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Acetaminophen (APAP)-induced liver injury is associated with activation of coagulation and fibrinolysis. In mice, both tissue factor-dependent thrombin generation and plasmin activity have been shown to promote liver injury after APAP overdose. However, the contribution of the contact and intrinsic coagulation pathways has not been investigated in this model. Mice deficient in individual factors of the contact (factor XII [FXII] and prekallikrein) or intrinsic coagulation (FXI) pathway were administered a hepatotoxic dose of 400 mg/kg of APAP. Neither FXII, FXI, nor prekallikrein deficiency mitigated coagulation activation or hepatocellular injury. Interestingly, despite the lack of significant changes to APAP-induced coagulation activation, markers of liver injury and inflammation were significantly reduced in APAP-challenged high-molecular-weight kininogen-deficient (HK-/-) mice. Protective effects of HK deficiency were not reproduced by inhibition of bradykinin-mediated signaling, whereas reconstitution of circulating levels of HK in HK-/- mice restored hepatotoxicity. Fibrinolysis activation was observed in mice after APAP administration. Western blotting, enzyme-linked immunosorbent assay, and mass spectrometry analysis showed that plasmin efficiently cleaves HK into multiple fragments in buffer or plasma. Importantly, plasminogen deficiency attenuated APAP-induced liver injury and prevented HK cleavage in the injured liver. Finally, enhanced plasmin generation and HK cleavage, in the absence of contact pathway activation, were observed in plasma of patients with acute liver failure due to APAP overdose. In summary, extrinsic but not intrinsic pathway activation drives the thromboinflammatory pathology associated with APAP-induced liver injury in mice. Furthermore, plasmin-mediated cleavage of HK contributes to hepatotoxicity in APAP-challenged mice independently of thrombin generation or bradykinin signaling.

Patients with biallelic loss-of-function variants of AIRE suffer from autoimmune polyendocrine syndrome type-1 (APS-1) and produce a broad range of autoantibodies (auto-Abs), including circulating auto-Abs neutralizing most type I interferons (IFNs). These auto-Abs were recently reported to account for at least 10% of cases of life-threatening COVID-19 pneumonia in the general population. We report 22 APS-1 patients from 21 kindreds in seven countries, aged between 8 and 48 yr and infected with SARS-CoV-2 since February 2020. The 21 patients tested had auto-Abs neutralizing IFN-alpha subtypes and/or IFN-w; one had anti-IFN-8 and another anti-IFN-E, but none had anti-IFN-K. Strikingly, 19 patients (86%) were hospitalized for COVID-19 pneumonia, including 15 (68%) admitted to an intensive care unit, 11 (50%) who required mechanical ventilation, and four (18%) who died. Ambulatory disease in three patients (14%) was possibly accounted for by prior or early specific interventions. Preexisting auto-Abs neutralizing type I IFNs in APS-1 patients confer a very high risk of life-threatening COVID-19 pneumonia at any age.

The NIH Virtual SARS-CoV-2 Antiviral Summit, held on 6 November 2020, was organized to provide an overview on the status and challenges in developing antiviral therapeutics for coronavirus disease 2019 (COVID-19), including combinations of antivirals. Scientific experts from the public and private sectors convened virtually during a live videocast to discuss severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) targets for drug discovery as well as the preclinical tools needed to develop and evaluate effective small-molecule antivirals. The goals of the Summit were to review the current state of the science, identify unmet research needs, share insights and lessons learned from treating other infectious diseases, identify opportunities for public-private partnerships, and assist the research community in designing and developing antiviral therapeutics. This report includes an overview of therapeutic approaches, individual panel summaries, and a summary of the discussions and perspectives on the challenges ahead for antiviral development.

