The purpose of this case-control study was to reveal miRNA expression patterns and predict the underlying biological pathways that are associated with clinical outcomes of low-grade CIN. METHODS: Women with low-grade CIN diagnosis and definitive clinical outcomes (n = 51) were identified retrospectively using electronic clinical records. Comprehensive miRNA expression profiling was performed on the low-grade CIN diagnostic cervical biopsies retrieved from pathology archives. Differential miRNA expression was analyzed by comparing women with CIN that progressed to women with CIN that resolved naturally. RESULTS: Differential expression of 29 miRNAs was observed in low-grade CIN that progressed to high-grade compared to low-grade CIN that resolved. Of these, 24 were significantly downregulated in progressive CIN, including miR-638, miR-3196, miR-4488, and miR-4508, while 5 miRNAs, including miR-1206a, were significantly upregulated. Computational gene ontology analysis based on the discovered miRNAs and their putative mRNA targets revealed biological processes associated with oncogenic phenotypes. CONCLUSION: Distinct miRNA expression profiles are associated with clinical outcomes of low-grade CIN. The functional effects of the differentially expressed miRNAs may be biological determinants of CIN progression or resolution.

NF-κB-Inducing Kinase Governs the Mitochondrial Respiratory Capacity, Differentiation, and Inflammatory Status of Innate Immune Cells.

NF-κB-inducing kinase (NIK), which is essential for the activation of the noncanonical NF-κB pathway, regulates diverse processes in immunity, development, and disease. Although recent studies have elucidated important functions of NIK in adaptive immune cells and cancer cell metabolism, the role of NIK in metabolic-driven inflammatory responses in innate immune cells remains unclear. In this study, we demonstrate that murine NIK-deficient bone marrow-derived macrophages exhibit defects in mitochondrial-dependent metabolism and oxidative phosphorylation, which impair the acquisition of a prorepair, anti-inflammatory phenotype. Subsequently, NIK-deficient mice exhibit skewing of myeloid cells characterized by aberrant eosinophil, monocyte, and macrophage cell populations in the blood, bone marrow, and adipose tissue. Furthermore, NIK-deficient blood monocytes display hyperresponsiveness to bacterial LPS and elevated TNF-α production ex vivo. These findings suggest that NIK governs metabolic rewiring, which is critical for balancing proinflammatory and anti-inflammatory myeloid immune cell function. Overall, our work highlights a previously unrecognized role for NIK as a molecular rheostat that fine-tunes immunometabolism in innate immunity, and suggests that metabolic dysfunction may be an important driver of inflammatory diseases caused by aberrant NIK expression or activity.

Contraceptive Use and Missed Opportunities for Family Planning Discussions in Women Living with Human Immunodeficiency Virus at an HIV Clinic.

The objective of this study was to examine contraception use and family planning discussions (FPD) in female people living with HIV (PLWH). A retrospective cohort study was conducted. Female PLWH were included if they were 18-44 years and received care in 2019 at an HIV clinic. 74 patients met inclusion; mean age was 35 years, 53% were white. All patients were prescribed antiretroviral therapy. 48.6% of patients had documented FPD. 64.9% of patients were using contraception; sterilization was most common (41.7%). Only five patients had a contraindication to hormonal contraception. No differences in contraception use were observed based on age, race, HIV viral load, number of visits, or past pregnancies. However, patients with documented FPD were more likely to use contraception (OR 4.55; 95% CI 1.35-15.29). Routine FPD and contraception use in female PLWH were low. Rates of sterilization were high in female PLWH. Providing quality family planning services is critical to increase contraception use and selection of the most appropriate contraception form.

Assessing Mitochondrial DNA Release into the Cytosol and Subsequent Activation of Innate Immune-related Pathways in Mammalian Cells.

Mitochondria have emerged as key drivers of mammalian innate immune responses, functioning as signaling hubs to trigger inflammation and orchestrating metabolic switches required for phagocyte activation. Mitochondria also contain damage-associated molecular patterns (DAMPs), molecules that share similarity with pathogen-associated molecular patterns (PAMPs) and can engage innate immune sensors to drive inflammation. The aberrant release of mitochondrial DAMPs during cellular stress and injury is an increasingly recognized trigger of inflammatory responses in human diseases. Mitochondrial DNA (mtDNA) is a particularly potent DAMP that engages multiple innate immune sensors, although mounting evidence suggests that cytosolic mtDNA is primarily detected via the cyclic GMP-AMP synthase-stimulator of interferon genes (cGAS-STING) pathway. cGAS and STING are widely expressed in mammalian cells and serve as key regulators of type I interferon and cytokine expression in both infectious and inflammatory diseases. Despite growing roles for the mtDNA-cGAS-STING axis in human disease, assays to quantify mtDNA release into the cytosol and approaches to link mtDNA to cGAS-STING signaling are not standardized, which increases the possibility for experimental artifacts and misinterpretation of data. Here, we present a series of protocols for assaying the release of mtDNA into the cytosol and subsequent activation of innate immune signaling in mammalian cells. We highlight genetic and pharmacological approaches to induce and inhibit mtDNA release from mitochondria. We also describe immunofluorescence microscopy and cellular fractionation assays to visualize morphological changes in mtDNA and quantify mtDNA accumulation in the cytosol. Finally, we include protocols to examine mtDNA-dependent cGAS-STING activation by RT-qPCR and western blotting. These methods can be performed with standard laboratory equipment and are highly adaptable to a wide range of mammalian cell types. They will permit researchers working across the spectrum of biological and biomedical sciences to accurately and reproducibly measure cytosolic mtDNA release and resulting innate immune responses. © 2022 Wiley Periodicals LLC. Basic Protocol 1: siRNA-mediated knockdown of TFAM to induce mtDNA instability, cytosolic release, and activation of the cGAS-STING pathway Alternate Protocol: Pharmacological induction of mtDNA release and cGAS-STING activation using ABT-737 and Q-VD-OPH Basic Protocol 2: Isolation and quantitation of DNA from cytosolic, mitochondrial, and nuclear fractions Basic Protocol 3: Pharmacological inhibition of mtDNA replication and release.