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Advances in multimodal single cell analysis can empower high-resolution dissection of human vaccination responses. The resulting data capture multiple layers of biological variations, including molecular and cellular states, vaccine formulations, inter- and intra-subject differences, and responses unfolding over time. Transforming such data into biological insight remains a major challenge. Here we present a systematic framework applied to multimodal single cell data obtained before and after influenza vaccination without adjuvants or pandemic H5N1 vaccination with the AS03 adjuvant. Our approach pinpoints responses shared across or unique to specific cell types and identifies adjuvant specific signatures, including pro-survival transcriptional states in B lymphocytes that emerged one day after vaccination. We also reveal that high antibody responders to the unadjuvanted vaccine have a distinct baseline involving a rewired network of cell type specific transcriptional states. Remarkably, the status of certain innate immune cells in this network in high responders of the unadjuvanted vaccine appear “naturally adjuvanted”: they resemble phenotypes induced early in the same cells only by vaccination with AS03. Furthermore, these cell subsets have elevated frequency in the blood at baseline and increased cell-intrinsic phospho-signaling responses after LPS stimulationex vivoin high compared to low responders. Our findings identify how variation in the status of multiple immune cell types at baseline may drive robust differences in innate and adaptive responses to vaccination and thus open new avenues for vaccine development and immune response engineering in humans.

Unbiased plasma proteomics in a matched case-control study of treated people with human immunodeficiency virus (PWH) revealed the complement cascade as being among the top pathways enriched in PWH. Specific complement components, namely C5, associated significantly with non-AIDS comorbidity prevalence, and did so more strongly than previously established predictive biomarkers.

The surgical ciliated cyst is an iatrogenic lesion occurring after surgeries in which the Schneiderian membrane has been exposed, such as in orthognathic surgery or maxillary sinus procedures. This lesion has been infrequently documented in western countries. In this case series, we present three cases of surgical ciliated cysts of the maxilla.

Loss of gut mucosal integrity and an aberrant gut microbiota are proposed mechanisms contributing to chronic inflammation and increased morbidity and mortality during antiretroviral-treated HIV disease. Sexual practice has recently been uncovered as a major source of microbiota variation, potentially confounding prior observations of gut microbiota alterations among persons with HIV (PWH). To overcome this and other confounding factors, we examine a well-powered subset of AGEhIV Cohort participants comprising antiretroviral-treated PWH and seronegative controls matched for age, body-mass index, sex, and sexual practice. We report significant gut microbiota differences in PWH regardless of sex and sexual practice including Gammaproteobacteria enrichment, Lachnospiraceae and Ruminococcaceae depletion, and decreased alpha diversity. Men who have sex with men (MSM) exhibit a distinct microbiota signature characterized by Prevotella enrichment and increased alpha diversity, which is linked with receptive anal intercourse in both males and females. Finally, the HIV-associated microbiota signature correlates with inflammatory markers including suPAR, nadir CD4 count, and prevalence of age-associated noncommunicable comorbidities., The role of sexual practice in HIV-associated gut microbiota remains poorly understood. Here, in a cohort of chronically treated HIV-infected people, the authors show microbiome signatures to be independent of sex and sexual practice and that the extent of dysbiosis correlates with nadir CD4, inflammatory markers, and comorbidities.

Pediatric COVID-19 (pCOVID-19) is rarely severe, however a minority of SARS-CoV-2-infected children may develop MIS-C, a multisystem inflammatory syndrome with significant morbidity. In this longitudinal multi-institutional study, we used multi-omics to identify novel time- and treatment-related immunopathological signatures in children with COVID-19 (n=105) and MIS-C (n=76). pCOVID-19 was characterized by enhanced type I IFN responses, and MIS-C by type II IFN- and NF-κB dependent responses, matrisome activation, and increased levels of Spike protein. Reduced levels of IL-33 in pCOVID-19, and of CCL22 in MIS-C suggested suppression of Th2 responses. Expansion of TRBV11-2 T-cell clonotypes in MIS-C was associated with inflammation and signatures of T-cell activation, and was reversed by glucocorticoids. The association of MIS-C with the combination of HLA A*02, B*35, C*04 alleles suggests genetic susceptibility. MIS-C B cells showed higher mutation load. Use of IVIG was identified as a confounding factor in the interpretation of autoantibody levels.

