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Background Although cardiovascular mortality (CVM) rates in the United States have been declining overall, our study evaluated whether this holds true for areas with increased social deprivation. Methods and Results We used county‐level cross‐sectional age‐adjusted CVM rates (aa‐CVM) (2000–2019) linked to the Centers for Disease Control and Prevention Social Vulnerability Index (SVI–2010). We grouped counties as per SVI (Groups I 0–0.2, II 0.21–0.4, III 0.41–0.6, IV 0.61–0.8, and V 0.81–1) and calculated the relative change in the aa‐CVM between 2000 to 2003 and 2016 to 2019. We used adjusted linear regression analyses to explore the association between a higher SVI and temporal aa‐CVM improvement; we studied this temporal change in aa‐CVM across subgroups of race, sex, and location. The median aa‐CVM rate (per 100 000) was 272.6 (interquartile range [IQR]: 237.5–311.7). The aa‐CVM was higher in men (315.6 [IQR: 273.4–363.9]) than women (221.3 [IQR: 189.6–256.7]), and in Black residents (347.2 [IQR: 301.1–391.1]; P <0.001) than White residents (258.9 [IQR: 226–299.1]; P <0.001). The aa‐CVM for SVI I (233.6 [IQR: 214.8–257.0]) was significantly lower than that of group V (323.6 [IQR: 277.2–359.2]; P <0.001). The relative reduction in CVM was significantly higher for SVI group I (32.2% [IQR: 24.2–38.4]) than group V (27.2% [IQR: 19–34.1]) counties. After multivariable adjustment, a higher SVI index was associated with lower relative improvement in the age‐adjusted CVM (model coefficient −3.11 [95% CI, −5.66 to −1.22]; P <0.001). Conclusions Socially deprived counties in the United States had higher aa‐CVM rates, and the improvement in aa‐CVM over the past 20 years was lower in these counties.

Abstract Introduction: While CT lung cancer screening reduces lung cancer-specific mortality, there are remaining challenges. Radiomic tools promiss to address these challenges, however, they are subject to interobserver variability if semi-automated segmentation techniques are used. Herein we report interobserver variability for two validated radiomic tools, BRODERS (Benign versus aggRessive nODule Evaluation using Radiomic Stratification) and CANARY (Computer-Aided Nodule Assessment and Risk Yield). Methods We retrospectively analyzed the CT images of 95 malignant lung nodules of the adenocarcinoma spectrum using BRODERS and CANARY. Cases were identified at Mayo Clinic (n = 45) and Vanderbilt University Medical Center and Nashville/Veteran Administration Tennessee Valley Health Care System (n = 50). Three observers with different training levels (medical student, internal medicine resident and thoracic radiology fellow) each performed lung nodule segmentation. All methods were carried out in accordance with relevant guidelines and regulations Results Interclass correlation coefficients (ICC) of 0.77, 0.98 and 0.97 for the average nodule volume, BRODERS cancer probability and Score Indicative of Lesion Aggression (SILA) which summarizes the distribution of the CANARY exemplars indicated good to excellent reliability, respectively. The dice similarity coefficient was 0.79 and 0.81 for the data sets from the two institutions. Conclusion: BRODERS and CANARY are robust radiomics tools with excellent interobserver variability. These tools are simple and reliable regardless the observer/operator’s level of training.

Autologous mesenchymal stem/stromal cells (MSCs) have demonstrated important therapeutic effects in several diseases. Cardiovascular risk factors may impair MSC mitochondrial structure and function, but the underlying mechanisms remain unknown. We hypothesized that metabolic syndrome (MetS) induces epigenetic alterations in mitochondria-related genes in swine MSCs. Pigs were fed a Lean or MetS diet (n = 6 each) for 16 weeks. MSCs were collected from subcutaneous abdominal fat, and DNA hydroxymethylation (5 hmC) profiles of mitochondria-related genes (MitoCarta-2.0) were analyzed by hydroxymethylated DNA immunoprecipitation and next-generation sequencing (hMeDIP-seq) in Lean- and MetS-MSCs untreated or treated with the epigenetic modulator vitamin (Vit)-C (n = 3 each). Functional analysis of genes with differential 5 hmC regions was performed using DAVID6.8. Mitochondrial structure (electron microscopy), oxidative stress, and membrane potential were assessed. hMeDIP-seq identified 172 peaks (associated with 103 mitochondrial genes) with higher and 416 peaks (associated with 165 mitochondrial genes) with lower 5 hmC levels in MetS-MSCs versus Lean-MSCs (≥2-fold, p < 0.05). Genes with higher 5 hmC levels in MetS + MSCs were primarily implicated in fatty acid metabolism, whereas those with lower 5 hmC levels were associated with electron transport chain activity. Vit-C increased 5 hmC levels in mitochondrial antioxidant genes, improved mitochondrial structure and membrane potential, and decreased oxidative stress. MetS alters 5 hmC levels of mitochondria-related genes in swine MSCs. Vit-C modulated 5 hmC levels in these genes and preserved mitochondrial structure and function in MetS-MSCs. These observations may contribute to development of strategies to overcome the deleterious effects of MetS on MSCs.

