Comparing machine learning screening approaches using clinical data and cytokine profiles for COVID-19 in resource-limited and resource-abundant settings.

Accurate screening of COVID-19 infection status for symptomatic patients is a critical public health task. Although molecular and antigen tests now exist for COVID-19, in resource-limited settings, screening tests are often not available. Furthermore, during the early stages of the pandemic tests were not available in any capacity. We utilized an automated machine learning (ML) approach to train and evaluate thousands of models on a clinical dataset consisting of commonly available clinical and laboratory data, along with cytokine profiles for patients (n = 150). These models were then further tested for generalizability on an out-of-sample secondary dataset (n = 120). We were able to develop a ML model for rapid and reliable screening of patients as COVID-19 positive or negative using three approaches: commonly available clinical and laboratory data, a cytokine profile, and a combination of the common data and cytokine profile. Of the tens of thousands of models automatically tested for the three approaches, all three approaches demonstrated > 92% sensitivity and > 88 specificity while our highest performing model achieved 95.6% sensitivity and 98.1% specificity. These models represent a potential effective deployable solution for COVID-19 status classification for symptomatic patients in resource-limited settings and provide proof-of-concept for rapid development of screening tools for novel emerging infectious diseases.

Complementary and alternative medicines and liver disease.

Complementary and alternative medicines (CAM) include conventional medical treatments. Patients worldwide use CAM at alarming rates; thus, reports of CAM-related DILI have been on the rise. The clinical presentations include asymptomatic liver test abnormalities, acute hepatitis with or without jaundice, acute cholestatic liver disease (bland or with hepatitis), acute liver failure, severe hepatitis with features of portal hypertension, and acute decompensation of known or unknown cirrhosis that can lead to acute-on-chronic liver failure. Acute hepatitis with or without necrosis, hepatocellular and canalicular cholestasis, herb-induced or CAM-triggered autoimmune hepatitis, granulomatous hepatitis, severe steatohepatitis, and vanishing bile duct syndrome are common liver biopsy findings in CAM-DILI. The presence of preexisting liver disease predicts severe liver injury, risk of progression to liver failure, and decreased transplant-free survival in patients with CAM-DILI. This review discusses global epidemiology and trends in CAM-DILI, clinical presentation, assessment and outcomes, commonly emerging threats in the context of hepatotoxic herbs, pragmatic assessment of "liver beneficial" herbs and health care myths, patient communication, regulatory framework, and future directions on research in CAM.

BCG as an Innovative Option for HCC Treatment: Repurposing and Mechanistic Insights.

This study investigates Bacillus Calmette-Guérin (BCG) as a potential treatment for hepatocellular carcinoma (HCC), a condition often associated with unfavorable treatment outcomes. Exploiting BCG's recognized immune-boosting properties, preclinical trials are conducted using HCC mice, with a single subcutaneous dose of BCG administered post-tumor formation. Results indicate that BCG treatment effectively diminishes tumor burden and extends survival in both male and female HCC mice. Positive influences on hepatic fibrosis and metabolism are observed, leading to a reduction in lipid levels. Spatial analysis underscores BCG's tumor-specific effects, inducing the enrichment of metabolic pathways and inhibiting various cancer-related pathways. Furthermore, BCG promotes immune cell infiltration, including CD4+, CD8+ T cells, and M1 macrophages, in both v-akt murine thymoma viral oncogene homolog 1(AKT)/neutoblastoma RAS viral oncogene homolog (RAS) and ß-catenin positive HCC models. Interestingly, blocking T cells, trained immunity, and Interferon-γ (IFN-γ) function reverses BCG's anti-HCC effects. In conclusion, BCG emerges as a promising treatment option for HCC, characterized by a favorable safety profile and efficacy in inhibiting fibrosis, improving metabolism, and engaging both trained immunity and T cells in therapeutic mechanisms.

Dynamics of temporal immune responses in nonhuman primates and humans immunized with COVID-19 vaccines.