X-linked immunodeficiency with magnesium defect, EBV infection, and neoplasia (XMEN) disease are caused by deficiency of the magnesium transporter 1 (MAGT1) gene. We studied 23 patients with XMEN, 8 of whom were EBV naive. We observed lymphadenopathy (LAD), cytopenias, liver disease, cavum septum pellucidum (CSP), and increased CD4-CD8-B220-TCRαβ+ T cells (αβDNTs), in addition to the previously described features of an inverted CD4/CD8 ratio, CD4+ T lymphocytopenia, increased B cells, dysgammaglobulinemia, and decreased expression of the natural killer group 2, member D (NKG2D) receptor. EBV-associated B cell malignancies occurred frequently in EBV-infected patients. We studied patients with XMEN and patients with autoimmune lymphoproliferative syndrome (ALPS) by deep immunophenotyping (32 immune markers) using time-of-flight mass cytometry (CyTOF). Our analysis revealed that the abundance of 2 populations of naive B cells (CD20+CD27-CD22+IgM+HLA-DR+CXCR5+CXCR4++CD10+CD38+ and CD20+CD27-CD22+IgM+HLA-DR+CXCR5+CXCR4+CD10-CD38-) could differentially classify XMEN, ALPS, and healthy individuals. We also performed glycoproteomics analysis on T lymphocytes and show that XMEN disease is a congenital disorder of glycosylation that affects a restricted subset of glycoproteins. Transfection of MAGT1 mRNA enabled us to rescue proteins with defective glycosylation. Together, these data provide new clinical and pathophysiological foundations with important ramifications for the diagnosis and treatment of XMEN disease.

COVID-19 exhibits extensive patient-to-patient heterogeneity. To link immune response variation to disease severity and outcome over time, we longitudinally assessed circulating proteins as well as 188 surface protein markers, transcriptome, and T-cell receptor sequence simultaneously in single peripheral immune cells from COVID-19 patients. Conditional-independence network analysis revealed primary correlates of disease severity, including gene expression signatures of apoptosis in plasmacytoid dendritic cells and attenuated inflammation but increased fatty acid metabolism in CD56dimCD16hi NK cells linked positively to circulating IL-15. CD8+ T cell activation was apparent without signs of exhaustion. While cellular inflammation was depressed in severe patients early after hospitalization, it became elevated by days 17-23 post symptom onset, suggestive of a late wave of inflammatory responses. Furthermore, circulating protein trajectories at this time were divergent between and predictive of recovery-fatal outcomes. Our findings stress the importance of timing in the analysis, clinical monitoring, and therapeutic intervention of COVID-19., A multiparameter metric developed by integrating circulating cytokines, multimodal single cell profiling, cell surface protein and immune cell analyses of COVID-19 patients over time, reveals a late wave of diverging inflammatory and host immune responses that predicts recovery versus fatality.

Multisystem inflammatory syndrome in children (MIS-C) is a life-threatening post-infectious complication occurring unpredictably weeks after mild or asymptomatic SARS-CoV-2 infection. We profiled MIS-C, adult COVID-19, and healthy pediatric and adult individuals using single-cell RNA sequencing, flow cytometry, antigen receptor repertoire analysis, and unbiased serum proteomics, which collectively identified a signature in MIS-C patients that correlated with disease severity. Despite having no evidence of active infection, MIS-C patients had elevated S100A-family alarmins and decreased antigen presentation signatures, indicative of myeloid dysfunction. MIS-C patients showed elevated expression of cytotoxicity genes in NK and CD8+ T cells and expansion of specific IgG-expressing plasmablasts. Clinically severe MIS-C patients displayed skewed memory T cell TCR repertoires and autoimmunity characterized by endothelium-reactive IgG. The alarmin, cytotoxicity, TCR repertoire, and plasmablast signatures we defined have potential for application in the clinic to better diagnose and potentially predict disease severity early in the course of MIS-C., Graphical abstract, Multisystem inflammatory syndrome in children (MIS-C) is a life-threatening and unpredictable condition of unknown etiology. Ramaswamy et al. use peripheral blood single-cell transcriptomic profiling along with other techniques to define key innate and adaptive signatures that characterize MIS-C.

