Clevelandkjeldsen2162

IL-10 is a potent anti-inflammatory cytokine capable of suppressing a number of proinflammatory signals associated with intestinal inflammatory diseases, such as ulcerative colitis and Crohn's disease. Clinical use of human IL-10 (hIL-10) has been limited by anemia and thrombocytopenia following systemic injection, side effects that might be eliminated by a gut-restricted distribution. We have identified a transcytosis pathway used by cholix, an exotoxin secreted by nonpandemic forms of the intestinal pathogen Vibrio cholerae A nontoxic fragment of the first 386 aa of cholix was genetically fused to hIL-10 to produce recombinant AMT-101. In vitro and in vivo characterization of AMT-101 showed it to efficiently cross healthy human intestinal epithelium (SMI-100) by a vesicular transcytosis process, activate hIL-10 receptors in an engineered U2OS osteosarcoma cell line, and increase cellular phospho-STAT3 levels in J774.2 mouse macrophage cells. AMT-101 was taken up by inflamed intestinal mucosa and activated pSTAT3 in the lamina propria with limited systemic distribution. AMT-101 administered to healthy mice by oral gavage or to cynomolgus monkeys (nonhuman primates) by colonic spray increased circulating levels of IL-1R antagonist (IL-1Ra). Oral gavage of AMT-101 in two mouse models of induced colitis prevented associated pathological events and plasma cytokine changes. Overall, these studies suggest that AMT-101 can efficiently overcome the epithelial barrier to focus biologically active IL-10 to the intestinal lamina propria.Iron is an essential element for Mycobacterium tuberculosis; it has at least 40 enzymes that require iron as a cofactor. Accessibility of iron at the phagosomal surface inside macrophage is crucial for survival and virulence of M. tuberculosis ESAT-6, a 6-kDa-secreted protein of region of difference 1, is known to play a crucial role in virulence and pathogenesis of M. tuberculosis In our earlier study, we demonstrated that ESAT-6 protein interacts with β-2-microglobulin (β2M) and affects class I Ag presentation through sequestration of β2M inside endoplasmic reticulum, which contributes toward inhibition of MHC class Iβ2Mpeptide complex formation. The 6 aa at C-terminal region of ESAT-6 are essential for ESAT6β2M interaction. β2M is essential for proper folding of HFE, CD1, and MHC class I and their surface expression. It is known that M. tuberculosis recruit holotransferrin at the surface of the phagosome. But the upstream mechanism by which it modulates holotransferrin-mediated iron uptake at the surface of macrophage is not well understood. In the current study, we report that interaction of the ESAT-6 protein with β2M causes downregulation of surface HFE, a protein regulating iron homeostasis via interacting with transferrin receptor 1 (TFR1). We found that ESAT-6β2M interaction leads to sequestration of HFE in endoplasmic reticulum, causing poorer surface expression of HFE and HFETFR1 complex (nonfunctional TFR1) in peritoneal macrophages from C57BL/6 mice, resulting in increased holotransferrin-mediated iron uptake in these macrophages. These studies suggest that M. tuberculosis probably targets the ESAT-6 protein to increase iron uptake.Malaria is associated with complicated immunopathogenesis. In this study, we provide evidence for an unexpected role of TLR3 in promoting the establishment of Plasmodium yoelii infection through delayed clearance of parasitemia in wild type C57BL/6jRj (B6) compared with TLR3 knockout mice. In this study, we confirmed an increased expression of Tlr3, Trif, Tbk1, and Irf7/Irf3 in the liver 42 h postinfection and the initiation of an early burst of proinflammatory response such as Ifng, NF-kB, and Tnfa in B6 mice that may promote parasite fitness. Interestingly, in the absence of TLR3, we showed the involvement of high IFN-γ and lower type I IFN response in the early clearance of parasitemia. In parallel, we observed an increase in splenic NK and NKT cells expressing TLR3 in infected B6 mice, suggesting a role for TLR sensing in the innate immune response. Finally, we find evidence that the increase in the frequency of CD19+TLR3+ B cells along with reduced levels of total IgG in B6 mice possibly suggests the initiation of TLR3-dependent pathway early during P. yoelii infection. Our results thus reveal a new mechanism in which a parasite-activated TLR3 pathway promotes blood stage infection along with quantitative and qualitative differences in Ab responses.Currently, there is a need for reliable tests that allow identification of individuals that have been infected with SARS-CoV-2 even if the infection was asymptomatic. To date, the vast majority of the serological tests for SARS-CoV-2-specific Abs are based on serum detection of Abs to either the viral spike glycoprotein (the major target for neutralizing Abs) or the viral nucleocapsid protein that is known to be highly immunogenic in other coronaviruses. Conceivably, exposure of Ags released from infected cells could stimulate Ab responses that might correlate with tissue damage and, hence, they may have some value as a prognostic indicator. We addressed whether other nonstructural viral proteins, not incorporated into the infectious viral particle, specifically the viral cysteine-like protease, might also be potent immunogens. Using ELISA tests, coating several SARS-CoV-2 proteins produced in vitro, we describe that COVID-19 patients make high titer IgG, IgM, and IgA Ab responses to the Cys-like protease from SARS-CoV-2, also known as 3CLpro or Mpro, and it can be used to identify individuals with positive serology against the coronavirus. Higher Ab titers in these assays associated with more-severe disease, and no cross-reactive Abs against prior betacoronavirus were found. Remarkably, IgG Abs specific for Mpro and other SARS-CoV-2 