Myotropic effects of FMRFamide containing peptides on the heart of the mosquito Anopheles gambiae.

FMRFamide-like peptides (FLPs) are produced by invertebrate and vertebrate animals, and regulate diverse physiological processes. In insects, several FLPs modulate heart physiology, with some increasing and others decreasing dorsal vessel contraction dynamics. Here, we describe the FMRFamide gene structure in the mosquito, Anopheles gambiae, quantify the developmental and spatial expression of FMRFamide and its putative receptor (FMRFamideR), and show that the peptides FMRFamide and SALDKNFMRFamide have complex myotropic properties. RACE sequencing showed that the FMRFamide gene encodes eight putative FLPs and is alternatively spliced. Of the eight FLPs, only one is shared by A. gambiae, Aedes aegypti and Culex quinquefasciatus: SALDKNFMRFamide. Quantitative PCR showed that peak expression of FMRFamide and FMRFamideR occurs in second instar larvae and around eclosion. In adults, FMRFamide is primarily transcribed in the head and thorax, and FMRFamideR is primarily transcribed in the thorax. Intravital video imaging of mosquitoes injected FMRFamide and SALDKNFMRFamide revealed that at low doses these peptides increase heart contraction rates. At high doses, however, these peptides decrease heart contraction rates and alter the proportional directionality of heart contractions. Taken altogether, these data describe the FMRFamide gene in A. gambiae, and show that FLPs are complex modulators of mosquito circulatory physiology.

Malaria-driven expansion of adaptive-like functional CD56-negative NK cells correlates with clinical immunity to malaria.

Natural killer (NK) cells likely play an important role in immunity to malaria, but the effect of repeated malaria on NK cell responses remains unclear. Here, we comprehensively profiled the NK cell response in a cohort of 264 Ugandan children. Repeated malaria exposure was associated with expansion of an atypical, CD56neg population of NK cells that differed transcriptionally, epigenetically, and phenotypically from CD56dim NK cells, including decreased expression of PLZF and the Fc receptor γ-chain, increased histone methylation, and increased protein expression of LAG-3, KIR, and LILRB1. CD56neg NK cells were highly functional and displayed greater antibody-dependent cellular cytotoxicity than CD56dim NK cells. Higher frequencies of CD56neg NK cells were associated with protection against symptomatic malaria and high parasite densities. After marked reductions in malaria transmission, frequencies of these cells rapidly declined, suggesting that continuous exposure to Plasmodium falciparum is required to maintain this modified, adaptive-like NK cell subset.

Availability of medications for opioid use disorder in outpatient and inpatient pharmacies in South Florida: a secret shopper survey.

BACKGROUND: Despite the proven efficacy of medications for opioid use disorder (MOUD) and recent reduction in barriers to prescribers, numerous obstacles exist for patients seeking MOUD. Prior studies have used telephone surveys to investigate pharmacy-related barriers to MOUD. We applied this methodology to evaluate inpatient and outpatient pharmacy barriers to MOUD in South Florida. METHODS: Randomly selected pharmacies in South Florida (Miami-Dade, Broward, and Palm Beach Counties) were called using a standardized script with a "secret shopper" approach until 200 successful surveys had been completed. The primary outcome was the availability of any buprenorphine products. Second, a list of all 48 acute care hospitals within the aforementioned counties was compiled, and hospitals were contacted by telephone using a second structured script. RESULTS: A total of 1374 outpatient pharmacies and 48 inpatient pharmacies were identified. 378 randomly selected outpatient pharmacies were contacted to accrue 200 successful calls (53% success rate). All 48 inpatient pharmacies were contacted to successfully complete 25 inpatient surveys (52%). Of the 200 outpatient pharmacies contacted, 38% had any buprenorphine available. There was a significant difference in buprenorphine availability by county, with Miami-Dade having the least availability and Palm Beach having the most availability (27% vs. 47%, respectively; p = 0.04). Of the 38% with buprenorphine available, 82% had a sufficient supply for a two-week prescription of buprenorphine 8 mg twice daily. Of the pharmacies that did not have buprenorphine, 55% would be willing to order with a median estimated time to receive an order of 2 days (IQR 1.25-3 days). Of the 25 surveyed inpatient pharmacies, 88% reported having buprenorphine on inpatient formulary, and 55% of hospitals had at least one restriction on ordering of buprenorphine beyond federal regulations. CONCLUSIONS: The results of this study highlight significant pharmacy-related barriers to comprehensive OUD treatment across the healthcare system including both acute care hospital pharmacies and outpatient community pharmacies. Despite efforts to increase the number of MOUD providers, there still remain downstream obstacles to MOUD access.