SUMMARYMultisystem inflammatory syndrome in children (MIS-C) is a life-threatening post-infectious complication occurring unpredictably weeks after mild or asymptomatic SARS-CoV2 infection in otherwise healthy children. Here, we define immune abnormalities in MIS-C compared to adult COVID-19 and pediatric/adult healthy controls using single-cell RNA sequencing, antigen receptor repertoire analysis, unbiased serum proteomics, andin vitroassays. Despite no evidence of active infection, we uncover elevated S100A-family alarmins in myeloid cells and marked enrichment of serum proteins that map to myeloid cells and pathways including cytokines, complement/coagulation, and fluid shear stress in MIS-C patients. Moreover, NK and CD8 T cell cytotoxicity genes are elevated, and plasmablasts harboring IgG1 and IgG3 are expanded. Consistently, we detect elevated binding of serum IgG from severe MIS-C patients to activated human cardiac microvascular endothelial cells in culture. Thus, we define immunopathology features of MIS-C with implications for predicting and managing this SARS-CoV2-induced critical illness in children.

Summary Ebola virus (EBOV) causes epidemics with high mortality yet remains understudied due to the challenge of experimentation in high-containment and outbreak settings. Here, we used single-cell transcriptomics and CyTOF-based single-cell protein quantification to characterize peripheral immune cells during EBOV infection in rhesus monkeys. We obtained 100,000 transcriptomes and 15,000,000 protein profiles, finding that immature, proliferative monocyte-lineage cells with reduced antigen-presentation capacity replace conventional monocyte subsets, while lymphocytes upregulate apoptosis genes and decline in abundance. By quantifying intracellular viral RNA, we identify molecular determinants of tropism among circulating immune cells and examine temporal dynamics in viral and host gene expression. Within infected cells, EBOV downregulates STAT1 mRNA and interferon signaling, and it upregulates putative pro-viral genes (e.g., DYNLL1 and HSPA5), nominating pathways the virus manipulates for its replication. This study sheds light on EBOV tropism, replication dynamics, and elicited immune response and provides a framework for characterizing host-virus interactions under maximum containment., Graphical Abstract, Highlights • Interferon response is suppressed in infected cells but activated in bystander cells • EBOV represses antiviral genes and upregulates pro-viral genes in infected cells • Proliferative CD14– CD16– monocyte precursors expand in circulation during EVD • Identification of expression markers of EBOV tropism for circulating cells in vivo, Single-cell profiling of circulating immune cells during Ebola virus (EBOV) infection in non-human primates resolves molecular correlates of viral tropism, characterizes replication dynamics within infected cells, and distinguishes expression changes that are mediated by viral infection from those due to cytokine signaling.

SummaryEbola virus (EBOV) causes epidemics with high case fatality rates, yet remains understudied due to the challenge of experimentation in high-containment and outbreak settings. To better understand EBOV infection in vivo, we used single-cell transcriptomics and CyTOF-based single-cell protein quantification to characterize peripheral immune cell activity during EBOV infection in rhesus monkeys. We obtained 100,000 transcriptomes and 15,000,000 protein profiles, providing insight into pathogenesis. We find that immature, proliferative monocyte-lineage cells with reduced antigen presentation capacity replace conventional circulating monocyte subsets within days of infection, while lymphocytes upregulate apoptosis genes and decline in abundance. By quantifying viral RNA abundance in individual cells, we identify molecular determinants of tropism and examine temporal dynamics in viral and host gene expression. Within infected cells, we observe that EBOV down-regulates STAT1 mRNA and interferon signaling, and up-regulates putative pro-viral genes (e.g., DYNLL1 and HSPA5), nominating cellular pathways the virus manipulates for its replication. Overall, this study sheds light on EBOV tropism, replication dynamics, and elicited immune response, and provides a framework for characterizing interactions between hosts and emerging viruses in a maximum containment setting.

