SIDT2 Transports Extracellular dsRNA into the Cytoplasm for Innate Immune Recognition.

Double-stranded RNA (dsRNA) is a common by-product of viral infections and acts as a potent trigger of antiviral immunity. In the nematode C. elegans, sid-1 encodes a dsRNA transporter that is highly conserved throughout animal evolution, but the physiological role of SID-1 and its orthologs remains unclear. Here, we show that the mammalian SID-1 ortholog, SIDT2, is required to transport internalized extracellular dsRNA from endocytic compartments into the cytoplasm for immune activation. Sidt2-deficient mice exposed to extracellular dsRNA, encephalomyocarditis virus (EMCV), and herpes simplex virus 1 (HSV-1) show impaired production of antiviral cytokines and-in the case of EMCV and HSV-1-reduced survival. Thus, SIDT2 has retained the dsRNA transport activity of its C. elegans ortholog, and this transport is important for antiviral immunity.

SIDT1 Localizes to Endolysosomes and Mediates Double-Stranded RNA Transport into the Cytoplasm.

dsRNA is a common by-product of viral replication and acts as a potent trigger of antiviral immunity. SIDT1 and SIDT2 are closely related members of the SID-1 transmembrane family. SIDT2 functions as a dsRNA transporter and is required to traffic internalized dsRNA from endocytic compartments into the cytosol for innate immune activation, but the role of SIDT1 in dsRNA transport and in the innate immune response to viral infection is unclear. In this study, we show that Sidt1 expression is upregulated in response to dsRNA and type I IFN exposure and that SIDT1 interacts with SIDT2. Moreover, similar to SIDT2, SIDT1 localizes to the endolysosomal compartment, interacts with the long dsRNA analog poly(I:C), and, when overexpressed, enhances endosomal escape of poly(I:C) in vitro. To elucidate the role of SIDT1 in vivo, we generated SIDT1-deficient mice. Similar to Sidt2-/- mice, SIDT1-deficient mice produced significantly less type I IFN following infection with HSV type 1. In contrast to Sidt2-/- mice, however, SIDT1-deficient animals showed no impairment in survival postinfection with either HSV type 1 or encephalomyocarditis virus. Consistent with this, we observed that, unlike SIDT2, tissue expression of SIDT1 was relatively restricted, suggesting that, whereas SIDT1 can transport extracellular dsRNA into the cytoplasm following endocytosis in vitro, the transport activity of SIDT2 is likely to be functionally dominant in vivo.

Should clinicians make chest surgery available to transgender male adolescents?

Bioethicists are increasingly engaged in considering the ethical issues associated with the care of transgender people. One such issue facing paediatric clinicians is requests for chest surgery from transgender male adolescents. For transgender young people who identify as male but have already progressed through the mid to late stages of puberty, hormone treatment will not reverse breast development. Some of these young people are distressed by their chest, and request surgery to remove this tissue. Demand for this surgery during adolescence is increasing. However, few paediatric hospitals make this intervention available to adolescents. This paper focuses on the following ethical question: Should clinicians make chest surgery available to transgender male adolescents? We argue that making chest surgery available to transgender male adolescents under some circumstances is ethically justifiable for three reasons, based on the concepts of beneficence, privacy, and non-discrimination. Firstly, the limited evidence to date suggests that chest surgery is beneficial to transgender male adolescents who seek this intervention. Secondly, chest surgery protects transgender adolescents' privacy by enabling them to better control disclosure of their transgender identity. Thirdly, chest surgery is already performed on other adolescent males for psychosocial reasons, such as in the case of gynaecomastia; non-discrimination thus provides a further reason for making chest surgery available to transgender male adolescents whose male gender identity is consistent. We suggest that the ethical justifiability of chest surgery in any specific transgender adolescent's case will depend on the individual patient's circumstances.

Predicting successful sperm retrieval in transfeminine adolescents after testicular biopsy.

