Mechanisms of CD40-dependent cDC1 licensing beyond costimulation.

CD40 signaling in classical type 1 dendritic cells (cDC1s) is required for CD8 T cell-mediated tumor rejection, but the underlying mechanisms are incompletely understood. Here, we identified CD40-induced genes in cDC1s, including Cd70, Tnfsf9, Ptgs2 and Bcl2l1, and examined their contributions to anti-tumor immunity. cDC1-specific inactivation of CD70 and COX-2, and global CD27 inactivation, only partially impaired tumor rejection or tumor-specific CD8 T cell expansion. Loss of 4-1BB, alone or in Cd27-/- mice, did not further impair anti-tumor immunity. However, cDC1-specific CD40 inactivation reduced cDC1 mitochondrial transmembrane potential and increased caspase activation in tumor-draining lymph nodes, reducing migratory cDC1 numbers in vivo. Similar impairments occurred during in vitro antigen presentation by Cd40-/- cDC1s to CD8+ T cells, which were reversed by re-expression of Bcl2l1. Thus, CD40 signaling in cDC1s not only induces costimulatory ligands for CD8+ T cells but also induces Bcl2l1 that sustains cDC1 survival during priming of anti-tumor responses.

Cis interactions in the Irf8 locus regulate stage-dependent enhancer activation.

Individual elements within a superenhancer can act in a cooperative or temporal manner, but the underlying mechanisms remain obscure. We recently identified an Irf8 superenhancer, within which different elements act at distinct stages of type 1 classical dendritic cell (cDC1) development. The +41-kb Irf8 enhancer is required for pre-cDC1 specification, while the +32-kb Irf8 enhancer acts to support subsequent cDC1 maturation. Here, we found that compound heterozygous Δ32/Δ41 mice, lacking the +32- and +41-kb enhancers on different chromosomes, show normal pre-cDC1 specification but, surprisingly, completely lack mature cDC1 development, suggesting cis dependence of the +32-kb enhancer on the +41-kb enhancer. Transcription of the +32-kb Irf8 enhancer-associated long noncoding RNA (lncRNA) Gm39266 is also dependent on the +41-kb enhancer. However, cDC1 development in mice remained intact when Gm39266 transcripts were eliminated by CRISPR/Cas9-mediated deletion of lncRNA promoters and when transcription across the +32-kb enhancer was blocked by premature polyadenylation. We showed that chromatin accessibility and BATF3 binding at the +32-kb enhancer were dependent on a functional +41-kb enhancer located in cis Thus, the +41-kb Irf8 enhancer controls the subsequent activation of the +32-kb Irf8 enhancer in a manner that is independent of associated lncRNA transcription.

High Amount of Transcription Factor IRF8 Engages AP1-IRF Composite Elements in Enhancers to Direct Type 1 Conventional Dendritic Cell Identity.

Development and function of conventional dendritic cell (cDC) subsets, cDC1 and cDC2, depend on transcription factors (TFs) IRF8 and IRF4, respectively. Since IRF8 and IRF4 can each interact with TF BATF3 at AP1-IRF composite elements (AICEs) and with TF PU.1 at Ets-IRF composite elements (EICEs), it is unclear how these factors exert divergent actions. Here, we determined the basis for distinct effects of IRF8 and IRF4 in cDC development. Genes expressed commonly by cDC1 and cDC2 used EICE-dependent enhancers that were redundantly activated by low amounts of either IRF4 or IRF8. By contrast, cDC1-specific genes relied on AICE-dependent enhancers, which required high IRF concentrations, but were activated by either IRF4 or IRF8. IRF8 was specifically required only by a minority of cDC1-specific genes, such as Xcr1, which could distinguish between IRF8 and IRF4 DNA-binding domains. Thus, these results explain how BATF3-dependent Irf8 autoactivation underlies emergence of the cDC1-specific transcriptional program.

Enhanced in vitro type 1 conventional dendritic cell generation via the recruitment of hematopoietic stem cells and early progenitors by Kit ligand.

