A phase I prospective, non-randomized trial of autologous dendritic cell-based cryoimmunotherapy in patients with metastatic castration-resistant prostate cancer.

Metastatic castration-resistant prostate cancer (mCRPC) is an immunologically cold disease with dismal outcomes. Cryoablation destroys cancer tissue, releases tumor-associated antigens and creates a pro-inflammatory microenvironment, while dendritic cells (DCs) activate immune responses through processing of antigens. Immunotherapy combinations could enhance the anti-tumor efficacy. This open-label, single-arm, single-center phase I trial determined the safety and tolerability of combining cryoablation and autologous immature DC, without and with checkpoint inhibitors. Immune responses and clinical outcomes were evaluated. Patients with mCRPC, confirmed metastases and intact prostate gland were included. The first participants underwent prostate cryoablation with intratumoral injection of autologous DCs in a 3 + 3 design. In the second part, patients received cryoablation, the highest acceptable DC dose, and checkpoint inhibition with either ipilimumab or pembrolizumab. Sequentially collected information on adverse events, quality of life, blood values and images were analyzed by standard descriptive statistics. Neither dose-limiting toxicities nor adverse events > grade 3 were observed in the 18 participants. Results indicate antitumor activity through altered T cell receptor repertoires, and 33% durable (> 46 weeks) clinical benefit with median 40.7 months overall survival. Post-treatment pain and fatigue were associated with circulating tumor cell (CTC) presence at inclusion, while CTC responses correlated with clinical outcomes. This trial demonstrates that cryoimmunotherapy in mCRPC is safe and well tolerated, also for the highest DC dose (2.0 × 108) combined with checkpoint inhibitors. Further studies focusing on the biologic indications of antitumor activity and immune system activation could be considered through a phase II trial focusing on treatment responses and immunologic biomarkers.

Persistent Immune Activation in Human Immunodeficiency Virus-Infected Pregnant Women Starting Combination Antiretroviral Therapy After Conception.

This study evaluated the impact of human immunodeficiency virus (HIV) and combination antiretroviral therapy (cART) on immune activation during pregnancy in a Zambian cohort of HIV-exposed but uninfected children followed up from birth. Activated CD8+ T cells (CD38+ and HLA-DR+) were compared among HIV-uninfected (n = 95), cART experienced HIV-infected (n = 111), and cART-naive HIV-infected (n = 21) pregnant women. Immune activation was highest among HIV-infected/cART-naive women but decreased during pregnancy. Immune activation HIV-infected women who started cART during pregnancy was reduced but not to levels similar to those in HIV-uninfected women. The effects of elevated maternal immune activation in pregnancy on subsequent infant health and immunity remain to be determined.

Lack of Trex1 Causes Systemic Autoimmunity despite the Presence of Antiretroviral Drugs.

Biallelic mutations of three prime repair exonuclease 1 (TREX1) cause the lupus-like disease Aicardi-Goutières syndrome in which accumulation of a yet unknown endogenous DNA substrate of TREX1 triggers a cyclic GMP-AMP synthase-dependent type I IFN response and systemic autoimmunity. Products of reverse transcription originating from endogenous retroelements have been suggested to be a major substrate for TREX1, and reverse transcriptase inhibitors (RTIs) were proposed as a therapeutic option in autoimmunity ensuing from defects of TREX1. In this study, we treated Trex1-/- mice with RTIs. The serum RTI levels reached were sufficient to block retrotransposition of endogenous retroelements. However, the treatment did not reduce the spontaneous type I IFN response and did not ameliorate lethal inflammation. Furthermore, long interspersed nuclear elements 1 retrotransposition was not enhanced in the absence of Trex1. Our data do not support the concept of retroelement-derived cDNA as key triggers of systemic autoimmunity in Trex1-deficient humans and mice and motivate the continuing search for the pathogenic IFN-inducing Trex1 substrate.

GM-CSF, Flt3-L and IL-4 affect viability and function of conventional dendritic cell types 1 and 2.

