Melissa Herbst-Kralovetz

Interests

Research

Dr. Herbst-Kralovetz’s laboratory is broadly interested in understanding innate mucosal immune responses to resident bacteria, pathogens, and microbial products at mucosal sites, including the female reproductive tract (FRT). Her lab is interested in studying the mucosal barrier function of the FRT and its role in host defense and maintaining mucosal homeostasis, which is widely relevant to infection, immunity, reproduction and even cancer. She has a long-standing interest in women’s health

Courses

Scholarly Contributions

Chapters

• Łaniewski, P., & Herbst-Kralovetz, M. M. (2019). Analysis of host responses to Neisseria gonorrhoeae using a human three-dimensional endometrial epithelial cell model.. In Neisseria gonorrhoeae. Springer Nature.
• Łaniewski, P., & Herbst-Kralovetz, M. (2018). Vagina. In Encyclopedia of Reproduction. doi:10.1016/B978-0-12-801238-3.64406-9
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  The vagina functions as a female copulative organ and a canal for childbirth and menstrual secretions and is composed of smooth muscles, connective tissue and mucosa lined by a nonkeratinized, stratified squamous epithelium covered by mucus. Sex hormones impact the physiology and structure of the vaginal epithelium and the vaginal microbiota (VMB) at this site. Estrogen facilitates glycogen production and promotion of lactobacilli growth. Lactobacilli acidify the microenvironment which protects the vagina against invading pathogens. Lactobacilli dominance is associated with vaginal health, whereas depletion of lactobacilli results in dysbiosis. The health outcomes of VMB disruption will be discussed.
• Łaniewski, P., & Herbst-Kralovetz, M. M. (2018). Chapter 52: Vagina. In Encyclopedia of Reproduction.
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  Chapter within the Encyclopedia of Reproduction

Journals/Publications

• Crossley, P., Sutar, Y., Tsoy, I., Mukkirwar, S., Łaniewski, P., Herbst-Kralovetz, M., & Date, A. (2024). Development of phenyllactic acid ionic liquids and evaluation of cytotoxicity to human cervical epithelial cells. RSC Advances, 14(23). doi:10.1039/d4ra01812e
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  Phenyllactic acid (PLA), is a naturally produced, broad-spectrum antimicrobial compound with activity against bacteria and fungi. PLA can be produced by a variety of lactic acid bacteria, including vaginal Lactobacillus species, which are healthy constituents of the vaginal microbiome with a protective role against invading pathogenic bacteria and/or fungi. Additionally, PLA has been shown to exhibit anti-inflammatory and immunomodulatory properties, overall indicating its therapeutic potential as an intravaginally delivered compound for modulation of the vaginal microbiome. However, PLA has low kinetic solubility in water. Hence, strategies to improve the solubility of PLA are necessary to facilitate its intravaginal delivery. Using biocompatible cations, choline and carnitine, we successfully transformed both d- and l-enantiomers of crystalline PLA into amorphous low-melting ionic liquids (ILs) with high water solubility. We further evaluated the in vitro cytotoxicity of PLA ILs to human cervical epithelial cells. Microscopic visualisation of cellular morphology using crystal violet staining and MTT cell proliferation assay revealed that PLA ILs result in minimal morphological changes and low cytotoxicity to human cervical epithelial cells. Overall, we successfully demonstrated that transforming PLA into ILs efficiently enhances its solubility in water and these formulations are not toxic to human epithelial cells. This investigation lays the groundwork for future testing of PLA ILs for their antimicrobial properties and metabolic activity within the cervicovaginal microenvironment.
• Harris, H., Łaniewski, P., Cui, H., Roe, D., Chase, D., & Herbst-Kralovetz, M. (2024). Cervicovaginal lavages uncover growth factors as key biomarkers for early diagnosis and prognosis of endometrial cancer. Molecular Biomedicine, 5(1). doi:10.1186/s43556-024-00219-6
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  Endometrial cancer (EC) rates are continuing to rise and it remains the most common gynecologic cancer in the US. Existing diagnostic methods are invasive and can cause pain and anxiety. Hence, there is a need for less invasive diagnostics for early EC detection. The study objective was to evaluate the utility of growth factors collected through minimally invasive cervicovaginal lavage (CVL) sampling as diagnostic and prognostic biomarkers for EC. CVL samples from 192 individuals undergoing hysterectomy for benign or malignant conditions were collected and used to quantify the concentrations of 19 growth and angiogenic factors using multiplex immunoassays. Patients were categorized based on disease groups: benign conditions (n = 108), endometrial hyperplasia (n = 18), and EC (n = 66). EC group was stratified into grade 1/2 endometrial endometrioid cancer (n = 53) and other EC subtypes (n = 13). Statistical associations were assessed using receiver operating characteristics, Spearman correlations and hierarchical clustering. Growth and angiogenic factors: angiopoietin-2, endoglin, fibroblast activation protein (FAP), melanoma inhibitory activity, and vascular endothelial growth factor-A (VEGF-A) were significantly (p < 0.0001) elevated in EC patients. A multivariate model combining 11 proteins with patient age and body mass index exhibited excellent discriminatory potential (area under curve = 0.918) for EC, with a specificity of 90.7% and a sensitivity of 87.8%. Moreover, angiopoietin-2, FAP and VEGF-A significantly (p < 0.05–0.001) associated with tumor grade, size, myometrial invasion, and mismatch repair status. Our results highlight the innovative use of growth and angiogenic factors collected through CVL sampling for the detecting endometrial cancer, showcasing not only their diagnostic potential but also their prognostic value.
• Jimenez, N., Norton, T., Diadala, G., Bell, E., Valenti, M., Farland, L., Mahnert, N., & Herbst-Kralovetz, M. (2024). Vaginal and rectal microbiome contribute to genital inflammation in chronic pelvic pain. BMC Medicine, 22(1). doi:10.1186/s12916-024-03500-1
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  Background: Chronic pelvic pain (CPP) is a multifactorial syndrome that can substantially affect a patient’s quality of life. Endometriosis is one cause of CPP, and alterations of the immune and microbiome profiles have been observed in patients with endometriosis. The objective of this pilot study was to investigate differences in the vaginal and gastrointestinal microbiomes and cervicovaginal immune microenvironment in patients with CPP and endometriosis diagnosis compared to those with CPP without endometriosis and no CPP. Methods: Vaginal swabs, rectal swabs, and cervicovaginal lavages (CVL) were collected among individuals undergoing gynecologic laparoscopy. Participants were grouped based on patients seeking care for chronic pain and/or pathology results: CPP and endometriosis (CPP-Endo) (n = 35), CPP without endometriosis (n = 23), or patients without CPP or endometriosis (controls) (n = 15). Sensitivity analyses were performed on CPP with endometriosis location, stage, and co-occurring gynecologic conditions (abnormal uterine bleeding, fibroids). 16S rRNA sequencing was performed to profile the microbiome, and a panel of soluble immune mediators was quantified using a multiplex assay. Statistical analysis was conducted with SAS, R, MicrobiomeAnalyst, MetaboAnalyst, and QIIME 2. Results: Significant differences were observed between participants with CPP alone, CPP-Endo, and surgical controls for body mass index, ethnicity, diagnosis of ovarian cysts, and diagnosis of fibroids. In rectal microbiome analysis, both CPP alone and CPP-Endo exhibited lower alpha diversity than controls, and both CPP groups revealed enrichment of irritable bowel syndrome-associated bacteria. CPP-Endo exhibited an increased abundance of vaginal Streptococcus anginosus and rectal Ruminococcus. Patients with CPP and endometrioma (s) demonstrated increased vaginal Streptococcus, Lactobacillus, and Prevotella compared to other endometriosis sites. Further, abnormal uterine bleeding was associated with an increased abundance of bacterial vaginosis-associated bacteria. Immunoproteomic profiles were distinctly clustered by CPP alone and CPP-Endo compared to controls. CPP-Endo was enriched in TNF⍺, MDC, and IL-1⍺. Conclusions: Vaginal and rectal microbiomes were observed to differ between patients with CPP alone and CPP with endometriosis, which may be useful in personalized treatment for individuals with CPP and endometriosis from those with other causes of CPP. Further investigation is warranted in patients with additional co-occurring conditions, such as AUB/fibroids, which add additional complexity to these conditions and reveal the enrichment of distinct pathogenic bacteria in both mucosal sites. This study provides foundational microbiome-immunoproteomic knowledge related to chronic pelvic pain, endometriosis, and co-occurring gynecologic conditions that can help improve the treatment of patients seeking care for pain.
• Lorentzen, G., Łaniewski, P., Cui, H., Mahnert, N., Mourad, J., Borst, M., Willmott, L., Chase, D., Roe, D., & Herbst-Kralovetz, M. (2024). Cervicovaginal Metabolome and Tumor Characteristics for Endometrial Cancer Detection and Risk Stratification. Clinical Cancer Research, 30(14). doi:10.1158/1078-0432.CCR-23-2934
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  Purpose: Endometrial cancer is highly prevalent and lacking noninvasive diagnostic techniques. Diagnosis depends on histological investigation of biopsy samples. Serum biomarkers for endometrial cancer have lacked sensitivity and specificity. The objective of this study was to investigate the cervicovaginal environment to improve the understanding of metabolic reprogramming related to endometrial cancer and identify potential biomarker candidates for noninvasive diagnostic and prognostic tests. Experimental Design: Cervicovaginal lavages were collected from 192 participants with endometrial cancer (n = 66) and nonmalignant conditions (n = 108), and global untargeted metabolomics was performed. Using the metabolite data (n = 920), we completed a multivariate biomarker discovery analysis. Results: We analyzed grade 1/2 endometrioid carcinoma (n = 53) and other endometrial cancer subtypes (n = 13) to identify shared and unique metabolic signatures between the subtypes. When compared to non-malignant conditions, downregulation of proline (P < 0.0001), tryptophan (P < 0.0001), and glutamate (P < 0.0001) was found among both endometrial cancer groups, relating to key hallmarks of cancer including immune suppression and redox balance. Upregulation (q < 0.05) of sphingolipids, fatty acids, and glycerophospholipids was observed in endometrial cancer in a type-specific manner. Furthermore, cervicovaginal metabolites related to tumor characteristics, including tumor size and myometrial invasion. Conclusions: Our findings provide insights into understanding the endometrial cancer metabolic landscape and improvement in diagnosis. The metabolic dysregulation described in this article linked specific metabolites and pathophysiological mechanisms including cellular proliferation, energy supply, and invasion of neighboring tissues. Furthermore, cervicovaginal metabolite levels related to tumor characteristics, which are used for risk stratification. Overall, development of noninvasive diagnostics can improve both the acceptability and accessibility of diagnosis.
• Mancilla, V., Jimenez, N., Bishop, N., Flores, M., & Herbst-Kralovetz, M. (2024). The Vaginal Microbiota, Human Papillomavirus Infection, and Cervical Carcinogenesis: A Systematic Review in the Latina Population. Journal of Epidemiology and Global Health, 14(2). doi:10.1007/s44197-024-00201-z
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  Background: Latina women experience disproportionately higher rates of HPV infection, persistence, and progression to cervical dysplasia and cancer compared to other racial–ethnic groups. This systematic review explores the relationship between the cervicovaginal microbiome and human papillomavirus infection, cervical dysplasia, and cervical cancer in Latinas. Methods: The review abides by the Preferred Reporting Items for Systematic Reviews and Meta-analyses guidelines. PubMed, EMBASE, and Scopus databases were searched from January 2000 through November 11, 2022. The review included observational studies reporting on the cervicovaginal microbiota in premenopausal Latina women with human papillomavirus infection, cervical dysplasia, and cervical cancer. Results: Twenty-five articles were eligible for final inclusion (N = 131,183). Forty-two unique bacteria were reported in the cervicovaginal microbiome of Latinas. Seven bacteria: Lactobacillus crispatus, Lactobacillus iners, Chlamydia trachomatis, Prevotella spp., Prevotella amnii, Fusobacterium spp. and Sneathia spp. were enriched across multiple stages of cervical carcinogenesis in Latinas. Therefore, the total number of reported bacteria includes four bacteria associated with the healthy state, 16 bacteria enriched in human papillomavirus outcomes, 24 unique bacteria associated with abnormal cytology/dysplasia, and five bacteria associated with cervical cancer. Furthermore, three studies reported significantly higher alpha and beta diversity in Latinas with cervical dysplasia and cancer compared to controls. Lactobacillus depletion and an increased abundance of L. iners in Latinas compared to non-Latinas, regardless of human papillomavirus status or lesions, were observed. Conclusions: The identification of 42 unique bacteria and their enrichment in cervical carcinogenesis can guide future cervicovaginal microbiome research to better inform cervical cancer prevention strategies in Latinas.
• Łaniewski, P., Joe, T., Jimenez, N., Eddie, T., Bordeaux, S., Quiroz, V., Peace, D., Cui, H., Roe, D., Caporaso, J., Lee, N., & Herbst-Kralovetz, M. (2024). Viewing Native American Cervical Cancer Disparities through the Lens of the Vaginal Microbiome: A Pilot Study. Cancer Prevention Research, 17(11). doi:10.1158/1940-6207.CAPR-24-0286
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  Vaginal dysbiosis is implicated in persistent human papillomavirus (HPV) infection and cervical cancer. Yet, there is a paucity of data on the vaginal microbiome in Native American communities. Here, we aimed to elucidate the relationships between microbiome, HPV, sociodemographic, and behavioral risk factors to better understand an increased cervical cancer risk in Native American women. In this pilot study, we recruited 31 participants (16 Native American and 15 non-Native women) in Northern Arizona and examined vaginal microbiota composition, HPV status, and immune mediators. We also assessed individuals’ sociodemographic information and physical, mental, sexual, and reproductive health. Overall, microbiota profiles were dominated by common Lactobacillus species (associated with vaginal health) or a mixture of bacterial vaginosis–associated bacteria. Only 44% of Native women exhibited Lactobacillus dominance, compared with 58% of nonNative women. Women with vaginal dysbiosis also had elevated vaginal pH and were more frequently infected with high-risk HPV. Furthermore, we observed associations of multiple people in a household, lower level of education, and high parity with vaginal dysbiosis and abundance of specific bacterial species. Finally, women with dysbiotic microbiota presented with elevated vaginal levels of proinflammatory cytokines. Altogether, these findings indicate an interplay between HPV, vaginal microbiota, and host defense, which may play a role in the cervical cancer disparity among Native American women. Future longitudinal studies are needed to determine the mechanistic role of vaginal microbiota in HPV persistence in the context of social determinants of health toward the long-term goal of reducing health disparities between non-Hispanic White and Native American populations.
• Chase, D. M., McCann, L. D., Treuth, A., Cui, H., Laniewski, P., Jimenez, N. R., Mahnert, N. D., Roe, D. J., & Herbst-Kralovetz, M. M. (2023). Preoperative quality of life at time of gynecologic surgery: considerations for postoperative management. AJOG global reports, 3(4), 100275.
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  Patients presenting for gynecologic surgery are a heterogeneous group. Preoperative quality of life may be a useful tool to guide postoperative management.
• Elnaggar, J. H., Ardizzone, C. M., Cerca, N., Toh, E., Łaniewski, P., Lillis, R. A., Herbst-Kralovetz, M. M., Quayle, A. J., Muzny, C. A., & Taylor, C. M. (2023). A novel , , and standard that improves accuracy in quantifying bacterial burden in vaginal microbial communities. Frontiers in cellular and infection microbiology, 13, 1198113.
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  Bacterial vaginosis (BV) is the most common vaginal dysbiosis. In this condition, a polymicrobial biofilm develops on vaginal epithelial cells. Accurately quantifying the bacterial burden of the BV biofilm is necessary to further our understanding of BV pathogenesis. Historically, the standard for calculating total bacterial burden of the BV biofilm has been based on quantifying 16S rRNA gene copy number. However, is improper for measuring the bacterial burden of this unique micro-environment. Here, we propose a novel qPCR standard to quantify bacterial burden in vaginal microbial communities, from an optimal state to a mature BV biofilm. These standards consist of different combinations of vaginal bacteria including three common BV-associated bacteria (BVAB) spp. (G), spp. (P), and spp. (F) and commensal spp. (L) using the 16S rRNA gene (G:P:F:L, G:P:F, G:P:L and 1G:9L). We compared these standards to the traditional (E) reference standard using known quantities of mock vaginal communities and 16 vaginal samples from women. The E standard significantly underestimated the copy numbers of the mock communities, and this underestimation was significantly greater at lower copy numbers of these communities. The G:P:L standard was the most accurate across all mock communities and when compared to other mixed vaginal standards. Mixed vaginal standards were further validated with vaginal samples. This new G:P:L standard can be used in BV pathogenesis research to enhance reproducibility and reliability in quantitative measurements of BVAB, spanning from the optimal to non-optimal (including BV) vaginal microbiota.
• Farland, L., Khan, S., Shilen, A., Heslin, K., Ishimwe, P., Allen, A., Herbst-Kralovetz, M., Mahnert, N., Pogreba-Brown, K., Ernst, K., & Jacobs, E. (2023). COVID-19 vaccination and changes in the menstrual cycle among vaccinated persons. Fertility and Sterility, 119(3). doi:10.1016/j.fertnstert.2022.12.023
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  Objective: To describe the characteristics of people who experience changes to their menstrual cycle after COVID-19 vaccination. Design: Longitudinal study. Patient(s): We recruited a volunteer sample with and without a history of SARS-CoV-2 infection who enrolled in the Arizona COVID-19 Cohort (CoVHORT) study and participated in a reproductive sub-cohort who were pre-menopausal, not pregnant, and had received a COVID-19 vaccine in 2021 (n = 545). Exposure(s): Demographic and reproductive characteristics were collected via self-reports. Main Outcome Measure(s): Information on self-reported changes in the menstrual cycle after COVID-19 vaccination was collected from May 2021 to December 2021. We looked at demographic and reproductive characteristics as predictors of menstrual cycle change. Result(s): The majority of our vaccinated sample received the Pfizer-BioNTech vaccine (58%), and were 26-35 years old (51%), non-Hispanic (84%), and White (88%). Approximately 25% of vaccinated participants reported a change in their menstrual cycle after vaccination; the majority reported changes after their second dose (56%) as compared with their first (18%) and third (14%) doses. The most commonly reported changes were irregular menstruation (43%), increased premenstrual symptoms (34%), increased menstrual pain or cramps (30%), and abnormally heavy or prolonged bleeding (31%). High self-reported perceived stress levels compared with low perceived stress (OR, 2.22; 95% CI 1.12-4.37) and greater body mass index (OR, 1.04; 95% CI 1.00-1.07) were associated with greater odds of experiencing the menstrual cycle changes after the vaccination. Participants having a history of SARS-CoV-2 infection were less likely to report changes in their menstrual cycle after vaccination compared with the participants with no history of SARS-CoV-2 infection (OR, 0.58; 95% CI 0.32-1.04). Conclusion(s): Among vaccinated participants, approximately 25% of them reported predominantly temporary changes in the menstrual cycle, however, we are unable to determine whether these changes are due to normal cycle variability. The COVID-19 vaccines are safe and effective for everyone, including pregnant people and people trying to conceive; hence, these findings should not discourage vaccination.
• Jimenez, N. R., Maarsingh, J. D., Łaniewski, P., & Herbst-Kralovetz, M. M. (2023). Commensal Lactobacilli Metabolically Contribute to Cervical Epithelial Homeostasis in a Species-Specific Manner. mSphere, e0045222.
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  In reproductive-age women, the vaginal microbiome is typically dominated by one or a few species, including Lactobacillus crispatus, Lactobacillus iners, Lactobacillus paragasseri, Lactobacillus mulieris, and Lactobaccillus crispatus, has been associated with optimal cervicovaginal health; however, much is still unknown about how other lactobacilli metabolically contribute to cervicovaginal health. We hypothesized that metabolites of each species differ and uniquely contribute to health and homeostasis. To address this hypothesis, we utilized a human three-dimensional (3D) cervical epithelial cell model in conjunction with genomics analyses and untargeted metabolomics to determine the metabolic contributions of less-studied vaginal lactobacilli-, , and . Our study validated that vaginal lactobacilli exhibit a close phylogenetic relationship. Genomic findings from publicly available strains and those used in our study indicated that is metabolically distinct from other species of lactobacilli, likely due to a reduced genome size. Lactobacilli and mock controls were distinguishable based on global metabolic profiles. We identified 95 significantly altered metabolites (0.05) between individual lactobacilli and mock controls. Metabolites related to amino acid metabolism were shared among the lactobacilli. -Acetylated amino acids with potential antimicrobial properties were significantly elevated in a species-specific manner. and shared aromatic, but not carbohydrate-derived, lactic acid metabolites with potential antimicrobial properties that may contribute to homeostasis of the cervicovaginal environment. Additionally, uniquely altered lipid metabolism, which may be a sign of adaptation to the cervicovaginal niche. Overall, these findings further elucidate the metabolic contributions of three key vaginal species in gynecological health. species contribute to cervicovaginal health by their production of lactic acid and other antimicrobial compounds. Yet, much is still unknown regarding the metabolic potential of lesser-studied but common vaginal lactobacilli. Here, we used untargeted metabolomics coupled with our 3D cervical epithelial cell model to identify metabolic differences among vaginal species (Lactobacillus iners, and ) and how those differences related to maintaining homeostasis of the cervical epithelium. Human 3D cell models are essential tools for studying host-bacteria interactions and reducing confounding factors inherent in clinical studies. Therefore, these unique models allowed us to decipher the putative lactobacilli mechanisms that contribute to their roles in health or disease. Metabolic analyses revealed distinct profiles of each species but also shared metabolic contributions associated with antimicrobial activity: amino acid metabolism, -acetylated amino acids, and aromatic lactic acids. These patterns provided validation of metabolites associated with health in clinical studies and provided novel targets, including immunomodulatory and antimicrobial metabolites, for postbiotic therapies.
• Bokulich, N. A., Łaniewski, P., Adamov, A., Chase, D. M., Caporaso, J. G., & Herbst-Kralovetz, M. M. (2022). Multi-omics data integration reveals metabolome as the top predictor of the cervicovaginal microenvironment. PLoS computational biology, 18(2), e1009876.
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  Emerging evidence suggests that host-microbe interaction in the cervicovaginal microenvironment contributes to cervical carcinogenesis, yet dissecting these complex interactions is challenging. Herein, we performed an integrated analysis of multiple "omics" datasets to develop predictive models of the cervicovaginal microenvironment and identify characteristic features of vaginal microbiome, genital inflammation and disease status. Microbiomes, vaginal pH, immunoproteomes and metabolomes were measured in cervicovaginal specimens collected from a cohort (n = 72) of Arizonan women with or without cervical neoplasm. Multi-omics integration methods, including neural networks (mmvec) and Random Forest supervised learning, were utilized to explore potential interactions and develop predictive models. Our integrated analyses revealed that immune and cancer biomarker concentrations were reliably predicted by Random Forest regressors trained on microbial and metabolic features, suggesting close correspondence between the vaginal microbiome, metabolome, and genital inflammation involved in cervical carcinogenesis. Furthermore, we show that features of the microbiome and host microenvironment, including metabolites, microbial taxa, and immune biomarkers are predictive of genital inflammation status, but only weakly to moderately predictive of cervical neoplastic disease status. Different feature classes were important for prediction of different phenotypes. Lipids (e.g. sphingolipids and long-chain unsaturated fatty acids) were strong predictors of genital inflammation, whereas predictions of vaginal microbiota and vaginal pH relied mostly on alterations in amino acid metabolism. Finally, we identified key immune biomarkers associated with the vaginal microbiota composition and vaginal pH (MIF), as well as genital inflammation (IL-6, IL-10, MIP-1α).
• Bordeaux, S., Caporaso, G., Herbst-Kralovetz, M., Laniewski, P., Lee, N., Martinez, E., Metz, N., Peace, D., & Quioz, V. (2022).