In vitro culture of bone marrow (BM) with Fms-like tyrosine kinase 3 ligand (Flt3L) is widely used to study development and function of type 1 conventional dendritic cells (cDC1). Hematopoietic stem cells (HSCs) and many progenitor populations that possess cDC1 potential in vivo do not express Flt3 and thus may not contribute to Flt3L-mediated cDC1 production in vitro. Here, we present a KitL/Flt3L protocol that recruits such HSCs and progenitors into the production of cDC1. Kit ligand (KitL) is used to expand HSCs and early progenitors lacking Flt3 expression into later stage where Flt3 is expressed. Following this initial KitL phase, a second Flt3L phase is used to support the final production of DCs. With this two-stage culture, we achieved approximately tenfold increased production of both cDC1 and cDC2 compared to Flt3L culture. cDC1 derived from this culture are similar to in vivo cDC1 in their dependence on IRF8, ability to produce IL-12, and induction of tumor regression in cDC1-deficient tumor-bearing mice. This KitL/Flt3L system for cDC1 production will be useful in further analysis of cDC1 that rely on in vitro generation from BM.

Klf4 expression in conventional dendritic cells is required for T helper 2 cell responses.

The two major lineages of classical dendritic cells (cDCs) express and require either IRF8 or IRF4 transcription factors for their development and function. IRF8-dependent cDCs promote anti-viral and T-helper 1 (Th1) cell responses, whereas IRF4-expressing cDCs have been implicated in controlling both Th2 and Th17 cell responses. Here, we have provided evidence that Kruppel-like factor 4 (Klf4) is required in IRF4-expressing cDCs to promote Th2, but not Th17, cell responses in vivo. Conditional Klf4 deletion within cDCs impaired Th2 cell responses during Schistosoma mansoni infection, Schistosoma egg antigen (SEA) immunization, and house dust mite (HDM) challenge without affecting cytotoxic T lymphocyte (CTL), Th1 cell, or Th17 cell responses to herpes simplex virus, Toxoplasma gondii, and Citrobacter rodentium infections. Further, Klf4 deletion reduced IRF4 expression in pre-cDCs and resulted in selective loss of IRF4-expressing cDCs subsets in several tissues. These results indicate that Klf4 guides a transcriptional program promoting IRF4-expressing cDCs heterogeneity.

Serological assays to measure dimeric IgA antibodies in SARS-CoV-2 infections.

Current serological tests cannot differentiate between total immunoglobulin A (IgA) and dimeric IgA (dIgA) associated with mucosal immunity. Here, we describe two new assays, dIgA-ELISA and dIgA-multiplex bead assay (MBA), that utilize the preferential binding of dIgA to a chimeric form of secretory component, allowing the differentiation between dIgA and monomeric IgA. dIgA responses elicited through severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection were measured in (i) a longitudinal panel, consisting of 74 samples (n = 20 individuals) from hospitalized cases of coronavirus disease 2019 (COVID-19); (ii) a longitudinal panel, consisting of 96 samples (n = 10 individuals) from individuals with mild COVID-19; (iii) a cross-sectional panel with PCR-confirmed SARS-CoV-2 infection with mild COVID-19 (n = 199) and (iv) pre-COVID-19 samples (n = 200). The dIgA-ELISA and dIgA-MBA demonstrated a specificity for dIgA of 99% and 98.5%, respectively. Analysis of dIgA responses in the longitudinal panels revealed that 70% (ELISA) and 50% (MBA) of patients elicited a dIgA response by day 20 after PCR diagnosis with a SARS-CoV-2 infection. Individuals with mild COVID-19 displayed increased levels of dIgA within the first 3 weeks after diagnosis but responses appeared to be short lived, compared with sustained IgA levels. However, in samples from hospitalized patients with COVID-19 we observed high and sustained levels of dIgA, up to 245 days after PCR diagnosis. Our results suggest that severe COVID-19 infections are associated with sustained levels of plasma dIgA compared with mild cases.

The MYCL and MXD1 transcription factors regulate the fitness of murine dendritic cells.