Conventional type 1 dendritic cells (cDC1) and conventional type 2 dendritic cells (cDC2) have attracted increasing attention as alternatives to monocyte-derived dendritic cells (moDCs) in cancer immunotherapy. Use of cDCs for therapy has been hindered by their low numbers in peripheral blood. In the present study, we found that extensive spontaneous apoptosis and cDC death in culture within 24hrs represent an additional challenge. Different media conditions that maintain cDC viability and function were investigated. CD141+ cDC1 and CD1c+ cDC2 were isolated from healthy blood donor buffy coats. Low viabilities were found with CellGenix DC, RPMI-1640, and X-VIVO 15 standard culture media and with several supplements at 24hrs and 48hrs. Among multiple factors it was found that GM-CSF improved both cDC1 and cDC2 viability, whereas Flt3-L and IL-4 only increased viability of cDC1 and cDC2, respectively. Combinations of these three cytokines improved viability of both cDCs further, both at 24hrs and 48hrs time points. Although these cytokines have been extensively investigated for their role in myeloid cell differentiation, and are also used clinically, their effects on mature cDCs remain incompletely known, in particular effects on pro-inflammatory or tolerogenic cDC features. HLA-DR, CD80, CD83, CD86, PD-L1 and PD-L2 cDC membrane expressions were relatively little affected by GM-CSF, IL-4 and Flt3-L cytokine supplements compared to the strong induction following Toll-like receptor (TLR) stimulation for 24hrs. With minor exceptions the three cytokines appeared to be permissive to the TLR-induced marker expression. Allogeneic mixed leukocyte reaction showed that the cytokines promoted T-cell proliferation and revealed a potential to boost both Th1 and Th2 polarizing cytokines. GM-CSF and Flt3-L and their combination improved the capability of cDC1 for dextran uptake, while in cDC2, dextran capture was improved by GM-CSF. The data suggest that GM-CSF, IL-4 and Flt3-L and combinations might be beneficial for DC viability and function in vitro. Limited viability of cDCs could be a confounding variable experimentally and in immunotherapy.

Evaluation of ß-Catenin Inhibition of Axitinib and Nitazoxanide in Human Monocyte-Derived Dendritic Cells.

Modulation of ß-catenin signaling has attractive therapeutic potential in cancer immunotherapy. Several studies have found that ß-catenin can mediate immune evasion in cancer and promote anti-inflammatory features of antigen-presenting dendritic cells. Many small molecular compounds that inhibit Wnt/ß-catenin signaling are currently in clinical development, but none have entered routine clinical use. New inhibitors of ß-catenin signaling are consequently desirable. Here, we have tested, in monocyte-derived dendritic cells, the effects of two small molecular compounds, axitinib and nitazoxanide, that previously have been discovered to inhibit ß-catenin signaling in colon cancer cells. Immature and lipopolysaccharide-matured dendritic cells prepared from healthy blood donor buffy coats were stimulated with 6-bromoindirubin-3'-oxime (6-BIO) to boost basal ß-catenin activity, and the effects of axitinib and nitazoxanide were compared with the commercial ß-catenin inhibitor ICG-001. Assays, including genome-wide RNA-sequencing, indicated that neither axitinib nor nitazoxanide demonstrated considerable ß-catenin inhibition. Both compounds were found to be less toxic to monocyte-derived dendritic cells than either 6-BIO or ICG-001. Axitinib stimulated several aspects of dendritic cell function, such as IL12-p70 secretion, and counteracted IL-10 secretion, according to the present study. However, neither axitinib nor nitazoxanide were found to be efficient ß-catenin inhibitors in monocyte-derived dendritic cells.

Transcriptional and clonal characterization of B cell plasmablast diversity following primary and secondary natural DENV infection.

Antibody-mediated humoral immunity is thought to play a central role in mediating the immunopathogenesis of acute DENV infection, but limited data are available on the diversity, specificity, and functionality of the antibody response at the molecular level elicited by primary or secondary DENV infection. In order to close this functional gap in our understanding of DENV-specific humoral immunity, we utilized high-throughput single cell RNA sequencing to investigate B cells circulating in both primary and secondary natural DENV infections. We captured full-length paired immunoglobulin receptor sequence data from 9,027 B cells from a total of 6 subjects, including 2,717 plasmablasts. In addition to IgG and IgM class-switched cells, we unexpectedly found a high proportion of the DENV-elicited plasmablasts expressing IgA, principally in individuals with primary DENV infections. These IgA class-switched cells were extensively hypermutated even in individuals with a serologically confirmed primary DENV infection. Utilizing a combination of conventional biochemical assays and high-throughput shotgun mutagenesis, we determined that DENV-reactive IgA class-switched antibodies represent a significant fraction of DENV-reactive Igs generated in response to DENV infection, and that they exhibit a comparable epitope specificity to DENV-reactive IgG antibodies. These results provide insight into the molecular-level diversity of DENV-elicited humoral immunity and identify a heretofore unappreciated IgA plasmablast response to DENV infection.

Dissecting the heterogeneity of DENV vaccine-elicited cellular immunity using single-cell RNA sequencing and metabolic profiling.

Generating effective and durable T cell immunity is a critical prerequisite for vaccination against dengue virus (DENV) and other viral diseases. However, understanding the molecular mechanisms of vaccine-elicited T cell immunity remains a critical knowledge gap in vaccinology. In this study, we utilize single-cell RNA sequencing (scRNAseq) and longitudinal TCR clonotype analysis to identify a unique transcriptional signature present in acutely activated and clonally-expanded T cells that become committed to the memory repertoire. This effector/memory-associated transcriptional signature is dominated by a robust metabolic transcriptional program. Based on this transcriptional signature, we are able to define a set of markers that identify the most durable vaccine-reactive memory-precursor CD8+ T cells. This study illustrates the power of scRNAseq as an analytical tool to assess the molecular mechanisms of host control and vaccine modality in determining the magnitude, diversity and persistence of vaccine-elicited cell-mediated immunity.

