Indian herb Tinospora cordifolia and Tinospora species: Phytochemical and therapeutic application.

Clinical investigations are increasingly focusing on natural materials with medical benefits because, in contrast to medicines, they have extremely few adverse effects. Tinospora species of the Menispermaceae family has many bioactive principles for plant nutraceuticals. A thorough assessment of the existing literature revealed that Indian Tinospora species are an important group of medicinal herbs used for a variety of pharmacological activities. While, Tinospora cordifolia is widely recognized as a significant herb in the Indian System of Medicines (ISM) due to its bioactive components and has been used in the treatment of diabetes, cancer, urinary problems, fever, jaundice, helminthiasis, leprosy, dysentery, skin diseases, and many more. Using the search phrases "phytochemistry," "traditional uses," and "pharmacological evaluation of Indian Tinospora species," appropriate articles were carefully extracted from the MEDLINE/PubMed, Scopus, and WOS databases. Around 180 articles, related to the India Tinospora species, were selected from a pool of 200 papers published between 1991 and 2023. T. cordifolia has received a lot of scientific attention because of its diverse therapeutic characteristics in treating various diseases. Our present study in this review encompasses 1.) Phytochemistry, traditional uses and pharmacological potential of T. cordifolia as well as other Indian Tinospora species. 2.) Safety and toxicity study and available marketed formulation of T. cordifolia for the treatment of various diseases. The chemical constitution and pharmacological characteristics of other Tinospora species must also be investigated, indicating a need for further scientific research.

Data analytics improves the diagnostic accuracy of serum free light chain results for detecting monoclonal gammopathy.

OBJECTIVES: To evaluate the real-world performance and reference intervals of the Binding Site Freelite serum free light chain (SFLC) assay (Thermo Fisher Scientific), a global standard for diagnosis, prognostication, and response assessment for monoclonal gammopathies. METHODS: An informatics-based approach was used to retrospectively evaluate concordance between SFLC and the orthogonal Sebia HYDRASYS immunofixation assay results in a large clinical data set consecutively reported between 2010 and 2020. RESULTS: Among patients with monoclonal-negative results by both SFLC and Sebia HYDRASYS immunofixation assays, 25% (1226/5057) had κ/λ ratios (KLRs) outside the manufacturer-defined and International Myeloma Working Group-cited normal reference interval of 0.26 to 1.65. These results were consistent over the study period and were not affected by sex, age, impaired kidney function, or assay antisera lot variation. Assay drift, in addition to other potential factors, affected the KLR distribution. Using International Statistical Classification of Diseases (ICD) codes, kidney function data, and the central 95% of KLR values generated on the Optilite platform (Thermo Fisher Scientific), we derived a new reference interval of 0.67 to 2.13, reducing the KLR false-positive rate to 8%. However, normal KLR persisted among 16% (14/85) of samples with free λ chains by immunofixation, warranting caution during interpretation. CONCLUSIONS: Our analysis indicated that revision of Freelite SFLC reference intervals improves assay interpretation and should prompt reconsideration of Freelite reference intervals worldwide.

Identification of Potential Antitubulin Agents with Anticancer Assets from a Series of Imidazo[1,2-a]quinoxaline Derivatives: In Silico and In Vitro Approaches.

Computer-aided drug design is a powerful and promising tool for drug design and development, with a reduced cost and time. In the current study, we rationally selected a library of 34 fused imidazo[1,2-a]quinoxaline derivatives and performed virtual screening, molecular docking, and molecular mechanics for a lead identification against tubulin as an anticancer molecule. The computational analysis and pharmacophoric features were represented as 1A2; this was a potential lead against tubulin, with a maximized affinity and binding score at the colchicine-binding site of tubulin. The efficiency of this lead molecule was further identified using an in vitro assay on a tubulin enzyme and the anticancer potential was established using an MTT assay. Compound 1A2 (IC50 = 4.33-6.11 µM against MCF-7, MDA-MB-231, HCT-116, and A549 cell lines) displayed encouraging results similar to the standard drug colchicine in these in vitro studies, which further confirmed the effectiveness of CADD in new drug developments. Thus, we successfully applied the utility of in silico techniques to identify the best plausible leads from the fused azaheterocycles.