  Abstract PO-238: Implementing a culturally-appropriate biospecimen collection protocol during the COVID-19 pandemic to address cervical cancer disparities among Native American women

  . Cancer Epidemiology, Biomarkers & Prevention, 31(1_Supplement), PO-238-PO-238. doi:10.1158/1538-7755.disp21-po-238
• Farland, L. V., Khan, S. M., Shilen, A., Heslin, K. M., Ishimwe, P., Allen, A. M., Herbst-Kralovetz, M. M., Mahnert, N. D., Pogreba-Brown, K., Ernst, K. C., & Jacobs, E. T. (2022). COVID-19 Vaccination and Changes in the Menstrual Cycle Among Vaccinated Persons. Fertility and sterility.
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  To describe characteristics of people who experience changes to their menstrual cycle following COVID-19 vaccination.
• Herbst-Kralovetz, M., Laniewski, P., Maarsingh, J., & McKenzie, R. (2022).

  Immunometabolic Analysis of Mobiluncus mulieris and Eggerthella sp. Reveals Novel Insights into Their Pathogenic Contributions to the Hallmarks of Bacterial Vaginosis

  . American Journal of Obstetrics and Gynecology. doi:10.1016/j.ajog.2021.11.1323
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  Bacterial vaginosis (BV) is a polymicrobial condition in which depletion of health-associated Lactobacillus spp. in the female reproductive tract (FRT) results in a detrimental shift to a more diverse community of anaerobic bacterial species that contribute to adverse health outcomes. These sequelae can include preterm birth (strongly linked to Mobiluncus) and an increased risk of contracting sexually transmitted infections (STIs). Eggerthella sp. may also enter the bloodstream through the FRT to cause bacteremia.
• Kaelin, E. A., Kaelin, E. A., Skidmore, P. T., Skidmore, P. T., Łaniewski, P., Łaniewski, P., Holland, L. A., Holland, L. A., Chase, D. M., Chase, D. M., Herbst-Kralovetz, M. M., Herbst-Kralovetz, M. M., Lim, E. S., & Lim, E. S. (2022). Cervicovaginal DNA Virome Alterations Are Associated with Genital Inflammation and Microbiota Composition. mSystems, 7(2), e0006422.
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  While the link between the cervicovaginal bacterial microbiome, human papillomavirus (HPV) infection, and cervical cancer is recognized (P. Łaniewski, D. Barnes, A. Goulder, H. Cui, et al., Sci. Rep. 8:7593, 2018, http://dx.doi.org/10.1038/s41598-018-25879-7; A. Mitra, D. A. MacIntyre, Y. S. Lee, A. Smith, et al., Sci. Rep. 5:16865, 2015, http://dx.doi.org/10.1038/srep16865; A. Mitra, D. A. MacIntyre, J. R. Marchesi, Y. S. Lee, et al., Microbiome 4:58, 2016, http://dx.doi.org/10.1186/s40168-016-0203-0; J. Norenhag, J. Du, M. Olovsson, H. Verstraelen, et al., BJOG, 127:171-180, 2020, http://dx.doi.org/10.1111/1471-0528.15854; E. O. Dareng, B. Ma, A. O. Famooto, S. N. Adebamowo, et al., Epidemiol. Infect. 144:123-137, 2016, http://dx.doi.org/10.1017/S0950268815000965; A. Audirac-Chalifour, K. Torres-Poveda, M. Bahena-Roman, J. Tellez-Sosa et al., PLoS One 11:e0153274, 2016, http://dx.doi.org/10.1371/journal.pone.0153274; M. Di Paola, C. Sani, A. M. Clemente, A. Iossa, et al., Sci. Rep. 7:10200, 2017, http://dx.doi.org/10.1038/s41598-017-09842-6), the role of the cervicovaginal virome remains poorly understood. In this pilot study, we conducted metagenomic next-generation sequencing of cervicovaginal lavage specimens to investigate the relationship between the cervicovaginal DNA virome, bacterial microbiome, genital inflammation, and HPV infection. Specific virome alterations were associated with features of the local microenvironment related to HPV persistence and progression to cervical cancer. Cervicovaginal viromes clustered distinctly by genital inflammation state. Genital inflammation was associated with decreased virome richness and alpha diversity and an increased abundance of species from the genus . bacteriophages were closely associated with increased abundance, consistent with phage-host relationships. Interestingly, bacteria-bacteriophage transkingdom interactions were linked to genital inflammation and showed specific interactions with bacterial vaginosis-associated bacteria, including and . Taken together, our results reveal prominent virome interactions with features of the cervicovaginal microenvironment that are associated with HPV and cervical cancer. These findings expand our understanding of the cervicovaginal host-microbiome interactions in women's health. HPV infection is an established risk factor for cervical cancer. However, more broadly, the role of the cervicovaginal virome in cervical cancer progression is not well understood. Here, we identified cervicovaginal DNA virome alterations associated with local microenvironment factors (vaginal microbiota and genital inflammation) that influence HPV persistence and progression to cervical cancer. These findings indicate that the cervicovaginal virome plays an important role in women's health.
• Lorentzen, G. M., Łaniewski, P., Cui, H., Roe, D. J., Mourad, J., Mahnert, N. D., Farland, L. V., & Herbst-Kralovetz, M. M. (2022). Immunometabolic profiling of cervicovaginal lavages identifies key signatures associated with adenomyosis. iScience, 25(12), 105508.
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  Adenomyosis is a burdensome gynecologic condition that is associated with pelvic pain, dysmenorrhea, and abnormal uterine bleeding, leading to a negative impact on quality of life; and yet is often left undiagnosed. We recruited 108 women undergoing hysterectomy for benign gynecologic conditions and collected non-invasive cervicovaginal lavage samples for immunometabolic profiling. Patients were grouped according to adenomyosis status. We investigated the levels of 72 soluble immune proteins and >900 metabolites using multiplex immunoassays and an untargeted global metabolomics platform. There were statistically significant alterations in the levels of several immune proteins and a large quantity of metabolites, particularly cytokines related to type II immunity and amino acids, respectively. Enrichment analysis revealed that pyrimidine metabolism, carnitine synthesis, and histidine/histamine metabolism were significantly upregulated pathways in adenomyosis. This study demonstrates utility of non-invasive sampling combined with immunometabolic profiling for adenomyosis detection and a greater pathophysiological understanding of this enigmatic condition.
• Maarsingh, J. D., Łaniewski, P., & Herbst-Kralovetz, M. M. (2022). Immunometabolic and potential tumor-promoting changes in 3D cervical cell models infected with bacterial vaginosis-associated bacteria. Communications biology, 5(1), 725.
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  Specific bacteria of the human microbiome influence carcinogenesis at diverse anatomical sites. Bacterial vaginosis (BV) is the most common vaginal disorder in premenopausal women that is associated with gynecologic sequelae, including cervical cancer. BV-associated microorganisms, such as Fusobacterium, Lancefieldella, Peptoniphilus, and Porphyromonas have been associated with gynecologic and other cancers, though the pro-oncogenic mechanisms employed by these bacteria are poorly understood. Here, we integrated a multi-omics approach with our three-dimensional (3-D) cervical epithelial cell culture model to investigate how understudied BV-associated bacteria linked to gynecologic neoplasia influence hallmarks of cancer in vitro. Lancefieldella parvulum and Peptoniphilus lacrimalis elicited robust proinflammatory responses in 3-D cervical cells. Fusobacterium nucleatum and Fusobacterium gonidiaformans modulated metabolic hallmarks of cancer corresponding to accumulation of 2-hydroxyglutarate, pro-inflammatory lipids, and signs of oxidative stress and genotoxic hydrogen sulfide. This study provides mechanistic insights into how gynecologic cancer-associated bacteria might facilitate a tumor-promoting microenvironment in the human cervix.
• Morales, C. G., Jimenez, N. R., Herbst-Kralovetz, M. M., & Lee, N. R. (2022). Novel Vaccine Strategies and Factors to Consider in Addressing Health Disparities of HPV Infection and Cervical Cancer Development among Native American Women. Medical sciences (Basel, Switzerland), 10(3).
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  Cervical cancer is the 4th most common type of cancer in women world-wide. Many factors play a role in cervical cancer development/progression that include genetics, social behaviors, social determinants of health, and even the microbiome. The prevalence of HPV infections and cervical cancer is high and often understudied among Native American communities. While effective HPV vaccines exist, less than 60% of 13- to 17-year-olds in the general population are up to date on their HPV vaccination as of 2020. Vaccination rates are higher among Native American adolescents, approximately 85% for females and 60% for males in the same age group. Unfortunately, the burden of cervical cancer remains high in many Native American populations. In this paper, we will discuss HPV infection, vaccination and the cervicovaginal microbiome with a Native American perspective. We will also provide insight into new strategies for developing novel methods and therapeutics to prevent HPV infections and limit HPV persistence and progression to cervical cancer in all populations.
• Łaniewski, P., & Herbst-Kralovetz, M. M. (2022). Connecting microbiome and menopause for healthy ageing. Nature microbiology, 7(3), 354-358.
• Łaniewski, P., Cui, H., Mahnert, N. D., Mourad, J., Borst, M. P., Willmott, L., Chase, D. M., Roe, D. J., & Herbst-Kralovetz, M. M. (2022). Protein biomarkers in cervicovaginal lavages for detection of endometrial cancer. Biomarker research, 10(1), 88.
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  Rates of endometrial cancer (EC) are increasing. For a definitive diagnosis, women undergo various time-consuming and painful medical procedures, such as endometrial biopsy with or without hysteroscopy, and dilation and curettage, which may create a barrier to early detection and treatment, particularly for women with inadequate healthcare access. Thus, there is a need to develop robust EC diagnostics based on non- or minimally-invasive sampling. The objective of this study was to quantify a broad range of immuno-oncology proteins in cervicovaginal lavage (CVL) samples and investigate these proteins as predictive diagnostic biomarkers for EC.
• Bordeaux, S. J., Baca, A. W., Begay, R. L., Gachupin, F. C., Caporaso, J. G., Herbst-Kralovetz, M. M., & Lee, N. R. (2021). Designing Inclusive HPV Cancer Vaccines and Increasing Uptake among Native Americans-A Cultural Perspective Review. Current oncology (Toronto, Ont.), 28(5), 3705-3716.
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  Despite a global and nationwide decrease, Native Americans continue to experience high rates of cancer morbidity and mortality. Vaccination is one approach to decrease cancer incidence such as the case of cervical cancer. However, the availability of vaccines does not guarantee uptake, as evident in the Coronavirus 2019 pandemic. Therefore, as we consider current and future cancer vaccines, there are certain considerations to be mindful of to increase uptake among Native Americans such as the incidence of disease, social determinants of health, vaccine hesitancy, and historical exclusion in clinical trials. This paper primarily focuses on human papillomavirus (HPV) and potential vaccines for Native Americans. However, we also aim to inform researchers on factors that influence Native American choices surrounding vaccination and interventions including cancer therapies. We begin by providing an overview of the historical distrust and trauma Native Americans experience, both past and present. In addition, we offer guidance and considerations when engaging with sovereign Tribal Nations in vaccine development and clinical trials in order to increase trust and encourage vaccine uptake.
• McKenzie, R., Maarsingh, J. D., Łaniewski, P., & Herbst-Kralovetz, M. M. (2021). Immunometabolic Analysis of and sp. Reveals Novel Insights Into Their Pathogenic Contributions to the Hallmarks of Bacterial Vaginosis. Frontiers in cellular and infection microbiology, 11, 759697.
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  The cervicovaginal microbiome plays an important role in protecting women from dysbiosis and infection caused by pathogenic microorganisms. In healthy reproductive-age women the cervicovaginal microbiome is predominantly colonized by protective spp. The loss of these protective bacteria leads to colonization of the cervicovaginal microenvironment by pathogenic microorganisms resulting in dysbiosis and bacterial vaginosis (BV). and sp. are two of the many anaerobes that can contribute to BV, a condition associated with multiple adverse obstetric and gynecological outcomes. has been linked to high Nugent scores (relating to BV morphotypes) and preterm birth (PTB), whilst some bacterial members of the family are highly prevalent in BV, and identified in ~85-95% of cases. The functional impact of and sp. in BV is still poorly understood. To determine the individual immunometabolic contributions of sp. and within the cervicovaginal microenvironment, we utilized our well-characterized human three-dimensional (3-D) cervical epithelial cell model in combination with multiplex immunoassays and global untargeted metabolomics approaches to identify key immune mediators and metabolites related to and sp. infections. We found that infection with significantly elevated multiple proinflammatory markers (IL-6, IL-8, TNF-α and MCP-1) and altered metabolites related to energy metabolism (nicotinamide and succinate) and oxidative stress (cysteinylglycine, cysteinylglycine disulfide and 2-hydroxygluatrate). sp. infection significantly elevated multiple sphingolipids and glycerolipids related to epithelial barrier function, and biogenic amines (putrescine and cadaverine) associated with elevated vaginal pH, vaginal amine odor and vaginal discharge. Our study elucidated that elevated multiple proinflammatory markers relating to PTB and STI acquisition, as well as altered energy metabolism and oxidative stress, whilst sp. upregulated multiple biogenic amines associated with the clinical diagnostic criteria of BV. Future studies are needed to evaluate how these bacteria interact with other BV-associated bacteria within the cervicovaginal microenvironment.
• Salliss, M. E., Farland, L. V., Mahnert, N. D., & Herbst-Kralovetz, M. M. (2021). The role of gut and genital microbiota and the estrobolome in endometriosis, infertility and chronic pelvic pain. Human reproduction update, 28(1), 92-131.
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  Endometriosis is a chronic, burdensome condition that is historically understudied. Consequently, there is a lack of understanding of the etiology of the disease and its associated symptoms, including infertility and chronic pelvic pain (CPP). Endometriosis development is influenced by estrogen metabolism and inflammation, which are modulated by several factors including the microbiome and the estrobolome (the collection of genes encoding estrogen-metabolizing enzymes in the gut microbiome). Therefore, there is increasing interest in understanding the role of microbiota in endometriosis etiology.
• Salliss, M. E., Salliss, M. E., Maarsingh, J. D., Maarsingh, J. D., Garza, C., Garza, C., Łaniewski, P., Łaniewski, P., Herbst-Kralovetz, M. M., & Herbst-Kralovetz, M. M. (2021). Veillonellaceae family members uniquely alter the cervical metabolic microenvironment in a human three-dimensional epithelial model. NPJ biofilms and microbiomes, 7(1), 57.
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  Bacterial vaginosis (BV) is a gynecologic disorder characterized by a shift in cervicovaginal microbiota from Lactobacillus spp. dominance to a polymicrobial biofilm composed of diverse anaerobes. We utilized a well-characterized human three-dimensional cervical epithelial cell model in conjunction with untargeted metabolomics and immunoproteomics analyses to determine the immunometabolic contribution of three members of the Veillonellaceae family: Veillonella atypica, Veillonella montpellierensis and Megasphaera micronuciformis at this site. We found that Veillonella spp. infections induced significant elevation of polyamines. M. micronuciformis infections significantly increased soluble inflammatory mediators, induced moderate levels of cell cytotoxicity, and accumulation of cell membrane lipids relative to Veillonella spp. Notably, both V. atypica and V. montpellierensis infections resulted in consumption of lactate, a key metabolite linked to gynecologic and reproductive health. Collectively our approach and data provide unique insights into the specific contributions of Veillonellaceae members to the pathogenesis of BV and women's health.
• Łaniewski, P., & Herbst-Kralovetz, M. M. (2021). Bacterial vaginosis and health-associated bacteria modulate the immunometabolic landscape in 3D model of human cervix. NPJ biofilms and microbiomes, 7(1), 88.
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  Bacterial vaginosis (BV) is an enigmatic polymicrobial condition characterized by a depletion of health-associated Lactobacillus and an overgrowth of anaerobes. Importantly, BV is linked to adverse gynecologic and obstetric outcomes: an increased risk of sexually transmitted infections, preterm birth, and cancer. We hypothesized that members of the cervicovaginal microbiota distinctly contribute to immunometabolic changes in the human cervix, leading to these sequelae. Our 3D epithelial cell model that recapitulates the human cervical epithelium was infected with clinical isolates of cervicovaginal bacteria, alone or as a polymicrobial community. We used Lactobacillus crispatus as a representative health-associated commensal and four common BV-associated species: Gardnerella vaginalis, Prevotella bivia, Atopobium vaginae, and Sneathia amnii. The immunometabolic profiles of these microenvironments were analyzed using multiplex immunoassays and untargeted global metabolomics. A. vaginae and S. amnii exhibited the highest proinflammatory potential through induction of cytokines, iNOS, and oxidative stress-associated compounds. G. vaginalis, P. bivia, and S. amnii distinctly altered physicochemical barrier-related proteins and metabolites (mucins, sialic acid, polyamines), whereas L. crispatus produced an antimicrobial compound, phenyllactic acid. Alterations to the immunometabolic landscape correlate with symptoms and hallmarks of BV and connected BV with adverse women's health outcomes. Overall, this study demonstrated that 3D cervical epithelial cell colonized with cervicovaginal microbiota faithfully reproduce the immunometabolic microenvironment previously observed in clinical studies and can successfully be used as a robust tool to evaluate host responses to commensal and pathogenic bacteria in the female reproductive tract.
• Łaniewski, P., Owen, K. A., Khnanisho, M., Brotman, R. M., & Herbst-Kralovetz, M. M. (2021). Clinical and Personal Lubricants Impact the Growth of Vaginal Lactobacillus Species and Colonization of Vaginal Epithelial Cells: An in Vitro Study. Sexually transmitted diseases, 48(1), 63-70.
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  Vaginal lubricants are commonly used during gynecological examinations, during sexual activities, or to alleviate vaginal dryness. Many lubricants contain potentially bacteriostatic or bactericidal agents (parabens, chlorhexidine gluconate, nonoxynol-9). Our objective was to evaluate the impact of lubricants that vary in formulation on the growth and viability of vaginal Lactobacillus species and vaginal epithelial cell (VEC) colonization in an in vitro model.
• Gardner, J. K., Łaniewski, P., Knight, A., Haddad, L. B., Swaims-Kohlmeier, A., & Herbst-Kralovetz, M. M. (2020). Interleukin-36γ Is Elevated in Cervicovaginal Epithelial Cells in Women With Bacterial Vaginosis and In Vitro After Infection With Microbes Associated With Bacterial Vaginosis. The Journal of infectious diseases, 221(6), 983-988.
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  In recent studies, the interleukin (IL)-36 cytokines were shown to be elevated in women with non-Lactobacillus-dominated vaginal microbiomes. In this study, we evaluated IL36G expression in clinical samples from women with and without bacterial vaginosis (BV) and a human 3-dimensional cervical epithelial cell model. IL36G expression was significantly elevated in cervicovaginal epithelial cells isolated from BV-positive women and corresponded with increased neutrophil counts relative to BV-negative women. In addition, specific BV-associated bacterial species as well as a polymicrobial cocktail significantly induced IL36G expression in vitro. These findings suggest that IL-36γ may exhibit an important function in the host response to BV and other sexually transmitted infections.
• Ilhan, Z. E., Łaniewski, P., Tonachio, A., & Herbst-Kralovetz, M. M. (2020). Members of Prevotella Genus Distinctively Modulate Innate Immune and Barrier Functions in a Human Three-Dimensional Endometrial Epithelial Cell Model. The Journal of infectious diseases, 222(12), 2082-2092.
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  Prevotella species are commonly isolated from the reproductive tract of women with obstetric/gynecologic health complications. However, contributions of this genus to changes in local microenvironment are not well characterized. Our objective was to evaluate species-specific effects of Prevotella on the human endometrial epithelium.
• Jackson, R., Maarsingh, J. D., Herbst-Kralovetz, M. M., & Van Doorslaer, K. (2020). 3D Oral and Cervical Tissue Models for Studying Papillomavirus Host-Pathogen Interactions. Current protocols in microbiology, 59(1), e129.
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  Human papillomavirus (HPV) infection occurs in differentiating epithelial tissues. Cancers caused by high-risk types (e.g., HPV16 and HPV18) typically occur at oropharyngeal and anogenital anatomical sites. The HPV life cycle is differentiation-dependent, requiring tissue culture methodology that is able to recapitulate the three-dimensional (3D) stratified epithelium. Here we report two distinct and complementary methods for growing differentiating epithelial tissues that mimic many critical morphological and biochemical aspects of in vivo tissue. The first approach involves growing primary human epithelial cells on top of a dermal equivalent consisting of collagen fibers and living fibroblast cells. When these cells are grown at the liquid-air interface, differentiation occurs and allows for epithelial stratification. The second approach uses a rotating wall vessel bioreactor. The low-fluid-shear microgravity environment inside the bioreactor allows the cells to use collagen-coated microbeads as a growth scaffold and self-assemble into 3D cellular aggregates. These approaches are applied to epithelial cells derived from HPV-positive and HPV-negative oral and cervical tissues. The second part of the article introduces potential downstream applications for these 3D tissue models. We describe methods that will allow readers to start successfully culturing 3D tissues from oral and cervical cells. These tissues have been used for microscopic visualization, scanning electron microscopy, and large omics-based studies to gain insights into epithelial biology, the HPV life cycle, and host-pathogen interactions. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Establishing human primary cell-derived 3D organotypic raft cultures Support Protocol 1: Isolation of epithelial cells from patient-derived tissues Support Protocol 2: Growth and maintenance of primary human epithelial cells in monolayer culture Support Protocol 3: PCR-based HPV screening of primary cell cultures Basic Protocol 2: Establishing human 3D cervical tissues using the rotating wall vessel bioreactor Support Protocol 4: Growth and maintenance of human A2EN cells in monolayer culture Support Protocol 5: Preparation of the slow-turning lateral vessel bioreactor Support Protocol 6: Preparation of Cytodex-3 microcarrier beads Basic Protocol 3: Histological assessment of 3D organotypic raft tissues Basic Protocol 4: Spatial analysis of protein expression in 3D organotypic raft cultures Basic Protocol 5: Immunofluorescence imaging of RWV-derived 3D tissues Basic Protocol 6: Ultrastructural visualization and imaging of RWV-derived 3D tissues Basic Protocol 7: Characterization of gene expression by RT-qPCR.
• Muzny, C. A., Łaniewski, P., Schwebke, J. R., & Herbst-Kralovetz, M. M. (2020). Host-vaginal microbiota interactions in the pathogenesis of bacterial vaginosis. Current opinion in infectious diseases, 33(1), 59-65.
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  The cause of bacterial vaginosis, the most common cause of vaginal discharge in women, remains controversial. We recently published an updated conceptual model on bacterial vaginosis pathogenesis, focusing on the roles of Gardnerella vaginalis and Prevotella bivia as early colonizers and Atopobium vaginae and other bacterial vaginosis-associated bacteria (BVAB) as secondary colonizers in this infection. In this article, we extend the description of our model to include a discussion on the role of host-vaginal microbiota interactions in bacterial vaginosis pathogenesis.