We previously found that MYCL is required by a Batf3-dependent classical dendritic cell subset (cDC1) for optimal CD8 T cell priming, but the underlying mechanism has remained unclear. The MAX-binding proteins encompass a family of transcription factors with overlapping DNA-binding specificities, conferred by a C-terminal basic helix-loop-helix domain, which mediates heterodimerization. Thus, regulation of transcription by these factors is dependent on divergent N-terminal domains. The MYC family, including MYCL, has actions that are reciprocal to the MXD family, which is mediated through the recruitment of higher-order activator and repressor complexes, respectively. As potent proto-oncogenes, models of MYC family function have been largely derived from their activity at supraphysiological levels in tumor cell lines. MYC and MYCN have been studied extensively, but empirical analysis of MYCL function had been limited due to highly restricted, lineage-specific expression in vivo. Here we observed that Mycl is expressed in immature cDC1s but repressed on maturation, concomitant with Mxd1 induction in mature cDC1s. We hypothesized that MYCL and MXD1 regulate a shared, but reciprocal, transcriptional program during cDC1 maturation. In agreement, immature cDC1s in Mycl-/- -deficient mice exhibited reduced expression of genes that regulate core biosynthetic processes. Mature cDC1s from Mxd1-/- mice exhibited impaired ability to inhibit the transcriptional signature otherwise supported by MYCL. The present study reveals LMYC and MXD1 as regulators of a transcriptional program that is modulated during the maturation of Batf3-dependent cDC1s.

Parasites under the Spotlight: Applications of Vibrational Spectroscopy to Malaria Research.

New technologies to diagnose malaria at high sensitivity and specificity are urgently needed in the developing world where the disease continues to pose a huge burden on society. Infrared and Raman spectroscopy-based diagnostic methods have a number of advantages compared with other diagnostic tests currently on the market. These include high sensitivity and specificity for detecting low levels of parasitemia along with ease of use and portability. Here, we review the application of vibrational spectroscopic techniques for monitoring and detecting malaria infection. We discuss the role of vibrational (infrared and Raman) spectroscopy in understanding the processes of parasite biology and its application to the study of interactions with antimalarial drugs. The distinct molecular phenotype that characterizes malaria infection and the high sensitivity enabling detection of low parasite densities provides a genuine opportunity for vibrational spectroscopy to become a front-line tool in the elimination of this deadly disease and provide molecular insights into the chemistry of this unique organism.

Deficiency of transcription factor RelB perturbs myeloid and DC development by hematopoietic-extrinsic mechanisms.

RelB is an NF-κB family transcription factor activated in the noncanonical pathway downstream of NF-κB-inducing kinase (NIK) and TNF receptor family members including lymphotoxin-ß receptor (LTßR) and CD40. Early analysis suggested that RelB is required for classical dendritic cell (cDC) development based on a severe reduction of cDCs in Relb-/- mice associated with profound myeloid expansion and perturbations in B and T cells. Subsequent analysis of radiation chimeras generated from wild-type and Relb-/- bone marrow showed that RelB exerts cell-extrinsic actions on some lineages, but it has remained unclear whether the impact of RelB on cDC development is cell-intrinsic or -extrinsic. Here, we reevaluated the role of RelB in cDC and myeloid development using a series of radiation chimeras. We found that there was no cell-intrinsic requirement for RelB for development of most cDC subsets, except for the Notch2- and LTßR-dependent subset of splenic CD4+ cDC2s. These results identify a relatively restricted role of RelB in DC development. Moreover, the myeloid expansion in Relb-/- mice resulted from hematopoietic-extrinsic actions of RelB. This result suggests that there is an unrecognized but critical role for RelB within the nonhematopoietic niche that controls normal myelopoiesis.

Field Performance and Diagnostic Accuracy of a Low-Cost Instrument-Free Point-of-Care CD4 Test (Visitect CD4) Performed by Different Health Worker Cadres among Pregnant Women.