Cutting Edge: Effect of Disease-Modifying Therapies on SARS-CoV-2 Vaccine-Induced Immune Responses in Multiple Sclerosis Patients.

Multiple sclerosis (MS) is a demyelinating inflammatory disease of the CNS treated by diverse disease-modifying therapies that suppress the immune system. Severe acute respiratory syndrome coronavirus 2 mRNA vaccines have been very effective in immunocompetent individuals, but whether MS patients treated with modifying therapies are afforded the same protection is not known. This study determined that dimethyl fumarate caused a momentary reduction in anti-Spike (S)-specific Abs and CD8 T cell response. MS patients treated with B cell-depleting (anti-CD20) or sphingosine 1-phosphate receptor agonist (fingolimod) therapies lack significant S-specific Ab response. Whereas S-specific CD4 and CD8 T cell responses were largely compromised by fingolimod treatment, T cell responses were robustly generated in anti-CD20-treated MS patients, but with a reduced proportion of CD4+CXCR5+ circulating follicular Th cells. These data provide novel information regarding vaccine immune response in patients with autoimmunity useful to help improve vaccine effectiveness in these populations.

Anti-Nuclear Antibody Quantitation: Calibration and Harmonization Adjustment via Population Interrogation.

BACKGROUND: The 2019 classification criteria for systemic lupus erythematosus (SLE) includes an initial criterion requiring the presence of an antinuclear antibody (ANA), positive at a titer of at least 1:80 on HEp-2 cells, or equivalent. However, results of ANA tests performed on HEp-2 cells vary when tested in different laboratories. Calibration of ANA assays by achieving a common specificity in healthy control populations offers the possibility of achieving harmonization via population interrogation, but the expected specificity in a healthy control population is not known. METHODS: The studies used to determine the use of ANAs performed by immunofluorescence microscopy on HEp-2 cells as the entry criterion for classification of SLE were reanalyzed by a meta-analysis to determine the expected frequency of positive ANAs in healthy control populations at serum dilutions of 1:40 and 1:80. RESULTS: Our meta-analysis demonstrated that the expected specificity in a healthy control population of ANA performed using serum diluted 1:80 is 91.3% (CI 86.1-94.7%). The expected specificity of ANA performed at 1:40 serum dilution is 79.2% (CI 72.3-84.8%). CONCLUSION: One approach to achieving harmonization of ANA assays from different laboratories with each other and with expected performance would involve adjusting assays so that about 10% of a healthy control population has a positive ANA when tested at 1:80 dilution, and about 20% of the healthy control population has a positive ANA when tested at 1:40 dilution. This pragmatic approach to calibration and harmonization adjustment via population interrogation offers an opportunity for individual laboratories to be aligned with each other and with ANA performance expected for consistent categorization of patients with SLE.

Self-collection of capillary blood using Tasso-SST devices for Anti-SARS-CoV-2 IgG antibody testing.

BACKGROUND: Efforts to minimize COVID-19 exposure during the current SARS-CoV-2 pandemic have led to limitations in access to medical care and testing. The Tasso-SST kit includes all of the components necessary for remote, capillary blood self-collection. In this study, we sought to investigate the accuracy and reliability of the Tasso-SST device as a self-collection device for measurement of SARS-CoV-2 IgG antibodies. METHODS: Capillary blood was obtained via unsupervised and supervised application of the Tasso-SST device, and venous blood was collected by standard venipuncture. Unsupervised self-collected blood samples underwent either extreme summer or winter-simulated shipping conditions prior to testing. Sera obtained by all three methods were tested concurrently using the EuroImmun anti-SARS-CoV-2 S1 IgG assay in a CLIA-certified clinical laboratory. RESULTS: Successful Tasso-SST capillary blood collection by unsupervised and supervised administration was completed by 93.4% and 94.5% of participants, respectively. Sera from 56 participants, 55 with documented (PCR+) COVID-19, and 33 healthy controls were then tested for anti-SARS-CoV-2 IgG antibodies. Compared to venous blood results, Tasso-SST-collected (unstressed) and the summer- and winter-stressed blood samples demonstrated Deming regression slopes of 1.00 (95% CI: 0.99-1.02), 1.00 (95% CI: 0.98-1.01), and 0.99 (95% CI: 0.97-1.01), respectively, with an overall accuracy of 98.9%. CONCLUSIONS: Capillary blood self-collection using the Tasso-SST device had a high success rate. Moreover, excellent concordance was found for anti-SARS-CoV-2 IgG results between Tasso-SST capillary and standard venous blood-derived sera. The Tasso-SST device should enable widespread collection of capillary blood for testing without medical supervision, facilitating epidemiologic studies.