PURPOSE: Increasing numbers of transgender adolescents are receiving gender-affirming treatments (GAT). Given GAT can impair reproductive function, clinical guidelines advise prior counselling regarding fertility preservation (FP). For transgender adults assigned male at birth, FP is usually achieved via a masturbatory sample and sperm cryopreservation. This is less straightforward in transgender adolescents, since they may not be developmentally ready to masturbate and/or masturbation may cause unacceptable gender dysphoria. Testicular biopsy represents an alternative method for sperm retrieval in these adolescents, but for those in early/mid puberty, it is difficult to predict whether sperm will be found. The purpose of this study was therefore to identify factors that predict successful sperm retrieval for cryopreservation via testicular biopsy. METHODS: A retrospective cohort study was undertaken at a tertiary-referral pediatric gender service. Subjects were included if they'd received a testicular biopsy in association with the commencement of GAT between 2010 and 2019. The primary outcome measure was successful sperm retrieval, and potential predictors included age, testicular volume and serum testosterone, LH and FSH levels. RESULTS: Of 25 subjects who received a biopsy prior to starting any GAT, 17 had successful sperm retrieval. While age, testosterone, LH and FSH levels showed minimal differences, testicular volume was significantly higher in those with successful sperm retrieval, and a threshold of ≥ 10 mL showed 92% sensitivity and 71% specificity in predicting successful retrieval. An additional 6 patients received a biopsy after starting puberty suppression and before commencement of oestrogen, and one of these individuals had sperm successfully retrieved despite > 2 years of regular puberty suppression. CONCLUSION: These findings suggest that testicular volume is most useful in predicting successful sperm retrieval following testicular biopsy in transgender adolescents and are likely to be of relevance to other young people undertaking FP, including those with cancer.

Extracellular Vesicles in Synovial Fluid from Rheumatoid Arthritis Patients Contain miRNAs with Capacity to Modulate Inflammation.

In rheumatoid arthritis (RA), extracellular vesicles (EVs) are associated with both the propagation and attenuation of joint inflammation and destruction. However, the specific EV content responsible for these processes is largely unknown. Investigations into identifying EV content are confounded by the challenges in obtaining high-quality EV preparations from synovial fluid. Implementing a size exclusion chromatography-based method of EV isolation, coupled with small RNA sequencing, we accurately characterised EV miRNAs in synovial fluid obtained from RA patients and investigated the differences between joints with high- and low-grade inflammation. Synovial fluid was obtained from the joints of 12 RA patients and, based on leukocyte counts, classified as either high (n = 7)- or low (n = 5)-grade inflammation. Using size exclusion chromatography, EVs were purified and small RNA was extracted and sequenced on a NextSeq 500. Sequencing reads were aligned to miRBase v21, and differences in miRNA profiles between RA patients with high- and low-grade joint inflammation were analysed. In total, 1972 distinct miRNAs were identified from RA synovial fluid EVs. miRNAs with less than five reads in fewer than five patients were filtered out, leaving 318 miRNAs for analysis. Analysis of the most abundant miRNAs suggested that they negatively regulate multiple genes relevant to inflammation, including signal transducer and activator of transcription 3 (STAT3), which lies downstream of IL-6 and has a pro-inflammatory role in RA. Synovial fluid from joints with high-grade inflammation contained 3.5-fold more EV miRNA per mL of synovial fluid (p = 0.0017). Seventy-eight EV miRNAs were differentially expressed between RA joints with high- and low-grade inflammation, and pathway analysis revealed that their target genes were commonly involved a variety of processes, including cellular apoptosis, proliferation and migration. Of the 49 miRNAs that were elevated in joints with high-grade inflammation, pathway analysis revealed that genes involved in cytokine-mediated signalling pathways were significantly enriched targets. In contrast, genes associated with reactive oxygen species signalling were significantly enriched as targets of the 29 miRNAs elevated in joints with low-grade inflammation. Our study identified an abundance of EV miRNAs from the synovial fluid of RA patients with the potential to modulate inflammation. In doing so, we defined potential mechanisms by which synovial fluid EVs may contribute to RA pathophysiology.