Measuring CD4 counts remains an important component of HIV care. The Visitect CD4 is the first instrument-free low-cost point-of-care CD4 test with results interpreted visually after 40 min, providing a result of ≥350 CD4 cells/mm3 The field performance and diagnostic accuracy of the test was assessed among HIV-infected pregnant women in South Africa. A nurse performed testing at the point-of-care using both venous and finger-prick blood, and a counselor and laboratory staff tested venous blood in the clinic laboratory (four Visitect CD4 tests/participant). Performance was compared to the mean CD4 count from duplicate flow cytometry tests on venous blood (FACSCalibur Trucount). In 2017, 156 patients were enrolled, providing a total of 624 Visitect CD4 tests (468 venous and 156 finger-prick samples). Of 624 tests, 28 (4.5%) were inconclusive. Generalized linear mixed modeling showed better performance of the test on venous blood (sensitivity = 81.7%; 95% confidence interval [CI] = 72.3 to 91.1]; specificity = 82.6%, 95% CI = 77.1 to 88.1) than on finger-prick specimens (sensitivity = 60.7%; 95% CI = 45.0 to 76.3; specificity = 89.5%, 95% CI = 83.2 to 95.8; P = 0.001). No difference in performance was detected by cadre of health worker (P = 0.113) or between point-of-care versus laboratory-based testing (P = 0.108). Adequate performance of Visitect CD4 with different operators and at the point of care, with no need of electricity or instrument, shows the potential utility of this device, especially for facilitating decentralization of CD4 testing services in rural areas.

Performance of a Novel Low-Cost, Instrument-Free Plasma Separation Device for HIV Viral Load Quantification and Determination of Treatment Failure in People Living with HIV in Malaysia: a Diagnostic Accuracy Study.

HIV viral load (VL) testing is the recommended method for monitoring the response of people living with HIV and receiving antiretroviral therapy (ART). The availability of standard plasma VL testing in low- and middle-income countries (LMICs), and access to this testing, are limited by the need to use fresh plasma. Good specimen collection methods for HIV VL testing that are applicable to resource-constrained settings are needed. We assessed the diagnostic performance of the filtered dried plasma spot (FDPS), created using the newly developed, instrument-free VLPlasma device, in identifying treatment failure at a VL threshold of 1,000 copies/ml in fresh plasma. Performance was compared with that of the conventional dried blood spot (DBS). Venous blood samples from 201 people living with HIV and attending an infectious disease clinic in Malaysia were collected, and HIV VL was quantified using fresh plasma (the reference standard), FDPS, and DBS specimens. VL testing was done using the Roche Cobas AmpliPrep/Cobas TaqMan v2.0 assay. At a threshold of 1,000 copies/ml, the diagnostic performance of the FDPS was superior (sensitivity, 100% [95% confidence interval {CI}, 89.1 to 100%]; specificity, 100% [95% CI, 97.8 to 100%]) to that of the DBS (sensitivity, 100% [95% CI, 89.4 to 100%]; specificity, 36.8% [95% CI, 29.4 to 44.7%]) (P < 0.001). A stronger correlation was observed between the FDPS VL and the plasma VL (r = 0.94; P < 0.001) than between the DBS VL and the plasma VL (r = 0.85; P < 0.001). The mean difference in VL measures between the FDPS and plasma (plasma VL minus FDPS VL) was 0.127 log10 copies/ml (standard deviation [SD], 0.32), in contrast to -0.95 log10 copies/ml (SD, 0.84) between the DBS and plasma. HIV VL measurement using the FDPS outperformed that with the DBS in identifying treatment failure at a threshold of 1,000 copies/ml and compared well with the quantification of VL in plasma. The FDPS can be an attractive alternative to fresh plasma for improving access to HIV VL monitoring among people living with HIV on ART in LMICs.

Revisiting the specificity of the MHC class II transactivator CIITA in classical murine dendritic cells in vivo.