Anti-SARS-CoV-2 Antibody Levels Measured by the AdviseDx SARS-CoV-2 Assay Are Concordant with Previously Available Serologic Assays but Are Not Fully Predictive of Sterilizing Immunity.

With the availability of widespread SARS-CoV-2 vaccination, high-throughput quantitative anti-spike protein serological testing will likely become increasingly important. Here, we investigated the performance characteristics of the recently FDA-authorized semiquantitative anti-spike protein AdviseDx SARS-CoV-2 IgG II assay compared to the FDA-authorized anti-nucleocapsid protein Abbott Architect SARS-CoV-2 IgG, Roche Elecsys anti-SARS-CoV-2-S, EuroImmun anti-SARS-CoV-2 enzyme-linked immunosorbent assay (ELISA), and GenScript surrogate virus neutralization assays and examined the humoral response associated with vaccination, natural protection, and vaccine breakthrough infection. The AdviseDx assay had a clinical sensitivity at 14 days after symptom onset or 10 days after PCR detection of 95.6% (65/68; 95% confidence interval [CI], 87.8 to 98.8%), with two discrepant individuals seroconverting shortly thereafter. The AdviseDx assay demonstrated 100% positive percent agreement with the four other assays examined using the same symptom onset or PCR detection cutoffs. Using a recently available WHO international standard for anti-SARS-CoV-2 antibody, we provide assay unit conversion factors to international units for each of the assays examined. We performed a longitudinal survey of healthy vaccinated individuals, finding that median AdviseDx immunoglobulin levels peaked 7 weeks after first vaccine dose at approximately 4,000 IU/ml. Intriguingly, among the five assays examined, there was no significant difference in antigen binding level or neutralizing activity between two seropositive patients protected against SARS-CoV-2 infection in a previously described fishing vessel outbreak and five health care workers who experienced vaccine breakthrough of SARS-CoV-2 infection, all with variants of concern. These findings suggest that protection against SARS-CoV-2 infection cannot currently be predicted exclusively using in vitro antibody assays against wild-type SARS-CoV-2 spike. Further work is required to establish protective correlates for SARS-CoV-2 infection.

Clinical, laboratory, and temporal predictors of neutralizing antibodies against SARS-CoV-2 among COVID-19 convalescent plasma donor candidates.

