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Description of western blot methodology for supplemental Figure and sources of gene expression data.

Supplemental Figure 1: The ability of 34.5ENVE to infect and replicate in DB-7 and Met-1 BC cells

Supplemental Figure S7. TNFα induces apoptosis in oHSV infected glioma cells.

Supplemental Figure S3. Representative fluorescent images of uninfected and infected co-cultures 12 hours post infection.

Supplemental Figure S2. Species specificity of murine TNFα ELISA kit for murine and not human TNFα

Co-culture model of endogenous Vstat120 impact on microglia response to oHSV-infected glioma.

Supplemental Figure S4. oHSV treatment increases secreted TNFα.

Supplementary Materials and Methods

Supplemental Figure S1. Percoll gradient isolated CD11b+CD45hi cells are predominantly infiltrating macrophages

Supplemental Figure S6. TNFα increases cell proliferation and is not cytotoxic to uninfected GB cells.

Ewing sarcoma subcutaneous tumors stained for CD68+ macrophages, with hematoxylin counterstain.

List of genes differentially induced ({greater than or equal to} 1.5 fold) in macrophages cultured with rHSVQ1 (Q) vs. RAMBO (R) infected glioma cells (UI, uninfected).

Supplemental Figure Legends from The Impact of Macrophage- and Microglia-Secreted TNFα on Oncolytic HSV-1 Therapy in the Glioblastoma Tumor Microenvironment

Supplemental Figure S5. Murine TNFα reduces viral replication in infected human glioma cells.

Purpose: Oncolytic herpes simplex viruses (oHSV) represent a promising therapy for glioblastoma (GBM), but their clinical success has been limited. Early innate immune responses to viral infection reduce oHSV replication, tumor destruction, and efficacy. Here, we characterized the antiviral effects of macrophages and microglia on viral therapy for GBM.Experimental Design: Quantitative flow cytometry of mice with intracranial gliomas (±oHSV) was used to examine macrophage/microglia infiltration and activation. In vitro coculture assays of infected glioma cells with microglia/macrophages were used to test their impact on oHSV replication. Macrophages from TNFα-knockout mice and blocking antibodies were used to evaluate the biologic effects of TNFα on virus replication. TNFα blocking antibodies were used to evaluate the impact of TNFα on oHSV therapy in vivo.Results: Flow-cytometry analysis revealed a 7.9-fold increase in macrophage infiltration after virus treatment. Tumor-infiltrating macrophages/microglia were polarized toward a M1, proinflammatory phenotype, and they expressed high levels of CD86, MHCII, and Ly6C. Macrophages/microglia produced significant amounts of TNFα in response to infected glioma cells in vitro and in vivo. Using TNFα-blocking antibodies and macrophages derived from TNFα-knockout mice, we discovered TNFα-induced apoptosis in infected tumor cells and inhibited virus replication. Finally, we demonstrated the transient blockade of TNFα from the tumor microenvironment with TNFα-blocking antibodies significantly enhanced virus replication and survival in GBM intracranial tumors.Conclusions: The results of these studies suggest that FDA approved TNFα inhibitors may significantly improve the efficacy of oncolytic virus therapy. Clin Cancer Res; 21(14); 3274–85. ©2015 AACR.

List of genes significantly up-regulated (>1.5 fold) in macrophages upon culture with glioma cells infected with RAMBO (R) or rHSVQ1 (Q) relative to uninfected (UI) cells.

The purpose of this study is to qualitatively explore transgender and nonbinary (TGNB) individuals’ experiences with bathroom use in neighborhoods and public spaces in the context of navigating experiences of harassment, lack of safety, and discrimination in New York City. Forty diverse TGNB individuals were recruited in the summer of 2017 in New York City for semi-structured qualitative interviews on health and neighborhoods. Inductive thematic analysis coding process was used to determine themes. Themes related to public bathroom use were compiled and analyzed. A total of 31 participant interviews identified bathroom use as an experience that shapes their health and wellbeing. Major themes included avoiding restroom use or planning travel around perceived bathroom access; planning bathroom use compromised mental health and self-image; delaying bathroom use to avoid harassment that leads to negative physical health effects; perceived gender presentation limited bathroom use; and bathrooms advertised as gender-neutral promoted safety and comfort. In a diverse, urban sample of TGNB individuals, bathroom use was identified as relevant to physical and mental health. Despite progressive bathroom laws in the state and city in which the study was conducted, respondents described how bathroom use influences how they move throughout their day. We demonstrate the continued importance of nondiscriminatory access to public spaces such as restrooms to the health of TGNB populations and the need for further prospective research elucidating the impact of nondiscrimination legislation.