Non-Binary and Binary Gender Identity in Australian Trans and Gender Diverse Individuals.

Many trans and gender diverse (TGD) people have gender identities that are not exclusively male or female but instead fall in-between or outside of the gender binary (non-binary). It remains unclear if and how those with non-binary gender identity differ from TGD individuals with binary identities. We aimed to understand the sociodemographic and mental health characteristics of people with non-binary identities compared with binary TGD identities. We performed a retrospective audit of new consultations for gender dysphoria between 2011 and 2016 in three clinical settings in Melbourne, Australia; (1) Equinox Clinic, an adult primary care clinic, (2) an adult endocrine specialist clinic, and (3) the Royal Children's Hospital, a child and adolescent specialist referral clinic. Age (grouped by decade), gender identity, sociodemographic, and mental health conditions were recorded. Of 895 TGD individuals, 128 (14.3%) had a non-binary gender. Proportions differed by clinical setting; 30.4% of people attending the adult primary care clinic, 7.4% attending the adult endocrine specialist clinic, and 8.0% attending the pediatric clinic identified as non-binary. A total of 29% of people in the 21-30-year-old age-group had a non-binary gender identity, higher than all other age-groups. Compared to TGD people with a binary gender identity, non-binary people had lower rates of gender-affirming interventions, and a higher prevalence of depression, anxiety, and illicit drug use. Tailoring clinical services to be inclusive of non-binary people and strategies to support mental health are required. Further research to better understand health needs and guide evidence-based gender-affirming interventions for non-binary people are needed.

Forever young? The ethics of ongoing puberty suppression for non-binary adults.

In this article, we analyse the novel case of Phoenix, a non-binary adult requesting ongoing puberty suppression (OPS) to permanently prevent the development of secondary sex characteristics, as a way of affirming their gender identity. We argue that (1) the aim of OPS is consistent with the proper goals of medicine to promote well-being, and therefore could ethically be offered to non-binary adults in principle; (2) there are additional equity-based reasons to offer OPS to non-binary adults as a group; and (3) the ethical defensibility of facilitating individual requests for OPS from non-binary adults also depends on other relevant considerations, including the balance of potential benefits over harms for that specific patient, and whether the patient's request is substantially autonomous. Although the broadly principlist ethical approach we take can be used to analyse other cases of non-binary adults requesting OPS apart from the case we evaluate, we highlight that the outcome will necessarily depend on the individual's context and values. However, such clinical provision of OPS should ideally be within the context of a properly designed research study with long-term follow-up and open publication of results.

Australian standards of care and treatment guidelines for transgender and gender diverse children and adolescents.

INTRODUCTION: The Australian standards of care and treatment guidelines aim to maximise quality care provision to transgender and gender diverse (TGD) children and adolescents across Australia, while recognising the unique circumstances of providing such care to this population. Recommendations are made based on available empirical evidence and clinician consensus, and have been developed in consultation with Australian professionals from multiple disciplines working with the TGD population, TGD support organisations, as well as TGD children and adolescents and their families. Main recommendations: Recommendations include general principles for supporting TGD children and adolescents using an affirmative approach, separate guidelines for the care of pre-pubertal children and TGD adolescents, as well as discipline-based recommendations for mental health care, medical and surgical interventions, fertility preservation, and speech therapy. Changes in management as a result of this statement: Although published international treatment guidelines currently exist, challenges in accessing and providing TGD health care specific to Australia have not been addressed to date. In response to this, these are the first guidelines to be developed for TGD children and adolescents in Australia. These guidelines also move away from treatment recommendations based on chronological age, with recommended timing of medical transition and surgical interventions dependent on the adolescent's capacity and competence to make informed decisions, duration of time on puberty suppression, coexisting mental health and medical issues, and existing family support.

A pro-inflammatory role for Th22 cells in Helicobacter pylori-associated gastritis.