We assessed the humoral immune responses to a COVID-19 vaccine in a well-controlled rhesus macaque model compared to humans immunized with two mRNA vaccines over several months post-second dose. The plasma IgG levels against seven coronaviruses (including SARS-CoV-2) and antibody subtypes (IgG 1-4 and IgM) against SARS-CoV-2 were evaluated using multiplex assays. The neutralization capacity of plasma antibodies against the original SAR-CoV-2 isolate and nine variants was evaluated in vaccinated humans and non-human primates. Immunization of macaques and humans with SARS-CoV-2 vaccines induced a robust neutralizing antibody response. In non-SIV-infected adult macaques immunized with an adenoviral vector expressing S-RBD (n = 7) or N protein (n = 3), elevated levels of IgG and neutralizing antibodies were detected 2 weeks post-second dose. Immune responses to the S-RBD vaccine in SIV-infected adult macaques (n = 2) were similar to the non-SIV-infected animals. Adult humans immunized with Pfizer (n = 35) or Moderna (n = 18) vaccines developed IgG and neutralizing antibodies at 4 weeks post-second dose. In both vaccine groups, IgG 1 was the predominant subtype, followed by IgG 3. The IgG levels, including total and IgG 1,2,3 elicited by the Moderna vaccine, were significantly higher than the corresponding levels elicited by the Pfizer vaccine at 4 weeks post-second dose. A significant correlation was observed between the plasma total IgG antibody levels and neutralization titers in both macaques and humans. Furthermore, broad-spectrum neutralization antibodies against several variants of SARS-CoV-2 were detected in the plasma of both macaques and humans after two vaccinations.

Lipid Mediators and Cytokines/Chemokines Display Differential Profiles in Severe versus Mild/Moderate COVID-19 Patients.

Host immune responses play a key role in COVID-19 pathogenesis. The underlying phenomena are orchestrated by signaling molecules such as cytokines/chemokines and lipid mediators. These immune molecules, including anti-SARS-CoV-2 antibodies, interact with immune cells and regulate host responses, contributing to inflammation that drives the disease. We investigated 48 plasma cytokines/chemokines, 21 lipid mediators, and anti-S protein (RBD) antibodies in COVID-19 patients (n = 56) and non-COVID-19 respiratory disease controls (n = 49), to identify immune-biomarker profiles. Cytokines/chemokines (IL-6, CXCL-10 (IP-10), HGF, MIG, MCP-1, and G-CSF) and lipid mediators (TxB2, 11-HETE, 9-HODE, 13-HODE, 5-HETE, 12-HETE, 15-HETE, 14S-HDHA, 17S-HDHA, and 5-oxo ETE) were significantly elevated in COVID-19 patients compared to controls. In patients exhibiting severe disease, pro-inflammatory cytokines/chemokines (IL-6, CXCL-10, and HGF) and anti-SARS-CoV-2 antibodies were significantly elevated. In contrast, lipid mediators involved in the reduction/resolution of inflammation, in particular, 5-HETE, 11-HETE, and 5-oxoETE, were significantly elevated in mild/moderate disease. Taken together, these immune-biomarker profiles provide insight into immune responses related to COVID-19 pathogenesis. Importantly, our findings suggest that elevation in plasma concentrations of IL-6, CXCL-10, HGF, and anti-SARS-CoV-2 antibodies can predict severe disease, whereas elevation in lipid mediators peaks early (compared to cytokines) and includes induction of mechanisms leading to reduction of inflammation, associated complications, and maintenance of homeostasis.

Ashwagandha-induced liver injury-A case series from India and literature review.

BACKGROUND: Ashwagandha herb is commonly used in Ayurveda and a "fad" dietary supplement for a host of indications based on low levels of evidence. Recently, ashwagandha was implicated in multiple reports of herb-induced liver injury (HILI), mainly from the United States. We present the first, and currently largest, series of ashwagandha-HILI from multiple centers in India. METHODS: We retrospectively analyzed the respective institutional electronic medical records for ashwagandha-HILI. Patients consuming ashwagandha as part of multiherbal formulations or along with other known hepatotoxic supplements or medicines were excluded. All patients underwent a detailed diagnostic workup to exclude competing causes reasonably. Where possible, the implicated herbal formulation was retrieved and subjected to chemical analysis. RESULTS: Out of 23 patients with liver injury from ashwagandha (January 2019 to December 2022), we report 8 patients with single-ingredient formulation-related HILI. Study cohort was male predominant, and cholestatic hepatitis was the commonest presentation. Five patients had underlying chronic liver disease; 3 presented with acute-on-chronic liver failure, and all 3 died on follow-up. In others, the liver injury was prolonged, nonetheless self-limiting. Liver biopsy revealed cholestatic features predominantly with hepatocellular necrosis and lymphocyte/eosinophil predominant portal-based inflammation. One patient progressed to chronic HILI. Chemical analysis revealed only natural phytochemicals without adulteration or contamination. CONCLUSIONS: Ashwagandha-HILI presents with cholestatic hepatitis and can lead to the syndrome of acute-on-chronic liver failure with high mortality in those with pre-existing liver disease. Educating the public on avoiding the use of potentially toxic and unrecommended herbal supplements can help mitigate the avoidable liver disease burden in the community.