Background There are specific issues regarding sexual orientation (SO) collection and analysis among transgender and nonbinary patients. A limitation to meaningful SO and gender identity (GI) data collection is their consideration as a fixed trait or demographic data point. Methods A de-identified patient database from a single electronic health record (EHR) that allows for searching any discrete data point in the EHR was used to query demographic data (sex assigned at birth and current GI) for transgender individuals from January 2011 to March 2020 at a large urban tertiary care academic health center. Results A cohort of transgender individuals were identified by using EHR data from a two-step demographic question. Almost half of male identified (46.70%, n = 85) and female identified (47.51%, n = 86) individuals had “heterosexual/straight” input for SO. Overall, male and female identified (i.e., binary) GI aggregate categories had similar SO responses. Assigned male at birth (AMAB) nonbinary individuals (n = 6) had “homosexual/gay” SO data input. Assigned female at birth (AFAB) nonbinary individuals (n = 56) had almost half “something else” SO data input (41.67%, n = 15). Individuals with “choose not to disclose” for GI (n = 249) almost all had “choose not to disclose” SO data (96.27%, n = 232). Conclusion Current SO categories do not fully capture transgender individuals' identities and experiences, and limit the clinical and epidemiological utility of collecting this data in the current form. Anatomical assumptions based on SO should be seen as a potential shortcoming in over-reliance on SO as an indicator of screening needs and risk factors.

Background: A vast amount of research has demonstrated the numerous adverse health risks of short sleep duration and poor sleep health among the general population, and increasing studies have been conducted among lesbian, gay, and bisexual individuals. However, although poor sleep health is disproportionately experienced by sexual and gender minority populations, little research has examined sleep quality and associated factors among transgender and gender nonbinary (TGNB) individuals. This study qualitatively explored the relationship that factors such as gender identity, mental health, and substance use have with sleep health among a sample of TGNB individuals in New York City. Methods: Forty in-depth interviews were conducted among an ethnically diverse sample who identified as transgender male, transgender female, and gender nonbinary from July to August 2017. All interviews were transcribed, coded, and thematically analyzed for domains affecting overall sleep, including mental health, gender identity, and various coping mechanisms to improve overall sleep. Results: TGNB interview participants frequently described one or more problems with sleeping. Some (15%) participants suggested that mental health issues caused them to have difficulty falling asleep, but that psychiatric medication was effective in reducing mental health issues and allowing them to sleep. An even larger number (35%) told us that their gender identity negatively impacted their sleep. Specifically, participants described that the presence of breasts, breast binding, stress and anxiety about their identity, and concerns about hormonal therapy and gender-affirming surgery were all reported as contributing to sleep problems. Given these sleep challenges, it is not surprising that most (60%) participants used various strategies to cope with and manage their sleep problems, including prescription and over-the-counter sleep medications (33%) and marijuana (18%). Conclusions: Our findings document that sleep health is frequently an issue for TGNB individuals, and they also offer insight into the various ways that TGNB individuals attempt to cope with these sleep problems. Sleep health promotion interventions should be developed for TGNB people, which would promote positive mental health, reduce the risk of pharmaceutical adverse events, and help alleviate psychosocial stress in this target population.

PURPOSE: Earlier literature has reported on the utility of diagnostic codes and demographic information for identifying transgender patients. We aim to assess which method identifies the most transgender patients utilizing readily available tools from within the electronic health record (EHR). METHODS: A de-identified patient database from a single EHR that allows for searching any discrete data point in the EHR was used to query International Statistical Classification of Diseases and Related Health Problems, 10th Revision (ICD-10) diagnostic codes and demographic data specific to transgender patients from January 2011 to April 2019. RESULTS: Demographic data and ICD-10 codes yielded 1494 individual EHRs with transgender-specific data domains. ICD-10 diagnostic codes alone identified 942 (63.05%) unique EHRs. Demographics alone identified 218 (14.59%) unique EHRs. A total of 334 (22.36%) unique EHRs had both ICD-10 and demographic identifiers. Of those identified by transgender-specific demographic data (552), 294 (53.26%) were trans masculine, 215 (38.95%) were trans feminine, and 43 (7.79%) were nonbinary. Of the 552 demographic-identified transgender patients, 141 (25.86%) were identified by a two-part gender identity demographic question. CONCLUSIONS: ICD-10 diagnostic codes, not demographic data, identified the most transgender patient records, but neither diagnostic codes alone nor demographic data captured the full population. Only 26.36% of the charts identified as transgender patients had both ICD-10 codes and demographic data. We recommend that when identifying transgender populations through EHR domains, a combination of diagnostic codes and demographic data be used. Furthermore, research is needed to optimize disclosure and collection of demographic information for gender minority populations.