• Roe, D. J., Laniewski, P., Ilhan, Z. E., Herbst-kralovetz, M. M., Cui, H., Chase, D. M., Caporaso, J. G., & Bokulich, N. A. (2020). Abstract A094: Integrative multi-omics approach reveals complex interplay between HPV, host and microbiome during cervical carcinogenesis in Hispanic and non-Hispanic women. Cancer Epidemiology, Biomarkers & Prevention. doi:10.1158/1538-7755.disp19-a094
• Łaniewski, P., Cui, H., Roe, D. J., Chase, D. M., & Herbst-Kralovetz, M. M. (2020). Vaginal microbiota, genital inflammation, and neoplasia impact immune checkpoint protein profiles in the cervicovaginal microenvironment. NPJ precision oncology, 4, 22.
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  Emerging evidence suggests that the vaginal microbiota play a role in HPV persistence and cervical neoplasia development and progression. Here we examine a broad range of immune checkpoint proteins in the cervicovaginal microenvironment across cervical carcinogenesis and explore relationships among these key immunoregulatory proteins, the microbiota composition, and genital inflammation. First, we demonstrate that immune checkpoint molecules can be measured in cervicovaginal lavages. Secondly, we identify CD40, CD27, and TIM-3 to specifically discriminate cervical cancer from other groups and CD40, CD28, and TLR2 to positively correlate to genital inflammation. Finally, PD-L1 and LAG-3 levels negatively, whereas TLR2 positively correlate to health-associated dominance. Overall, our study identifies immune checkpoint signatures associated with cervical neoplasm and illuminates the multifaceted microbiota-host immunity network in the local microenvironment. This study provides a foundation for future mechanistic studies and highlights the utility of cervicovaginal lavage profiling for predicting and monitoring response to cancer therapy.
• Łaniewski, P., Ilhan, Z. E., & Herbst-Kralovetz, M. M. (2020). The microbiome and gynaecological cancer development, prevention and therapy. Nature reviews. Urology, 17(4), 232-250.
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  The female reproductive tract (FRT), similar to other mucosal sites, harbours a site-specific microbiome, which has an essential role in maintaining health and homeostasis. In the majority of women of reproductive age, the microbiota of the lower FRT (vagina and cervix) microenvironment is dominated by Lactobacillus species, which benefit the host through symbiotic relationships. By contrast, the upper FRT (uterus, Fallopian tubes and ovaries) might be sterile in healthy individuals or contain a low-biomass microbiome with a diverse mixture of microorganisms. When dysbiosis occurs, altered immune and metabolic signalling can affect hallmarks of cancer, including chronic inflammation, epithelial barrier breach, changes in cellular proliferation and apoptosis, genome instability, angiogenesis and metabolic dysregulation. These pathophysiological changes might lead to gynaecological cancer. Emerging evidence shows that genital dysbiosis and/or specific bacteria might have an active role in the development and/or progression and metastasis of gynaecological malignancies, such as cervical, endometrial and ovarian cancers, through direct and indirect mechanisms, including modulation of oestrogen metabolism. Cancer therapies might also alter microbiota at sites throughout the body. Reciprocally, microbiota composition can influence the efficacy and toxic effects of cancer therapies, as well as quality of life following cancer treatment. Modulation of the microbiome via probiotics or microbiota transplant might prove useful in improving responsiveness to cancer treatment and quality of life. Elucidating these complex host-microbiome interactions, including the crosstalk between distal and local sites, will translate into interventions for prevention, therapeutic efficacy and toxic effects to enhance health outcomes for women with gynaecological cancers.
• Gardner, J. K., Swaims-Kohlmeier, A., & Herbst-Kralovetz, M. M. (2019). IL-36γ Is a Key Regulator of Neutrophil Infiltration in the Vaginal Microenvironment and Limits Neuroinvasion in Genital HSV-2 Infection. Journal of immunology (Baltimore, Md. : 1950), 203(10), 2655-2664.
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  HSV-2 is a neurotropic virus that causes a persistent, lifelong infection that increases risk for other sexually transmitted infections. The vaginal epithelium is the first line of defense against HSV-2 and coordinates the immune response through the secretion of immune mediators, including the proinflammatory cytokine IL-36γ. Previously, we showed that IL-36γ treatment promoted transient polymorphonuclear cell infiltration to the vaginal cavity and protected against lethal HSV-2 challenge. In this report, we reveal that IL-36γ specifically induces transient neutrophil infiltration but does not impact monocyte and macrophage recruitment. Using IL-36γ mice in a lethal HSV-2 challenge model, we show that neutrophil counts are significantly reduced at 1 and 2 d postinfection and that KC-mediated mature neutrophil recruitment is impaired in IL-36γ mice. Additionally, IL-36γ mice develop genital disease more rapidly, have significantly reduced survival time, and exhibit an increased incidence of hind limb paralysis that is linked to productive HSV-2 infection in the brain stem. IL-36γ mice also exhibit a significant delay in clearance of the virus from the vaginal epithelium and a more rapid spread of HSV-2 to the spinal cord, bladder, and colon. We further show that the decreased survival time and increased virus spread observed in IL-36γ mice are not neutrophil-dependent, suggesting that IL-36γ may function to limit HSV-2 spread in the nervous system. Ultimately, we demonstrate that IL-36γ is a key regulator of neutrophil recruitment in the vaginal microenvironment and may function to limit HSV-2 neuroinvasion.
• Gardner, J., Laniewski, P., Knight, A., Haddad, L., Swaims-Kohlmeier, A., & Herbst-Kralovetz, M. M. (2019). IL-36gamma is elevated in cervicovaginal epithelial cells from women with bacterial vaginosis and in vitro after infection with microbes associated with bacterial vaginosis. Journal of Infectious Diseases.
• Herbst-Kralovetz, M. M. (2019). Chronic Immune Barrier Dysregulation among Women with a History of Violence Victimization.. JCI Insight.
• Herbst-Kralovetz, M. M., Gardner, J., & Swaims-Kohlmeier, A. (2019). IL-36gamma is a key regulator of neutrophil infiltration in the vaginal microenvironment and limits neuroinvasion to protect against genital HSV-2 disease pathogenesis.. Journal of Immunology.
• Ilhan, E., Łaniewski, P., Cui, H., Roe, D. J., Chase, D. M., & Herbst-Kralovetz, M. M. (2019). Deciphering the complex interplay between microbiota, HPV, inflammation and cancer through metabolic profiling. EBioMedicine.
• Ilhan, Z. E., Łaniewski, P., Thomas, N., Roe, D. J., Chase, D. M., & Herbst-Kralovetz, M. M. (2019). Deciphering the complex interplay between microbiota, HPV, inflammation and cancer through cervicovaginal metabolic profiling. EBioMedicine, 44, 675-690.
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  Dysbiotic vaginal microbiota have been implicated as contributors to persistent HPV-mediated cervical carcinogenesis and genital inflammation with mechanisms unknown. Given that cancer is a metabolic disease, metabolic profiling of the cervicovaginal microenvironment has the potential to reveal the functional interplay between the host and microbes in HPV persistence and progression to cancer.
• Munzy, C., Łaniewski, P., Schwebke, J., & Herbst-Kralovetz, M. M. (2019). Host-Vaginal Microbiota Interactions in the Pathogenesis of Bacterial Vaginosis. Current Opinions in Infectious Diseases.
• Owen, K. A., Laniewski, P., Herbst-kralovetz, M. M., & Brotman, R. M. (2019). Clinical and personal lubricants impact growth of vaginal lactobacillus species and colonization of vaginal epithelial cells. American Journal of Obstetrics and Gynecology, 221(6), 694. doi:10.1016/j.ajog.2019.10.059
• Roe, D. J., Laniewski, P., Herbst-kralovetz, M. M., Cui, H., & Chase, D. M. (2019). Immune checkpoint proteins in women with cervical neoplasm associate with features of the local cervicovaginal microenvironment. American Journal of Obstetrics and Gynecology, 221(6), 691. doi:10.1016/j.ajog.2019.10.053
• Swaims-Kohlmeier, A., Haddad, L. B., Li, Z. T., Brookmeyer, K. A., Baker, J. M., Widom, C. S., Lamousin, J. C., Chi, K. H., Chen, C. Y., Kersh, E. N., Johnson, J. A., Herbst-Kralovetz, M. M., Hogben, M., Ofotokun, I., & Kohlmeier, J. E. (2019). Chronic immune barrier dysregulation among women with a history of violence victimization. JCI insight, 4(10).
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  We explored the association between violence victimization and increased risk for acquiring sexually transmitted infections (STIs) in women by measuring cellular immune barrier properties from the female reproductive tract. STI-negative participants reporting repeated prior victimization occurrences through the lifetime trauma and victimization history (LTVH) instrument were more likely to exhibit alterations in barrier homeostasis and the composition of critical immune mediators irrespective of demographic parameters or presence of bacterial vaginosis. By combining cellular data with mixed-effect linear modeling, we uncovered differences in local T cells, MHCII+ antigen-presenting cells, and epithelial cells indicative of altered trafficking behavior, increased immunosuppressive function, and decreased barrier integrity at sites of STI exposure that correlate most strongly with LTVH score. These data evidence a biological link between a history of violence victimization and risk of STI acquisition through immune dysregulation in the female reproductive tract.
• Thomas, N., Roe, D. J., Laniewski, P., Ilhan, Z. E., Herbst-kralovetz, M. M., & Chase, D. M. (2019). O05.6 Cervicovaginal metabolic profiling reveals the interplay between HPV, microbiota and inflammation in cervical carcinogenesis. Sexually Transmitted Infections, 95. doi:10.1136/sextrans-2019-sti.133
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  Background Vaginal dysbiosis has emerged as a key risk factor in HPV acquisition, persistence, and potentially cervical carcinogenesis. However, the biological mechanisms driving persistence and carcinogenesis have not been elucidated. Hence, our objective was to perform metabolic profiling of the cervicovaginal microenvironment to identify interactions between virus, host and microbes in the context of genital inflammation, dysplasia, and cancer. Methods In a multicenter study, metabolic profiles of 78 premenopausal, non-pregnant women with low-grade (LSIL) and high-grade squamous intraepithelial lesions (HSIL), invasive cervical cancer (ICC), or healthy controls (HPV-positive and -negative Ctrl) were analyzed using gas chromatography-mass spectrometry. Metabolome and vaginal microbiome datasets were integrated using state-of-the-art bioinformatic tools (PICRUSt, AMON, and MIMOSA). Hierarchical clustering analysis (HCA) and principal component analysis (PCA) were employed to reveal the influence of genital inflammation, patient groups, and microbiota on metabolic profiles. Receiver Operating Characteristics (ROC) analysis was used to discriminate metabolites for each patient group. Statistical differences were tested using ANOVA or Mann-Whitney U test. Results Metabolomes of ICC patients (n=468 metabolites) formed a distinct cluster on PCA and HCA plots, due to enrichment of membrane lipids. Amino acid and nucleotide metabolites were depleted in HPV-positive Ctrl, LSIL and HSIL groups (P 0.9, P 0.7). Anti-inflammatory nucleotides, adenosine and cytosine positively correlated with Lactobacillus abundance (Spearman’s rho>0.5) and negatively correlated with genital inflammation (Spearman’s rho Conclusion The complex virus-host-microbe interplay within the cervicovaginal microenvironment lead to unique metabolic fingerprints that could be exploited for future development of diagnostics, preventatives or treatments to positively impact women’s health outcomes. Disclosure No significant relationships.
• Wilkinson, E. M., Łaniewski, P., Herbst-Kralovetz, M. M., & Brotman, R. M. (2019). Personal and Clinical Vaginal Lubricants: Impact on Local Vaginal Microenvironment and Implications for Epithelial Cell Host Response and Barrier Function. The Journal of infectious diseases, 220(12), 2009-2018.
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  A majority of US women report past use of vaginal lubricants to enhance the ease and comfort of intimate sexual activities. Lubricants are also administered frequently in clinical practice. We sought to investigate if hyperosmolar lubricants are toxic to the vaginal mucosal epithelia.
• Wilkinson, E., Łaniewski, P., Herbst-Kralovetz, M. M., & Brotman, R. (2019). Personal and Clinical Vaginal Lubricants: Impact on Local Vaginal Microenvironment and Implications for Epithelial Cell Host Response and Barrier Function. Journal of Infectious Diseases.
• Łaniewski, P., & Herbst-Kralovetz, M. M. (2019). Analysis of Host Responses to Neisseria gonorrhoeae Using a Human Three-Dimensional Endometrial Epithelial Cell Model. Methods in molecular biology (Clifton, N.J.), 1997, 347-361.
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  Neisseria gonorrhoeae infections have been associated with complications including chronic endometritis and pelvic inflammatory disease. Robust in vitro models of the female reproductive tract are urgently needed to better understand the biological mechanisms leading to these pathophysiological changes. Our human three-dimensional (3D) endometrial epithelial cell (EEC) model, which is generated using the HEC-1A cell line and rotating wall vessel (RWV) bioreactor technology, replicates several hallmarks of endometrial tissue in vivo. Studying the interactions of N. gonorrhoeae with the host using this newly characterized human 3D EEC model allows for the investigation of unique mechanisms of gonococcal pathogenesis in the upper female reproductive tract. In this chapter, we describe methodologies that can be used to investigate the interactions of N. gonorrhoeae with the human 3D endometrial epithelium. Protocols for generating the human 3D EEC model using the RWV technology and assessing the host response (including morphological/ultrastructural changes to the epithelial cells; cytokine/chemokine secretion or gene expression changes) following infection with N. gonorrhoeae are presented.
• Łaniewski, P., Cui, H., Roe, D. J., Barnes, D., Goulder, A., Chase, D. M., Monk, B., Greenspan, D., & Herbst-Kralovetz, M. M. (2019). Features of the cervicovaginal microenvironment drive cancer biomarker signatures in patients across cervical carcinogenesis. Scientific reports.
• Łaniewski, P., Cui, H., Roe, D. J., Barnes, D., Goulder, A., Monk, B. J., Greenspan, D. L., Chase, D. M., & Herbst-Kralovetz, M. M. (2019). Features of the cervicovaginal microenvironment drive cancer biomarker signatures in patients across cervical carcinogenesis. Scientific reports, 9(1), 7333.
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  Persistent human papillomavirus (HPV) infection is the vital factor driving cervical carcinogenesis; however, other features of the local cervicovaginal microenvironment (CVM) may play a critical role in development of precancerous cervical dysplasia and progression to invasive cervical carcinoma (ICC). Here we investigated relationships between locally secreted cancer biomarkers and features of the local CVM to better understand the complex interplay between host, virus and vaginal microbiota (VMB). We enrolled women with ICC, high- and low-grade squamous intraepithelial lesions, as well as, HPV-positive and healthy HPV-negative controls. A broad range of cancer biomarkers was present in the local CVM and specifically elevated in ICC patients. The majority of cancer biomarkers were positively correlated to other biomarkers and linked to genital inflammation. Several cancer biomarkers were also negatively correlated to Lactobacillus abundance and positively correlated with abnormal vaginal pH. Finally, a hierarchical clustering analysis of cancer biomarkers and immune mediators revealed three patient clusters, which varied in levels of cancer biomarkers, genital inflammation, vaginal pH and VMB composition. Specific cancer biomarkers discriminated patients with features of the CVM, such as high genital inflammation, elevated vaginal pH and dysbiotic non-Lactobacillus-dominant VMB, that have been associated with HPV persistence, dysplasia and progression to ICC.
• Baker, J. M., Chase, D. M., & Herbst-Kralovetz, M. M. (2018). Uterine Microbiota: Residents, Tourists, or Invaders?. Frontiers in immunology, 9, 208.
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  Uterine microbiota have been reported under various conditions and populations; however, it is uncertain the level to which these bacteria are residents that maintain homeostasis, tourists that are readily eliminated or invaders that contribute to human disease. This review provides a historical timeline and summarizes the current status of this topic with the aim of promoting research priorities and discussion on this controversial topic. Discrepancies exist in current reports of uterine microbiota and are critically reviewed and examined. Established and putative routes of bacterial seeding of the human uterus and interactions with distal mucosal sites are discussed. Based upon the current literature, we highlight the need for additional robust clinical and translational studies in this area. In addition, we discuss the necessity for investigating host-microbiota interactions and the physiologic and functional impact of these microbiota on the local endometrial microenvironment as these mechanisms may influence poor reproductive, obstetric, and gynecologic health outcomes and sequelae.
• Gardner, J. K., & Herbst-Kralovetz, M. M. (2018). IL-36γ induces a transient HSV-2 resistant environment that protects against genital disease and pathogenesis. Cytokine, 111, 63-71.
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  Herpes simplex virus 2 (HSV-2) causes a persistent, lifelong infection that increases risk for sexually transmitted infection acquisition. Both the lack of a vaccine and the need for chronic suppressive therapies to control infection presents the need to further understand immune mechanisms in response to acute HSV-2 infection. The IL-36 cytokines are recently identified members of the IL-1 family and function as inflammatory mediators at epithelial sites. Here, we first used a well-characterized three-dimensional (3-D) human vaginal epithelial cell (VEC) model to understand the role of IL-36γ in the context of HSV-2 infection. In 3-D VEC, IL-36γ is induced by HSV-2 infection, and pretreatment with exogenous IL-36γ significantly reduced HSV-2 replication. To assess the impact of IL-36γ treatment on HSV-2 disease pathogenesis, we employed a lethal genital infection model. We showed that IL-36γ treatment in mice prior to lethal intravaginal challenge significantly limited vaginal viral replication, delayed disease onset, decreased disease severity, and significantly increased survival. We demonstrated that IL-36γ treatment transiently induced pro-inflammatory cytokines, chemokines, and antimicrobial peptides in murine lower female reproductive tract (FRT) tissue and vaginal lavages. Induction of the chemokines CCL20 and KC in IL-36γ treated mice also corresponded with increased polymorphonuclear (PMN) leukocyte infiltration observed in vaginal smears. Altogether, these studies demonstrate that IL-36γ drives the transient production of immune mediators and promotes PMN recruitment in the vaginal microenvironment that increases resistance to HSV-2 infection and disease. Our data indicate that IL-36γ may participate as a key player in host defense mechanisms against invading pathogens in the FRT.
• Wilkinson, E. M., Herbst-kralovetz, M. M., & Brotman, R. M. (2018). Clinical and personal lubricants alter cell viability, cytotoxicity and mucin production in human vaginal epithelial cell culture models. American Journal of Obstetrics and Gynecology, 219(6), 638. doi:10.1016/j.ajog.2018.10.076
• Wilkinson, E. M., Ilhan, Z. E., & Herbst-Kralovetz, M. M. (2018). Microbiota-drug interactions: Impact on metabolism and efficacy of therapeutics. Maturitas, 112, 53-63.
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  The microbiome not only represents a vital modifier of health and disease, but is a clinically important drug target. Therefore, study of the impact of the human microbiome on drug metabolism, toxicity and efficacy is urgently needed. This review focuses on gut and vaginal microbiomes, and the effect of those microbiomes or components thereof on the pharmacokinetics of specific chemotherapeutic agents, immunotherapies, anti-inflammatory and antimicrobial drugs. In some cases, the presence of specific bacterial species within the microbiome can alter the metabolism of certain drugs, such as chemotherapeutic agents and antiviral drugs. These microbiota-drug interactions are identified mostly through studies using germ-free or microbiome-depleted animal models, or by the administration of specific bacterial isolates. The biotransformation of drugs can cause drug-related toxicities; however, biotransformation also provides a mechanism by which drug developers could exploit host microbiota to create more site-specific drugs. Within this review we consider the importance of the route of drug administration and interactions with microbiota at various mucosal sites. Notably, we discuss the potential utility of bacterial therapeutics in altering the microbiome to enhance therapeutic efficacy and clinical outcomes in a personalized fashion. Based on the data to date, there is a clinically important relationship between microbiota and drug metabolism throughout the lifespan; therefore, profiling of the human microbiome will be essential in order to understand the mechanisms by which these microbiota-drug interactions occur and the degree to which this complex interplay affects drug efficacy.
• Łaniewski, P., Barnes, D., Goulder, A., Cui, H., Roe, D. J., Chase, D. M., & Herbst-Kralovetz, M. M. (2018). Linking cervicovaginal immune signatures, HPV and microbiota composition in cervical carcinogenesis in non-Hispanic and Hispanic women. Scientific reports, 8(1), 7593.
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  While high-risk human papillomavirus (HPV) infection is a well-established risk factor for cervical cancer, there are likely other factors within the local microenvironment that contribute to cervical carcinogenesis. Here we investigated relationships between HPV, vaginal pH, vaginal microbiota (VMB) composition, level of genital immune mediators and severity of cervical neoplasm. We enrolled women with low- and high-grade cervical dysplasia (LGD, HGD), invasive cervical carcinoma (ICC), and healthy controls. HPV16, HPV45, HPV58, and HPV31 were the most prevalent in our cohort with HPV16 and HPV31 genotypes more prevalent in Hispanics. Vaginal pH was associated with ethnicity and severity of cervical neoplasm. Lactobacillus dominance decreased with the severity of cervical neoplasm, which correlated with elevated vaginal pH. Hispanic ethnicity was also associated with decreased Lactobacillus dominance. Furthermore, Sneathia was enriched in all precancerous groups, ICC, abnormal pH and Hispanic origin. Patients with ICC, but not LGD and HGD, exhibited increased genital inflammatory scores and elevated specific immune mediators. Notably, IL-36γ was significantly associated with ICC. Our study revealed local, host immune and microbial signatures associated with cervical carcinogenesis and provides an initial step to understanding the complex interplay between mucosal inflammation, HPV persistence and the VMB.
• Baker, J. M., Al-Nakkash, L., & Herbst-Kralovetz, M. M. (2017). Estrogen-gut microbiome axis: Physiological and clinical implications. Maturitas, 103, 45-53.
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  Low levels of gonadal circulating estrogen observed in post-menopausal women can adversely impact a diverse range of physiological factors, with clinical implications for brain cognition, gut health, the female reproductive tract and other aspects of women's health. One of the principal regulators of circulating estrogens is the gut microbiome. This review aims to shed light on the role of the gut microbiota in estrogen-modulated disease. The gut microbiota regulates estrogens through secretion of β-glucuronidase, an enzyme that deconjugates estrogens into their active forms. When this process is impaired through dysbiosis of gut microbiota, characterized by lower microbial diversity, the decrease in deconjugation results in a reduction of circulating estrogens. The alteration in circulating estrogens may contribute to the development of conditions discussed herein: obesity, metabolic syndrome, cancer, endometrial hyperplasia, endometriosis, polycystic ovary syndrome, fertility, cardiovascular disease (CVD) and cognitive function. The bi-directional relationship between the metabolic profile (including estrogen levels) and gut microbiota in estrogen-driven disease will also be discussed. Promising therapeutic interventions manipulating the gut microbiome and the metabolic profile of estrogen-driven disease, such as bariatric surgery and metformin, will be detailed. Modulation of the microbiome composition subsequently impacts the metabolic profile, and vice versa, and has been shown to alleviate many of the estrogen-modulated disease states. Last, we highlight promising research interventions in the field, such as dietary therapeutics, and discuss areas that provide exciting unexplored topics of study.