Early immune responses have long-term associations with clinical, virologic, and immunologic outcomes in patients with COVID-19.

The great majority of SARS-CoV-2 infections are mild and uncomplicated, but some individuals with initially mild COVID-19 progressively develop more severe symptoms. Furthermore, there is substantial heterogeneity in SARS-CoV-2-specific memory immune responses following infection. There remains a critical need to identify host immune biomarkers predictive of clinical and immunologic outcomes in SARS-CoV-2-infected patients. Leveraging longitudinal samples and data from a clinical trial in SARS-CoV-2 infected outpatients, we used host proteomics and transcriptomics to characterize the trajectory of the immune response in COVID-19 patients within the first 2 weeks of symptom onset. We identify early immune signatures, including plasma RIG-I levels, early interferon signaling, and related cytokines (CXCL10, MCP1, MCP-2 and MCP-3) associated with subsequent disease progression, control of viral shedding, and the SARS-CoV-2 specific T cell and antibody response measured up to 7 months after enrollment. We found that several biomarkers for immunological outcomes are shared between individuals receiving BNT162b2 (Pfizer-BioNTech) vaccine and COVID-19 patients. Finally, we demonstrate that machine learning models using 7-10 plasma protein markers measured early within the course of infection are able to accurately predict disease progression, T cell memory, and the antibody response post-infection in a second, independent dataset.

Age-dependent changes in circulating Tfh cells influence development of functional malaria antibodies in children.

T-follicular helper (Tfh) cells are key drivers of antibodies that protect from malaria. However, little is known regarding the host and parasite factors that influence Tfh and functional antibody development. Here, we use samples from a large cross-sectional study of children residing in an area of high malaria transmission in Uganda to characterize Tfh cells and functional antibodies to multiple parasites stages. We identify a dramatic re-distribution of the Tfh cell compartment with age that is independent of malaria exposure, with Th2-Tfh cells predominating in early childhood, while Th1-Tfh cell gradually increase to adult levels over the first decade of life. Functional antibody acquisition is age-dependent and hierarchical acquired based on parasite stage, with merozoite responses followed by sporozoite and gametocyte antibodies. Antibodies are boosted in children with current infection, and are higher in females. The children with the very highest antibody levels have increased Tfh cell activation and proliferation, consistent with a key role of Tfh cells in antibody development. Together, these data reveal a complex relationship between the circulating Tfh compartment, antibody development and protection from malaria.

TNF-α+ CD4+ T cells dominate the SARS-CoV-2 specific T cell response in COVID-19 outpatients and are associated with durable antibodies.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-specific CD4+ T cells are likely important in immunity against coronavirus 2019 (COVID-19), but our understanding of CD4+ longitudinal dynamics following infection and of specific features that correlate with the maintenance of neutralizing antibodies remains limited. Here, we characterize SARS-CoV-2-specific CD4+ T cells in a longitudinal cohort of 109 COVID-19 outpatients enrolled during acute infection. The quality of the SARS-CoV-2-specific CD4+ response shifts from cells producing interferon gamma (IFNγ) to tumor necrosis factor alpha (TNF-α) from 5 days to 4 months post-enrollment, with IFNγ-IL-21-TNF-α+ CD4+ T cells the predominant population detected at later time points. Greater percentages of IFNγ-IL-21-TNF-α+ CD4+ T cells on day 28 correlate with SARS-CoV-2-neutralizing antibodies measured 7 months post-infection (⍴ = 0.4, p = 0.01). mRNA vaccination following SARS-CoV-2 infection boosts both IFNγ- and TNF-α-producing, spike-protein-specific CD4+ T cells. These data suggest that SARS-CoV-2-specific, TNF-α-producing CD4+ T cells may play an important role in antibody maintenance following COVID-19.