Institutional guidelines for transgender children and adolescent minors fail to adequately address a critical juncture of care of this population: how to proceed if a minor and their parents have disagreements concerning their gender-affirming medical care. Through arguments based on ethical, paediatric, adolescent and transgender health research, we illustrate ethical dilemmas that may arise in treating transgender and gender diverse youth. We discuss three potential avenues for providing gender-affirming care over parental disagreement: legal carve-outs to parental consent, the mature minor doctrine and state intervention for neglect. Our discussion approaches this parent–child disagreement in a manner that prioritises the developing autonomy of transgender youth in the decision-making process surrounding medically assisted gender affirmation. We base our arguments in the literature surrounding the risks and benefits of gender-affirming therapy in transgender children and the existing legal basis for recognising minors’ decision-making authority in certain medical situations.

Medicine sometimes fails to address social, economic, and political determinants of health. But how far beyond clinical encounters should intervention efforts extend? Because prevention efforts can marginalize patients by stigmatizing certain behaviors, interrogating the scope of medicine's prevention obligations is important. Additionally, it is important to distinguish clinician preferences regarding patients' personal behaviors (presumably based on clinicians' hopes for patients' positive health outcomes) from clinician biases expressed (consciously or unconsciously) about those behaviors. I illustrate the urgency of asking how far medicine should be expected to go to prevent disease by sharing how my own medical training has stoked my personal fear of acquiring HIV.

Background: The published literature on education about transgender health within health professions curricula was previously found to be sporadic and fragmented. Recently, more inclusive and holistic approaches have been adopted. We summarize advances in transgender health education. Methods: A 5-stage scoping review framework was followed, including a literature search for articles relevant to transgender health care interventions in 5 databases (Education Source, LGBT Source, MedEd Portal, PsycInfo, PubMed) from January 2017 to September 2019. Search results were screened to include original articles reporting outcomes of educational interventions with a transgender health component that included MD/DO students in the United States and Canada. A gray literature search identified continuing medical education (CME) courses from 12 health professional associations with significant transgender-related content. Results: Our literature search identified 966 unique publications published in the 2 years since our prior review, of which 10 met inclusion criteria. Novel educational formats included interdisciplinary interventions, post-residency training including CME courses, and online web modules, all of which were effective in improving competencies related to transgender health care. Gray literature search resulted 15 CME courses with learning objectives appropriate to the 7 professional organizations who published them. Conclusions: Current transgender health curricula include an expanding variety of educational intervention formats driven by their respective educational context, learning objectives, and placement in the health professional curriculum. Notable limitations include paucity of objective educational intervention outcomes measurements, absence of long-term follow-up data, and varied nature of intervention types. A clear best practice for transgender curricular development has not yet been identified in the literature.

Sex tourism among men who have sex with men (MSM) has been associated with increased risk for human immunodeficiency virus (HIV) due to sexually scripted environments characterized by multiple sexual partners, increased availability of alcohol and drugs, and limited availability of HIV-prevention services. The current study examined the knowledge of and likelihood of using different modalities of pre-exposure prophylaxis (PrEP), an important biomedical HIV-prevention strategy, among MSM in Paris who have engaged in sex tourism. A sample of 580 MSM from a highly popular geosocial-networking smartphone application in Paris, France, participated in the survey. Of the 580 MSM, 444 participants reported an HIV-negative status and represent the analytic sample for this study. Approximately 27% reported engaging in sexual tourism. MSM who engaged in sex tourism were more likely to aware of on-demand PrEP and more likely to express interest in using on-demand PrEP (adjusted risk ratio [aRR] = 1.26; 95% confidence interval [CI] = 1.03-1.53, aRR = 1.29; 95% CI = 1.04-1.61, respectively) than MSM who never engaged in sex tourism. Moreover, participants who engaged in sex tourism were more likely to express interest in rectal microbicides or both rectal and penile microbicides (aRR = 1.34; 95% CI = 1.13-1.59, aRR = 1.26; 95% CI = 1.03-1.55, respectively) than participants who had not engaged in sex tourism. With the high likelihood of interest in using alternative forms of PrEP in MSM who engage in sex tourism, this study suggests potential benefits for these alternative forms of PrEP for this specific population and underscores the importance of their continued development.