BACKGROUNDSARS-CoV-2-specific antibodies may protect from reinfection and disease, providing rationale for administration of plasma containing SARS-CoV-2-neutralizing antibodies (nAbs) as a treatment for COVID-19. Clinical factors and laboratory assays to streamline plasma donor selection, and the durability of nAb responses, are incompletely understood.METHODSPotential convalescent plasma donors with virologically documented SARS-CoV-2 infection were tested for serum IgG against SARS-CoV-2 spike protein S1 domain and against nucleoprotein (NP), and for nAb.RESULTSAmong 250 consecutive persons, including 27 (11%) requiring hospitalization, who were studied a median of 67 days since symptom onset, 97% were seropositive on 1 or more assays. Sixty percent of donors had nAb titers ≥1:80. Correlates of higher nAb titers included older age (adjusted OR [AOR] 1.03 per year of age, 95% CI 1.00-1.06), male sex (AOR 2.08, 95% CI 1.13-3.82), fever during illness (AOR 2.73, 95% CI 1.25-5.97), and disease severity represented by hospitalization (AOR 6.59, 95% CI 1.32-32.96). Receiver operating characteristic analyses of anti-S1 and anti-NP antibody results yielded cutoffs that corresponded well with nAb titers, with the anti-S1 assay being slightly more predictive. nAb titers declined in 37 of 41 paired specimens collected a median of 98 days (range 77-120) apart (P < 0.001). Seven individuals (2.8%) were persistently seronegative and lacked T cell responses.CONCLUSIONnAb titers correlated with COVID-19 severity, age, and sex. SARS-CoV-2 IgG results can serve as useful surrogates for nAb testing. Functional nAb levels declined, and a small proportion of convalescent individuals lacked adaptive immune responses.FUNDINGThe project was supported by the Frederick National Laboratory for Cancer Research with support from the NIAID under contract number 75N91019D00024, and was supported by the Fred Hutchinson Joel Meyers Endowment, Fast-Grants, a New Investigator award from the American Society for Transplantation and Cellular Therapy, and NIH contracts 75N93019C0063, 75N91019D00024, and HHSN272201800013C, and NIH grants T32-AI118690, T32-AI007044, K08-AI119142, and K23-AI140918.

SARS-CoV-2 Viral Load on Admission Is Associated With 30-Day Mortality.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viral load on admission was associated with a significantly increased 30-day mortality (odds ratio [OR], 4.20; 95% CI, 1.62-10.86), and anti-SARS-CoV-2 nucleocapisid IgG seropositivity on admission trended toward a reduced 30-day mortality (OR, 0.43; 95% CI, 0.15-1.26). Reporting of quantitative SARS-CoV-2 viral load and serologic assays may offer prognostic clinical information.

Prevalence of autoantibody responses in acute coronavirus disease 2019 (COVID-19).

Immunopathology may play a significant role in the pathogenesis of Coronavirus-Induced Disease-19 (COVID-19). Immune-mediated tissue damage could result from development of rapid autoimmune responses, characterized by production of self-reactive autoantibodies. In this study, we tested specimens from acutely ill patients hospitalized with COVID-19 for autoantibodies against nuclear, vasculitis-associated, and phospholipid antigens. Detectable autoantibodies were present in 30% of the patients in our cohort, with the majority of reactive specimens demonstrating antibodies to nuclear antigens. However, antinuclear antibodies were only weakly reactive and directed to single antigens, as is often seen during acute infection. We identified strongly reactive antibodies to nuclear antigens only in patients with a prior history of autoimmune disease. In our cohort, the prevalence of antiphospholipid antibodies was low, and we did not detect any vasculitis-associated autoantibodies. We found similar levels of inflammatory markers and total immunoglobulin levels in autoantibody positive versus negative patients, but anti-SARS-CoV-2 antibody levels were increased in autoantibody positive patients. Together, our results suggest that acute COVID-19 is not associated with a high prevalence of clinically significant autoantibody responses of the type usually associated with autoimmune rheumatic disease.

Clinical, laboratory, and temporal predictors of neutralizing antibodies to SARS-CoV-2 after COVID-19.

BACKGROUND: SARS-CoV-2-specific antibodies may protect from reinfection and disease, providing the rationale for administration of plasma containing SARS-CoV-2 neutralizing antibodies (nAb) as a treatment for COVID-19. The clinical factors and laboratory assays to streamline plasma donor selection, and the durability of nAb responses, are incompletely understood. METHODS: Adults with virologically-documented SARS-CoV-2 infection in a convalescent plasma donor screening program were tested for serum IgG to SARS-CoV-2 spike protein S1 domain, nucleoprotein (NP), and for nAb. RESULTS: Amongst 250 consecutive persons studied a median of 67 days since symptom onset, 243/250 (97%) were seropositive on one or more assays. Sixty percent of donors had nAb titers ≥1:80. Correlates of higher nAb titer included older age (adjusted OR [AOR] 1.03/year of age, 95% CI 1.00-1.06), male sex (AOR 2.08, 95% CI 1.13-3.82), fever during acute illness (AOR 2.73, 95% CI 1.25-5.97), and disease severity represented by hospitalization (AOR 6.59, 95% CI 1.32-32.96). Receiver operating characteristic (ROC) analyses of anti-S1 and anti-NP antibody results yielded cutoffs that corresponded well with nAb titers, with the anti-S1 assay being slightly more predictive. NAb titers declined in 37 of 41 paired specimens collected a median of 98 days (range, 77-120) apart (P<0.001). Seven individuals (2.8%) were persistently seronegative and lacked T cell responses. CONCLUSIONS: Nab titers correlated with COVID-19 severity, age, and sex. Standard commercially available SARS-CoV-2 IgG results can serve as useful surrogates for nAb testing. Functional nAb levels were found to decline and a small proportion of COVID-19 survivors lack adaptive immune responses.