Early immune markers of clinical, virological, and immunological outcomes in patients with COVID-19: a multi-omics study.

Background: The great majority of severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) infections are mild and uncomplicated, but some individuals with initially mild COVID-19 progressively develop more severe symptoms. Furthermore, there is substantial heterogeneity in SARS-CoV-2-specific memory immune responses following infection. There remains a critical need to identify host immune biomarkers predictive of clinical and immunological outcomes in SARS-CoV-2-infected patients. Methods: Leveraging longitudinal samples and data from a clinical trial (N=108) in SARS-CoV-2-infected outpatients, we used host proteomics and transcriptomics to characterize the trajectory of the immune response in COVID-19 patients. We characterized the association between early immune markers and subsequent disease progression, control of viral shedding, and SARS-CoV-2-specific T cell and antibody responses measured up to 7 months after enrollment. We further compared associations between early immune markers and subsequent T cell and antibody responses following natural infection with those following mRNA vaccination. We developed machine-learning models to predict patient outcomes and validated the predictive model using data from 54 individuals enrolled in an independent clinical trial. Results: We identify early immune signatures, including plasma RIG-I levels, early IFN signaling, and related cytokines (CXCL10, MCP1, MCP-2, and MCP-3) associated with subsequent disease progression, control of viral shedding, and the SARS-CoV-2-specific T cell and antibody response measured up to 7 months after enrollment. We found that several biomarkers for immunological outcomes are shared between individuals receiving BNT162b2 (Pfizer-BioNTech) vaccine and COVID-19 patients. Finally, we demonstrate that machine-learning models using 2-7 plasma protein markers measured early within the course of infection are able to accurately predict disease progression, T cell memory, and the antibody response post-infection in a second, independent dataset. Conclusions: Early immune signatures following infection can accurately predict clinical and immunological outcomes in outpatients with COVID-19 using validated machine-learning models. Funding: Support for the study was provided from National Institute of Health/National Institute of Allergy and Infectious Diseases (NIH/NIAID) (U01 AI150741-01S1 and T32-AI052073), the Stanford's Innovative Medicines Accelerator, National Institutes of Health/National Institute on Drug Abuse (NIH/NIDA) DP1DA046089, and anonymous donors to Stanford University. Peginterferon lambda provided by Eiger BioPharmaceuticals.

Emerging role of γδ T cells in vaccine-mediated protection from infectious diseases.

γδ T cells are fascinating cells that bridge the innate and adaptive immune systems. They have long been known to proliferate rapidly following infection; however, the identity of the specific γδ T cell subsets proliferating and the role of this expansion in protection from disease have only been explored more recently. Several recent studies have investigated γδ T-cell responses to vaccines targeting infections such as Mycobacterium, Plasmodium and influenza, and studies in animal models have provided further insight into the association of these responses with improved clinical outcomes. In this review, we examine the evidence for a role for γδ T cells in vaccine-induced protection against various bacterial, protozoan and viral infections. We further discuss results suggesting potential mechanisms for protection, including cytokine-mediated direct and indirect killing of infected cells, and highlight remaining open questions in the field. Finally, building on current efforts to integrate strategies targeting γδ T cells into immunotherapies for cancer, we discuss potential approaches to improve vaccines for infectious diseases by inducing γδ T-cell activation and cytotoxicity.