Background A growing body of research continues to elucidate health inequities experienced by transgender individuals and further underscores the need for medical providers to be appropriately trained to deliver care to this population. Medical education in transgender health can empower physicians to identify and change the systemic barriers to care that cause transgender health inequities as well as improve knowledge about transgender-specific care. Methods We conducted structured searches of five databases to identify literature related to medical education and transgender health. Of the 1272 papers reviewed, 119 papers were deemed relevant to predefined criteria, medical education, and transgender health topics. Citation tracking was conducted on the 119 papers using Scopus to identify an additional 12 relevant citations (a total of 131 papers). Searches were completed on October 15, 2017 and updated on December 11, 2017. Results Transgender health has yet to gain widespread curricular exposure, but efforts toward incorporating transgender health into both undergraduate and graduate medical educations are nascent. There is no consensus on the exact educational interventions that should be used to address transgender health. Barriers to increased transgender health exposure include limited curricular time, lack of topic-specific competency among faculty, and underwhelming institutional support. All published interventions proved effective in improving attitudes, knowledge, and/or skills necessary to achieve clinical competency with transgender patients. Conclusion Transgender populations experience health inequities in part due to the exclusion of transgender-specific health needs from medical school and residency curricula. Currently, transgender medical education is largely composed of one-time attitude and awareness-based interventions that show significant short-term improvements but suffer methodologically. Consensus in the existing literature supports educational efforts to shift toward pedagogical interventions that are longitudinally integrated and clinical skills based, and we include a series of recommendations to affirm and guide such an undertaking.

The functional plasticity and anti-tumor potential of human γδ T cells have been widely studied. However, the epigenetic regulation of γδ T-cell/tumor cell interactions has been poorly investigated. In the present study, we show that treatment with the histone deacetylase inhibitor Valproic acid (VPA) significantly enhanced the expression and/or release of the NKG2D ligands MICA, MICB and ULBP-2, but not ULBP-1 in the pancreatic carcinoma cell line Panc89 and the prostate carcinoma cell line PC-3. Under in vitro tumor co-culture conditions, the expression of full length and the truncated form of the NKG2D receptor in γδ T cells was significantly downregulated. Furthermore, using a newly established flow cytometry-based method to analyze histone acetylation (H3K9ac) in γδ T cells, we showed constitutive H3K9aclow and inducible H3K9achigh expression in Vδ2 T cells. The detailed analysis of H3K9aclow Vδ2 T cells revealed a significant reversion of TEMRA to TEM phenotype during in vitro co-culture with pancreatic ductal adenocarcinoma cells. Our study uncovers novel mechanisms of how epigenetic modifiers modulate γδ T-cell differentiation during interaction with tumor cells. This information is important when considering combination therapy of VPA with the γδ T-cell-based immunotherapy for the treatment of certain types of cancer.

Low perception of HIV risk is a challenge to PrEP implementation. We analyzed associations between perceptions of PrEP candidacy, behavioral indications for PrEP, and sexual behaviors. We recruited a sample of 580 MSM from a geosocial-networking smartphone application in Paris, France. A modified Poisson regression model was conducted to examine associations between perceived candidacy for PrEP and behavioral indications for PrEP, and relationships among engagement in group sex, transactional sex, HIV test history, and indications for PrEP. Adjusted risk ratios (aRR) and 95% confidence intervals (CIs) were calculated. For the outcome of perceived candidacy for PrEP, a multinomial logistic regression was performed, and adjusted relative risk ratios (aRRR) were calculated. Multivariate analyses were adjusted for socio-demographics. Respondents who considered themselves PrEP candidates were more likely to meet PrEP eligibility criteria compared to those who did not consider themselves candidates (aRR 1.65; 95% CI 1.34-2.03). Those who had engaged in group or transactional sex were more likely to have behavioral indications for PrEP (aRR 1.27; 95% CI 1.07-1.50, aRR 1.32; 95% CI 1.13-1.56, respectively), whereas HIV test history was not significantly associated with behavioral indications for PrEP. Respondents who had engaged in group sex or transactional sex were more likely to perceive themselves as candidates for PrEP (aRRR 2.24; 95% CI 1.21-4.16, aRRR 2.58; 95% CI 1.09-6.13, respectively), although those never tested for HIV were less likely to perceive themselves as candidates for PrEP (aRRR 0.18; 95% CI 0.03-0.91). The elucidation of candidacy perceptions and risk behaviors is key to furthering the effective implementation of PrEP engagement interventions.