Ags can also be detected in saliva. In conclusion, Mpro is a potent Ag in infected patients that can be used in serological tests, and its detection in saliva could be the basis for a rapid, noninvasive test for COVID-19 seropositivity.The chronic inflammatory autoimmune disease rheumatoid arthritis (RA) is characterized by an infiltration of activated proinflammatory immune cells into the joint that is accompanied by an overproduction of various mediators, leading to destruction of cartilage and bone erosion. Angiotensin II type 2 receptor (AT2R) is involved in antioxidative, anti-inflammatory, and antifibrotic responses. Synovial macrophages (SMs) are a type of tissue macrophages that are derived from bone marrow cells. SMs plays a central role in synovial regional immunization, which is significantly increased in both collagen-induced mice with arthritis mice and RA patients. AT2R activation caused a reversal of the polarization of SMs in the joint from the proinflammatory M1 SM to the tolerogenic, benign M2 SM. In consequence, this switch resulted in an attenuated form of the joint pathology in a rat model of collagen-induced arthritis. These results were mechanistically linked to the observation that GRK2 was translocated into cytoplasm, and ERK1/2 and NF-κB activation were inhibited. These findings open the way to a new therapeutic approach using an activation of AT2R to subvert joint inflammation in RA.Emerging evidence indicates that metabolic programs regulate B cell activation and Ab responses. However, the metabolic mediators that support the durability of the memory B cell and long-lived plasma cell populations are not fully elucidated. Adenosine monophosphate-activated protein kinase (AMPK) is an evolutionary conserved serine/threonine kinase that integrates cellular energy status and nutrient availability to intracellular signaling and metabolic pathways. In this study, we use genetic mouse models to show that loss of ΑMPKα1 in B cells led to a weakened recall Ab response associated with a decline in the population of memory-phenotype B cells. AMPKα1-deficient memory B lymphocytes exhibited aberrant mitochondrial activity, decreased mitophagy, and increased lipid peroxidation. Moreover, loss of AMPKα1 in B lymphoblasts was associated with decreased mitochondrial spare respiratory capacity. Of note, AMPKα1 in B cells was dispensable for stability of the bone marrow-resident, long-lived plasma cell population, yet absence of this kinase led to increased rates of Ig production and elevated serum Ab concentrations elicited by primary immunization. Collectively, our findings fit a model in which AMPKα1 in B cells supports recall function of the memory B cell compartment by promoting mitochondrial homeostasis and longevity but restrains rates of Ig production.Bacterial vaginosis (BV) is caused by the excessive and imbalanced growth of bacteria in vagina, affecting 30 to 50% of women. Gram staining followed by Nugent scoring based on bacterial morphotypes under the microscope is considered the gold standard for BV diagnosis; this method is often labor-intensive and time-consuming, and results vary from person to person. We developed and optimized a convolutional neural network (CNN) model and evaluated its ability to automatically identify and classify three categories of Nugent scores from microscope images. The CNN model was first established with a panel of microscopic images with Nugent scores determined by experts. The model was trained by minimizing the cross-entropy loss function and optimized by using a momentum optimizer. The separate test sets of images collected from three hospitals were evaluated by the CNN model. The CNN model consisted of 25 convolutional layers, 2 pooling layers, and a fully connected layer. The model obtained 82.4% sensitivity and 9s with proper supporting hardware.Oropharyngeal Chlamydia trachomatis (CT) infections and, especially, Neisseria gonorrhoeae (NG) infections are common, but few commercial nucleic acid amplification tests (NAATs) specify extragenital samples for intended use. The test characteristics of the cobas 4800 CT/NG assay were evaluated for oropharyngeal swabs. The technical validation included analysis of the specificity, sensitivity, dynamic range, linearity, efficiency, and precision. The probability of detection curve combined with historical data enabled the estimation of potentially missed diagnoses. A clinical evaluation was performed on a subset of 2,798 clinical samples available from routine diagnostics. Results of the cobas 4800 were compared with those from in-house C. trachomatis/N. gonorrhoeae PCR assays. Discrepant samples were tested with resolver assays, and these results were considered decisive. No cross-reactivity was seen in the analytical specificity analysis. High linearity (R2 ≥ 0.983), efficiency (89% to 99%), and precision (cycle threshold [CT ] value of 0.1 to 0.9) were seen for both C. trachomatis and N. gonorrhoeae The limit of detection in oropharyngeal samples was 3.2 × 102 inclusion-forming units (IFU)/ml for C. CWI1-2 solubility dmso trachomatis and 6.7 × 102 CFU/ml for N. gonorrhoeae Estimates on potentially missed diagnoses were up to 7.2% for C. trachomatis and up to 24.7% for N. gonorrhoeae Clinical sensitivity and specificity were evaluated with 25 C. trachomatis-positive, 86 N. gonorrhoeae-positive, and 264 negative samples, resulting in 100% and 99.6% for C. trachomatis and 100% and 96.7% for N. gonorrhoeae, respectively. The findings in this study demonstrate the utility of the cobas 4800 CT/NG assay for oropharyngeal samples. Despite its being a highly accurate test, the range of reported CT values, especially for N. gonorrhoeae, suggests relatively low oropharyngeal loads. Hence, consistent detection over the full range of oropharyngeal loads could be impaired.