Ciita was discovered for its role in regulating transcription of major histocompatibility complex class II (MHCII) genes. Subsequently, CIITA was predicted to control many other genes based on reporter and ChIP-seq analysis but few such predictions have been verified in vivo using Ciita-/- mice. Testing these predictions for classical dendritic cells (cDCs) has been particularly difficult, since Ciita-/- mice lack MHCII expression required to identify cDCs. However, recent identification of the cDC-specific transcription factor Zbtb46 allows the identification of cDCs independently of MHCII expression. We crossed Zbtb46gfp mice onto the Ciita-/- background and found that all cDC lineages developed in vivo in the absence of Ciita. We then compared the complete transcriptional profile of wild-type and Ciita-/- cDCs to define the physiological footprint of CIITA for both immature and activated cDCs. We find that CIITA exerts a highly restricted control over only the MHCII, H2-DO and H2-DM genes, in DC1 and DC2 cDC subsets, but not over other proposed targets, including Ii. These findings emphasize the caveats needed in interpreting transcription factor binding sites identified by in-vitro reporter analysis, or by ChIP-seq, which may not necessarily indicate their functional activity in vivo.

Molecular test for chlamydia and gonorrhoea used at point of care in remote primary healthcare settings: a diagnostic test evaluation.

OBJECTIVES: A new molecular test for Chlamydia trachomatis (CT) and Neisseria gonorrhoeae (NG) (GeneXpert CT/NG) has been demonstrated to be as accurate as conventional nucleic acid amplification tests (NAAT), but performance has not been evaluated in routine primary care, performed at the point of care by clinicians. We aimed to examine its diagnostic performance when used by clinicians in remote community health services in Australia with high prevalences of CT and NG infection. The trial was registered with the Australian and New Zealand Clinical Trials Registry (#12613000808741) METHODS: At 12 health services, training was provided to 99 clinicians in the use of the GeneXpert CT/NG assay who tested specimens from all patients undergoing STI screening. Specimens were also sent in parallel for conventional laboratory-based NAATs and the concordance of results was evaluated. RESULTS: Clinicians conducted 2486 tests: CT concordance was 99.4% (95% CI 99.1 to 99.7) with a positive concordance of 98.6% (95% CI 95.9 to 99.7) and negative concordance of 99.5% (95% CI 99.1 to 99.8); NG concordance was 99.9% (95% CI 99.7 to 100.0) with a positive concordance of 100.0% (95% CI 97.5 to 100.0) and negative concordance of 99.9% (95% CI 99.7 to 100.0). CONCLUSIONS: In this first study reporting routine point-of-care use of GeneXpert CT/NG by primary care clinicians, we found excellent concordance with conventional NAATs. The use of the GeneXpert CT/NG at the point of care could potentially transform management and control of these infections in many endemic settings, including low/middle-income countries.

Feasibility of antiretroviral treatment monitoring in the era of decentralized HIV care: a systematic review.

BACKGROUND: Regular monitoring of HIV patients who are receiving antiretroviral therapy (ART) is required to ensure patient benefits and the long-term effectiveness and sustainability of ART programs. Prompted by WHO recommendations for expansion and decentralization of HIV treatment and care in low and middle income countries, we conducted a systematic review to assess the feasibility of treatment monitoring in these settings. METHODS: A comprehensive search strategy was developed using a combination of MeSH and free text terms relevant to HIV treatment and care, health service delivery, health service accessibility, decentralization and other relevant terms. Five electronic databases and two conference websites were searched to identify relevant studies conducted in LMICs, published in English between Jan 2006 and Dec 2015. Outcomes of interest included the proportion of patients who received treatment monitoring and health system factors related to monitoring of patients on ART under decentralized HIV service delivery models. RESULTS: From 5363 records retrieved, twenty studies were included in the review; all but one was conducted in sub-Saharan African countries. The majority of studies (15/20) had relatively short follow-up duration (≤24 months), and only two studies were specifically designed to assess treatment monitoring practices. The most frequently studied follow-up period was 12 months and a wide range of treatment monitoring coverage was observed. The reported proportions of patients on ART who received CD4 monitoring ranged from very low (6%; N = 2145) to very high (95%; N = 488). The median uptake of viral load monitoring was 86% with studies in program settings reporting coverage as low as 14%. Overall, the longer the follow-up period, the lower the proportion of patients who received regular monitoring tests; and programs in rural areas reported low coverage of laboratory monitoring. Moreover, uptake in the context of research had significantly better where monitoring was done by dedicated research staff. In the absence of point of care (POC) testing, the limited capacity for blood sample transportation between clinic and laboratory and poor quality of nursing staff were identified as a major barrier for treatment monitoring practice. CONCLUSIONS: There is a paucity of data on the uptake of treatment monitoring, particularly with longer-term follow-up. Wide variation in access to both virological and immunological regular monitoring was observed, with some clinics in well-resourced settings supported by external donors achieving high coverage. The feasibility of treatment monitoring, particularly in decentralized settings of HIV treatment and care may thus be of concern and requires further study. Significant investment in POC diagnostic technologies and, improving the quality of and training for nursing staff is required to ensure effective scale up of ART programs towards the targets of 90-90-90 by the year 2020.