Analysis of the TCR Repertoire in HIV-Exposed but Uninfected Infants.

Maternal human immunodeficiency virus (HIV) infection has been shown to leave profound and lasting impacts on the HIV-exposed uninfected (HEU) infant, including increased mortality and morbidity, immunological changes, and developmental delays compared to their HIV-unexposed (HU) counterparts. Exposure to HIV or antiretroviral therapy may influence immune development, which could increase morbidity and mortality. However, a direct link between the increased mortality and morbidity and the infant's immune system has not been identified. To provide a global picture of the neonatal T cell repertoire in HEU versus HU infants, the diversity of the T cell receptor beta chain (TRB) expressed in cord blood samples from HEU infants was determined using next-generation sequencing and compared to healthy (HU) infants collected from the same community. While the TRB repertoire of HU infants was broadly diverse, in line with the expected idea of a naïve T cell repertoire, samples of HEU infants showed a significantly reduced TRB diversity. This study is the first to demonstrate differences in TRB diversity between HEU and HU cord blood samples and provides evidence that maternal HIV, in the absence of transmission, influences the adaptive immune system of the unborn child.

HIV-exposed uninfected infants: elevated cord blood Interleukin 8 (IL-8) is significantly associated with maternal HIV infection and systemic IL-8 in a Kenyan cohort.

BACKGROUND: In low and middle income countries, human immunodeficiency virus (HIV) exposed, uninfected (HEU) infants demonstrate higher morbidity and mortality than their unexposed counterparts. To determine possible immune correlates of this effect, we investigated the impact of in utero HIV exposure on the uninfected neonatal immune milieu and maternal factors mediating these abnormalities in a cohort of vaginally delivered mother-infants. Samples of delivery and cord blood plasma were selected from 22 Kenyan HIV-infected women and their HIV exposed uninfected (HEU) infants drawn from the pre-ARV era, while 19 Kenyan HIV-uninfected (HU) women and their infants were selected from a control cohort. RESULTS: Compared to HU cord plasma, HEU cord plasma contained significantly higher levels of pro-inflammatory cytokines interleukins (IL)-6 and -8 (both p < 0.001) and significantly lower levels of CXC motif chemokine 11 (CXC11) (p < 0.001). Mediation analysis demonstrated that maternal HIV infection status was a significant determinant of infant IL-8 responses: HEU status was associated with a ninefold higher infant:mother (cord:delivery) plasma levels of IL-8 (p < 0.005), whereas maternal viral load was negatively associated with HEU IL-8 levels (p = 0.04) and not associated with HEU IL-6 levels. CONCLUSIONS: Exposure to maternal HIV infection drives an increase in prenatal IL-8 that is partially mediated by maternal cytokine levels. Differences between maternal and infant cytokine levels strongly suggest independent modulation in utero, consistent with prenatal immune activation. Elevated pro-inflammatory signals at birth may interfere with T cell responses at birth and subsequently influence immune maturation and the risk of morbidity and mortality in HEU infants.

Suppressing active replication of a live attenuated simian immunodeficiency virus vaccine does not abrogate protection from challenge.

Although safety concerns preclude the use of live attenuated HIV vaccines in humans, they provide a useful system for identifying the elusive correlates of protective immunity in the SIV/macaque animal model. However, a number of pieces of evidence suggest that protection may result from prior occupancy of susceptible target cells by the vaccine virus rather than the immune response. To address this, we developed a Nef-deletion variant of an RT-SHIV whose active replication could be shut off by treatment with RT-inhibitors. Groups of macaques were inoculated with the ∆Nef-RT-SHIV and immune responses allowed to develop before antiretroviral treatment and subsequent challenge with wild-type SIVmac239. Vaccinated animals either resisted infection fully or significantly controlled the subsequent viremia. However, there was no difference between animals undergoing replication of the vaccine virus and those without. This strongly suggests that competition for available target cells does not play a role in protection.

