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Previously published digital autoradiography of H-3-labeled capecitabine reveals a near-uniform distribution of activity throughout a murine pancreatic model. This is in contrast both to C-14-labeled gemcitabine, and established expectations, as the dense stroma of pancreatic cancer is understood to inhibit drug penetration. Capecitabine is a pro-drug for 5 FU. The positioning of the radiolabel on capecitabine leaves open the possibility that much of the autoradiographic signal is generated by nontoxic compounds. Studies were performed on tumors derived via organoid culture from a murine KPC tumor. As before, we performed autoradiography comparing H-3 capecitabine to the gemcitabine analog F-18-FAC. The metabolism of capecitabine in this model was studied through LC-MS of tumor tissue. The autoradiographs confirmed that the H-3 label from capecitabine was much more uniformly distributed through the tumor than the F-18 from the gemcitabine analog. LC-MS revealed that approximately 75% of the molar mass of capecitabine had been converted into 5 FU or pre-5 FU compounds. The remainder had been converted into nontoxic species. Therapeutically relevant capecitabine metabolites achieve a relatively even distribution in this pancreatic cancer model, in contrast to the gemcitabine analog F-18-FAC. In a human xenograft model, (BxPC3), the H-3 label from capecitabine was also uniformly spread across the tumor autoradiographs. However, at 2 h post-administration the metabolism of capecitabine had proceeded further and the bulk of the agent was in the form of nontoxic species.

Objectives: We describe the clinical, mycological, immunological, and genetic characteristics of six HIV-negative patients presenting with invasive cryptococcosis.& nbsp;Methods: Patients with cryptococcosis without any of the classical risk factors, such as HIV infection, followed at Cayenne Hospital, were prospectively included. An immunologic and genetic assessment was performed.& nbsp;Results: Five male patients and one female patient, 5 adults and one child, were investigated. All presented a neuromeningeal localization. Cryptococcus neoformans var. gattii and C. neoformans var. grubii were isolated in two and three patients, respectively, whereas one patient could not be investigated. Overall, we did not observe any global leukocyte defect. Two patients were found with high levels of circulating autoantibodies against Granulocyte macrophage-colony stimulating factor (GM-CSF), and none had detectable levels of autoantibodies against Interferon gamma (IFN-gamma) Sequencing of STAT1 exons and flanking regions performed for four patients was wild type.& nbsp;Conclusion: To better understand cryptococcosis in patients with cryptococcosis but otherwise healthy, further explorations are needed with repeated immune checkups and strain virulence studies.

Background: Previous research has detailed the impor-tance of a racially and ethnically diverse physician workforce in improving access to care, communication, patient satisfaction, and health outcomes, particularly for underserved and systemi-cally marginalized patients (1). Despite this need, disparities in representation within medicine, including internal medicine (IM), persist. Many identities fall under the umbrella of diversity; here, references to diversity pertain to race/ethnicity. Objective: To elucidate trends in representation for IM resi-dency applicants and matriculants who identify as underrepre-sented in medicine (UIM). Methods and Findings: Data about applicants and matriculants to Accreditation Council for Graduate Medical Education IM resi-dency programs between July 2010 and July 2018 were acquired from the Association of American Medical Colleges. Institutional review board review was not required for the deidentified, publicly available data. Analysis was performed using Prism (version 9.2.0; GraphPad Software) and Excel (version 16.43; Microsoft). Between 2010 and 2018, a total of 214656 unique persons applied to IM residency programs and 87489 matriculated; 28222 of the applicants (13.2%) and 9269 of the matriculants (10.6%) identified as a UIM race/ethnicity (Figure, A). The proportion of aggregate UIM applicants grew minimally but significantly (slope, 0.34 [95% CI, 0.29 to 0.39]; Figure, B). On stratified analysis, a statistically significant increase was seen only for applicants who were Black or African American (slope, 0.11 [CI, 0.05 to 0.17]) and those who were Hispanic, Latino, or of Spanish origin (slope, 0.22 [CI, 0.15 to 0.29]). Trends for the proportion of aggregate UIM matriculants similarly had minimal but significant growth (slope, 0.11 [CI, 0.02 to 0.20]; Figure, C). In examining disaggregated UIM matriculant data, only the proportion of matriculants who were Hispanic, Latino, or of Spanish origin significantly changed (slope, 0.13 [CI, 0.05 to 0.21]).

