Myeloid dendritic cells (DCs) have the innate capacity to sense pathogens and orchestrate immune responses. However, DCs do not mount efficient immune responses to HIV-1, primarily due to restriction of virus reverse transcription, which prevents accumulation of viral cDNA and limits its detection through the cGAS-STING pathway. By allowing reverse transcription to proceed, we find that DCs detect HIV-1 in distinct phases, before and after virus integration. Blocking integration suppresses, but does not abolish, activation of the transcription factor IRF3, downstream interferon (IFN) responses, and DC maturation. Consistent with two stages of detection, HIV-1 "primes" chromatin accessibility of innate immune genes before and after integration. Once primed, robust IFN responses can be unmasked by agonists of the innate adaptor protein, MyD88, through a process that requires cGAS, STING, IRF3, and nuclear factor κB. Thus, HIV-1 replication increases material available for sensing, and discrete inflammatory inputs tune cGAS signaling to drive DC maturation.

To facilitate dynamic imaging of neural crest (NC) lineages and discrimination of individual cells in the enteric nervous system (ENS) where close juxtaposition often complicates viewing, we generated a mouse BAC transgenic line that drives a Histone2BVenus (H2BVenus) reporter from Sox10 regulatory regions. This strategy does not alter the endogenous Sox10 locus and thus facilitates analysis of normal NC development. Our Sox10-H2BVenus BAC transgene exhibits temporal, spatial, and cell-type specific expression that reflects endogenous Sox10 patterns. Individual cells exhibiting nuclear–localized fluorescence of the H2BVenus reporter are readily visualized in both fixed and living tissue and are amenable to isolation by fluorescence activated cell sorting (FACS). FACS-isolated H2BVenus+ enteric NC-derived progenitors (ENPs) exhibit multi-potency, readily form neurospheres, self-renew in vitro and express a variety of stem cell genes. Dynamic live imaging as H2BVenus+ ENPs migrate down the fetal gut reveals cell fragmentation suggesting that apoptosis occurs at a low frequency during normal development of the ENS. Confocal imaging both during population of the fetal intestine and in post-natal gut muscle strips revealed differential expression between individual cells consistent with down-regulation of the transgene as progression towards non-glial fates occurs. The expression of the Sox10-H2BVenus transgene in multiple regions of the peripheral nervous system will facilitate future studies of NC lineage segregation as this tool is expressed in early NC progenitors and maintained in enteric glia.

During development neural crest (NC)-derived neuronal progenitors migrate away from the neural tube to form autonomic ganglia in visceral organs like the intestine and lower urinary tract. Both during development and in mature tissues these cells are often widely dispersed throughout tissues so that isolation of discrete populations using methods like laser capture micro-dissection is difficult. They can however be directly visualized by expression of fluorescent reporters driven from regulatory regions of neuron-specific genes like Tyrosine hydroxylase (TH). We describe a method optimized for high yields of viable TH+ neuronal progenitors from fetal mouse visceral tissues, including intestine and lower urogenital tract (LUT), based on dissociation and fluorescence-activated cell sorting (FACS).The Th gene encodes the rate-limiting enzyme for production of catecholamines. Enteric neuronal progenitors begin to express TH during their migration in the fetal intestine 1 and TH is also present in a subset of adult pelvic ganglia neurons 2-4 . The first appearance of this lineage and the distribution of these neurons in other aspects of the LUT, and their isolation has not been described. Neuronal progenitors expressing TH can be readily visualized by expression of EGFP in mice carrying the transgene construct Tg(Th-EGFP)DJ76Gsat/Mmnc 1 . We imaged expression of this transgene in fetal mice to document the distribution of TH+ cells in the developing LUT at 15.5 days post coitus (dpc), designating the morning of plug detection as 0.5 dpc, and observed that a subset of neuronal progenitors in the coalescing pelvic ganglia express EGFP.To isolate LUT TH+ neuronal progenitors, we optimized methods that were initially used to purify neural crest stem cells from fetal mouse intestine [2][3][4][5][6] . Prior efforts to isolate NC-derived populations relied upon digestion with a cocktail of collagenase and trypsin to obtain cell suspensions for flow cytometry. In our hands these methods produced cell suspensions from the LUT with relatively low viability. Given the already low incidence of neuronal progenitors in fetal LUT tissues, we set out to optimize dissociation methods such that cell survival in the final dissociates would be increased. We determined that gentle dissociation in Accumax (Innovative Cell Technologies, Inc), manual filtering, and flow sorting at low pressures allowed us to achieve consistently greater survival (>70% of total cells) with subsequent yields of neuronal progenitors sufficient for downstream analysis. The method we describe can be broadly applied to isolate a variety of neuronal populations from either fetal or adult murine tissues.

IntroductionA ‘Sleep-On-Side When Baby’s Inside’ public health campaign was initiated in New Zealand in 2018. This was in response to evidence that maternal supine going-to-sleep position was an independent risk factor for stillbirth from 28 weeks’ gestation. We evaluated the success of the campaign on awareness and modification of late pregnancy going-to-sleep position through nationwide surveys.Methods and analysisTwo web-based cross-sectional surveys were conducted over 12 weeks in 2019–2020 in a sample of (1) pregnant women ≥28 weeks, primary outcome of going-to-sleep position; and (2) health professionals providing pregnancy care, primary outcome of knowledge of going-to-sleep position and late stillbirth risk. Univariable logistic regression was performed to identify factors associated with supine going-to-sleep position.DiscussionThe survey of pregnant women comprised 1633 eligible participants. Going-to-sleep position last night was supine (30, 1.8%), non-supine (1597, 97.2%) and no recall (16, 1.0%). Supine position had decreased from 3.9% in our previous New Zealand-wide study (2012–2015). Most women (1412, 86.5%) had received sleep-on-side advice with no major resultant worry (1276, 90.4%). Two-thirds (918, 65.0%) had changed their going-to-sleep position based on advice, with most (611 of 918, 66.5%) reporting little difficulty. Supine position was associated with Māori (OR 5.05, 95% CI 2.10 to 12.1) and Asian-non-Indian (OR 4.20, 95% CI 1.27 to 13.90) ethnicity; single (OR 10.98, 95% CI 4.25 to 28.42) and cohabitating relationship status (OR 2.69, 95% CI 1.09 to 6.61); hospital-based maternity provider (OR 2.55, 95% CI 1.07 to 6.10); education overseas (OR 3.92, 95% CI 1.09 to 14.09) and primary-secondary level (OR 2.80, 95% CI 1.32 to 6.08); and not receiving sleep-on-side advice (OR 6.70, 95% CI 3.23 to 13.92). The majority of health professionals (709 eligible participants) reported awareness of supine going-to-sleep position and late stillbirth risk (543, 76.6%).ConclusionMost pregnant women had received and implemented sleep-on-side advice without major difficulty or concern. Some groups of women may need a tailored approach to acquisition of going-to-sleep position information.