OBJECTIVE: Helper T (Th) cell responses are critical for the pathogenesis of Helicobacter pylori-induced gastritis. Th22 cells represent a newly discovered Th cell subset, but their relevance to H. pylori-induced gastritis is unknown. DESIGN: Flow cytometry, real-time PCR and ELISA analyses were performed to examine cell, protein and transcript levels in gastric samples from patients and mice infected with H. pylori. Gastric tissues from interleukin (IL)-22-deficient and wild-type (control) mice were also examined. Tissue inflammation was determined for pro-inflammatory cell infiltration and pro-inflammatory protein production. Gastric epithelial cells and myeloid-derived suppressor cells (MDSC) were isolated, stimulated and/or cultured for Th22 cell function assays. RESULTS: Th22 cells accumulated in gastric mucosa of both patients and mice infected with H. pylori. Th22 cell polarisation was promoted via the production of IL-23 by dendritic cells (DC) during H. pylori infection, and resulted in increased inflammation within the gastric mucosa. This inflammation was characterised by the CXCR2-dependent influx of MDSCs, whose migration was induced via the IL-22-dependent production of CXCL2 by gastric epithelial cells. Under the influence of IL-22, MDSCs, in turn, produced pro-inflammatory proteins, such as S100A8 and S100A9, and suppressed Th1 cell responses, thereby contributing to the development of H. pylori-associated gastritis. CONCLUSIONS: This study, therefore, identifies a novel regulatory network involving H. pylori, DCs, Th22 cells, gastric epithelial cells and MDSCs, which collectively exert a pro-inflammatory effect within the gastric microenvironment. Efforts to inhibit this Th22-dependent pathway may therefore prove a valuable strategy in the therapy of H. pylori-associated gastritis.

T-cell immunity to influenza A viruses.

Influenza infection remains a global threat to human health. Influenza viruses are normally controlled by antibodies specific for the surface glycoproteins hemagglutinin (HA) and neuraminidase (NA). Standard influenza vaccines are aimed at inducing these antibodies, but they must be administered annually and can be rendered ineffective since different strains circulate from year to year and vary considerably in their individual HA and NA profiles. Influenza-specific T cells have been shown to be protective in animal models and typically recognize the more conserved internal influenza proteins. Improving our understanding of influenza-specific T-cell responses, including immunodominance, specific epitope sequences, strain-related epitope variation, host/virus interaction, and the balance between immunity versus immunopathology, will be important to improve future T-cell-based vaccines, which promise broader strain coverage and longer-lasting protection than current standard vaccines.

Increasing viral dose causes a reversal in CD8+ T cell immunodominance during primary influenza infection due to differences in antigen presentation, T cell avidity, and precursor numbers.

T cell responses are characterized by the phenomenon of immunodominance (ID), whereby peptide-specific T cells are elicited in a reproducible hierarchy of dominant and subdominant responses. However, the mechanisms that give rise to ID are not well understood. We investigated the effect of viral dose on primary CD8(+) T cell (T(CD8+)) ID by injecting mice i.p. with various doses of influenza A virus and assessing the primary T(CD8+) response to five dominant and subdominant peptides. Increasing viral dose enhanced the overall strength of the T(CD8+) response, and it altered the ID hierarchy: specifically, NP(366-374) T(CD8+) were dominant at low viral doses but were supplanted by PA(224-233) T(CD8+) at high doses. To understand the basis for this reversal, we mathematically modeled these T(CD8+) responses and used Bayesian statistics to obtain estimates for Ag presentation, T(CD8+) precursor numbers, and avidity. Interestingly, at low viral doses, Ag presentation most critically shaped ID hierarchy, enabling T(CD8+) specific to the more abundantly presented NP(366-374) to dominate. By comparison, at high viral doses, T(CD8+) avidity and precursor numbers appeared to be the major influences on ID hierarchy, resulting in PA(224-233) T(CD8+) usurping NP(366-374) cells as the result of higher avidity and precursor numbers. These results demonstrate that the nature of primary T(CD8+) responses to influenza A virus is highly influenced by Ag dose, which, in turn, determines the relative importance of Ag presentation, T(CD8+) avidity, and precursor numbers in shaping the ID hierarchy. These findings provide valuable insights for future T(CD8+)-based vaccination strategies.