• Łaniewski, P., Gomez, A., Hire, G., So, M., & Herbst-Kralovetz, M. M. (2017). Human three-dimensional endometrial epithelial cell model to study host interactions with vaginal bacteria and Neisseria gonorrhoeae. Infection and immunity.
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  Colonization of the endometrium by pathogenic bacteria, ascending from the lower female reproductive tract (FRT), is associated with many gynecologic and obstetric health complications. To study these host-microbe interactions in vitro, we developed a human three-dimensional (3-D) endometrial epithelial cell (EEC) model using the HEC-1A cell line and rotating wall vessel (RWV) bioreactor technology. Our model, composed of 3-D EEC aggregates, recapitulates several functional/structural characteristics of human endometrial epithelial tissue including cell differentiation, junctional complexes/desmosomes, microvilli, membrane-associated mucins and Toll-like receptors (TLRs). TLR function was evaluated by exposing the EEC aggregates to viral and bacterial products. Treatment with polyinosinic-polycytidylic acid (poly(I:C)) and flagellin, but not with synthetic lipoprotein (FSL-1) or lipopolysaccharide (LPS), significantly induced proinflammatory mediators in a dose dependent manner. To simulate ascending infection, we infected EEC aggregates with commensal and pathogenic bacteria: Lactobacillus crispatus, Gardnerella vaginalis and Neisseria gonorrhoeae All vaginal microbiota and N. gonorrhoeae efficiently colonized the 3-D surface localizing to crevices of the EEC model and interacting with multiple adjacent cells simultaneously. However, only infection with pathogenic N. gonorrhoeae significantly induced proinflammatory mediators and significant ultrastructural changes to the host cells relative to other bacteria tested. This latter observation is consistent with clinical findings and illustrated the functional specificity of our system. Additionally, we highlighted the utility of 3-D EEC model to study N. gonorrhoeae pathogenesis using a well-characterized ΔpilT mutant. Overall, this study demonstrates that the human 3-D EEC model is a robust tool for studying host-microbe interactions and bacterial pathogenesis in the upper FRT.
• Herbst-Kralovetz, M. M., Pyles, R. B., Ratner, A. J., Sycuro, L. K., & Mitchell, C. (2016). New Systems for Studying Intercellular Interactions in Bacterial Vaginosis. The Journal of infectious diseases, 214 Suppl 1, S6-S13.
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  Bacterial vaginosis (BV) affects almost a quarter of US women, making it a condition of major public health relevance. Key questions remain regarding the etiology of BV, mechanisms for its association with poor reproductive health outcomes, and reasons for high rates of treatment failure. New model systems are required to answer these remaining questions, elucidate the complex host-microbe and microbe-microbe interactions, and develop new, effective interventions. In this review, we cover the strengths and limitations of in vitro and in vivo model systems to study these complex intercellular interactions. Furthermore, we discuss advancements needed to maximize the translational utility of the model systems. As no single model can recapitulate all of the complex physiological and environmental conditions of the human vaginal microenvironment, we conclude that combinatorial approaches using in vitro and in vivo model systems will be required to address the remaining fundamental questions surrounding the enigma that is BV.
• Herbst-kralovetz, M. M., & Gardner, J. K. (2016). Three-Dimensional Rotating Wall Vessel-Derived Cell Culture Models for Studying Virus-Host Interactions.. Viruses, 8(11), 304. doi:10.3390/v8110304
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  The key to better understanding complex virus-host interactions is the utilization of robust three-dimensional (3D) human cell cultures that effectively recapitulate native tissue architecture and model the microenvironment. A lack of physiologically-relevant animal models for many viruses has limited the elucidation of factors that influence viral pathogenesis and of complex host immune mechanisms. Conventional monolayer cell cultures may support viral infection, but are unable to form the tissue structures and complex microenvironments that mimic host physiology and, therefore, limiting their translational utility. The rotating wall vessel (RWV) bioreactor was designed by the National Aeronautics and Space Administration (NASA) to model microgravity and was later found to more accurately reproduce features of human tissue in vivo. Cells grown in RWV bioreactors develop in a low fluid-shear environment, which enables cells to form complex 3D tissue-like aggregates. A wide variety of human tissues (from neuronal to vaginal tissue) have been grown in RWV bioreactors and have been shown to support productive viral infection and physiological meaningful host responses. The in vivo-like characteristics and cellular features of the human 3D RWV-derived aggregates make them ideal model systems to effectively recapitulate pathophysiology and host responses necessary to conduct rigorous basic science, preclinical and translational studies.
• Muhleisen, A. L., & Herbst-Kralovetz, M. M. (2016). Menopause and the vaginal microbiome. Maturitas, 91, 42-50.
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  For over a century it has been well documented that bacteria in the vagina maintain vaginal homeostasis, and that an imbalance or dysbiosis may be associated with poor reproductive and gynecologic health outcomes. Vaginal microbiota are of particular significance to postmenopausal women and may have a profound effect on vulvovaginal atrophy, vaginal dryness, sexual health and overall quality of life. As molecular-based techniques have evolved, our understanding of the diversity and complexity of this bacterial community has expanded. The objective of this review is to compare the changes that have been identified in the vaginal microbiota of menopausal women, outline alterations in the microbiome associated with specific menopausal symptoms, and define how hormone replacement therapy impacts the vaginal microbiome and menopausal symptoms; it concludes by considering the potential of probiotics to reinstate vaginal homeostasis following menopause. This review details the studies that support the role of Lactobacillus species in maintaining vaginal homeostasis and how the vaginal microbiome structure in postmenopausal women changes with decreasing levels of circulating estrogen. In addition, the associated transformations in the microanatomical features of the vaginal epithelium that can lead to vaginal symptoms associated with menopause are described. Furthermore, hormone replacement therapy directly influences the dominance of Lactobacillus in the microbiota and can resolve vaginal symptoms. Oral and vaginal probiotics hold great promise and initial studies complement the findings of previous research efforts concerning menopause and the vaginal microbiome; however, additional trials are required to determine the efficacy of bacterial therapeutics to modulate or restore vaginal homeostasis.
• Winkle, S. M., Throop, A. L., & Herbst-Kralovetz, M. M. (2016). IL-36γ Augments Host Defense and Immune Responses in Human Female Reproductive Tract Epithelial Cells. Frontiers in microbiology, 7, 955.
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  IL-36γ is a proinflamatory cytokine which belongs to the IL-1 family of cytokines. It is expressed in the skin and by epithelial cells (ECs) lining lung and gut tissue. We used human 3-D organotypic cells, that recapitulate either in vivo human vaginal or cervical tissue, to explore the possible role of IL-36γ in host defense against pathogens in the human female reproductive tract (FRT). EC were exposed to compounds derived from virus or bacterial sources and induction and regulation of IL-36γ and its receptor was determined. Polyinosinic-polycytidylic acid (poly I:C), flagellin, and synthetic lipoprotein (FSL-1) significantly induced expression of IL-36γ in a dose-dependent manner, and appeared to be TLR-dependent. Recombinant IL-36γ treatment resulted in self-amplification of IL-36γ and its receptor (IL-36R) via increased gene expression, and promoted other inflammatory signaling pathways. This is the first report to demonstrate that the IL-36 receptor and IL-36γ are present in the human FRT EC and that they are differentially induced by microbial products at this site. We conclude that IL-36γ is a driver for epithelial and immune activation following microbial insult and, as such, may play a critical role in host defense in the FRT.
• Chase, D., Goulder, A., Zenhausern, F., Monk, B., & Herbst-Kralovetz, M. (2015). The vaginal and gastrointestinal microbiomes in gynecologic cancers: a review of applications in etiology, symptoms and treatment. Gynecologic oncology, 138(1), 190-200.
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  The human microbiome is the collection of microorganisms in the body that exist in a mutualistic relationship with the host. Recent studies indicate that perturbations in the microbiome may be implicated in a number of diseases, including cancer. More specifically, changes in the gut and vaginal microbiomes may be associated with a variety of gynecologic cancers, including cervical cancer, uterine cancer, and ovarian cancer. Current research and gaps in knowledge regarding the association between the gut and vaginal microbiomes and the development, progression, and treatment of gynecologic cancers are reviewed here. In addition, the potential use of probiotics to manage symptoms of these gynecologic cancers is discussed. A better understanding of how the microbiome composition is altered at these sites and its interaction with the host may aid in prevention, optimization of current therapies, development of new therapeutic agents and/or dosing regimens, and possibly limit the side effects associated with cancer treatment.
• Winkle, S. M., Throop, A. L., & Herbst-kralovetz, M. M. (2015). P06.12 Human il-36 gamma as an indicator of vaginal infection and promoter of mucosal inflammation. Sexually Transmitted Infections, 91(Suppl 2), A118.3-A119. doi:10.1136/sextrans-2015-052270.313
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  Introduction IL-36γ (also designated as IL-1F9) has been recently identified and belongs to the IL-1 family of cytokines. Despite expression of IL-36γ at other mucosal sites, it has not previously been reported in the vaginal or cervical epithelium. Overall, there is a paucity of information regarding the induction and physiological function of IL-36γ. Methods Utilising our human 3-D vaginal EC model, that more accurately recapitulates in vivo human vaginal tissue, we tested the hypothesis that IL-36γ induction in the vaginal epithelium is microbe-dependent by testing a panel of STI microbes and microbial products. To further investigate the induction and regulation of IL-36γ, 3-D vaginal EC were treated with poly (I:C), flagellin or FSL-1 for 24 h. Human 3-D cells were analysed by real-time qPCR analysis. Cell pellets and culture supernatants were also collected and analysed by IL-36γ ELISA, Western blot and cytometric bead array. Results Following exposure to STI pathogens (herpes simplex virus and bacterial vaginosis (BV)-associated bacteria) and specific microbial products, IL-36γ expression was significantly increased relative to untreated and Lactobacilli spp. bacteria in the vaginal EC model. All microbial products tested significantly (p Conclusion We show that human 3-D vaginal EC express IL-36γ and this cytokine is elicited in a microbe-dependent manner at this mucosal site. Furthermore, we demonstrate that IL-36γ is an important driver for epithelial activation and inflammation following infection with STI-related pathogens and BV-associated bacteria, as such this novel cytokine may play an important role in host defense in the vaginal epithelium. Disclosure of interest statement No pharmaceutical grants were received in the development of this study.
• Yarbrough, V. L., Winkle, S. M., & Herbst-kralovetz, M. M. (2015). Antimicrobial peptides in the female reproductive tract: a critical component of the mucosal immune barrier with physiological and clinical implications.. Human reproduction update, 21(3), 353-77. doi:10.1093/humupd/dmu065
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  At the interface of the external environment and the mucosal surface of the female reproductive tract (FRT) lies a first-line defense against pathogen invasion that includes antimicrobial peptides (AMP). Comprised of a unique class of multifunctional, amphipathic molecules, AMP employ a wide range of functions to limit microbial invasion and replication within host cells as well as independently modulate the immune system, dampen inflammation and maintain tissue homeostasis. The role of AMP in barrier defense at the level of the skin and gut has received much attention as of late. Given the far reaching implications for women's health, maternal and fetal morbidity and mortality, and sexually transmissible and polymicrobial diseases, we herein review the distribution and function of key AMP throughout the female reproductive mucosa and assess their role as an essential immunological barrier to microbial invasion throughout the reproductive cycle of a woman's lifetime..A comprehensive search in PubMed/Medline was conducted related to AMP general structure, function, signaling, expression, distribution and barrier function of AMP in the FRT, hormone regulation of AMP, the microbiome of the FRT, and AMP in relation to implantation, pregnancy, fertility, pelvic inflammatory disease, complications of pregnancy and assisted reproductive technology..AMP are amphipathic peptides that target microbes for destruction and have been conserved throughout all living organisms. In the FRT, several major classes of AMP are expressed constitutively and others are inducible at the mucosal epithelium and by immune cells. AMP expression is also under the influence of sex hormones, varying throughout the menstrual cycle, and dependent on the vaginal microbiome. AMP can prevent infection with sexually transmissible and opportunistic pathogens of the female reproductive tissues, although emerging understanding of vaginal dysbiosis suggests induction of a unique AMP profile with increased susceptibility to these pathogens. During pregnancy, AMP are key immune effectors of the fetal membranes and placenta and are dysregulated in states of intrauterine infection and other complications of pregnancy..At the level of the FRT, AMP serve to inhibit infection by sexually and vertically transmissible as well as by opportunistic bacteria, fungi, viruses, and protozoa and must do so throughout the hormone flux of menses and pregnancy. Guarding the exclusive site of reproduction, AMP modulate the vaginal microbiome of the lower FRT to aid in preventing ascending microbes into the upper FRT. Evolving in parallel with, and in response to, pathogenic insults, AMP are relatively immune to the resistance mechanisms employed by rapidly evolving pathogens and play a key role in barrier function and host defense throughout the FRT.
• Yarbrough, V., Winkle, S., & Herbst-Kralovetz, M. (2013). Antimicrobial Peptides in the Female Reproductive Tract: A critical component of the mucosal immune barrier with physiological and clinical implications. Human Reproduction Update.
• Doerflinger, S. Y., Throop, A. L., & Herbst-Kralovetz, M. M. (2014). Bacteria in the vaginal microbiome alter the innate immune response and barrier properties of the human vaginal epithelia in a species-specific manner. The Journal of infectious diseases, 209(12), 1989-99.
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  Bacterial vaginosis increases the susceptibility to sexually transmitted infections and negatively affects women's reproductive health.
• Herbst-Kralovetz, M. M. (2014). Overcoming barriers in the mucosal delivery of virus-like particle-based vaccines. Therapeutic delivery, 5(7), 741-4.
• Kilbourne, J., Hjelm, B. E., & Herbst-kralovetz, M. M. (2014). TLR7 and 9 agonists are highly effective mucosal adjuvants for norovirus virus-like particle vaccines.. Human vaccines & immunotherapeutics, 10(2), 410-6. doi:10.4161/hv.27147
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  Virus-like particles (VLPs) are an active area of vaccine research, development and commercialization. Mucosal administration of VLPs provides an attractive avenue for delivery of vaccines with the potential to produce robust immune responses. Nasal and oral delivery routes are particularly intriguing due to differential activation of mucosa-associated lymphoid tissues. We compared both intranasal and oral administration of VLPs with a panel of toll-like receptor (TLR) agonists (TLR3, 5, 7, 7/8, and 9) to determine the mucosal adjuvant activity of these immunomodulators. We selected Norwalk virus (NV) VLPs because it is an effective model antigen and an active area of research and commercialization. To prioritize these adjuvants, VLP-specific antibody production in serum (IgG, IgG1, IgG2a), vaginal lavages (IgG, IgA), and fecal pellets (IgA) were measured across a longitudinal timeseries in vaccinated mice. Additional distal mucosal sites (nasal, brochoalveolar, salivary, and gastrointestinal) were evaluated for VLP-specific responses (IgA). Intranasal co-delivery of VLPs with TLR7 or TLR9 agonists produced the most robust and broad-spectrum immune responses, systemically and at distal mucosal sites inducing VLP-specific antibodies at all sites evaluated. In addition, these VLP-specific antibodies blocked binding of NV VLPs to histo-blood group antigen (H type 1), supporting their functionality. Oral administration and/or other TLR agonists tested in the panel did not consistently enhance VLP-specific immune responses. This study demonstrates that intranasal co-delivery of VLPs with TLR7 or TLR9 agonists provides dose-sparing advantages for induction of specific and functional antibody responses against VLPs (i.e., non-replicating antigens) in the respiratory, gastrointestinal, and reproductive tract.
• Mathew, L. G., Herbst-Kralovetz, M. M., & Mason, H. S. (2014). Norovirus Narita 104 virus-like particles expressed in Nicotiana benthamiana induce serum and mucosal immune responses. BioMed research international, 2014, 807539.
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  Narita 104 virus is a human pathogen belonging to the norovirus (family Caliciviridae) genogroup II. Noroviruses cause epidemic gastroenteritis worldwide. To explore the potential of developing a plant-based vaccine, a plant optimized gene encoding Narita 104 virus capsid protein (NaVCP) was expressed transiently in Nicotiana benthamiana using a tobacco mosaic virus expression system. NaVCP accumulated up to approximately 0.3 mg/g fresh weight of leaf at 4 days postinfection. Initiation of hypersensitive response-like symptoms followed by tissue necrosis necessitated a brief infection time and was a significant factor limiting expression. Transmission electron microscopy of plant-derived NaVCP confirmed the presence of fully assembled virus-like particles (VLPs). In this study, an optimized method to express and partially purify NaVCP is described. Further, partially purified NaVCP was used to immunize mice by intranasal delivery and generated significant mucosal and serum antibody responses. Thus, plant-derived Narita 104 VLPs have potential for use as a candidate subunit vaccine or as a component of a multivalent subunit vaccine, along with other genotype-specific plant-derived VLPs.
• Herbst-Kralovetz, M. M., Radtke, A. L., Lay, M. K., Hjelm, B. E., Bolick, A. N., Sarker, S. S., Atmar, R. L., Kingsley, D. H., Arntzen, C. J., Estes, M. K., & Nickerson, C. A. (2013). Lack of norovirus replication and histo-blood group antigen expression in 3-dimensional intestinal epithelial cells. Emerging Infectious Diseases, 19(3).
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  Noroviruses (NoVs) are a leading cause of gastroenteritis worldwide. An in vitro model for NoV replication remains elusive, making study of the virus difficult. A previous study, which used a 3-dimensional (3-D) intestinal model derived from INT-407 cells reported NoV replication and extensive cytopathic effects (CPE). Using the same 3-D model, but with highly purified Norwalk virus (NV), we attempted to replicate this study. Our results showed no evidence of NV replication by real-time PCR of viral RNA or by immunocytochemical detection of viral structural and nonstructural proteins. Immunocytochemical analysis of the 3-D cultures also showed no detectable presence of histo-blood group antigens that participate in NV binding and host tropism. To determine the potential cause of CPE observed in the previous study, we exposed 3-D cultures to lipopolysaccharide concentrations consistent with contaminated stool samples and observed morphologic features similar to CPE. We conclude that the 3-D INT-407 model does not support NV replication.
• Hjelm, B. E., Kilbourne, J., & Herbst-Kralovetz, M. M. (2013). TLR7 and 9 agonists are highly effective mucosal adjuvants for norovirus virus-like particle vaccines. Human vaccines & immunotherapeutics, 10(3).
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  Virus-like particles (VLPs) are an active area of vaccine research, development and commercialization. Mucosal administration of VLPs provides an attractive avenue for delivery of vaccines with the potential to produce robust immune responses. Nasal and oral delivery routes are particularly intriguing due to differential activation of mucosa-associated lymphoid tissues. We compared both intranasal and oral administration of VLPs with a panel of toll-like receptor (TLR) agonists (TLR3, 5, 7, 7/8, and 9) to determine the mucosal adjuvant activity of these immunomodulators. We selected Norwalk virus (NV) VLPs because it is an effective model antigen and an active area of research and commercialization. To prioritize these adjuvants, VLP-specific antibody production in serum (IgG, IgG1, IgG2a), vaginal lavages (IgG, IgA), and fecal pellets (IgA) were measured across a longitudinal timeseries in vaccinated mice. Additional distal mucosal sites (nasal, brochoalveolar, salivary, and gastrointestinal) were evaluated for VLP-specific responses (IgA). Intranasal co-delivery of VLPs with TLR7 or TLR9 agonists produced the most robust and broad-spectrum immune responses, systemically and at distal mucosal sites inducing VLP-specific antibodies at all sites evaluated. In addition, these VLP-specific antibodies blocked binding of NV VLPs to histo-blood group antigen (H type 1), supporting their functionality. Oral administration and/or other TLR agonists tested in the panel did not consistently enhance VLP-specific immune responses. This study demonstrates that intranasal co-delivery of VLPs with TLR7 or TLR9 agonists provides dose-sparing advantages for induction of specific and functional antibody responses against VLPs (i.e., non-replicating antigens) in the respiratory, gastrointestinal, and reproductive tract.
• McGowin, C. L., Radtke, A. L., Abraham, K., Martin, D. H., & Herbst-Kralovetz, M. (2013). Mycoplasma genitalium infection activates cellular host defense and inflammation pathways in a 3-dimensional human endocervical epithelial cell model. The Journal of Infectious Diseases, 207(12).
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  Because Mycoplasma genitalium is a prevalent and emerging cause of sexually transmitted infections, understanding the mechanisms by which M. genitalium elicits mucosal inflammation is an essential component to managing lower and upper reproductive tract disease syndromes in women.
• Radtke, A. J., & Herbst-kralovetz, M. M. (2013). IL-36{gamma} induction in the human female reproductive tract is microbe-dependent (P4009). Journal of Immunology, 190.
• Herbst-Kralovetz, M., Jackson, E. M., & Herbst-Kralovetz, M. -. (2012). Intranasal vaccination with murabutide enhances humoral and mucosal immune responses to a virus-like particle vaccine. PloS one, 7(7).
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  Murabutide (MB) is a synthetic immunomodulator recognized by the nucleotide-binding oligomerization domain-containing protein 2 (NOD2) receptor on mammalian cells. MB has previously been approved for testing in multiple human clinical trials to determine its value as an antiviral therapeutic, and as an adjuvant for injected vaccines. We have found a new use for this immunomodulator; it functions as a mucosal adjuvant that enhances immunogenicity of virus-like particles (VLP) administered intranasally. MB enhanced Norwalk virus (NV) VLP-specific IgG systemically and IgA production at distal mucosal sites following intranasal (IN) vaccination. A dose escalation study identified 100 µg as the optimal MB dosage in mice, based on the magnitude of VLP-specific IgG, IgG1, IgG2a and IgA production in serum and VLP-specific IgA production at distal mucosal sites. IN vaccination using VLP with MB was compared to IN delivery VLP with cholera toxin (CT) or gardiquimod (GARD) and to parenteral VLP delivery with alum; the MB groups were equivalent to CT and GARD and superior to alum in inducing mucosal immune responses and stimulated equivalent systemic VLP-specific antibodies. These data support the further testing of MB as a potent mucosal adjuvant for inducing robust and durable antibody responses to non-replicating subunit vaccines.