B cells, which are critical for intestinal homeostasis, remain understudied in ulcerative colitis (UC). In this study, we recruited three cohorts of patients with UC (primary cohort, n = 145; validation cohort 1, n = 664; and validation cohort 2, n = 143) to comprehensively define the landscape of B cells during UC-associated intestinal inflammation. Using single-cell RNA sequencing, single-cell IgH gene sequencing and protein-level validation, we mapped the compositional, transcriptional and clonotypic landscape of mucosal and circulating B cells. We found major perturbations within the mucosal B cell compartment, including an expansion of naive B cells and IgG(+) plasma cells with curtailed diversity and maturation. Furthermore, we isolated an auto-reactive plasma cell clone targeting integrin alpha v beta 6 from inflamed UC intestines. We also identified a subset of intestinal CXCL13-expressing TFH-like T peripheral helper cells that were associated with the pathogenic B cell response. Finally, across all three cohorts, we confirmed that changes in intestinal humoral immunity are reflected in circulation by the expansion of gut-homing plasmablasts that correlates with disease activity and predicts disease complications. Our data demonstrate a highly dysregulated B cell response in UC and highlight a potential role of B cells in disease pathogenesis. Multi-modal profiling reveals major alterations in colonic B cells in patients with ulcerative colitis, including reduced clonal diversity of plasma cells, and suggests that circulating gut-homing plasmablasts could serve as a biomarker for disease activity.

The photoreceptor rhodopsin (Rho) becomes active when a tethered inverse agonist ligand (11CR) is photoconverted to an agonist (ATR). The ligand-binding pocket of inactive rhodopsin is completely enclosed, whereas active rhodopsin displays pores accessible from the lipid bilayer. Stabilization of active rhodopsin impedes 11CR binding and photoreceptor dark adaptation. Here, we used genetic code expansion and bioorthogonal labeling to engineer Rho mutants that serve as FRET sensors for measuring 11CR binding kinetics and energetics. We found that mutations that alter a channel between transmembrane helices 5 and 6 (TM5/6) dramatically affect 11CR binding kinetics but not agonist release kinetics. Our data provide direct experimental evidence for 11CR entry between TM5/6 in Rho that involves dynamic allosteric control of the ligand entry channel. Our findings provide a conceptual framework for understanding the function of G protein-coupled receptors with hydrophobic ligands that are hypothesized to enter their binding pockets through transmembrane pores.

Background. Acute rejection (AR) and recurrent hepatitis C virus (R-HCV) are significant complications in liver allograft recipients. Noninvasive diagnosis of intragraft pathologies may improve their management. Methods. We performed small RNA sequencing and microRNA (miRNA) microarray profiling of RNA from sera matched to liver allograft biopsies from patients with nonimmune, nonviral (NINV) native liver disease. Absolute levels of informative miRNAs in 91 sera matched to 91 liver allograft biopsies were quantified using customized real-time quantitative PCR (RT-qPCR) assays: 30 biopsy-matched sera from 26 unique NINV patients and 61 biopsy-matched sera from 41 unique R-HCV patients. The association between biopsy diagnosis and miRNA abundance was analyzed by logistic regression and calculating the area under the receiver operating characteristic curve. Results. Nine miRNAs-miR-22, miR-34a, miR-122, miR-148a, miR-192, miR-193b, miR-194, miR-210, and miR-885-5p-were identified by both sRNA-seq and TLDA to be associated with NINV-AR. Logistic regression analysis of absolute levels of miRNAs and goodness-of-fit of predictors identified a linear combination of miR-34a + miR-210 (P < 0.0001) as the best statistical model and miR-122 + miR-210 (P < 0.0001) as the best model that included miR-122. A different linear combination of miR-34a + miR-210 (P < 0.0001) was the best model for discriminating NINV-AR from R-HCV with intragraft inflammation, and miR-34a + miR-122 (P < 0.0001) was the best model for discriminating NINV-AR from R-HCV with intragraft fibrosis. Conclusions. Circulating levels of miRNAs, quantified using customized RT-qPCR assays, may offer a rapid and noninvasive means of diagnosing AR in human liver allografts and for discriminating AR from intragraft inflammation or fibrosis due to R-HCV.