Evaluation of a codesigned group cognitive-behavioural therapy intervention for trans young people (TAG TEAM): protocol for a feasibility trial and a subsequent pilot RCT.

INTRODUCTION: Trans young people are at a higher risk of mental health difficulties such as depression, anxiety and suicidality than their cisgender peers, due in part to their experiences of minority stress. This protocol describes a feasibility trial and subsequent pilot randomised controlled trial (RCT) of a codesigned group cognitive-behavioural therapy intervention for trans young people, named Trans Adolescent Group ThErapy for Alleviating Minority stress (TAG TEAM). METHODS AND ANALYSIS: To evaluate TAG TEAM, we will conduct a feasibility trial followed by a pilot RCT with trans young people aged 14-16 years who have been referred to the Royal Children's Hospital Gender Service in Melbourne, Australia. In the feasibility trial, we aim to enrol 32 participants who will be randomised at a 1:1 ratio to either in-person or online intervention arms. Participants will be assessed at baseline and post-treatment, with a nested qualitative evaluation post-treatment. Primary outcomes are the feasibility and acceptability of the intervention and the study design and associated procedures, including comparison of the in-person and online delivery modes. In the subsequent pilot RCT, we aim to enrol 64 participants who will be randomised at a 1:1 ratio to an intervention or waitlist control arm, with delivery mode determined by the feasibility trial. Participants will complete assessments at baseline, post-treatment and 3-month follow-up. Primary outcomes are the feasibility and acceptability of the RCT study design. In both the feasibility trial and pilot RCT, participants will complete assessments related to mood, anxiety, suicidality, quality of life, minority stress, family support and social transition. Quantitative data will be analysed using descriptive statistics. Qualitative data will be analysed using thematic and interpretive analysis. ETHICS AND DISSEMINATION: The Royal Children's Hospital Human Research Ethics Committee has approved this study (#91162). Informed consent will be obtained in writing from all participants and a legal guardian. Findings will inform the development of a full-scale RCT to evaluate the efficacy of TAG TEAM and will be disseminated through conferences and peer-reviewed journals. TRIAL REGISTRATION NUMBER: ACTRN12623000302651, ACTRN12623000318684.

Pelvic pain in transmasculine adolescents receiving testosterone therapy.

Background: Pelvic pain is a common complaint among individuals assigned female at birth. However, few studies have explored pelvic pain among transmasculine patients on gender-affirming testosterone treatment, and most of these were performed in adult populations. Aims: The aim of our study was to investigate the prevalence, risk factors, nature and treatment of pelvic pain among trans adolescents on testosterone. Methods: A retrospective cohort study was performed on all trans adolescents started on gender-affirming testosterone treatment at our institution between 2007 and 2020. Results: Among 158 trans adolescents who were started on testosterone therapy and followed-up for at least six months, 37 (23.4%) reported pelvic pain, with a median interval between testosterone initiation and reported onset of pain of 1.6 months (range 0.3-6.4). The prevalence of pelvic pain was higher in patients who were receiving menstrual suppression (n = 36, 26.3%) compared to those who were not (n = 1, 4.8%), giving a risk difference of 21.5% (95% CI 9.8% to 33.2%, p = 0.028). The most common descriptive terms were "cramps" (n = 17, 45.9%) and "similar to previous period pain" (n = 8, 21.6%). A range of different pharmacological strategies were employed, including paracetamol, NSAIDs, danazol, norethisterone, medroxyprogesterone, etonogestrel implant, intra-uterine device, goserelin and pelvic floor physiotherapy, with variable outcomes. Conclusion: In conclusion, we report here - in what is to our knowledge the first time - the prevalence rate of pelvic pain in trans adolescents on gender-affirming testosterone treatment, and observe that a quarter of them described pelvic pain. Limitations of our study include its retrospective nature, which is likely to be associated with under-reporting of pelvic pain, and the limited documentation of the nature and likely causes of this pain within the medical records. Prospective longitudinal studies to better understand the nature, etiology and optimal management of testosterone-associated pelvic pain are therefore warranted.