• Herbst-Kralovetz, M., Radtke, A. L., & Herbst-Kralovetz, M. -. (2012). Culturing and applications of rotating wall vessel bioreactor derived 3D epithelial cell models. Journal of visualized experiments : JoVE.
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  Cells and tissues in the body experience environmental conditions that influence their architecture, intercellular communications, and overall functions. For in vitro cell culture models to accurately mimic the tissue of interest, the growth environment of the culture is a critical aspect to consider. Commonly used conventional cell culture systems propagate epithelial cells on flat two-dimensional (2-D) impermeable surfaces. Although much has been learned from conventional cell culture systems, many findings are not reproducible in human clinical trials or tissue explants, potentially as a result of the lack of a physiologically relevant microenvironment. Here, we describe a culture system that overcomes many of the culture condition boundaries of 2-D cell cultures, by using the innovative rotating wall vessel (RWV) bioreactor technology. We and others have shown that organotypic RWV-derived models can recapitulate structure, function, and authentic human responses to external stimuli similarly to human explant tissues (1-6). The RWV bioreactor is a suspension culture system that allows for the growth of epithelial cells under low physiological fluid shear conditions. The bioreactors come in two different formats, a high-aspect rotating vessel (HARV) or a slow-turning lateral vessel (STLV), in which they differ by their aeration source. Epithelial cells are added to the bioreactor of choice in combination with porous, collagen-coated microcarrier beads (Figure 1A). The cells utilize the beads as a growth scaffold during the constant free fall in the bioreactor (Figure 1B). The microenvironment provided by the bioreactor allows the cells to form three-dimensional (3-D) aggregates displaying in vivo-like characteristics often not observed under standard 2-D culture conditions (Figure 1D). These characteristics include tight junctions, mucus production, apical/basal orientation, in vivo protein localization, and additional epithelial cell-type specific properties. The progression from a monolayer of epithelial cells to a fully differentiated 3-D aggregate varies based on cell type(1, 7-13). Periodic sampling from the bioreactor allows for monitoring of epithelial aggregate formation, cellular differentiation markers and viability (Figure 1D). Once cellular differentiation and aggregate formation is established, the cells are harvested from the bioreactor, and similar assays performed on 2-D cells can be applied to the 3-D aggregates with a few considerations (Figure 1E-G). In this work, we describe detailed steps of how to culture 3-D epithelial cell aggregates in the RWV bioreactor system and a variety of potential assays and analyses that can be executed with the 3-D aggregates. These analyses include, but are not limited to, structural/morphological analysis (confocal, scanning and transmission electron microscopy), cytokine/chemokine secretion and cell signaling (cytometric bead array and Western blot analysis), gene expression analysis (real-time PCR), toxicological/drug analysis and host-pathogen interactions. The utilization of these assays set the foundation for more in-depth and expansive studies such as metabolomics, transcriptomics, proteomics and other array-based applications. Our goal is to present a non-conventional means of culturing human epithelial cells to produce organotypic 3-D models that recapitulate the human in vivo tissue, in a facile and robust system to be used by researchers with diverse scientific interests.
• Herbst-Kralovetz, M., Radtke, A. L., Quayle, A. J., & Herbst-Kralovetz, M. -. (2012). Microbial products alter the expression of membrane-associated mucin and antimicrobial peptides in a three-dimensional human endocervical epithelial cell model. Biology of reproduction, 87(6).
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  Our understanding of the mechanisms that regulate tissue-specific mucosal defense can be limited by the lack of appropriate human in vitro models. The endocervix lies between the microbe-rich vaginal cavity and the relatively sterile endometrium and is a major portal of entry for Chlamydia trachomatis, Neisseria gonorrhoeae, Mycoplasma genitalium, human immunodeficiency virus (HIV), and herpes simplex virus (HSV) infection in women. The endocervix is lined with a simple epithelium, and these cells produce mucus, which plays a key role in immune defense and reproduction. Here we describe the development of a human three-dimensional endocervical epithelial cell model generated by rotating wall vessel bioreactor technology. The model is composed of cellular aggregates that recapitulate major structural and barrier properties essential for the function and protection of the endocervix, including junctional complexes, microvilli, innate immune receptors, antimicrobial peptides, and mucins, the major structural component of mucus. Using this model, we also report, for the first time, that the membrane-associated mucin genes MUC1, MUC4, and MUC16 are differentially regulated in these aggregates by different bacterial and viral products. Differential induction of antimicrobial peptides was also observed with these products. Together these data define unique and flexible innate endocervical immune signatures that follow exposure to microbial products and that likely play a critical role in the outcome of pathogen challenge at this site.
• Jackson, E., & Herbst-Kralovetz, M. -. (2012). Intranasal vaccination with murabutide enhances humoral and mucosal immune responses to a virus-like particle vaccine. PLoS ONE, 7.
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  e41529. Epub 2012 Jul 25
• Mason, H. S., & Herbst-Kralovetz, M. M. (2012). Plant-derived antigens as mucosal vaccines. Current topics in microbiology and immunology, 354, 101-20.
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  During the last two decades, researchers have developed robust systems for recombinant subunit vaccine production in plants. Stably and transiently transformed plants have particular advantages that enable immunization of humans and animals via mucosal delivery. The initial goal to immunize orally by ingestion of plant-derived antigens has proven difficult to attain, although many studies have demonstrated antibody production in both humans and animals, and in a few cases, protection against pathogen challenge. Substantial hurdles for this strategy are low-antigen content in crudely processed plant material and limited antigen stability in the gut. An alternative is intranasal delivery of purified plant-derived antigens expressed with robust viral vectors, especially virus-like particles. The use of pattern recognition receptor agonists as adjuvants for mucosal delivery of plant-derived antigens can substantially enhance serum and mucosal antibody responses. In this chapter, we briefly review the methods for recombinant protein expression in plants, and describe progress with human and animal vaccines that use mucosal delivery routes. We do not attempt to compile a comprehensive list, but focus on studies that progressed to clinical trials or those that showed strong indications of efficacy in animals. Finally, we discuss some regulatory concerns regarding plant-based vaccines.
• Radtke, A. L., & Herbst-Kralovetz, M. -. (2012). Culturing and applications of rotating wall vessel bioreactor derived 3D epithelial cell models. J Vis Exp.
• Radtke, A. L., Quayle, A. J., & Herbst-Kralovetz, M. -. (2012). Microbial products alter the expression of membrane-associated mucin and antimicrobial peptides in a 3-D human endocervical epithelial cell model. Biology of Reproduction, 6(87), 132.
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  PMID: 23053434
• Phoolcharoen, W., Dye, J. M., Kilbourne, J., Piensook, K., Pratt, W. D., Arntzen, C. A., Mason, H., & Herbst-Kralovetz, M. -. (2011). Ebola immune complex co-delivered with poly (I:C) protects mice against lethal Ebola challenge. Proc Natl Acad Sci U S A., 51(108), 20695-20700.
• Phoolcharoen, W., Dye, J. M., Kilbourne, J., Piensook, K., Pratt, W. D., Arntzen, C. J., Chen, Q., Mason, H. S., & Herbst-Kralovetz, M. M. (2011). A nonreplicating subunit vaccine protects mice against lethal Ebola virus challenge. Proceedings of the National Academy of Sciences of the United States of America, 108(51).
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  Ebola hemorrhagic fever is an acute and often deadly disease caused by Ebola virus (EBOV). The possible intentional use of this virus against human populations has led to design of vaccines that could be incorporated into a national stockpile for biological threat reduction. We have evaluated the immunogenicity and efficacy of an EBOV vaccine candidate in which the viral surface glycoprotein is biomanufactured as a fusion to a monoclonal antibody that recognizes an epitope in glycoprotein, resulting in the production of Ebola immune complexes (EICs). Although antigen-antibody immune complexes are known to be efficiently processed and presented to immune effector cells, we found that codelivery of the EIC with Toll-like receptor agonists elicited a more robust antibody response in mice than did EIC alone. Among the compounds tested, polyinosinic:polycytidylic acid (PIC, a Toll-like receptor 3 agonist) was highly effective as an adjuvant agent. After vaccinating mice with EIC plus PIC, 80% of the animals were protected against a lethal challenge with live EBOV (30,000 LD(50) of mouse adapted virus). Surviving animals showed a mixed Th1/Th2 response to the antigen, suggesting this may be important for protection. Survival after vaccination with EIC plus PIC was statistically equivalent to that achieved with an alternative viral vector vaccine candidate reported in the literature. Because nonreplicating subunit vaccines offer the possibility of formulation for cost-effective, long-term storage in biothreat reduction repositories, EIC is an attractive option for public health defense measures.
• Velasquez, L. S., Shira, S., Berta, A. N., Kilbourne, J., Medi, B. M., Tizard, I., Ni, Y., Arntzen, C. J., & Herbst-Kralovetz, M. M. (2011). Intranasal delivery of Norwalk virus-like particles formulated in an in situ gelling, dry powder vaccine. Vaccine, 29(32).
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  The development of a vaccine to prevent norovirus infections has been focused on immunization at a mucosal surface, but has been limited by the low immunogenicity of self-assembling Norwalk virus-like particles (NV VLPs) delivered enterically or at nasal surfaces. Nasal immunization, which offers the advantage of ease of immunization, faces obstacles imposed by the normal process of mucociliary clearance, which limits residence time of applied antigens. Herein, we describe the use of a dry powder formulation (GelVac) of an inert in situ gelling polysaccharide (GelSite) extracted from Aloe vera for nasal delivery of NV VLP antigen. Powder formulations, with or without NV VLP antigen, were similar in structure in dry form or when rehydrated in simulated nasal fluids. Immunogenicity of the dry powder VLP formulation was compared to equivalent antigen/adjuvant liquid formulations in animals. For the GelVac powder, we observed superior NV-specific serum and mucosal (aerodigestive and reproductive tracts) antibody responses relative to liquid formulations. Incorporation of the TLR7 agonist gardiquimod in dry powder formulations did not enhance antibody responses, although its inclusion in liquid formulations did enhance VLP immunogenicity irrespective of the presence or absence of GelSite. We interpret these data as showing that GelSite-based dry powder formulations (1) stabilize the immunogenic structural properties of VLPs and (2) induce systemic and mucosal antibody titers which are equal or greater than those achieved by VLPs plus adjuvant in a liquid formulation. We conclude that in situ gelation of the GelVac dry powder formulation at nasal mucosal surfaces delays mucociliary clearance and thereby prolongs VLP antigen exposure to immune effector sites.
• Barrila, J., Radtke, A. L., Crabbé, A., Sarker, S. F., Herbst-Kralovetz, M. M., Ott, C. M., & Nickerson, C. A. (2010). Organotypic 3D cell culture models: using the rotating wall vessel to study host-pathogen interactions. Nature reviews. Microbiology, 8(11).
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  Appropriately simulating the three-dimensional (3D) environment in which tissues normally develop and function is crucial for engineering in vitro models that can be used for the meaningful dissection of host-pathogen interactions. This Review highlights how the rotating wall vessel bioreactor has been used to establish 3D hierarchical models that range in complexity from a single cell type to multicellular co-culture models that recapitulate the 3D architecture of tissues observed in vivo. The application of these models to the study of infectious diseases is discussed.
• Herbst-Kralovetz, M., Mason, H. S., & Chen, Q. (2010). Norwalk virus-like particles as vaccines. Expert review of vaccines, 9(3).
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  Noroviruses (NoV) cause the great majority of epidemic nonbacterial gastroenteritis in humans. Expression of the capsid protein in recombinant systems, including insect and plant cells, yields assembly of virus-like particles (VLPs) that mimic the antigenic structure of authentic virions, and are relatively acid- and heat-stable. Norwalk virus (NV), the prototype NoV, has been studied extensively, and Norwalk virus-like particles (NVLPs) produced in insect cells and plants are immunogenic in mice and humans when delivered orally, stimulating the production of systemic and mucosal anti-NV antibodies. NVLPs are also highly immunogenic when delivered intranasally, provoking antibodies at levels similar to orally delivered VLP at much lower doses. Oral and nasal delivery of NVLPs efficiently produces antibodies at distal mucosal sites, which suggests that NVLPs could be used to deliver heterologous peptide antigens by production of genetic fusion chimeric capsid proteins. Examination of norovirus VLP surface structures and receptor binding motifs facilitates identification of potential sites for insertion of foreign peptides that will minimally affect the efficiency of VLP assembly and receptor binding. Thus, there is strong potential to use norovirus VLPs as vaccine-delivery vehicles.
• Hjelm, B. E., Berta, A. N., Nickerson, C. A., Arntzen, C. J., & Herbst-Kralovetz, M. M. (2010). Development and characterization of a three-dimensional organotypic human vaginal epithelial cell model. Biology of reproduction, 82(3).
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  We have developed an in vitro human vaginal epithelial cell (EC) model using the innovative rotating wall vessel (RWV) bioreactor technology that recapitulates in vivo structural and functional properties, including a stratified squamous epithelium with microvilli, tight junctions, microfolds, and mucus. This three-dimensional (3-D) vaginal model provides a platform for high-throughput toxicity testing of candidate microbicides targeted to combat sexually transmitted infections, effectively complementing and extending existing testing systems such as surgical explants or animal models. Vaginal ECs were grown on porous, collagen-coated microcarrier beads in a rotating, low fluid-shear environment; use of RWV bioreactor technology generated 3-D vaginal EC aggregates. Immunofluorescence and scanning and transmission electron microscopy confirmed differentiation and polarization of the 3-D EC aggregates among multiple cell layers and identified ultrastructural features important for nutrient absorption, cell-cell interactions, and pathogen defense. After treatment with a variety of toll-like receptor (TLR) agonists, cytokine production was quantified by cytometric bead array, confirming that TLRs 2, 3, 5, and 6 were expressed and functional. The 3-D vaginal aggregates were more resistant to nonoxynol-9 (N-9), a contraceptive and previous microbicide candidate, when compared to two-dimensional monolayers of the same cell line. A dose-dependent production of tumor necrosis factor-related apoptosis-inducing ligand and interleukin-1 receptor antagonist, biomarkers of cervicovaginal inflammation, correlated to microbicide toxicity in the 3-D model following N-9 treatment. These results indicate that this 3-D vaginal model could be used as a complementary tool for screening microbicide compounds for safety and efficacy, thus improving success in clinical trials.
• Velasquez, L. S., Hjelm, B. E., Arntzen, C. J., & Herbst-Kralovetz, M. M. (2010). An intranasally delivered Toll-like receptor 7 agonist elicits robust systemic and mucosal responses to Norwalk virus-like particles. Clinical and vaccine immunology : CVI, 17(12).
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  Norwalk virus (NV) is an enteric pathogen from the genus Norovirus and a major cause of nonbacterial gastroenteritis in humans. NV virus-like particles (VLPs) are known to elicit systemic and mucosal immune responses when delivered nasally; however, the correlates of immune protection are unknown, and codelivery with a safe and immunogenic mucosal adjuvant may enhance protective anti-NV immune responses. Resiquimod (R848), an imidazoquinoline-based Toll-like receptor 7 and/or 8 (TLR7/8) agonist, is being evaluated as an adjuvant in FDA-approved clinical vaccine trials. As such, we evaluated the adjuvant activity of two imidazoquinoline-based TLR7 and TLR7/8 agonists when codelivered intranasally with plant-derived NV VLPs. We also compared the activity of these agonists to the gold standard mucosal adjuvant, cholera toxin (CT). Our results indicate that codelivery with the TLR7 agonist, gardiquimod (GARD), induces NV VLP-specific serum IgG and IgG isotype responses and mucosal IgA responses in the gastrointestinal, respiratory, and reproductive tracts that are superior to those induced by R848 and comparable to those induced by the mucosal adjuvant CT. This study supports the continued investigation of GARD as a mucosal adjuvant for NV VLPs and possible use for other VLP-based vaccines for which immune responses at distal mucosal sites (e.g., respiratory and reproductive tracts) are desired.
• Herbst-Kralovetz, M. M., Quayle, A. J., Ficarra, M., Greene, S., Rose, W. A., Chesson, R., Spagnuolo, R. A., & Pyles, R. B. (2008). Quantification and comparison of toll-like receptor expression and responsiveness in primary and immortalized human female lower genital tract epithelia. American journal of reproductive immunology (New York, N.Y. : 1989), 59(3).
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  To better understand innate immune responses to sexually-transmitted infection (STI) and the appropriateness of epithelial cell (EC) models of the vaginal and cervical mucosa, quantified toll-like receptor (TLR) expression from a population of women is needed.
• Herbst-Kralovetz, M. -., & Pyles, R. B. (2006). Toll-like Receptors, Innate Immunity and Herpes Simplex Virus Pathogenesis. Herpes, 37-41.
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  PMID: 16895653
• Herbst-Kralovetz, M. M., & Pyles, R. B. (2006). Quantification of poly(I:C)-mediated protection against genital herpes simplex virus type 2 infection. Journal of virology, 80(20).
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  Alternative strategies for controlling the growing herpes simplex virus type 2 (HSV-2) epidemic are needed. A novel class of immunomodulatory microbicides has shown promise as antiherpetics, including intravaginally applied CpG-containing oligodeoxynucleotides that stimulate toll-like receptor 9 (TLR9). In the current study, we quantified protection against experimental genital HSV-2 infection provided by an alternative nucleic acid-based TLR agonist, polyinosine-poly(C) (PIC) (TLR3 agonist). Using a protection quantification paradigm, groups of mice were PIC treated and then subdivided into groups challenged with escalating doses of HSV-2. Using this paradigm, a temporal window of PIC efficacy for single applications was defined as 1 day prior to (prophylactic) through 4 h after (therapeutic) viral challenge. PIC treatment within this window protected against 10-fold-higher HSV-2 challenges, as indicated by increased 50% infectious dose values relative to those for vehicle-treated controls. Disease resolution and survival were significantly enhanced by repetitive PIC doses. Using optimal PIC regimens, cytokine induction was evaluated in murine vaginal lavages and in human vaginal epithelial cells. Similar induction patterns were observed, with kinetics that explained the limited durability of PIC-afforded protection. Daily PIC delivery courses did not generate sustained cytokine levels in murine vaginal fluids that would be indicative of local immunotoxicity. No evidence of immunotoxicity was observed in selected organs that were analyzed following repetitive vaginal PIC doses. Animal and in vitro data indicate that PIC may prove to be a valuable preventative microbicide and/or therapeutic agent against genital herpes by increasing resistance to HSV-2 and enhancing disease resolution following a failure of prevention.
• Herbst-Kralovetz, M., & Pyles, R. (2006). Toll-like receptors, innate immunity and HSV pathogenesis. Herpes : the journal of the IHMF, 13(2), 37-41.
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  In the last decade, substantial progress has been made in understanding the molecular mechanisms involved in initial host responses to viral infections, and how viral recognition leads to the innate responses that ultimately shape the adaptive immune response. Viruses, including herpes simplex virus (HSV) types 1 and 2, trigger toll-like receptors (TLRs) that elicit cytokine and chemokine production. In turn, this can create local resistance and modulate T- and B-cell-mediated immunity. TLR activation by HSV-produced molecules (or other synthetic agonists) leads to the remodelling of draining lymph nodes. This enhances the screening of naive T-cells, from which antigen-specific lymphocytes can be selected and expanded. The innate response thereby serves to direct a timely and effective acquired immune response, through the initial TLR recognition of viral pathogen-associated molecular patterns that can limit or possibly exacerbate viral pathogenesis. Recently, these findings have been exploited by strategies that utilize synthetic TLR agonists as prophylactic or therapeutic devices. Such devices prime innate immune responses, enhancing host resistance to viral infections, including experimental HSV infections.
• Green, R., Herbst-Kralovetz, M. -., & Schountz, T. (2004). Genomic Organization of Deer Mouse (Peromyscus maniculatus) Tumor Necrosis Factor. Bios., 12-17.
• Herbst, M. M., & Pyles, R. B. (2003). Immunostimulatory CpG treatment for genital HSV-2 infections. The Journal of antimicrobial chemotherapy, 52(6).
• Herbst, M. M., Prescott, J., Palmer, A. D., & Schountz, T. (2002). Sequence and expression analysis of deer mouse interferon-gamma, interleukin-10, tumor necrosis factor, and lymphotoxin-alpha. Cytokine, 17(4).
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  Deer mice (Peromyscus maniculatus) are the principal host species of Sin Nombre (SN) virus, the primary etiologic agent of hantavirus cardiopulmonary syndrome in North America. The disease is a cytokine-mediated immunopathology characterized by pulmonary mononuclear infiltrates without discernible viral pathology. Infected deer mice remain life-long carriers and virus is found in many organs, including the lungs, but without pathology. It is unclear how deer mice respond to SN virus because no tools exist to examine the immune response in infected animals. As an initial step in examining host responses to SN virus, we have cloned partial cDNAs of deer mouse interferon-gamma (IFN-gamma), interleukin-10 (IL-10), tumor necrosis factor (TNF) and lymphotoxin-alpha (LTalpha). IL-10, TNF and LTalpha sequences are highly conserved compared to orthologs of other mammalian species, while IFN-gamma is substantially less conserved. Phylogenetic analyses indicate that the amino acid sequences of IFN-gamma and TNF may be useful in resolving relationships at the subfamily level within the rodent family Muridae. While all four sets of analyses were able to reconstruct clade Rodentia, they were not able to resolve the relationships among the mammalian orders represented in this study. Reverse transcriptase polymerase chain reaction (RT-PCR) analysis of concanavalin A-stimulated splenocytes determined that maximal IFN-gamma and TNF expression occurred rapidly while IL-10 and LTalpha expression was maximal at 24 h.