Firefighter perceptions of cancer risk: Results of a qualitative study.

BACKGROUND: Recent epidemiological research on firefighters indicates an increased incidence of specific types of cancer. Intervention is needed in the fire service yet little is known about how firefighters perceive their cancer risk. METHODS: Participant observation (150 h, n = 100) and focus group (n = 17) data were collected from 15 fire stations in South Florida. Firefighters had at least 3 years of experience, ranks included drivers, captains, lieutenants, and specialty captains, with a median age of 51 years. RESULTS: From the qualitative analysis, two major categories (direct and indirect factors) for cancer risks emerged based on participant notions of cancer risk and cancer prevention behaviors as they relate to firefighting. CONCLUSIONS: Firefighters perceive cancer risks as the result of performing essential job tasks and from indirect job factors related to being a firefighter. The two categories of cancer risks suggest different points of entry for intervention.

An evaluation of a novel dual treponemal/nontreponemal point-of-care test for syphilis as a tool to distinguish active from past treated infection.

BACKGROUND: Most syphilis point-of-care (POC) tests detect treponemal antibodies, which persist after successful treatment. Subsequent POC tests are positive, despite no active infection, and can lead to unnecessary treatment. We evaluated a new POC test, incorporating a nontreponemal component, to distinguish active from past infection. METHODS: Sera stored at 2 Australian laboratories were tested with DPP Screen and Confirm Assay. Treponemal and nontreponemal test lines were compared to corresponding conventional treponemal and nontreponemal reference test results: immunoassays and rapid plasma reagin (RPR), respectively, with RPR quantification by endpoint titration. POC test outcome concordance with conventional test results was assessed according to serological and clinical categories. RESULTS: Among 1005 serum samples tested, DPP treponemal line sensitivity was 89.8% (95% confidence interval [CI], 87.3%-91.9%) and specificity was 99.3% (95% CI, 97.0%-99.9%). DPP nontreponemal line sensitivity was 94.2% (95% CI, 91.8%-96.0%) and specificity was 62.2% (95% CI, 57.5%-66.6%). DPP test outcome (pair of test lines) was concordant with both reference test results for 94.3% of 404 high-titer infections, 90.1% of 121 low-titer infections, 27.5% of 211 past/treated infections, and 78.1% of 242 infections classified as not syphilis. Among 211 past/treated infections, 49.8% were incorrectly identified as active infection and a further 22.8% as not syphilis. CONCLUSIONS: DPP test use would result in identification of >93% of active syphilis infections, whereas just over half of past infections would be diagnosed as past or not syphilis, avoiding unnecessary treatment compared with other POC tests. This may be at the expense of missing some active infections; thus, its potential benefits will depend on the prevalence of past vs active infection in a population.

Dimeric immunoglobulin A as a novel diagnostic marker of measles infection.