Peripheral γδ T Cells Regulate Neutrophil Expansion and Recruitment in Experimental Psoriatic Arthritis.

OBJECTIVE: This study was undertaken to identify the mechanistic role of γδ T cells in the pathogenesis of experimental psoriatic arthritis (PsA). METHODS: In this study, we performed interleukin-23 (IL-23) gene transfer in wild-type (WT) and T cell receptor δ-deficient (TCRδ-/- ) mice and conducted tissue phenotyping in the joint, skin, and nails to characterize the inflammatory infiltrate. We further performed detailed flow cytometry, immunofluorescence staining, RNA sequencing, T cell repertoire analysis, and in vitro T cell polarization assays to identify regulatory mechanisms of γδ T cells. RESULTS: We demonstrated that γδ T cells support systemic granulopoiesis, which is critical for murine PsA-like pathology. Briefly, γδ T cell ablation inhibited the expression of neutrophil chemokines CXCL1 and CXCL2 and neutrophil CD11b+Ly6G+ accumulation in the aforementioned PsA-related tissues. Although significantly reduced expression of granulocyte-macrophage colony-stimulating factor (GM-CSF) and IL-17A was detected systemically in TCRδ-/- mice, no GM-CSF+/IL-17A+ γδ T cells were detected locally in the inflamed skin or bone marrow in WT mice. Our data showed that nonresident γδ T cells regulate the expansion of an CD11b+Ly6G+ neutrophil population and their recruitment to joint and skin tissues, where they develop hallmark pathologic features of human PsA. CONCLUSION: Our findings do not support the notion that tissue-resident γδ T cells initiate the disease but demonstrate a novel role of γδ T cells in neutrophil regulation that can be exploited therapeutically in PsA patients.

Diagnostic performance of blood inflammatory markers for tuberculosis screening in people living with HIV.

BACKGROUND: Approaches to screening for active tuberculosis (TB) among people living with HIV are inadequate, leading to missed diagnoses and poor implementation of preventive therapy. METHODS: Consecutive HIV-infected adults hospitalized at Mulago Hospital (Kampala, Uganda) between June 2011 and July 2013 with a cough ≥ 2 weeks were enrolled. Patients underwent extensive evaluation for pulmonary TB. Concentrations of 43 cytokines/chemokines were measured at the same time point as C-reactive protein (CRP) in banked plasma samples using commercially-available multiplex kits. Advanced classification algorithms were used to rank cytokines/chemokines for their ability to identify TB, and to model the specificity of the top-ranked cytokines/chemokines individually and in combination with sensitivity constrained to ≥ 90% as recommended for TB screening. RESULTS: The median plasma level of 5 biomarkers (IL-6, INF-γ, MIG, CRP, IL-18) was significantly different between patients with and without TB. With sensitivity constrained to 90%, all had low specificity with IL-6 showing the highest specificity (44%; 95% CI 37.4-49.5). Biomarker panels were found to be more valuable than any biomarker alone. A panel combining IFN-γ and IL-6 had the highest specificity (50%; 95% CI 46.7-53.3). Sensitivity remained high (>85%) for all panels among sputum smear-negative TB patients. CONCLUSIONS: Direct measurement of unstimulated plasma cytokines/chemokines in peripheral blood is a promising approach to TB screening. Cytokine/chemokine panels retained high sensitivity for smear-negative TB and achieved improved specificity compared to individual cytokines/chemokines. These markers should be further evaluated in outpatient settings where most TB screening occurs and where other illnesses associated with systematic inflammation are less common.

Gender biased immune-biomarkers in active tuberculosis and correlation of their profiles to efficacy of therapy.

Active pulmonary TB is an inflammatory disease and is increasingly viewed as an imbalance of immune responses to Mycobacterium tuberculosis (M. tb.) infection. In addition, this immune imbalance may be gender biased (males have a higher prevalence of TB) but reasons for such bias are uncertain. We hypothesized that studies on profiles of immune-biomarkers will not only provide insight into molecular basis of gender bias but may also help identify biomarkers to monitor efficacy of TB therapy. We examined 10 plasma cytokine/chemokine/growth-factor and 8 antibody (against 8 M. tb. antigens) biomarkers (elevated in TB patients) by multiplex microbead immunoassays. In addition, we examined these biomarkers in patients under anti-tuberculosis therapy (ATT). The results showed that female patients contained significantly higher levels of CXCL9 (MIG) and CXCL10 (IP-10), while males contained higher levels of PDGF-BB. In contrast, more males than females contained antibodies against several antigens. Our results also show that there are progressive and substantial decreases in plasma levels of CXCL9, CXCL10, PDGF-BB, IFNγ, and IL-18, correlating with treatment success. Our results suggest that studies on gender bias in immunebiomarkers will enhance understanding of host responses in TB and would be valuable as biomarkers for monitoring efficacy of ATT.