Presentations

• Laniewski, P., & Herbst-Kralovetz, M. -. (2021, Summer). Cervicovaginal microbiota species distinctly modulate the immunometabolic microenvironment in a human three-dimensional cervical model. STI and HIV 2021 World Congress. Virtual—Amsterdam; Oral presentation..
• Gardner, J., & Herbst-Kralovetz, M. (2019, spring). IL-36g is a crucial regulator of HSV-2-induced neutrophil recruitment and limits neuroinvasion in genital HSV-2 pathogenesis. American Society for Microbiology Arizona/Southern Nevada Branch Meeting, Flagstaff, AZ Oral Presentation.. Phoenix: UA.
• Gardner, J., & Herbst-Kralovetz, M. (2019, spring). IL-36g is crucial for HSV-2-mediated neutrophil infiltration and limits neuroinvasion in genital HSV-2 disease pathogenesis. Arizona Biomedical Research Commission Research Conference, Phoenix, AZ. Oral presentation. Phoenix: UA.
• Gardner, J., & Herbst-Kralovetz, M. (2019, spring). IL-36g regulates neutrophil infiltration and limits neuroinvasion in genital HSV-2 pathogenesis. AAI Immunology 2019, San Diego, CA. Oral presentation.. Phoenix: UA.
• Gardner, J., & Herbst-Kralovetz, M. (2019, summer). The role of IL-36g in genital HSV-2 disease pathogenesis. Viroholics, Tempe, AZ. Oral presentation. Phoenix: UA.
• Herbst-Kralovetz, M., Łaniewski, P., Thomas, N., Roe, D., Chase, D., & Ilhan, E. (2019, spring). Vaginal microbiota, genital Inflammation, and HPV infection modulate cervicovaginal metabolomes in cervical carcinogenesis. Frontiers in Immunobiology & Immunopathogenesis Symposium, 2019, Tucson, AZ. Phoenix: UA.
• Ilhan, E., Laniewski, P., & Herbst-Kralovetz, M. (2019, spring). Clinical Prevotella isolates exhibit species-specific inflammatory and cytotoxic effects on human three-dimensional endometrial epithelial cell model.. American Society for Microbiology Arizona/Southern Nevada Branch Meeting, Flagstaff, AZ Oral Presentation.. Phoenix: UA.
• Laniewski, P., Tonachio, A., & Herbst-Kralovetz, M. (2019, spring). . Identifying mechanisms of host defense to Sneathia amnii infection using human three-dimensional female reproductive tract epithelium models. American Society for Microbiology Arizona/Southern Nevada Branch Meeting, Flagstaff, AZ Oral Presentation.. Phoenix: UA.
• Herbst-Kralovetz, M. (2018, August). Clinical and Personal Lubricants Alter Cell Viability, Cytotoxicity and Mucin Production in Human Vaginal Epithelial Cell Culture Models. Infectious Diseases Society of Gynecology, Annual Meeting 2018. Philadelphia, PA..
• Herbst-Kralovetz, M. (2018, Fall). Human In Vitro Systems to Study Genital Tract Mucosal Defenses. Global Health International Keystone Symposia: Role of the Genital Tract Microbiome in Sexual and Reproductive Health. Cape Town, South Africa.
• Herbst-Kralovetz, M. (2018, Spring). Depo Provera and b-estradiol regulate IL-36g in the vaginal microenvironment and impact HSV-2 pathogenesis. Frontiers in Immunobiology and Immunopathogenesis Symposium 2018. Tucson, AZ.
• Herbst-Kralovetz, M. (2018, summer). Investigating HPV, vaginal microbiota, and the immune microenvironment in cervical carcinogenesis. Microlunch Seminar Series. Tucson, AZ.
• Gardner, J., & Herbst-Kralovetz, M. (2017, Spring). IL-36g treatment induces a transient HSV-2 resistant environment in the female reproductive tract.. American Society for Microbiology Arizona/Southern Nevada Branch Meeting. Tucson, AZ.
• Laniewski, P., Goulder, A., Barnes, D., Cui, H., Roe, D., Chase, D., & Herbst-Kralovetz, M. (2017, Fall). Association between local immune signatures and vaginal microbiota composition in cervical carcinogenesis in Non-Hispanic and Hispanic population. AZ Wellbeing Commons: Viruses, Microbiome, Immunity and Infectious Diseases. Tempe, AZ.
• Laniewski, P., Goulder, A., Barnes, D., Cui, H., Roe, D., Chase, D., & Herbst-Kralovetz, M. (2017, Summer). Linking cervicovaginal immune signatures and microbiota composition in cervical carcinogenesis. International Congress of Mucosal Immunology (ICMI). Washington, D.C..
• Gardner, J., & Herbst-Kralovetz, M. (2016, Spring). Herpes simplex virus replication is limited by IL-36gamma in human vaginal epithelial cells. American Society of Microbiology, AZ/NV Regional Meeting. Tempe, AZ.
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  Selected for oral presentation. Jameson gave oral presentation and was awarded "Best Oral Presentation" in Viral Immunology Session
• Herbst-Kralovetz, M., & Gardner, J. (2016, Spring). IL-36gamma limits herpes simplex virus replication in human vaginal epithelial cells. 11th Annual Frontiers in Immunopathogenesis and Immunobiology Symposium. Tucson, AZ.
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  Jameson Gardner's poster was selected for oral presentation.
• Laniewski, P., Gomez, A., Hire, G., & Herbst-Kralovetz, M. (2016, Spring). Ascending vaginal microbiota impacts human endometrial epithelial barrier function and integrity. American Society of Microbiology, AZ/NV Regional Meeting. Tempe, AZ.
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  Dr. Laniewski was selected for an oral presentation at this meeting.
• Herbst-Kralovetz, M. (2015, April 24, 2015). Epithelial Cell Modeling and Microbiome Analysis to Study Cancer Development and Treatment. UACC-Phoenix, Dignity Health/PBC Cancer Symposium. UA Cancer Center Phoenix: UA Cancer Center Phoenix and Dignity Health.
• Herbst-Kralovetz, M. (2015, June). Host-Microbiota Interactions at the Human Vaginal and Cervical Epithelial Cell Interfaces. BMS Faculty Retreat. Phoenix: BMS Department.
• Herbst-Kralovetz, M. (2015, November 13, 2015). The Genital Microbiome, Cancer and Beyond. UA Cancer Center Phoenix Board Meeting Presentation. UA Cancer Center Phoenix: UA Cancer Center Phoenix.
• Herbst-Kralovetz, M. (2015, October 22, 2015). Vaginal Microbiota, Host Immunity and Women's Health. BMS Seminar Series. UA-COM Phoenix: BMS Department.
• Herbst-Kralovetz, M. -. (2014, August). Progress in Norovirus Vaccine Development. International Association of Food Protection, Indianapolis, IN.
• Herbst-Kralovetz, M. -. (2014, June). Generating optimal VLP-specific antibody responses using mucosal delivery routes and formulations.. International VLPNPV Meeting, Salk Institute, La Jolla, CA..
• Herbst-Kralovetz, M. -. (2014, June). Pathways to Science (9th and 10th graders). MedStart-Phoenix.
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  1.5hr Session for 9th and 10th grade high school students for the MedStart-Phoenix Program
• Herbst-Kralovetz, M. -. (2014, June). Vaginal microbiota alter the innate immune barrier of the human vaginal epithelia in a species-specific fashion.. Vaginal Microbiome/Bacterial Vaginosis Conference..
• Herbst-Kralovetz, M. -. (2014, May). Hands On Interactive Laboratory Session: Cell culture and microscopy. MedStart-Phoenix.
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  Hands on demonstration session for 11th and 12th grade high school students for the MedStart-Phoenix Program on cell culture and light microscopy. 1.5 hr session that began with a presentation and followed by 2 interactive, hands-on stations in the laboratory.
• Herbst-Kralovetz, M. -. (2014, May). Pathways to Science (11th and 12th graders). MedStart-Phoenix.
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  Session for 11th and 12th grade high school students for the MedStart-Phoenix Program
• Herbst-Kralovetz, M. -. (2014, May). Pathways to Science--Panel Session. MedStart-Phoenix.
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  Panel Session for 11th and 12th grade high school students for the MedStart-Phoenix Program, followed my introductory presentation.
• Herbst-Kralovetz, M. -. (2014, November). Opportunties for Collaboration to Study Host-Pathogen Interactions in the Female Reproductive Tract. Banner Health OB/GYN rounds.
• Radtke, A., Doerflinger, S., & Herbst-Kralovetz, M. -. (2013, Spring). Discrimination of Commensal vs. Pathogenic Bacteria in the Human Female Reproductive Tract.. 8th Annual Frontiers in Immunobiology and Immunopathogenesis Symposium, University of Arizona, Tucson, AZ.
• Herbst-Kralovetz, M. -. (2012, Spring). Optimizing mucosal delivery of VLPs.. VLPNPV Meeting, Cannes, France.
• McGowin, C. L., Abraham, K., Quayle, A. J., Herbst-Kralovetz, M. -., & Martin, D. H. (2011, Spring). Mycoplasma genitalium elicits acute-phase inflammation from human endocervical epithelial cells: findings from a novel 3-dimensional organotypic model. ASM General Meeting, New Orleans, LA.
• Arntzen, C., Kingsley, D., Herbst-Kralovetz, M. -., Dienhalt, C., & Nickerson, C. (2010, Spring). Novel Platform Technologies for Analysis of Norovirus Contamination of Seafood. Washington, D.C. USDA Meeting.
• Herbst-Kralovetz, M. -., Shira, S., Berta, A., Kilbourne, J., Ni, Y., & Arntzen, C. (2010, Spring). Optimizing Intranasal Immunization with Norwalk VLP. 4th International Calicivirus Conference. Santa Cruz, Chile.
• Herbst-Kralovetz, M. -., Slater, K., Berta, A., Valaquez, L., Caine, B., & Arntzen, C. (2010, Spring). In Vitro Human Female Reproductive Tract Models for Microbicide Screening. Dublin, Ireland.
• Herbst-Kralovetz, M. -., Berta, A., Shira, S., Kilbourne, J., & Arntzen, C. (2009, Spring). Utilizing Multifactorial Pattern Recognition Receptor Agonist-based Adjuvants to Enhance Intranasal Immunization. International Conference on Modern Vaccines, Adjuvants and Delivery Systems. Vienna, Austria.
• Herbst-Kralovetz, M. -., Hjelm, B., LaVigne, D., Kilbourne, D., Arntzen, C., Chen, Q., & Mason, H. (2008, Spring). Achieving Vaginal Immune Responses through Mucosal Immunization. International Conference on Modern Mucosal Vaccines, Adjuvants and Microbicides, Porto, Portugal.
• Herbst-Kralovetz, M. -., Higgins, D., Van Nest, G., & Pyles, R. B. (2003, Spring). Optimization of Immunostimulatory Sequence-containing Oligonucleotides for the Treatment of Genital Herpes Simplex Virus Type 2 (HSV-2) Infection.. American Society for Virology.