Prevalence of Eating Disorder Symptoms in Transgender and Gender Diverse Adolescents Presenting for Gender-Affirming Care.

PURPOSE: To describe the prevalence of eating disorder symptoms among adolescents seeking gender-affirming care. METHODS: Cross-sectional study of 660 gender-diverse adolescents who completed the Branched Eating Disorder Test to measure anorexia and bulimia symptoms. RESULTS: 23.9% (95% CI 20.7-27.4) reported both anorexia symptoms, namely overvaluation of weight and fear of (or recurrent interference with) weight gain. 0.9% (95% CI 0.3-2.0) reported all bulimia symptoms, namely overvaluation of weight, recurrent binge eating, and recurrent compensatory behaviors (e.g., weekly purging). For all symptoms, prevalence was higher among i) adolescents assigned female at birth compared to those assigned male at birth, and ii) adolescents who felt unsure about their gender identity compared to those who identified as trans or nonbinary. DISCUSSION: Clinicians should monitor eating disorder symptoms among adolescents presenting for gender-affirming care, especially among those assigned female at birth or who are unsure about their gender identity.

A comparison of gender diversity in transgender young people with and without autistic traits from the Trans 20 cohort study.

Background: There is an elevated co-occurrence of autism in trans individuals, with recent meta-analyses suggesting that 11% of trans individuals are autistic. The presence of autism in trans young people can create clinical challenges by adding complexity to the presentation, assessment and management of those presenting to gender clinics. Although many trans young people display traits of autism, how these traits relate to the nature of their gender diversity is unclear. Methods: This study compared gender identity, gender expression and gender dysphoria (GD) in trans young people with and without autistic traits. Baseline data from a cohort study of trans children and adolescents who first attended the Royal Children's Hospital Gender Service (Victoria, Australia) between February 2017 and January 2020 were analysed cross-sectionally. Autistic traits were assessed via the Social Responsiveness Scale-2. Gender was assessed using tools that measure gender identity, social transition, GD, body dissatisfaction, voice dysphoria, and chest dysphoria. Findings: 522 participants were included, of whom 239 (45.8%) exhibited autistic traits (SRS total T-score ≥60). Those with and without autistic traits were similar in their age (mean (SD) age 14.0 (2.9) and 13.1 (3.6) years respectively) and gender identity: the majority (73.7% (n = 174) and 70.5% (n = 198) respectively) identified in a binary way. Higher rates of social transition (specifically, changing pronouns) were noted in those with autistic traits (Difference in proportion 11.7, 95% confidence interval [CI] 2.4-21.1, p = 0.014). GD was high in both groups with ∼95% displaying clinically relevant levels of GD. Chest dysphoria was similar between groups, while voice dysphoria was higher in those with autistic traits (standardised mean difference [SMD] = 0.3, 95% confidence interval [CI]: 0.1-0.5 p = 0.00087). Dissatisfaction with secondary gendered characteristics (SMD = 0.3, CI: 0.1-0.5 p = 0.0011) and hormonally unresponsive body characteristics (SMD = 0.2, CI: 0.1-0.4 p = 0.016) was higher in trans young people with autistic traits. Interpretation: The similarly high severity of GD in those with and without autistic traits reinforces the importance of trans young people with and without autistic traits being availed the same opportunities to access gender-affirming care. Subtle differences identified between the groups in other areas of gender diversity suggest trans young people with autistic traits may have distinct needs and that gender-affirming care may need to be tailored accordingly. Funding: The Royal Children's Hospital Foundation, Hugh D.T. Williamson Foundation; Australian National Health and Medical Research Council-Clinical Trials and Cohort Studies scheme (GNT 2006529).