SOMAscan is an aptamer-based proteomics assay capable of measuring 1,305 human protein analytes in serum, plasma, and other biological matrices with high sensitivity and specificity. In this work, we present a comprehensive meta-analysis of performance based on multiple serum and plasma runs using the current 1.3 k assay, as well as the previous 1.1 k version. We discuss normalization procedures and examine different strategies to minimize intra- and interplate nuisance effects. We implement a meta-analysis based on calibrator samples to characterize the coefficient of variation and signal-over-background intensity of each protein analyte. By incorporating coefficient of variation estimates into a theoretical model of statistical variability, we also provide a framework to enable rigorous statistical tests of significance in intervention studies and clinical trials, as well as quality control within and across laboratories. Furthermore, we investigate the stability of healthy subject baselines and determine the set of analytes that exhibit biologically stable baselines after technical variability is factored in. This work is accompanied by an interactive web-based tool, an initiative with the potential to become the cornerstone of a regularly updated, high quality repository with data sharing, reproducibility, and reusability as ultimate goals.

Ozcay, Figen/0000-0002-5214-516X; celikel, cigdem/0000-0002-9360-8116 WOS: 000604865800004 PubMed: 33398182 CHAPLE disease is a lethal syndrome caused by genetic loss of the complement regulatory protein CD55. Lenardo, Ozen and their colleagues report that blockade of C5 complement activation in a small cohort of pediatric patients with CHAPLE disease reduced gastrointestinal pathology and restored their immunity and growth. Complement hyperactivation, angiopathic thrombosis and protein-losing enteropathy (CHAPLE disease) is a lethal disease caused by genetic loss of the complement regulatory protein CD55, leading to overactivation of complement and innate immunity together with immunodeficiency due to immunoglobulin wasting in the intestine. We report in vivo human data accumulated using the complement C5 inhibitor eculizumab for the medical treatment of patients with CHAPLE disease. We observed cessation of gastrointestinal pathology together with restoration of normal immunity and metabolism. We found that patients rapidly renormalized immunoglobulin concentrations and other serum proteins as revealed by aptamer profiling, re-established a healthy gut microbiome, discontinued immunoglobulin replacement and other treatments and exhibited catch-up growth. Thus, we show that blockade of C5 by eculizumab effectively re-establishes regulation of the innate immune complement system to substantially reduce the pathophysiological manifestations of CD55 deficiency in humans. Division of Intramural Research, National Institute of Allergy and Infectious Diseases, NIH, BCBB Support Services [HHSN316201300006W/HHSN27200002]; Marmara University, Scientific Research Projects Committee (BAPKO)Marmara University [SAG-C-TUP-230119-0018]; NIAID Microbiome Program, NIAID, NIH; NIHUnited States Department of Health & Human ServicesNational Institutes of Health (NIH) - USA [R01-GM129325] This work was supported in part by the Division of Intramural Research, National Institute of Allergy and Infectious Diseases, NIH, BCBB Support Services Contract HHSN316201300006W/HHSN27200002, and The Marmara University, Scientific Research Projects Committee (BAPKO, grant no. SAG-C-TUP-230119-0018). We thank the Turkish National Society of Allergy and Clinical Immunology (TNSACI) for supporting travel expenses for the screening studies. We thank C. Kemper of the National Heart, Lung and Blood Institute for thoughtful editing of the final manuscript. We thank M. Quinones, the Center for Human Immunology and the NIAID Microbiome Program, NIAID, NIH, for research support. We also thank A. Kiykim for patient care, A. Dalga and I. Tatli for technical assistance and H. Su and X. He for advice and assistance; D. Comrie, S. Kubo and J. Ravell for critical reading of the manuscript; and R. Kissinger for artwork. We thank important colleagues at Regeneron: A. N. Thomas for sample processing; C. Huang for biomarker analysis; H. Qiu, and E. Sook Yen for eculizumab analysis; and C. H. Lai, L. DeStefano and K. Donohue for total C5 analysis. Molecular graphics and analyses were performed with UCSF ChimeraX, developed by the Resource for Biocomputing, Visualization and Informatics at the University of California, San Francisco, with support from NIH R01-GM129325 and the Office of Cyber Infrastructure and Computational Biology, NIAID.