Purpose: Oncolytic herpes simplex viruses (oHSV) represent a promising therapy for glioblastoma (GBM), but their clinical success has been limited. Early innate immune responses to viral infection reduce oHSV replication, tumor destruction, and efficacy. Here, we characterized the antiviral effects of macrophages and microglia on viral therapy for GBM. Experimental Design: Quantitative flow cytometry of mice with intracranial gliomas (±oHSV) was used to examine macrophage/microglia infiltration and activation. In vitro coculture assays of infected glioma cells with microglia/macrophages were used to test their impact on oHSV replication. Macrophages from TNFα-knockout mice and blocking antibodies were used to evaluate the biologic effects of TNFα on virus replication. TNFα blocking antibodies were used to evaluate the impact of TNFα on oHSV therapy in vivo. Results: Flow-cytometry analysis revealed a 7.9-fold increase in macrophage infiltration after virus treatment. Tumor-infiltrating macrophages/microglia were polarized toward a M1, proinflammatory phenotype, and they expressed high levels of CD86, MHCII, and Ly6C. Macrophages/microglia produced significant amounts of TNFα in response to infected glioma cells in vitro and in vivo. Using TNFα-blocking antibodies and macrophages derived from TNFα-knockout mice, we discovered TNFα-induced apoptosis in infected tumor cells and inhibited virus replication. Finally, we demonstrated the transient blockade of TNFα from the tumor microenvironment with TNFα-blocking antibodies significantly enhanced virus replication and survival in GBM intracranial tumors. Conclusions: The results of these studies suggest that FDA approved TNFα inhibitors may significantly improve the efficacy of oncolytic virus therapy. Clin Cancer Res; 21(14); 3274–85. ©2015 AACR.

The 2-year survival rate of patients with breast cancer brain metastases is less than 2%. Treatment options for breast cancer brain metastases are limited, and there is an unmet need to identify novel therapies for this disease. Brain angiogenesis inhibitor 1 (BAI1) is a GPCR involved in tumor angiogenesis, invasion, phagocytosis, and synaptogenesis. For the first time, we identify that BAI1 expression is significantly reduced in breast cancer and higher expression is associated with better patient survival. Nestin is an intermediate filament whose expression is upregulated in several cancers. We found that higher Nestin expression significantly correlated with breast cancer lung and brain metastases, suggesting both BAI1 and Nestin can be therapeutic targets for this disease. Here, we demonstrate the ability of an oncolytic virus, 34.5ENVE, to target and kill high Nestin-expressing cells and deliver Vstat120 (extracellular fragment of BAI1). Finally, we created two orthotopic immune-competent murine models of breast cancer brain metastases and demonstrated 34.5ENVE extended the survival of immune-competent mice bearing intracranial breast cancer tumors. Mol Cancer Ther; 14(1); 307–14. ©2014 AACR.

Purpose: Brain angiogenesis inhibitor (BAI1) facilitates phagocytosis and bacterial pathogen clearance by macrophages; however, its role in viral infections is unknown. Here, we examined the role of BAI1, and its N-terminal cleavage fragment (Vstat120) in antiviral macrophage responses to oncolytic herpes simplex virus (oHSV). Experimental Design: Changes in infiltration and activation of monocytic and microglial cells after treatment of glioma-bearing mice brains with a control (rHSVQ1) or Vstat120-expressing (RAMBO) oHSV was analyzed using flow cytometry. Co-culture of infected glioma cells with macrophages or microglia was used to examine antiviral signaling. Cytokine array gene expression and Ingenuity Pathway Analysis (IPA) helped evaluate changes in macrophage signaling in response to viral infection. TNFα-blocking antibodies and macrophages derived from Bai1−/− mice were used. Results: RAMBO treatment of mice reduced recruitment and activation of macrophages/microglia in mice with brain tumors, and showed increased virus replication compared with rHSVQ1. Cytokine gene expression array revealed that RAMBO significantly altered the macrophage inflammatory response to infected glioma cells via altered secretion of TNFα. Furthermore, we showed that BAI1 mediated macrophage TNFα induction in response to oHSV therapy. Intracranial inoculation of wild-type/RAMBO virus in Bai1−/− or wild-type non–tumor-bearing mice revealed the safety of this approach. Conclusions: We have uncovered a new role for BAI1 in facilitating macrophage anti-viral responses. We show that arming oHSV with antiangiogenic Vstat120 also shields them from inflammatory macrophage antiviral response, without reducing safety. Clin Cancer Res; 23(7); 1809–19. ©2016 AACR.