Abstract Bacterial superantigens (BSAg) are produced by Staphylococcus aureus and Streptococcus pyogenes. BSAg bind directly to the MHC class II molecules without undergoing any processing and subsequently activate T cells in an antigen non-specific manner by interacting with the TCR Vβ region. BSAg have been implicated primarily in acute diseases such as food poisoning, toxic shock syndrome, etc. Since carrier strains of S. aureus and S. pyogenes can elaborate superantigens, there is a distinct possibility of chronic exposure to low doses of BSAg and it is imperative to study its outcome. As BSAg bind more avidly to human (HLA) than to murine (H-2) MHC class II, we delivered low concentrations (total of 10 μg/mouse) of BSAg to HLA-DR3 [staphylococcal enterotoxin (SE) B] or HLA-DQ8 [streptococcal pyrogenic exotoxin (SPE) A] transgenic mice through subcutaneously implanted miniosmotic pumps for 7 days. Continuous exposure to low concentrations of BSAg resulted in a significant protracted expansion of CD4+ T cells expressing TCR Vβ8 (for SEB); Vβ8 and Vβ14 (for SPEA) with a concomitant decrease in percentage of CD8+ T cells expressing these TCR. T cells bearing TCR Vβ6 showed an inverse correlation (slight decrease in CD4+ and increase in CD8+ compartments with SEB and SPEA). Histological examinations of major organs showed intense perivascular infiltration in lungs, kidneys and liver along with inflammatory infiltrates in the cardiac valvular endothelium in mice bearing pumps delivering BSAg but not PBS. In conclusion, we have shown the immunological outcome of chronic exposure to low levels of BSAg using the HLA class II transgenic mouse model.

Abstract Staphylococcal Enterotoxin B (SEB) is a superantigen made by S. aureus. SEB can cause food poisoning and shock and its stability and ease of preparation make it a potential agent of bioterrorism. SEB-mediated activation induces T cell proliferation and secretion of cytokines. Antibodies that block the interaction between SEB and MHC or SEB and TCR, can neutralize the activating effects of SEB and provide protection against its debilitating effects. Human IVIG, a therapy for SEB intoxication is of low potency and sometimes, short supply. To surmount we have derived of mouse monoclonal antibodies that block the binding of SEB to MHC at separate and distinct sites. Combinations of these antibodies synergistically neutralize SEB mediated T cell activation. Genes encoding the VL and VH genes of synergistically neutralizing pairs of cell lines were cloned and grafted onto genes encoding either the constant region of human IgH or Igκ and transfected into SP2/0 cells. Chimeric human-mouse antibodies expressed by the transfectants were purified. Tests of these chimeric antibodies demonstrated that they were neutralizing and in combination with each other, still capable of synergistic neutralization of SEB. Supported by NIH grant # AI057652

Abstract TCR interactions with agonist pMHCs induce a conformational change in the tails of the associated CD3 complex (CD3Δc) that contributes to efficient TCR/CD3 triggering and productive T cell responses. In contrast, TCR engagement by antagonistic pMHCs does not induce either CD3Δc or full TCR/CD3 signaling in mature T cells. Here, we have used a monovalent Fab fragment (Mono-Fab) specific for the murine CD3 complex to provide induction of CD3Δc to mature T cells engaging pMHC ligands. This Mono-Fab does not interfere with TCR capacity to bind pMHC and fails to elicit signaling in the absence of TCR/MHC interactions. Using OT-I Tg T cells and a collection of altered peptide ligands derived of the agonist peptide OVA, we show that addition of CD3Δc to OT-I T cells using Mono-Fab enables T cell responses to antagonist ligands that fail to induce CD3Δc on their own. Moreover, using a mouse model of metastatic melanoma, we show that Mono-Fab promotes in vivo T cell dependent anti-melanoma responses that are antigen-specific. This Mono-Fab can also be combined with established therapies targeting signals 2 and 3 involved in T cell activation for a synergistic effect. Our data suggests that in-trans CD3Δc provision decreases the activation threshold of T cells. We propose that targeting the CD3 complex for the induction of CD3Δc could be exploited to achieve efficient TCR/CD3 triggering by poorly immunogenic antigens in the context of vaccination against cancer and infectious diseases.