Gender and Sexuality in the Federal Medical Schools in Brazil: an Analysis of the Curricular Pedagogical Projects / Gênero e Sexualidade nas Escolas Médicas Federais do Brasil: uma Análise de Projetos Pedagógicos Curriculares

Abstract Introduction: The Brazilian National Curriculum Guidelines (NCG) for undergraduate courses in Medicine since 2014 have highlighted the need to include gender and sexuality in the medical school curricula, aiming at better care for people. Method: Thus, the objective is to identify and analyze the approach of gender and sexuality in the Curriculum Pedagogical Projects (CPP) of the medical courses of the Federal Institutions of Brazil. This is a cross-sectional study of descriptive and documentary analysis, carried out through the analysis of CPP, of the undergraduate medical courses of the Federal Universities of Brazil. Data were obtained from the website of the undergraduate medical course of each higher education institution. In addition, emails were sent to the coordinators of these courses to obtain the updated CPPs. For the quantitative analysis, an initial description of the sample was performed followed by a statistical analysis using the chi-square test to assess whether or not there was an association between the presence of gender and / or sexuality debate in CPP, with the publication of the 2014 NCG and with the Brazilian geographic region, as well as between the nature of the curricular unit (compulsory or complementary / optional) and the perspective of the approach. The documentary analysis was used for the qualitative analysis with the content analysis technique, through the thematic analysis method. Results: It was found that 48 (69.56%) of the 69 analyzed CPP discuss the topics gender and / or sexuality in one or more subjects offered by the course. There is a predominance of the sexuality topic, of compulsory curricular units (88.32% of 137 curricular units) and an approach beyond the exclusively biological perspective (63.5% of 48 CPPs and 62.04% of curricular units). No statistical significance was evidenced by the chi-square test. Regarding the documentary analysis, two categories were constructed: "gender and sexuality associated with a biological perspective", "gender and sexuality associated with a predominantly broader perspective or social determinants in health". It was observed that the first category predominates in curricular units associated with organs, systems or life cycles of the human being, while the second, to social and human sciences applied to health and Primary Health Care. Conclusions: We conclude there is a significant number of CPPs and, consequently, also of curricular units that have a perspective beyond the exclusively biological approach. Nevertheless, there is difficulty with comprehensive health care that may be related to a selective visibility to pathology and technical aspects of medical practice and a difficulty in effectively promoting an interdisciplinary dialogue between the biological, exact and human sciences as proposed by the NCG.

Resumo: Introdução: Desde 2014, as Diretrizes Curriculares Nacionais (DCN) para os cursos de graduação em Medicina do Brasil destacam a necessidade da inclusão dos temas gênero e sexualidade nos currículos dos cursos de Medicina, almejando um melhor cuidado com as pessoas. Objetiva-se, com isso, identificar e analisar a abordagem de gênero e sexualidade nos Projetos Pedagógicos Curriculares (PPC) dos cursos de Medicina das Instituições Federais do Brasil. Método: Trata-se de um estudo transversal do tipo descritivo e de análise documental, realizado por meio da análise de PPC dos cursos de graduação em Medicina das Universidades Federais do Brasil. Os dados foram obtidos no sítio eletrônico do curso de graduação de Medicina de cada Instituição de Ensino Superior. Além disso, foram enviadas mensagens por correio eletrônico para as coordenações desses cursos de modo a obter os PPC atualizados. Para a análise quantitativa, realizou-se uma descrição inicial da amostra seguida de uma análise estatística utilizando o teste qui-quadrado para avaliar se haveria associação ou não entre a presença do debate de gênero e/ou sexualidade nos PCC, a publicação das DCN de 2014 e a região geográfica brasileira, bem como entre a natureza da unidade curricular (obrigatória ou complementar/optativa) e a perspectiva da abordagem. Para a análise qualitativa, foi utilizada a análise documental com a técnica de análise de conteúdo por meio do método de análise temática. Resultados: Identificou-se que 48 (69,56%) dos 69 PPC analisados discutem os temas gênero e/ou sexualidade em uma ou mais disciplinas oferecidas pelo curso. Há um predomínio da temática da sexualidade, de unidades curriculares de caráter obrigatório (88,32% das 137 unidades curriculares) e de uma abordagem para além da perspectiva exclusivamente biológica (63,5% dos 48 PPC e 62,04% das unidades curriculares). Não foram evidenciadas significâncias estatísticas pelo teste qui-quadrado. Em relação à análise documental, construíram-se duas categorias: "gênero e sexualidade associados a uma perspectiva biológica" e "gênero e sexualidade associados a uma perspectiva predominantemente ampliada ou dos determinantes sociais em saúde". Observou-se que a primeira categoria predomina em unidades curriculares associada a órgãos, sistemas ou ciclos de vida do ser humano, enquanto a segunda, às Ciências Sociais e Humanas aplicadas à Saúde e à Atenção Primária à Saúde. Conclusões: Há uma quantidade relevante de PPC e, consequentemente, de unidades curriculares que têm uma perspectiva para além da abordagem exclusivamente biológica. Apesar disso, há uma dificuldade para um cuidado integral em saúde que pode estar relacionado a uma visibilidade seletiva à patologia e aos aspectos técnicos da prática médica e a uma dificuldade de efetivamente promover um diálogo interdisciplinar entre as ciências biológicas, exatas e humanas como proposto pelas DCN.