Staphylococcus saprophyticus is a common pathogen of the urinary tract, a heavy metal-rich environment, but information regarding its heavy metal resistance is unknown. We investigated 422 S. saprophyticus isolates from human infection and colonization/contamination, animals, and environmental sources for resistance to copper, zinc, arsenic, and cadmium using the agar dilution method. To identify the genes associated with metal resistance and assess possible links to pathogenicity, we accessed the whole-genome sequence of all isolates and used in silico and pangenome-wide association approaches. The MIC values for copper and zinc were uniformly high (1,600mg/liter). Genes encoding copper efflux pumps (copA, copB, copZ, mco, and csoR) and zinc transporters (zinT, czrAB, znuBC, and zur) were abundant in the population (20 to 100%). Arsenic and cadmium showed various susceptibility levels. Genes encoding the ars operon (arsRDABC), an ABC transporter and a two-component permease, were linked to resistance to arsenic (MICs >= 1,600mg/liter; 14% [58/422]; P < 0.05). At least three cad genes (cadA or cadC and cadD-cadX or czrC) and genes encoding multidrug efflux pumps and hyperosmoregulation in acidified conditions were associated with resistance to cadmium (MICs >= 200mg/liter; 20% [85/422]; P < 0.05). These resistance genes were frequently carried by mobile genetic elements. Resistance to arsenic and cadmium were linked to human infection and a clonal lineage originating in animals (P < 0.05). Altogether, S. saprophyticus was highly resistant to heavy metals and accumulated multiple metal resistance determinants. The highest arsenic and cadmium resistance levels were associated with infection, suggesting resistance to these metals is relevant for S. saprophyticus pathogenicity.

Investigation of changes in the skin microbiome following treatment of atopic dermatitis (AD) with dupilumab may provide valuable insights into the skin microbiome as a therapeutic target. The aim of this study is to assess changes in the AD skin microbiome following treatment of AD with dupilumab (n = 27). E-swabs were collected from nose, lesional, and nonlesional skin before and after 16 weeks of dupilumab therapy, and the microbiome was analyzed by 16S rRNA and tuf gene sequencing. Data for 17 patients with milder disease receiving treatment with non-targeted therapies are also presented. The results show that both groups experienced clinical improvement (p < 0.001) following dupilumab therapy and that Shannon diversity increased and bacterial community structure changed. The relative abundance of the genus Staphylococcus (S.) and S. aureus decreased, while that of S. epidermidis and S. hominis increased. No significant changes were observed for patients receiving non-targeted treatments. The increases in S. epidermidis and S. hominis and the decrease in S. aureus correlated with clinical improvement. Furthermore, changes in S. hominis and S. epidermidis correlated inversely with S. aureus. In conclusion, treatment with dupilumab significantly changed the skin microbiome and decreased S. aureus. Our results suggest a favorable role of commensal staphylococci in AD.

Germinal centers (GCs) are the site of immunoglobulin somatic hypermutation and affinity maturation, processes essential to an effective antibody response. The formation of GCs has been studied in detail, but less is known about what leads to their regression and eventual termination, factors that ultimately limit the extent to which antibodies mature within a single reaction. We show that contraction of immunization-induced GCs is immediately preceded by an acute surge in GC-resident Foxp3(+) T cells, attributed at least partly to up-regulation of the transcription factor Foxp3 by T follicular helper (T-FH) cells. Ectopic expression of Foxp3 in T-FH cells is sufficient to decrease GC size, implicating the natural up-regulation of Foxp3 by T-FH cells as a potential regulator of GC lifetimes.

In contrast to the western honey bee, Apis mellifera, other honey bee species have been largely neglected despite their importance and diversity. The genetic basis of the evolutionary diversification of honey bees remains largely unknown. Here, we provide a genome-wide comparison of three honey bee species, each representing one of the three subgenera of honey bees, namely the dwarf (Apis florea), giant (A. dorsata), and cavity-nesting (A. mellifera) honey bees with bumblebees as an outgroup. Our analyses resolve the phylogeny of honey bees with the dwarf honey bees diverging first. We find that evolution of increased eusocial complexity in Apis proceeds via increases in the complexity of gene regulation, which is in agreement with previous studies. However, this process seems to be related to pathways other than transcriptional control. Positive selection patterns across Apis reveal a trade-off between maintaining genome stability and generating genetic diversity, with a rapidly evolving piRNA pathway leading to genomes depleted of transposable elements, and a rapidly evolving DNA repair pathway associated with high recombination rates in all Apis species. Diversification within Apis is accompanied by positive selection in several genes whose putative functions present candidate mechanisms for lineage-specific adaptations, such as migration, immunity, and nesting behavior.