IMPORTANCE: The world is facing a measles resurgence, and improved diagnostic tests for measles infection are an urgent World Health Organization research priority. Detection of measles-specific immunoglobulin M (IgM) as a standard diagnostic test has low positive predictive value in elimination settings, and there is a need for new biomarkers of measles infection to enable enhanced surveillance and response to outbreaks. We demonstrate the detection of measles-specific dimeric immunoglobulin A (dIgA) in patients with confirmed measles infections using a new indirect enzyme-linked immunosorbent assay protocol that selects for the dIgA fraction from total IgA in the blood. The magnitude of measles-specific dIgA responses showed a low correlation with IgM responses, and our results highlight the potential of dIgA for further development as an alternative and/or complementary biomarker to IgM for serological diagnosis of measles infection.

Occluded insertion loss from intracochlear pressure measurements during acoustic shock wave exposure.

INTRODUCTION: Injuries to the peripheral auditory system are among the most common results of high intensity impulsive noise exposure. Hearing protection can mitigate this injury, but careful assessment of the insertion loss they provide is necessary. Insertion loss is typically measured using microphone-based acoustic manikins to measure the decrease in sound pressure level transmitted into the ear canal, which precisely measure the change in air conducted sound, but neglect alternate pathways to the inner ear such as bone conduction. In a previous study we reported intracochlear pressures in cadaveric human specimens to acoustic shock waves, which revealed a substantial bone conducted component (Greene, et al., 2018). Here we evaluate insertion loss to several hearing protection devices (HPDs) in those same specimens using intracochlear pressure measurements. METHODS: Human cadaver heads were exposed to impulsive acoustic pressure waves with peak overpressures of 7 and 28 kPa (171 & 183 dB SPL). Ear canal (EAC), middle ear, and intracochlear sound pressure levels were measured bilaterally with fiber-optic pressure sensors. Surface-mounted sensors measured SPL and skull strain near the opening of each EAC and at the forehead. Responses were measured with specimen ears unoccluded, as reported previously, as well as fitted with four types of HPDs. Impulse peak insertion loss (IPIL) and impulse spectrum insertion loss (ISIL) were calculated for each HPD. RESULTS: For all HPDs, IPIL generally increases with exposure level, though ISIL tended to be more consistent, and the spectral characteristics across frequency appear to be highly dependent on exposure level. ISIL measured in the ear canal tended to overestimate insertion loss measured in the cochlea, particularly at frequencies > 1 kHz; however, low signal-to-noise in intracochlear pressures limited comparisons. As a proof of concept, 36 low-level unoccluded exposures, were averaged together, and the resulting signal-to-noise ratio was improved by up to 15 dB. CONCLUSIONS: Insertion loss measured in the cochlea was lower than in the ear canal, suggesting substantial contributions from transmission pathways in parallel with air conduction (e.g., bone conduction) were present, which will require novel strategies to mitigate. However, high variance was observed, and noise reduction strategies should be utilized in future studies to facilitate more precise insertion loss estimates.

IL-6 selectively suppresses cDC1 specification via C/EBPß.

Cytokines produced in association with tumors can impair antitumor immune responses by reducing the abundance of type 1 conventional dendritic cells (cDC1), but the mechanism remains unclear. Here, we show that tumor-derived IL-6 generally reduces cDC development but selectively impairs cDC1 development in both murine and human systems through the induction of C/EBPß in the common dendritic cell progenitor (CDP). C/EBPß and NFIL3 compete for binding to sites in the Zeb2 -165 kb enhancer and support or repress Zeb2 expression, respectively. At homeostasis, pre-cDC1 specification occurs upon Nfil3 induction and consequent Zeb2 suppression. However, IL-6 strongly induces C/EBPß expression in CDPs. Importantly, the ability of IL-6 to impair cDC development is dependent on the presence of C/EBPß binding sites in the Zeb2 -165 kb enhancer, as this effect is lost in Δ1+2+3 mutant mice in which these binding sites are mutated. These results explain how tumor-associated IL-6 suppresses cDC1 development and suggest therapeutic approaches preventing abnormal C/EBPß induction in CDPs may help reestablish cDC1 development to enhance antitumor immunity.