Poster Presentations

• Bordeaux, S., Martinez, E., Laniewski, P., Metz, N., Quiroz, V., Caporaso, G., Herbst-Kralovetz, M. -., & Lee, N. (2021, Fall). Implementing a culturally-appropriate biospecimen collection protocol during the COVID-19 pandemic to address cervical cancer disparities among Native American women.. 14th American Association for Cancer Research (AACR) Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved. Virtual.
• Jimenez, N., Maarsingh, J., Laniewski, P., & Herbst-Kralovetz, M. -. (2021, Summer). Immunometabolic Profiles Differ Amongst Lactobacilli in Human Cervical Epithelial Cell Models.. Arizona Postdoctoral Symposium. Phoenix, AZ.
• McKenzie, R., Maarsingh, J., Laniewski, P., & Herbst-Kralovetz, M. -. (2021, Summer). Immunometabolic Analysis of Mobiluncus mulieris and Eggerthella sp. Reveals Novel Insights into Their Pathogenic Contributions to the Hallmarks of Bacterial Vaginosis. Infectious Diseases Society of Obstetrics and Gynecology Annual Meeting. Virtual.
  More info

  Poster presentation and Ross McKenzie, my trainee applied for and was an IDSOG Scholar Award for his work.
• Gallitano-Mendel, A. L., Hale, T., Herbst-Kralovetz, M., Gonzales, R. J., Titelbaum, A. R., Federico, G., Mahnert, N., Garcia-Filion, P., Omalley, C. W., Nelson, L. R., Muhammad, S., Parrish, J., Lucio, F., Mallin, E., Hartmark-Hill, J. R., Gulati, M., & Thomas, T. C. (2019, November). . Strategic initiative to create a Women in Medicine and Science (WIMS) program at a recently established and independently accredited College of Medicine. AAMC-Learn, Serve, Lead. Phoenix: AAMC.
• Gallitano-Mendel, A. L., Hale, T., Herbst-Kralovetz, M., Gonzales, R. J., Titelbaum, A. R., Federico, G., Mahnert, N., Garcia-Filion, P., Omalley, C. W., Nelson, L. R., Muhammad, S., Parrish, J., Lucio, F., Mallin, E., Hartmark-Hill, J. R., Gulati, M., & Thomas, T. C. (2019, November). Strategic initiative to create a Women in Medicine and Science (WIMS) program at a recently established and independently accredited College of Medicine. AAMC-Learn, Serve, Lead. Phoenix: AAMC.
• Gardner, J., & Herbst-Kralovetz, M. (2019, spring). IL-36g is crucial for HSV-2-induced neutrophil recruitment and protects against neuroinvasion in genital HSV-2 pathogenesis. Frontiers in Immunobiology & Immunopathogenesis Symposium, 2019, Tucson, AZ. Phoenix: UA.
• Gardner, J., & Herbst-Kralovetz, M. (2019, spring). IL-36g is crucial for HSV-2-mediated neutrophil infiltration and limits neuroinvasion in genital HSV-2 disease pathogenesis. Arizona Biomedical Research Commission Research Conference, Phoenix, AZ. Oral presentation. Phoenix: UA.
• Herbst-Kralovetz, M., Thomas, T., Mallin, E., Gulati, M., Garcia, P. C., Hartmark-Hill, J. R., Lucio, F., Nelson, L. R., Gallitano-Mendel, A. L., Martinez, G. F., Herbst-Kralovetz, M., Thomas, T., Mallin, E., Gulati, M., Garcia, P. C., Hartmark-Hill, J. R., Lucio, F., Nelson, L. R., Gallitano-Mendel, A. L., & Martinez, G. F. (2019, November). Strategic initiative to create a Women in Medicine and Science (WIMS) program at a recently established and independently accredited College of Medicine. AAMC Learn Serve Lead. Phoenix, AZ: AAMC.
• Ilhan, E., Laniewski, P., Thomas, N., Roe, D., Chase, D., & Herbst-Kralovetz, M. (2019, spring). Vaginal microbiota, genital inflammation, and HPV infection modulate cervicovaginal metabolomes in cervical carcinogenesis. Arizona Biomedical Research Commission Research Conference, Phoenix, AZ. Oral presentation. Phoenix: UA.
• Karjala, S., Laniewski, P., & Herbst-Kralovetz, M. (2019, Fall). Differential cytotoxicity of bacteria and bacteria-free supernatants from members of fusobacteria on human endometrial epithelial cells. Re-imagine health: is my fate in my genes? 2019, Phoenix, AZ; poster presentation.. Phoenix: UA.
• Khnanisho, M., Laniewski, P., & Herbst-Kralovetz, M. (2019, Fall). Excipients in clinical and personal lubricants inhibit the growth of health-associated lactobacilli found in vaginal microbiome. Re-imagine health: is my fate in my genes? 2019, Phoenix, AZ; poster presentation.. Phoenix: UA.
• Krishna, G., Hair, C., Laubitz, D., Herbst-Kralovetz, M., & Thomas, T. (2019, Fall). Impact of diffuse traumatic brain injury (TBI) on gut microbiota: Preliminary sex-specific findings. Re-imagine health: is my fate in my genes? 2019, Phoenix, AZ; poster presentation.. Phoenix: UA.
• Laniewski, P., Cui, H., Roe, D., Chase, D., & Herbst-Kralovetz, M. (2019, Summer). Immune Checkpoint Proteins in Women with Cervical Neoplasm Associate with Features of the Local Cervicovaginal Microenvironment.. . Infectious Diseases Society of Obstetrics and Gynecology Annual Meeting, Big Sky, Montana; Poster presentation. Phoenix: UA.
• Laniewski, P., Cui, H., Roe, D., Chase, D., & Herbst-Kralovetz, M. (2019, spring). Evaluating immune checkpoint proteins in the local microenvironment in women with cervical neoplasm: potential implications for tailored immunotherapies?. Arizona Biomedical Research Commission Research Conference, Phoenix, AZ. Oral presentation. Phoenix: UA.
• Laniewski, P., Ilhan, E., Bokulich, N., Cui, H., Roe, D., Chase, D., Caporaso, G., & Herbst-Kralovetz, M. (2019, Summer). Integrative multi-omics approach reveals complex interplay between HPV, host and microbiome during cervical carcinogenesis in Hispanic and non-Hispanic women.. 12th AACR Conference on The Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved. San Francisco, CA; Poster presentation.. Phoenix: UA.
• Laniewski, P., Ludwig, R., Montero, R., Laubitz, D., Mourad, J., Mahnert, N., Chase, D., & Herbst-Kralovetz, M. (2019, spring). Characterization of the genital microbiota in obese and non-obese type I endometrial cancer patients. Arizona Biomedical Research Commission Research Conference, Phoenix, AZ. Oral presentation. Phoenix: UA.
• Laniewski, P., Owen, K., Brotman, R., & Herbst-Kralovetz, M. (2019, Summer). Clinical and Personal Lubricants Impact Growth of Vaginal Lactobacillus Species and Colonization of Vaginal Epithelial Cells. Infectious Diseases Society of Obstetrics and Gynecology Annual Meeting, Big Sky, Montana; Poster presentation. Phoenix: UA.
• Maarsingh, J., Laniewski, P., Roe, D., Chase, D., & Herbst-Kralovetz, M. (2019, Fall). Metabolic hallmarks of cancer altered in Hispanic women across HPV-mediated cervical carcinogenesis.. Re-imagine health: is my fate in my genes? 2019, Phoenix, AZ; poster presentation.. Phoenix: UA.
• Salliss, M., Laniewski, P., & Herbst-Kralovetz, M. (2019, Summer). Investigating the antimicrobial activity of the bacterial metabolite, glycochenodeoxycholate on vaginal bacteria as a novel therapeutic approach against bacterial vaginosis. Re-imagine health: is my fate in my genes? 2019, Phoenix, AZ; poster presentation.. Phoenix: UA.
• Herbst-Kralovetz, M. (2018, Fall). Cervicovaginal Metabolome Associated with HPV, Vaginal Microbiota, and Inflammation in Cervical Carcinogenesis. University of Arizona Research Symposium. Phoenix, AZ.
• Herbst-Kralovetz, M. (2018, Fall). DOES VAGINAL MICROBIOME COMPOSITION ALTER LOCAL CANCER BIOMARKER EXPRESSION IN CERVICAL DYSPLASIA AND CANCER?. University of Arizona Research Symposium. Phoenix, AZ.
• Herbst-Kralovetz, M. (2018, Fall). Site-specific cytotoxicity and epithelial host fefense mechanisms to Sneathia amnii infection in the female reproductive tract. Global Health International Keystone Symposia: Role of the Genital Tract Microbiome in Sexual and Reproductive Health. Cape Town, South Africa.
• Herbst-Kralovetz, M. (2018, May). Female sex hormones regulate the IL-36 family members in the female reproductive tract and impact HSV-2 pathogenesis.. Immunology 2018 (AAI). Austin, TX.
• Herbst-Kralovetz, M. (2018, September). DOES VAGINAL MICROBIOME COMPOSITION ALTER LOCAL CANCER BIOMARKER EXPRESSION IN CERVICAL DYSPLASIA AND CANCER?. International Gynecologic Cancer Society Symposia. Kyoto, Japan.
• Herbst-Kralovetz, M. (2018, Spring). Biogenic amines alter inflammatory mediators and mucin production but do not alter vaginal epithelial cell viability or morphology. Frontiers in Immunobiology and Immunopathogenesis Symposium 2018. Tucson, AZ.
• Herbst-Kralovetz, M. (2018, Spring). Functional impact of Sneathia amnii on the female reproductive tract epithelium. Frontiers in Immunobiology and Immunopathogenesis Symposium 2018. Tucson, AZ.
• Herbst-Kralovetz, M. (2018, Spring). Genital tract microbiota in obese and non-obese patients undergoing hysterectomy: a pilot study. 3rd Annual ABRC Research Conference 2018. Phoenix, AZ.
• Herbst-Kralovetz, M. (2018, Spring). Investigating lactobacilli species-specific differences and impact on immune barrier properties of the human vaginal and cervical epithelia. Frontiers in Immunobiology and Immunopathogenesis Symposium 2018. Tucson, AZ.
• Herbst-Kralovetz, M. (2018, Spring). Requirement for IL-36g and IL-36Ra in vaginal bacterial infection and inflammation. 3rd Annual ABRC Research Conference 2018. Phoenix, AZ.
• Herbst-Kralovetz, M. (2018, Summer). In-vitro and in-silico analyses reveal species-specific interplay between clinical Prevotella isolates and host epithelial vaginal cells. AZ Wellbeing Commons Meeting. Scottsdale, AZ.
• Herbst-Kralovetz, M. (2018, Summer). Investigating lactobacilli species-specific differences and impact on immune barrier properties of the human vaginal and cervical epithelia. AZ Wellbeing Commons Meeting. Scottsdale, AZ.
• Gardner, J., & Herbst-Kralovetz, M. (2017, Fall). IL-36g induces a HSV-2 resistant environment that protects against genital disease and pathogenesis. AZ Wellbeing Commons: Viruses, Microbiome, Immunity and Infectious Diseases Symposia. Tempe, AZ.
• Gardner, J., & Herbst-Kralovetz, M. (2017, Fall). IL-36g treatment induces a transient HSV-2 resistant environment in the female reproductive tract.. AZ Bio Awards. Phoenix, AZ.
• Gardner, J., & Herbst-Kralovetz, M. (2017, Spring). IL-36g creates a HSV-2 resistant environment in 3-D human vaginal epithelial cell and mouse models. 12th Annual Frontiers in Immunobiology & Immunopathogenesis Symposium. Tucson, AZ.
• Gardner, J., & Herbst-Kralovetz, M. (2017, Spring). IL-36g treatment induces a transient HSV-2 resistant environment in the female reproductive tract.. American Society for Microbiology Arizona/Southern Nevada Branch Meeting. Tucson, AZ.
• Gardner, J., & Herbst-Kralovetz, M. (2017, Summer). IL-36g induces a HSV-2 resistant environment in human vaginal epithelial cell and mouse models. International Congress of Mucosal Immunology (ICMI). Washington, D.C..
• Ito, M., & Herbst-Kralovetz, M. (2017, Fall). Investigating lactobacilli species-specific differences and impact on immune barrier properties of the human vaginal epithelia. AZ Wellbeing Commons: Viruses, Microbiome, Immunity and Infectious Diseases Symposia. Tempe, AZ.
• Laniewski, P., Baker, J., & Herbst-Kralovetz, M. (2017, Spring). IL-36gamma as a driver of mucosal immune response against pathogenic gonococci in human 3-D female reproductive tract models.. 12th Annual Frontiers in Immunobiology & Immunopathogenesis Symposium. Tucson, AZ.
• Laniewski, P., Baker, J., & Herbst-Kralovetz, M. (2017, Summer). IL-36gamma drives host defense response to Neisseria gonorrhoeae, but not Lactobacillus species, in human 3-D vaginal and endocervical models. ASM Microbe 2017. New Orleans, LA.
• Laniewski, P., Cui, H., Roe, D., Chase, D., & Herbst-Kralovetz, M. (2017, Fall). Vaginal microbiota, local inflammation and gynecologic cancers.. UA Cancer Center Retreat and Symposia, Phoenix, AZ. Phoenix, AZ.
• Laniewski, P., Goulder, A., Barnes, D., Cui, H., Roe, D., Chase, D., & Herbst-Kralovetz, M. (2017, Fall). Association between local immune signatures and vaginal microbiota composition in cervical carcinogenesis in Non-Hispanic and Hispanic population. AZ Wellbeing Commons: Viruses, Microbiome, Immunity and Infectious Diseases. Tempe, AZ.
• Laniewski, P., Goulder, A., Barnes, D., Cui, H., Roe, D., Chase, D., & Herbst-Kralovetz, M. (2017, Spring). Microbial drivers and host immune signatures of cervical carcinogenesis.. American Society for Microbiology Arizona/Southern Nevada Branch Meeting, Tucson, AZ. Tucson, AZ.
• Laniewski, P., Goulder, A., Barnes, D., Cui, H., Roe, D., Chase, D., & Herbst-Kralovetz, M. (2017, Summer). Signatures of cervicovaginal inflammation in patients over the course of cervical carcinogenesis. Keystone Symposia on Inflammation-driven Cancer. Keystone, CO.
• Smith, R., Gardner, J., & Herbst-Kralovetz, M. (2017, Spring). Effects of the pro-inflammatory cytokines IL-36g and IL-22 on innate immune signaling and antiviral response in murine female reproductive tract. American Society for Microbiology Arizona/Southern Nevada Branch Meeting. Tucson, AZ.
• Wilkinson, E., Gardner, J., Borgogna, J., Yeoman, C., & Herbst-Kralovetz, M. (2017, Fall). Vaginal biogenic amines increase mucin production but do not alter vaginal epithelial cell viability or morphology. AZ Wellbeing Commons: Viruses, Microbiome, Immunity and Infectious Diseases Symposia. Tempe, AZ.
• Gardner, J., & Herbst-Kralovetz, M. (2016, Spring). Herpes simplex virus replication is limited by IL-36gamma in human vaginal epithelial cells. American Society of Microbiology, AZ/NV Regional Meeting. Tempe, AZ.
• Gardner, J., & Herbst-Kralovetz, M. (2016, Spring). IL-36gamma limits herpes simplex virus replication in human vaginal epithelial cells. 11th Annual Frontiers in Immunopathogenesis/Immunobiology Symposia. Tucson.
• Gardner, J., & Herbst-Kralovetz, M. (2016, Summer). IL-36gamma limits herpes simplex virus replication in human vaginal epithelial cells. AZ Bio Awards. Phoenix, AZ.
• Gomez, A., Laniewski, P., Hire, G., & Herbst-Kralovetz, M. (2016, Spring). Epithelial barrier integrity is altered by vaginal microbiota in a site and species-specific manner in the female reproductive tract.. 11th Annual Frontiers in Immunobiology & Immunopathogenesis Symposium. Tucson, AZ.
• Herbst-Kralovetz, M., Laniewski, P., Barnes, D., Goulder, A., & Chase, D. (2016, Spring). The functional role of the vaginal microbiome in promotion of infection-associated cancer in high risk populations. Flinn Foundation and ABRC Awardee Research Conference. Phoenix, AZ.
• Laniewski, P., Barnes, D., Goulder, A., Chase, D., & Herbst-Kralovetz, M. (2016, Spring). . Evaluation of cervicovaginal mucosal immune responses in patients with human papilloma virus-associated cervical cancer. 11th Annual Frontiers in Immunobiology & Immunopathogenesis Symposium. Tucson, AZ.
• Laniewski, P., Hire, G., Gomez, A., & Herbst-Kralovetz, M. (2016, Summer). Human endometrial epithelial barrier integrity is altered by vaginal microbiota in a species-specific manner. Keystone Symposia: Gut Microbiota in Health and Disease. Newport, Rhode Island.
• Gomez, A., Laniewski, P., & Herbst-Kralovetz, M. (2015, June). Vaginal Microbiota Induce Site-Specific Changes in Barrier Function and Integrity in the Female Reproductive Tract. BMS Faculty Retreat. Phoenix: BMS Department.
• Herbst-Kralovetz, M., Winkle, S., & Throop, A. (2015, September). Human IL-36 gamma as an indicator of vaginal infection and promoter of mucosal inflammation. World STI and HIV Congress. Brisbane, Australia: IUSTI, ASHM.
• Hire, G., Laniewski, P., & Herbst-Kralovetz, M. (2015, June 2015). Site-specific Difference in Cytotoxicity Following Gardnerella vaginalis Infection of Human Female Reproductive Tract Epithelia. BMS Retreat. Phoenix, AZ: BMS Department.
• Winkle, S., Throw, A., & Herbst-Kralovetz, M. (2015, Summer). Microbe-dependent Induction of IL-36g at the Human Vaginal and Cervical Epithelial Cell Interfaces. American Society of Microbiology General Meeting. New Orleans, LA: American Society of Microbiology.
• Majewski, L., Winkle, S., & Herbst-Kralovetz, M. -. (2014, March). IL-17 and IL-22 Impact Immune and Epithelial Barrier Function in the Female Reproductive Tract. 9th Annual Immunobiology/Immunopathogenesis Symposium.
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  IL-17 and IL-22 Impact Immune and Epithelial Barrier Function in the Female Reproductive TractLauren Majewski, Sean Winkle, Melissa M. Herbst-KralovetzDepartment of Basic Medical Sciences, College of Medicine-Phoenix; The University of Arizona, Phoenix, AZ, USAThe T helper 17 (Th17) cell subset secrete IL-17 and IL-22. These cytokines act directly on epithelial cells by regulating homeostasis and mucosal epithelial barrier function in the skin, gut and respiratory tract. Despite active research on the function of Th17 cells in the female reproductive tract (FRT), few studies have examined the impact that Th17 cytokines have on the epithelial immune barrier at this mucosal site. To restrict pathogen invasion, IL-17 promotes cellular recruitment and activation mediating a strong proinflammatory response from epithelial cells. This cellular response induces secretion of IL-6, IL-8, and several growth factors linking IL-17 to numerous autoimmune disorders. IL-22 also has important functions in host defense of mucosal barriers as well as in tissue repair. In the gut, IL-22 has been shown to promote barrier function through induction of antimicrobial peptides and mucins, a major component of mucus. IL-22 can be beneficial to the host in protection against infection, but depending on the target tissue it can be damaging due to proinflammatory responses, which are enhanced when IL-22 is released together with IL-17. To investigate the impact of these Th17 cytokines on immune barrier function in the FRT, three-dimensional human FRT epithelial cells were treated with IL-17 and IL-22 at varying concentrations and cells were collected at 24 hours. Quantitative RT-PCR assays were utilized to analyze differences in mucin expression following treatment with these Th17 cytokines. In addition, epithelial-specific cytokine and chemokine (CCL20, IL-1β, IL-6, and IL-36γ) expression was measured. Cell-associated mucin and innate inflammatory mediator expression were measured to determine the impact of these Th17 cytokines on epithelial immune barrier function. We determined that individually both IL-17 and IL-22 induced expression of IL-1β, IL-6, CCL20, and IL-36γ by 3D FRT epithelial cells. Treatment with both IL-17 and IL-22 resulted in a significant increase in the expression of these cytokines, therefore demonstrating a synergistic activity that promoted inflammation. When compared to untreated cells, IL-17, IL-22 and IL-17/IL-22 treatments significantly increased expression of cell-associated mucins (MUC1 and MUC16) that could promote barrier function. Our data suggest that the immune epithelial barrier of the FRT is significantly modified following exposure to IL-17 or IL-22 and co-delivery of these Th17 cytokines amplifies this response. In conclusion, further investigation into the epithelial barrier function will reveal if these Th17 cytokines have similar or disparate roles in promoting inflammation that is protective or damaging to the reproductive mucosa.