SOMAscan™ is a complex proteomic platform created by SomaLogic. Experimental data resulting from the assay is provided by SomaLogic in a proprietary text-based format called ADAT. This manuscript describes a user-friendly point and click open source, platform-independent software tool designed to be used for navigating and plotting data from an ADAT file. This tool was used either alone or in conjunction with other tools as a first pass analysis of the data on several different on-going research projects. We have seen a need from our experience for a web interface to the ADAT file so that users can navigate, generate plots, perform QC and conduct statistical analysis on their own data in a point and click manner. After several rounds of interacting with biologists and their requirements with respect to data analysis, we present an online interactive Shiny Web Tool for Navigating and Plotting data contained within the ADAT file. Extensive video tutorials, example data, the tool and the source code are available online.

Systemic Autoinflammatory diseases (SAIDs) present with chronic sterile inflammation. NLRP3 mutation cause the IL-1 mediated SAID Neonatal-Onset Multisystem Inflammatory Disease (NOMID), that presents early in life with fever, rash, neutrophilic meningitis, hearing loss and eye inflammation. Chronic Atypical Neutrophilic Dermatosis, Lipodystrophy and Elevated Temperature (CANDLE) caused by additive loss-of-function mutations in proteasome genes including PSMB8, leads to chronic type 1 interferon signaling, characterized by early-onset fever, neutrophilic panniculitis, lipodystrophy, cytopenias, myositis and lymphocytic meningitis. IL-1 blocking treatments are approved for NOMID, and JAK-inhibitors show efficacy in CANDLE treatment. We used proteomic analysis to compare differentially-regulated proteins in active NOMID and CANDLE compared to healthy children before and after treatment to identify inflammatory pathways that distinguish NOMID and CANDLE and are uniquely responsive to either IL-1 or IFN signaling blockade. Serum samples from active NOMID (n=12) and CANDLE (n=7) before and after treatment and healthy controls (n=7) were subjected to proteomic profiling using SomaLogic platform (n=1129 proteins). Enriched pathways were identified in REACTOME and KEGG. Cytoscape was used for network extraction and visualization. Differentially expressed proteins in NOMID vs CANDLE suggested endothelial cell function, coagulation and complement cascades and inflammation related pathways dysregulation. Distinct protein markers (i.e. cytokine and chemokines) are consisted the different immune dysregulation in NOMID and CANDLE; and uniquely respond to either IL-1 or IFN signaling blockade.

This article carefully examines the constitutionality of a recent capital child rape statute in Louisiana. It specifically focuses on whether such a statute violates the Eighth Amendment to the Constitution by being a disproportionate and cruel and unusual application of the death penalty. The origins, interpretation, and evolving nature of the Eighth Amendment are closely explored. The Furman procedural test and Solem proportionality test are applied to properly assess the statute's constitutionality. The relevant literature on these legal applications is also reviewed. In addition, the role of forensic psychology is investigated to determine how psychologists and psychiatrists may play a role in this new policy toward child molesters. Research on pedophilia as a mental abnormality and effective approaches to treatment of pedophilia by professionals are briefly discussed.