Until 2010, the National Assisted Reproductive Technology Surveillance System (NASS) report, published annually by the Center for Disease Control and Prevention (CDC), demonstrated almost constantly improving live birth rates following fresh non-donor (fnd) in vitro fertilization (IVF) cycles. Almost unnoticed by profession and public, by 2016 they, however, reached lows not seen since 1996-1997. We here attempted to understand underlying causes for this decline. This study used publicly available IVF outcome data, reported by the CDC annually under Congressional mandate, involving over 90% of U.S. IVF centers and over 95% of U.S. IVF cycles. Years 2005, 2010, 2015 and 2016 served as index years, representing respectively, 27,047, 30,425, 21,771 and 19,137 live births in fnd IVF cycles. Concomitantly, the study associated timelines for introduction of new add-ons to IVF practice with changes in outcomes of fnd IVF cycles. Median female age remained at 36.0 years during the study period and center participation was surprisingly stable, thereby confirming reasonable phenotype stability. Main outcome measures were associations of specific IVF practice changes with declines in live IVF birth rates. Time associations were observed with increased utilization of "all-freeze" cycles (embryo banking), mild ovarian stimulation protocols, preimplantation genetic testing for aneuploidy (PGT-A) and increasing utilization of elective single embryo transfer (eSET). Among all add-ons, PGT-A, likely, affected fndIVF most profoundly. Though associations cannot denote causation, they can be hypothesis-generating. Here presented time-associations are compelling, though some of observed pregnancy and live birth loss may have been compensated by increases in frozen-thawed cycles and consequential pregnancies and live births not shown here. Pregnancies in frozen-thawed cycles, however, represent additional treatment cycles, time delays and additional costs. IVF live birth rates not seen since 1996-1997, and a likely continuous downward trend in U.S. IVF outcomes, therefore, mandate a reversal of current outcome trends, whatever ultimately the causes.

More than one year after its inception, the coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) remains difficult to control despite the availability of several working vaccines. Progress in controlling the pandemic is slowed by the emergence of variants that appear to be more transmissible and more resistant to antibodies(1,2). Here we report on a cohort of 63 individuals who have recovered from COVID-19 assessed at 1.3, 6.2 and 12 months after SARS-CoV-2 infection, 41% of whom also received mRNA vaccines(3,4). In the absence of vaccination, antibody reactivity to the receptor binding domain (RBD) of SARS-CoV-2, neutralizing activity and the number of RBD-specific memory B cells remain relatively stable between 6 and 12 months after infection. Vaccination increases all components of the humoral response and, as expected, results in serum neutralizing activities against variants of concern similar to or greater than the neutralizing activity against the original Wuhan Hu-1 strain achieved by vaccination of naive individuals(2,5-8). The mechanism underlying these broad-based responses involves ongoing antibody somatic mutation, memory B cell clonal turnover and development of monoclonal antibodies that are exceptionally resistant to SARS-CoV-2 RBD mutations, including those found in the variants of concern(4,9). In addition, B cell clones expressing broad and potent antibodies are selectively retained in the repertoire over time and expand markedly after vaccination. The data suggest that immunity in convalescent individuals will be very long lasting and that convalescent individuals who receive available mRNA vaccines will produce antibodies and memory B cells that should be protective against circulating SARS-CoV-2 variants.

Although mutations in DNA are the best-studied source of neoantigens that determine response to immune checkpoint blockade, alterations in RNA splicing within cancer cells could similarly result in neoepitope production. However, the endogenous antigenicity and clinical potential of such splicing-derived epitopes have not been tested. Here, we demonstrate that pharmacologic modulation of splicing via specific drug classes generates bona fide neoantigens and elicits anti-tumor immunity, augmenting checkpoint immunotherapy. Splicing modulation inhibited tumor growth and enhanced checkpoint blockade in a manner dependent on host T cells and peptides presented on tumor MHC class I. Splicing modulation induced stereotyped splicing changes across tumor types, altering the MHC I-bound immunopeptidome to yield splicing-derived neoepitopes that trigger an anti-tumor T cell response in vivo. These data definitively identify splicing modulation as an untapped source of immunogenic peptides and provide ameans to enhance response to checkpoint blockade that is readily translatable to the clinic.