These data identify a host pathway by which VPS29 and associated factors control the endosomal environment in a manner that influences susceptibility to viral infection. This pathway could serve as a pharmaceutical target for intervention in zoonotic viral diseases, including those caused by coronaviruses, influenza viruses, and filoviruses, all of which are pandemic threats. Emerging zoonotic viral pathogens threaten global health, and there is an urgent need to discover host and viral determinants influencing infection. We performed a loss-of-function genome-wide CRISPR screen in a human lung cell line using HCoV-OC43, a human betacoronavirus. One candidate gene, VPS29, a component of the retromer complex, was required for infection by HCoV-OC43, SARS-CoV-2, other endemic- and pandemic-threat coronaviruses, as well as ebolavirus. Notably, we observed a heightened requirement for VPS29 by the recently described Omicron variant of SARS-CoV-2 compared to the ancestral variant. However, VPS29 deficiency had no effect on certain other viruses that enter cells via endosomes and had an opposing, enhancing effect on influenza A virus infection. Deficiency in VPS29 or other retromer components caused changes in endosome morphology and acidity and attenuated the activity of endosomal proteases. These changes in endosome properties caused incoming coronavirus, but not influenza virus particles, to become entrapped therein. Overall, these data show how host regulation of endosome characteristics can influence cellular susceptibility to viral infection and identify a host pathway that could serve as a pharmaceutical target for intervention in zoonotic viral diseases. IMPORTANCE These data identify a host pathway by which VPS29 and associated factors control the endosomal environment in a manner that influences susceptibility to viral infection. This pathway could serve as a pharmaceutical target for intervention in zoonotic viral diseases, including those caused by coronaviruses, influenza viruses, and filoviruses, all of which are pandemic threats. Our findings show how host regulation of endosome characteristics can influence viral susceptibility in both a positive and negative manner.

Recent studies reported the presence of pre-existing autoantibodies (auto-Abs) neutralizing type I interferons (IFNs) in at least 15% of patients with critical COVID-19 pneumonia. In one study, these auto-Abs were found in almost 20% of deceased patients across all ages. We aimed to assess the prevalence and clinical impact of the auto-Abs to type I IFNs in the Seine-Saint-Denis district, which was one of the most affected areas by COVID-19 in France during the first wave. We tested for the presence of auto-Abs neutralizing type I IFNs in a cohort of patients admitted for critical COVID-19 pneumonia during the first wave in the spring of 2020 in the medicine departments at Robert Ballanger Hospital, Aulnay sous Bois. We found circulating auto-Abs that neutralized 100 pg/mL IFN-alpha 2 and/or IFN-omega in the plasma (diluted 1/10) of 7.9% (11 of 139) of the patients hospitalized for critical COVID-19. The presence of neutralizing auto-Abs was associated with an increased risk of mortality, as these auto-Abs were detected in 21% of patients who died from COVID-19 pneumonia. Deceased patients with and without auto-Abs did not present overt clinical differences. These results confirm both the importance of type I IFN immunity in host defense against SARS-CoV-2 infection and the usefulness of detection of auto-Abs neutralizing type I IFNs in the management of patients.

Globally, autosomal recessive IFNAR1 deficiency is a rare inborn error of immunity underlying susceptibility to live attenuated vaccine and wild-type viruses. We report seven children from five unrelated kindreds of western Polynesian ancestry who suffered from severe viral diseases. All the patients are homozygous for the same nonsense IFNAR1 variant (p.GLu386*). This allele encodes a truncated protein that is absent from the cell surface and is loss-of-function. The fibroblasts of the patients do not respond to type I IFNs (IFN-alpha 2, IFN-omega, or IFN-beta). Remarkably, this IFNAR1 variant has a minor allele frequency >1% in Samoa and is also observed in the Cook, Society, Marquesas, and Austral islands, as well as Fiji, whereas it is extremely rare or absent in the other populations tested, including those of the Pacific region. Inherited IFNAR1 deficiency should be considered in individuals of Polynesian ancestry with severe viral illnesses.

Interferon signaling mediates resistance to immune checkpoint blockade therapy, but the underlying mechanisms are poorly understood. In this issue of Immunity, Cucolo et al. identify RIPK1 as an interferon-stimulated gene with potent effects on cell extrinsic and intrinsic immunotherapy resistance.