NOD1 and NOD2 signalling links ER stress with inflammation.

Endoplasmic reticulum (ER) stress is a major contributor to inflammatory diseases, such as Crohn disease and type 2 diabetes. ER stress induces the unfolded protein response, which involves activation of three transmembrane receptors, ATF6, PERK and IRE1α. Once activated, IRE1α recruits TRAF2 to the ER membrane to initiate inflammatory responses via the NF-κB pathway. Inflammation is commonly triggered when pattern recognition receptors (PRRs), such as Toll-like receptors or nucleotide-binding oligomerization domain (NOD)-like receptors, detect tissue damage or microbial infection. However, it is not clear which PRRs have a major role in inducing inflammation during ER stress. Here we show that NOD1 and NOD2, two members of the NOD-like receptor family of PRRs, are important mediators of ER-stress-induced inflammation in mouse and human cells. The ER stress inducers thapsigargin and dithiothreitol trigger production of the pro-inflammatory cytokine IL-6 in a NOD1/2-dependent fashion. Inflammation and IL-6 production triggered by infection with Brucella abortus, which induces ER stress by injecting the type IV secretion system effector protein VceC into host cells, is TRAF2, NOD1/2 and RIP2-dependent and can be reduced by treatment with the ER stress inhibitor tauroursodeoxycholate or an IRE1α kinase inhibitor. The association of NOD1 and NOD2 with pro-inflammatory responses induced by the IRE1α/TRAF2 signalling pathway provides a novel link between innate immunity and ER-stress-induced inflammation.

Abnormal Mammary Development in 129:STAT1-Null Mice is Stroma-Dependent.

Female 129:Stat1-null mice (129S6/SvEvTac-Stat1(tm1Rds) homozygous) uniquely develop estrogen-receptor (ER)-positive mammary tumors. Herein we report that the mammary glands (MG) of these mice have altered growth and development with abnormal terminal end buds alongside defective branching morphogenesis and ductal elongation. We also find that the 129:Stat1-null mammary fat pad (MFP) fails to sustain the growth of 129S6/SvEv wild-type and Stat1-null epithelium. These abnormalities are partially reversed by elevated serum progesterone and prolactin whereas transplantation of wild-type bone marrow into 129:Stat1-null mice does not reverse the MG developmental defects. Medium conditioned by 129:Stat1-null epithelium-cleared MFP does not stimulate epithelial proliferation, whereas it is stimulated by medium conditioned by epithelium-cleared MFP from either wild-type or 129:Stat1-null females having elevated progesterone and prolactin. Microarrays and multiplexed cytokine assays reveal that the MG of 129:Stat1-null mice has lower levels of growth factors that have been implicated in normal MG growth and development. Transplanted 129:Stat1-null tumors and their isolated cells also grow slower in 129:Stat1-null MG compared to wild-type recipient MG. These studies demonstrate that growth of normal and neoplastic 129:Stat1-null epithelium is dependent on the hormonal milieu and on factors from the mammary stroma such as cytokines. While the individual or combined effects of these factors remains to be resolved, our data supports the role of STAT1 in maintaining a tumor-suppressive MG microenvironment.

Exploratory study on plasma immunomodulator and antibody profiles in tuberculosis patients.

Host immune responses to Mycobacterium tuberculosis are generally able to contain infection and maintain a delicate balance between protection and immunopathology. A shift in this balance appears to underlie active disease observed in about 10% of infected individuals. Effects of local inflammation, combined with anti-M. tuberculosis systemic immune responses, are directly detectable in peripheral circulation, without ex vivo stimulation of blood cells or biopsy of the affected organs. We studied plasma immunomodulator and antibody biomarkers in patients with active pulmonary tuberculosis (TB) by a combination of multiplex microbead immunoassays and computational tools for data analysis. Plasma profiles of 10 immunomodulators and antibodies against eight M. tuberculosis antigens (previously reported by us) were examined in active pulmonary TB patients in a country where TB is endemic, Pakistan. Multiplex analyses were performed on samples from apparently healthy individuals without active TB from the same community as the TB patients to establish the assay baselines for all analytes. Over 3,000 data points were collected from patients (n = 135) and controls (n = 37). The data were analyzed by multivariate and computer-assisted cluster analyses to reveal patterns of plasma immunomodulators and antibodies. This study shows plasma profiles that in most patients represented either strong antibody or strong immunomodulator biomarkers. Profiling of a combination of both immunomodulators and antibodies described here may be valuable for the analysis of host immune responses in active TB in countries where the disease is endemic.