Abstract CD4 and CD8 coreceptors play an important role in the development and maturation of αβ TCR+ T cells. However, T cells lacking CD4 and CD8 (double negative T or DNT cells) are present in the periphery and are significantly expanded in systemic lupus erythematosus (SLE) and Sjogren’s syndrome. The antigens that cause expansion of these DNT cells are unknown. As T cell activation by superantigens does not require coreceptors, we hypothesized that superantigens could activate DNT cells and contribute to autoimmunity. HLA-DQ8 and HLA-DR3 transgenic mice lacking both CD4 and CD8 were generated by intercrossing CD4 and CD8 single knockout mice. The CD4.CD8 double knockout (DKO) mice harbored a diverse repertoire of DNT cells in the periphery, albeit at reduced numbers compared to wild type (WT) mice. DNT cells from DKO mice responded robustly to the superantigen, staphylococcal enterotoxin B, but not to HLA restricted conventional antigens. Implantation of mini-osmotic pumps delivering 10 or 50 μg of SEB continuously over 7 or 28 days induced a lupus-like disease in WT as well as DKO DR3 and DQ8 mice. However, the immunopathology was significantly more pronounced in DKO mice. Immunostaining revealed extensive infiltration of multiple organs including lungs, liver and kidneys with CD3+ T cells in DKO mice. Overall, our study unraveled the role of CD4 and CD8 coreceptors in T cell development/function and the role of superantigens and DNT cells in the pathogenesis of lupus.

Abstract Menstrual or non-menstrual toxic shock syndrome (TSS), caused by the superantigen exotoxins of Staphylococcus aureus or Streptococcus pyogenes, is characterized by a robust activation of the immune system, systemic cytokine/chemokine storm, multiple organ failure and possibly death, if not treated promptly. We tested whether increasing the numbers of Treg in vivo by administration of IL-2-anti-IL-2 complexes (IL2C) could be beneficial in TSS. Administration of IL2C [prepared by incubating 1µg of IL-2 (Peprotech) and 5µg anti-IL-2 (eBioscience, clone-JES6-1A12) in a total 200 µl at 37oC for 30 min] on days 0, 1 and 2, caused a 3 to 4-fold increase in CD4+CD25+FoxP3+ Treg numbers in the spleens of HLA-DR3 transgenic mice by day 5. However, upon challenge with staphylococcal enterotoxin B (SEB, 50µg), compared to untreated HLA-DR3 transgenic mice, IL2C-treated mice produced significantly higher levels of IFN-γ (1714±1365 versus 6642±2253 pg/ml), MIP-1α (782±23 versus 2305±376 pg/ml,), MIP-1β, RANTES and TNF-α. SEB-induced mortality was also significantly increased in the IL2C treated group. While only 1/5 mice died in untreated group, 6/11 mice died in the IL2C treated group. In conclusion, even though administration of IL2C significantly increased the Treg numbers, such a treatment also increased their susceptibility to TSS. Hence, the benefits of T reg-promoting IL-2-anti-IL-2 complexes in TSS or related conditions need more stringent evaluation.

Abstract Bacterial superantigen-induced toxic shock syndrome (TSS) and sepsis are characterized by a profound systemic inflammatory response. Given the important role of NF¿B pathway in inflammation, we investigated whether a clinically approved proteasome inhibitor, Bortezomib, capable of inhibiting NF¿B pathway, be of therapeutic use in such conditions using HLA class II transgenic mice. Bortezomib significantly blocked staphylococcal enterotoxin B (SEB)-induced T cell activation and cytokine production in vitro. In SEB-induced TSS, in vivo administration of Bortezomib significantly reduced serum levels of several pro-inflammatory cytokines and chemokines. Nevertheless, 100% of SEB challenged mice treated with Bortezomib succumbed to TSS, and Bortezomib sensitized mice to TSS at concentrations of SEB which are otherwise non-lethal (i.e., as low as 2 μg/mouse), even when administered 24 hours after exposure to SEB. Sensitization to TSS by Bortezomib was not due to the pro-apoptotic effects of Bortezomib. Serum biochemical parameters and histopathological findings suggested acute liver failure as the possible cause of mortality. In sepsis model, whereas LPS alone (10 μg/mouse) was seldom lethal, it was uniformly lethal when administered along with Bortezomib. In conclusion, Bortezomib sensitizes mice to certain acute systemic inflammatory diseases. Supported by R01AI068741 and AI 72211