• Winkle, S., & Herbst-Kralovetz, M. -. (2014, March). IL-36γ Induces and Regulates Pro-inflammatory Cytokine Response to Inflammation in the Female Reproductive Tract. 9th Annual Immunobiology/Immunopathogenesis Symposium.
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  IL-36γ Induces and Regulates Pro-inflammatory Cytokine Response to Inflammation in the Female Reproductive Tract Sean Winkle, Melissa Herbst-KralovetzBasic Medical Sciences, College of Medicine-Phoenix,University of Arizona, Phoenix, ArizonaInflammation in the human female reproductive tract (FRT) is an important part of the complex immune response propagated by exposure to bacterial and viral agents. The IL-36 family is a recently identified and characterized group of IL-1 cytokines, which are well known for their involvement in the immune system and role in inflammation. IL-36γ, an isoform of the IL-36 family has been shown to promote inflammation and induce proinflammatory responses in the epithelial tissue. While IL-36γ has not previously been shown to be expressed in the FRT, it has been shown to be expressed at epithelial and mucosal sites throughout the body. We have previously shown that IL-36γ induction in the FRT is microbe-dependent using a variety of vaginotropic organisms. To further investigate the proinflammatory activity of this novel cytokine in the FRT and extend these findings, we treated our human 3-D organotypic FRT EC models with poly(I:C) (1,25, and 100ug/ml), flagellin (0.5 and 5ug/ml), and FSL-1 (0.1 and 1ug/ml) to determine differential inflammatory response in terms of IL-36γ expression. We found that poly(I:C), flagellin, and FSL-1 induced expression of IL-36γ in a dose dependent manner. While poly(I:C) and FSL-1 induced secretion of the cytokine, flagellin upregulated intracellular production of IL-36γ but did not induce secretion. In addition, poly(I:C) induced the IL-36γ receptor IL-1Rrp2 suggesting that IL-36γ exhibits autocrine behavior. To verify that IL-36γ bolsters autocrine activity we treated our 3-D FRT EC with recombinant IL-36γ (1, 10, and 100ng/ml). Both IL-36γ and IL-1Rrp2 were induced in dose dependent fashion signifying that autocrine activity was apparent. IL-36γ treatments also induced expression and secretion of inflammatory cytokines IL-6 and IL-8 and Th17 chemokine, CCL20. The observed induction of CCL20 prompted us to treat 3-D FRT EC with other cytokines (IL-17 (1, 10, and 100ng/ml)) alone and in combination with microbial products (poly(I:C) (25ug/ml) and flagellin 5ug/ml). IL-17 treatment resulted in the dose dependent induction of IL-36γ as well as upregulation of IL-36γ, IL-1β and CCL20 when IL-17 was co-delivered with poly(I:C) or flagellin in the FRT epithelium. IL-36γ is potentially an important biomarker of epithelial activation by pathogenic insult and inflammation in the FRT. Furthermore, IL-36γ plays a critical role in inflammation pathways in the FRT by promoting the expression of proinflammatory cytokines and antimicrobial peptides.
• Wright, K., Winkle, S., & Herbst-Kralovetz, M. -. (2014, March). Human Epididymis Protein 4 is a Potential Inflammatory Biomarker of Dysbiosis in the Female Reproductive Tract. 9th Annual Immunobiology/Immunopathogenesis Symposium.
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  Human Epididymis Protein 4 is a Potential Inflammatory Biomarker of Dysbiosis in the Female Reproductive Tract Kayla L. Wright, Sean Winkle, and Melissa M. Herbst-Kralovetz Department of Basic Medical Sciences, College of Medicine-PhoenixThe University of Arizona College of Medicine-Phoenix, Phoenix, AZHuman Epididymis Protein 4 (HE4) is a protease inhibitor known to have multiple functions, including that of an antimicrobial peptide. Recently, HE4 has been investigated as a specific early biomarker to detect and monitor ovarian epithelial cancers. In clinical samples, HE4 has been shown to positively correlate with particular bacterial species of the vaginal microbiota in women suffering from bacterial vaginosis (BV). HE4 could serve as a potential inflammatory biomarker for vaginal dysbiosis, including in women with BV. BV is a condition in which the normal vaginal microbiota is disturbed and replaced by an overgrowth of certain “unhealthy” bacteria. Due to the fact that BV can be caused by a multitude of different microbial species, it is an extremely difficult condition to diagnose. This condition is, however, associated with many obstetrical and gynecological complications, including preterm labor and delivery, pelvic inflammatory disease, and predisposition to sexually transmitted infections, including HIV. Bacteria from the Lactobacillus family such as Lactobacillus crispatus and Lactobacillus iners have been identified with a “healthy” vaginal microbial environment, whereas bacteria including the gram positive Atopobium vaginae and the gram negative Prevotella bivia are associated with BV and a “disrupted” vaginal environment. In order to further investigate the induction and regulation of this protease inhibitor as an inflammatory biomarker, we utilized our 3-D human female reproductive tract models colonized with either “healthy” or BV-associated bacteria (BV-AB) and compared to uninfected control cells. Through conventional and quantitative RT-PCR analysis, we have determined that HE4 is upregulated in both our 3-D vaginal and endocervical cells infected with BV-AB, indicating the potential of HE4 as a highly specific biomarker for BV-AB. In contrast, colonization with the “healthy” Lactobacillus spp. and uninfected controls did not induce expression of HE4. In addition, we treated 3-D vaginal cells with microbial products that trigger toll-like receptors (TLR): FSL-1 (a synthetic lipoprotein; TLR2/6 agonist), flagellin (TLR5 agonist), and poly(I:C), synthetic dsRNA (TLR3 agonist) to mimic viral infection, to determine the level of HE4 induction. Conventional RT-PCR analysis indicated that FSL-1 and flagellin treatment upregulated HE4 expression in our 3-D vaginal cells, whereas poly(I:C) did not alter expression. Therefore our data suggests that specific bacteria and bacterial components induce HE4 expression, whereas poly(I:C) does not. This data indicates that HE4 may serve as a specific and discriminating inflammatory biomarker for vaginal dysbiosis or bacterial exposure in the female reproductive tract and warrants further investigation.
• Doerflinger, S. Y., Radtke, A. L., Tsinajinnie, D., & Herbst-Kralovetz, M. -. (2013, Spring). Modeling the Vaginal Microflora and Microenviroment to More Accurately Predict Compound Toxicity. 8th Annual Frontiers in Immunobiology and Immunopathogenesis Symposium, UA, Tucson, AZ.
• McGowin, C. L., Radtke, A. L., Favaloro, S., Martin, D. H., & Herbst-Kralovetz, M. -. (2013, Spring). Mycoplasma genitalium infection activates host defense and inflammation pathways, and leads to recruitment of leukocytes to the cervical epithelium. ASM General Meeting. Denver, CO.
• Radtke, A. L., Winkle, S., & Herbst-Kralovetz, M. -. (2013, Spring). IL-36 induction in the human female reproductive tract is microbe-dependent. AAI General Meeting 2013. Honolulu, HI.
• Radtke, A., Doerflinger, S., & Herbst-Kralovetz, M. -. (2013, Spring). Host Discrimination of Commensal vs. Pathogenic Bacteria in the Human Vaginal Epithelium. 16th International Congress of Mucosal Immunology (ICMI 2013), Vancouver, Canada.
• Winkle, S., Herbst-Kralovetz, M. -., & Radtke, A. L. (2013, Spring). Induction and Regulation of IL-36 at the Female Reproductive Mucosa.. 8th Annual Frontiers in Immunobiology and Immunopathogenesis Symposium, UA, Tucson, AZ.
• McGowin, C. L., Annan, R. S., Radtke, A. L., Martin, D. H., & Herbst-Kralovetz, M. -. (2012, Spring). Characterization of antimicrobial peptides and inflammatory responses to acute and persistent Mycoplasma genitalium infection of the human endocervix.. ASM General Meeting, San Franscico, CA.
• Radtke, A. L., Quayle, A. J., & Herbst-Kralovetz, M. -. (2012, Spring). Investigating the Pathogen-Specific Innate Immune Responses of the Endocervix Using a Human 3-D Endocervical Epithelial Cell Model.. ASM General Meeting, San Franscico, CA..
• Radtke, A. L., Emmert, N., Abraham, K., Nickerson, C. A., & Herbst-Kralovetz, M. -. (2011, Spring). Role of Mucins in Limiting HSV-2 Infection in an Organotypic Human Vaginal Epithelial Model.. ASM General Meeting, New Orleans, LA.
• Radtke, A. L., Herbst-Kralovetz, M. -., & Jackson, E. (2011, Spring). . Evaluation of microbicide toxicity and efficacy using a novel 3D human organotypic co-culture vaginal cell culture model. 8th World Congress on Alternatives and Animal Use in the Life Sciences. Montreal, Canada.
• Herbst-Kralovetz, M. -., Berta, A., Hjelm, B., Kilbourne, J., & Arntzen, C. (2009, Spring). Enhancing Intranasal Immunization of Virus-like Particles with Multifactorial Pattern Recognition Receptor Agonist-based Adjuvants.. Keystone Symposia Pattern Recognition Molecules and Immune Sensors of Pathogens. Banff, Alberta, Canada.
• Hjelm, B., Berta, A., Nickerson, C., Arntzen, C., & Herbst-Kralovetz, M. -. (2009, Spring). An In Vitro Human Vaginal Epithelial Cell Model for High-Throughput Microbicide Screening.. The 1st International Symposium on Advancing Prevention Technologies for Sexual and Reproductive Health: A Strategy Symposium, Berkeley, CA..
• Hjelm, B., Berta, A., Nickerson, C., Arntzen, C., & Herbst-Kralovetz, M. -. (2009, Spring). Development and Characterization of a 3-D Human Vaginal Epithelia Model for Microbicide Screening.. Annual STI-CRC Meeting, Bethesda, MD.
• Herbst-Kralovetz, M. -. (2008, Spring). . Enhancing Mucosal Vaccination and Vaginal Immune Responses with Tobacco-derived Virus-like Particles Through TLR Signaling.. Annual STI-CRC meeting at University of North Carolina-Chapel Hill..
• Hjelm, B., Berta, A., Nickerson, C., Arntzen, C., & Herbst-Kralovetz, M. -. (2008, Spring). Development of a 3-D Model of Human Vaginal Epithelia for Microbicide Screening.. 7th Arizona Biosciences Leadership Symposium: Translational Medicine, Tucson, AZ..
• Herbst-Kralovetz, M. -., Hjelm, B., LaVigne, D., Kilbourne, J., Arntzen, C., Chen, Q., & Mason, H. (2007, Spring). Utilizing TLR Agonists to Augment Mucosal Vaccination with Tobacco-derived Norwalk Virus Capsid Protein Virus-like Particles.. Annual STI-CRC meeting at University of Washington.
• Herbst-Kralovetz, M. -., Hjelm, B., LaVigne, D., Kilbourne, J., Arntzen, C., Chen, Q., & Mason, H. (2007, Spring). Utilizing TLR Agonists to Augment Mucosal Vaccination. Keystone Symposia in Immunologic Memory.
• Herbst-Kralovetz, M. -., Hjelm, B., Straub, T., Richter, E., Arntzen, C., & Nickerson, C. (2007, Spring). Development of a Virus Neutralization Assay for Evaluation/Screening of Human Norovirus Vaccine Candidates.. 3rd International Calicivirus Conference, Cancun, Mexico.
• Tacket, C. O., Herbst-Kralovetz, M. -., Hjelm, B., LaVigne, D., Kilbourne, J., Arntzen, C., Chen, Q., & Mason, H. (2007, Spring). Tobacco-derived Norwalk Capsid Protein VLPs as a Platform Technology to Induce Systemic and Mucosal Immunity. Annual STI-CRC meeting at University of Washington.
• Herbst-Kralovetz, M. -., Arntzen, C. J., & Pyles, R. B. (2006, Spring). Adjuvant Activity of Mucosally Applied Nucleic Acid-Based TLR Agonists.. Modern Vaccines, Adjuvants and Delivery Systems.
• Herbst-Kralovetz, M. -., & Pyle, R. B. (2005, Spring). Innate Immune Mechanisms of the Genital Mucosa in Response to Nucleic Acid-based TLR agonists.. Keystone Symposia in Innate Immunity.
• Herbst-Kralovetz, M. -., & Pyles, R. B. (2004, Spring). Nucleic Acid-based TLR Agonists Protect Against Experimental Genital Herpes Simplex Virus Type 2 (HSV-2) Infection.. Pathology Trainee Research Session.
• Herbst-Kralovetz, M. -., & Pyles, R. B. (2004, Spring). Quantification of Protection Afforded by Immunostimulatory Sequence-containing Oligonucleotides During Genital Herpes Simplex Virus Type 2 (HSV-2) Infection. General American Society for Microbiology Meeting.
• Herbst-Kralovetz, M. -., & Pyles, R. B. (2004, Spring). Quantification of Protection Afforded by Nucleic Acid-based TLR Agonists Against Genital Herpes Simplex Virus Type 2 (HSV-2) Infection. James W. McLaughlin Fund Symposia.
• Herbst-Kralovetz, M. -., Greene, S., Quayle, A. J., & Pyles, R. B. (2004, Spring). Human Vaginal and Cervical Epithelial Cell TLR Expression and Activation to Prevent Infection by Herpes Simplex Virus Type 2 (HSV-2).. CIRWH Meeting.
• Herbst-Kralovetz, M. -., Higgins, D., Davis, M. M., Van Nest, G., & Pyles, R. B. (2003, Spring). Vaginal Treatment of Genital Herpes Simplex Virus Type 2 (HSV-2) Infection by Immunostimulatory Sequence-containing Oligonucleotides.. CIRWH Meeting.

Reviews

• Baker, J. M., Al-Nakkash, L., & Herbst-Kralovetz, M. M. (2017. Estrogen-gut microbiome axis: Physiological and clinical implications.(pp 45-53).
• Gardner, J. K., & Herbst-Kralovetz, M. M. (2016. Three-Dimensional Rotating Wall Vessel-Derived Cell Culture Models for Studying Virus-Host Interactions.
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  The key to better understanding complex virus-host interactions is the utilization of robust three-dimensional (3D) human cell cultures that effectively recapitulate native tissue architecture and model the microenvironment. A lack of physiologically-relevant animal models for many viruses has limited the elucidation of factors that influence viral pathogenesis and of complex host immune mechanisms. Conventional monolayer cell cultures may support viral infection, but are unable to form the tissue structures and complex microenvironments that mimic host physiology and, therefore, limiting their translational utility. The rotating wall vessel (RWV) bioreactor was designed by the National Aeronautics and Space Administration (NASA) to model microgravity and was later found to more accurately reproduce features of human tissue in vivo. Cells grown in RWV bioreactors develop in a low fluid-shear environment, which enables cells to form complex 3D tissue-like aggregates. A wide variety of human tissues (from neuronal to vaginal tissue) have been grown in RWV bioreactors and have been shown to support productive viral infection and physiological meaningful host responses. The in vivo-like characteristics and cellular features of the human 3D RWV-derived aggregates make them ideal model systems to effectively recapitulate pathophysiology and host responses necessary to conduct rigorous basic science, preclinical and translational studies.
• Yarbrough, V. L., Winkle, S., & Herbst-Kralovetz, M. M. (2014. Antimicrobial peptides in the female reproductive tract: a critical component of the mucosal immune barrier with physiological and clinical implications.
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  At the interface of the external environment and the mucosal surface of the female reproductive tract (FRT) lies a first-line defense against pathogen invasion that includes antimicrobial peptides (AMP). Comprised of a unique class of multifunctional, amphipathic molecules, AMP employ a wide range of functions to limit microbial invasion and replication within host cells as well as independently modulate the immune system, dampen inflammation and maintain tissue homeostasis. The role of AMP in barrier defense at the level of the skin and gut has received much attention as of late. Given the far reaching implications for women's health, maternal and fetal morbidity and mortality, and sexually transmissible and polymicrobial diseases, we herein review the distribution and function of key AMP throughout the female reproductive mucosa and assess their role as an essential immunological barrier to microbial invasion throughout the reproductive cycle of a woman's lifetime.

Others

• Herbst-Kralovetz, M., Gomez, A., & Laniewski, P. (2016, May 2016). Cover Image for JID supplemental.
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  JID Cover image: SEM of Gardnerella vaginalis on the surface of vaginal epithelial cells.
• Herbst-Kralovetz, M. -. (2014, November). Norovirus Grown in Lab, with Help from Bacteria. Science News.
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  Expert comment.
• Herbst-Kralovetz, M. -. (2013, September). Nanoparticle vaccine offers better protection Particles that deliver vaccines directly to mucosal surfaces could defend against many infectious diseases.. MIT News. http://web.mit.edu/newsoffice/2013/nanoparticle-vaccine-offers-better-protection-0925.html
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  I provided expert comment on the research published in a recent journal, this publication quotes my commentary
• Mason, H. S., & Herbst-Kralovetz, M. -. (2011, Spring). Plant-derived antigens as mucosal vaccines. In: Current Topics in Microbiology and Immunology; Mucosal Vaccines: Modern Concepts, Strategies, and Challenges. Curr Top Microbiol Immunol..
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  (Kozlowski, PA, ed.) [Epub ahead of print] PMID: 21811930