Rapid Screening Evaluation of SARS-CoV-2 IgG Assays Using Z-Scores to Standardize Results.

Many serologic tests are now available for measuring severe acute respiratory syndrome coronavirus 2 antibodies to evaluate potential protective immunity and for seroprevalence studies. We describe an approach to standardizing positivity thresholds and quantitative values for different assays that uses z-scores to enable rapid and efficient comparison of serologic test performance.

Performance Characteristics of the Abbott Architect SARS-CoV-2 IgG Assay and Seroprevalence in Boise, Idaho.

Coronavirus disease 2019 (COVID-19), the novel respiratory illness caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is associated with severe morbidity and mortality. The rollout of diagnostic testing in the United States was slow, leading to numerous cases that were not tested for SARS-CoV-2 in February and March 2020 and necessitating the use of serological testing to determine past infections. Here, we evaluated the Abbott SARS-CoV-2 IgG test for detection of anti-SARS-CoV-2 IgG antibodies by testing 3 distinct patient populations. We tested 1,020 serum specimens collected prior to SARS-CoV-2 circulation in the United States and found one false positive, indicating a specificity of 99.90%. We tested 125 patients who tested reverse transcription-PCR (RT-PCR) positive for SARS-CoV-2 for whom 689 excess serum specimens were available and found that sensitivity reached 100% at day 17 after symptom onset and day 13 after PCR positivity. Alternative index value thresholds for positivity resulted in 100% sensitivity and 100% specificity in this cohort. We tested specimens from 4,856 individuals from Boise, ID, collected over 1 week in April 2020 as part of the Crush the Curve initiative and detected 87 positives for a positivity rate of 1.79%. These data demonstrate excellent analytical performance of the Abbott SARS-CoV-2 IgG test as well as the limited circulation of the virus in the western United States. We expect that the availability of high-quality serological testing will be a key tool in the fight against SARS-CoV-2.

Xenophagy: Pathogen-Containing Vacuoles Are Hard to Digest.

Many intracellular pathogens reside in host-membrane-encased vacuoles, but the mechanism initiating xenophagic targeting of these vacuoles was unknown. A recent study identifies the host vacuolar-ATPase as essential to xenophagic clearance and the Salmonellae effector SopF that inhibits bacterial clearance by its ADP-ribosylation.

ß-Barrel outer membrane proteins suppress mTORC2 activation and induce autophagic responses.

The outer membranes of Gram-negative bacteria and mitochondria contain proteins with a distinct ß-barrel tertiary structure that could function as a molecular pattern recognized by the innate immune system. Here, we report that purified outer membrane proteins (OMPs) from different bacterial and mitochondrial sources triggered the induction of autophagy-related endosomal acidification, LC3B lipidation, and p62 degradation. Furthermore, OMPs reduced the phosphorylation and therefore activation of the multiprotein complex mTORC2 and its substrate Akt in macrophages and epithelial cells. The cell surface receptor SlamF8 and the DNA-protein kinase subunit XRCC6 were required for these OMP-specific responses in macrophages and epithelial cells, respectively. The addition of OMPs to mouse bone marrow-derived macrophages infected with Salmonella Typhimurium facilitated bacterial clearance. These data identify a specific cellular response mediated by bacterial and mitochondrial OMPs that can alter inflammatory responses and influence the killing of pathogens.