Stereological analysis of bacterial load and lung lesions in nonhuman primates (rhesus macaques) experimentally infected with Mycobacterium tuberculosis.

Infection with Mycobacterium tuberculosis primarily produces a multifocal distribution of pulmonary granulomas in which the pathogen resides. Accordingly, quantitative assessment of the bacterial load and pathology is a substantial challenge in tuberculosis. Such assessments are critical for studies of the pathogenesis and for the development of vaccines and drugs in animal models of experimental M. tuberculosis infection. Stereology enables unbiased quantitation of three-dimensional objects from two-dimensional sections and thus is suited to quantify histological lesions. We have developed a protocol for stereological analysis of the lung in rhesus macaques inoculated with a pathogenic clinical strain of M. tuberculosis (Erdman strain). These animals exhibit a pattern of infection and tuberculosis similar to that of naturally infected humans. Conditions were optimized for collecting lung samples in a nonbiased, random manner. Bacterial load in these samples was assessed by a standard plating assay, and granulomas were graded and enumerated microscopically. Stereological analysis provided quantitative data that supported a significant correlation between bacterial load and lung granulomas. Thus this stereological approach enables a quantitative, statistically valid analysis of the impact of M. tuberculosis infection in the lung and will serve as an essential tool for objectively comparing the efficacy of drugs and vaccines.

A biochemical study on the effect of proteolysis of beta-thromboglobulin proteins released from activated platelets on fibroblast proliferation.

beta-Thromboglobulin (beta-TG) proteins are a heterogeneous group released from platelet alpha-granules on activation and have an effect on fibroblast migration and proliferation. We have previously reported the action of a metal-dependent protease on platelet-released proteins, which generates low-molecular-weight proteins that could be inhibited by ethylenediaminetetraacetic acid (EDTA). To understand the physiological significance of the breakdown of proteins after release, their effect on fibroblast proliferation in vitro was studied. Platelet releasates were obtained without and with EDTA inhibition designated as R1 and R2, respectively, and proteins were affinity purified for testing. Cell proliferation was measured using [(3)H]-thymidine assay. Both R1 and R2 showed maximum activity at 100 microg/ml and R2 elicited significant proliferation compared to R1. When affinity-purified proteins were tested at 100 ng/ml, high-molecular-weight proteins showed significantly higher proliferation than low-molecular-weight proteins. We have shown that beta-TG is cleaved after being released from activated platelets, thereby becoming less mitogenic for fibroblasts.

Increased platelet cholesterol and decreased percentage volume of platelets as a secondary risk factor for coronary artery disease.

Platelet hyperactivity is likely to contribute to the progression of atherogenesis and organized thrombus formation on vascular surfaces. The purpose of this study was to examine the effect of hypercholesterolemia on the cholesterol content of platelets, on platelet responsiveness and other platelet indices using platelets from 5 groups of age-matched subjects (n = 30 each), which includes healthy controls. All groups except controls had a high plasma lipid profile. While subjects in group I had only hyperlipidemia, those in groups II and III had hyperlipidemia in conjunction with diabetes mellitus and hypertension, respectively. The fourth group consisted of patients with confirmed coronary artery disease (CAD). The parameters studied include packed cell volume of platelets (platelet crit), platelet distribution width (PDW), platelet cholesterol and platelet aggregation in response to adenosine diphosphate and collagen. All the patient groups showed increased platelet aggregation (p < 0.05) and low platelet crit compared with controls (p < 0.05). In addition, platelet cholesterol was increased in patients with coronary disease, hyperlipidemia and diabetes mellitus (p < 0.05) but not in patients with hypertension (p > 0.05); PDW was high only in CAD (p < 0.05). A higher PDW indicated a prothrombotic tendency in CAD patients. Our data suggest that hyperlipidemia increases the lipid content in platelets and enhances their reactivity. Hyperactive platelets with increased platelet cholesterol may contribute to accelerated atherogenesis associated with CAD.