Human Diversity in a Cell Surface Receptor that Inhibits Autophagy.

Mutations in genes encoding autophagy proteins have been associated with human autoimmune diseases, suggesting that diversity in autophagy responses could be associated with disease susceptibility or severity. A cellular genome-wide association study (GWAS) screen was performed to explore normal human diversity in responses to rapamycin, a microbial product that induces autophagy. Cells from several human populations demonstrated variability in expression of a cell surface receptor, CD244 (SlamF4, 2B4), that correlated with changes in rapamycin-induced autophagy. High expression of CD244 and receptor activation with its endogenous ligand CD48 inhibited starvation- and rapamycin-induced autophagy by promoting association of CD244 with the autophagy complex proteins Vps34 and Beclin-1. The association of CD244 with this complex reduced Vps34 lipid kinase activity. Lack of CD244 is associated with auto-antibody production in mice, and lower expression of human CD244 has previously been implicated in severity of human rheumatoid arthritis and systemic lupus erythematosus, indicating that increased autophagy as a result of low levels of CD244 may alter disease outcomes.

A Cellular GWAS Approach to Define Human Variation in Cellular Pathways Important to Inflammation.

An understanding of common human diversity in innate immune pathways should be beneficial in understanding autoimmune diseases, susceptibility to infection, and choices of anti-inflammatory treatment. Such understanding could also result in definition of currently unknown components of human inflammation pathways. A cellular genome-wide association studies (GWAS) platform, termed Hi-HOST (High-throughput human in vitro susceptibility testing), was developed to assay in vitro cellular phenotypes of infection in genotyped lymphoblastoid cells from genetically diverse human populations. Hi-HOST allows for measurement of multiple host and pathogen parameters of infection/inflammation including: bacterial invasion and intracellular replication, host cell death, and cytokine production. Hi-HOST has been used to successfully define a significant portion of the heritable human diversity in inflammatory cell death in response to Salmonella typhimurium. It also led to the discovery of genetic variants important to protection against systemic inflammatory response syndrome (SIRS) and protection against death and bacteremia in individuals with SIRS. Our laboratory is currently using this platform to define human diversity in autophagy and the NLPR3 inflammasome pathways, and to define new components that can impact the expression of phenotypes related to these pathways.

Salmonellae interactions with host processes.

Salmonellae invasion and intracellular replication within host cells result in a range of diseases, including gastroenteritis, bacteraemia, enteric fever and focal infections. In recent years, considerable progress has been made in our understanding of the molecular mechanisms that salmonellae use to alter host cell physiology; through the delivery of effector proteins with specific activities and through the modulation of defence and stress response pathways. In this Review, we summarize our current knowledge of the complex interplay between bacterial and host factors that leads to inflammation, disease and, in most cases, control of the infection by its animal hosts, with a particular focus on Salmonella enterica subsp. enterica serovar Typhimurium. We also highlight gaps in our knowledge of the contributions of salmonellae and the host to disease pathogenesis, and we suggest future avenues for further study.

A new protein-protein interaction sensor based on tripartite split-GFP association.

Monitoring protein-protein interactions in living cells is key to unraveling their roles in numerous cellular processes and various diseases. Previously described split-GFP based sensors suffer from poor folding and/or self-assembly background fluorescence. Here, we have engineered a micro-tagging system to monitor protein-protein interactions in vivo and in vitro. The assay is based on tripartite association between two twenty amino-acids long GFP tags, GFP10 and GFP11, fused to interacting protein partners, and the complementary GFP1-9 detector. When proteins interact, GFP10 and GFP11 self-associate with GFP1-9 to reconstitute a functional GFP. Using coiled-coils and FRB/FKBP12 model systems we characterize the sensor in vitro and in Escherichia coli. We extend the studies to mammalian cells and examine the FK-506 inhibition of the rapamycin-induced association of FRB/FKBP12. The small size of these tags and their minimal effect on fusion protein behavior and solubility should enable new experiments for monitoring protein-protein association by fluorescence.