Role of endogenous 4-1BB in the development of systemic lupus erythematosus

Systemic lupus erythematosus (SLE) is characterized by the production of autoantibodies directed against nuclear antigens including nucleosomes and DNA. To determine the role of T-cell costimulatory molecule 4-1BB in the regulation of SLE, MRL-Fas(lpr) (lpr) mice deficient in 4-1BB (lpr/4-1BB(-/-)) were generated and their disease phenotype was compared to that of control lpr mice. The main finding of this study is that the lpr/4-1BB(-/-) mice had more pronounced skin lesions which appeared earlier, increased lymphadenopathy, increased renal damage, and higher mortality than 4-1BB-intact control lpr mice. The increased severity of lesions in lpr/4-1BB(-/-) mice was closely associated with increases in CD4(+) T, CD3(+) B220(+) double-negative T cells, serum immunoglobulin, anti-dsDNA autoantibodies, and tissue immunoglobulin deposits. These data suggest that the 4-1BB-4-1BB ligand signalling pathway plays an important role in SLE and that deletion of 4-1BB confers susceptibility to lpr mice, leading to accelerated induction of disease and early mortality.     

Nano-purification of raw semen minimises oxidative stress with improvement in post-thaw quality of buffalo spermatozoa

The study consisted of application of anti-ubiquitin antibodies (Abs)-coated iron oxide-nanoparticles (IONPs) for minimisation of oxidative stress to contemporary live spermatozoa from the raw semen. Round-shaped IONPs (12.09 ± 0.91 nm) after two-stage functionalisation (silanisation and pegylation) were conjugated with Abs. Four aliquots from each of the 24 ejaculates (4 buffalo bulls) formed Control (Group I) and treatment (II, III and IV) groups; each containing 150 ± 25 million dead/damaged spermatozoa. IONPs-Abs complex were added at ratio of 1:1 (0.5 µg/ml), 1:2 (1.0 µg/ml) and 1:4 (2.0 µg/ml), respectively, in Groups II, III and IV. The semen quality parameters showed improvement at lag-stage (post-nano-purification before processing for cryopreservation). The mean post-thaw motility (%) in Group IV was found to be greater (p < .05) than Group I. Moreover, the overall DNA integrity (%) at post-thaw stage was improved in the nano-purified semen samples. The value of malondialdehyde was greater (p < .001) in Group I than Groups II, III and IV. The mean total antioxidant capacity and superoxide dismutase (U/mg protein) activity values in Group IV was greater (p < .05) than Group I. The study results show that IONPs conjugated with anti-ubiquitin Abs at 2.0 µg/ml can be an effective dose for depletion of dead/damaged spermatozoa from buffalo ejaculates to minimise oxidative stress.     

Development of a Cell-Based Luciferase Complementation Assay for Identification of SARS-CoV-2 3CLpro Inhibitors

The 3C-like protease (3CL^pro) of SARS-CoV-2 is considered an excellent target for COVID-19 antiviral drug development because it is essential for viral replication and has a cleavage specificity distinct from human proteases. However, drug development for 3CL^pro has been hindered by a lack of cell-based reporter assays that can be performed in a BSL-2 setting. Current efforts to identify 3CL^pro inhibitors largely rely upon in vitro screening, which fails to account for cell permeability and cytotoxicity of compounds, or assays involving replication-competent virus, which must be performed in a BSL-3 facility. To address these limitations, we have developed a novel cell-based luciferase complementation reporter assay to identify inhibitors of SARS-CoV-2 3CL^pro in a BSL-2 setting. The assay is based on a lentiviral vector that co-expresses 3CL^pro and two luciferase fragments linked together by a 3CL^pro cleavage site. 3CL^pro-mediated cleavage results in a loss of complementation and low luciferase activity, whereas inhibition of 3CL^pro results in 10-fold higher levels of luciferase activity. The luciferase reporter assay can easily distinguish true 3CL^pro inhibition from cytotoxicity, a powerful feature that should reduce false positives during screening. Using the assay, we screened 32 small molecules for activity against SARS-CoV-2 3CL^pro, including HIV protease inhibitors, HCV protease inhibitors, and various other compounds that have been reported to inhibit SARS-CoV-2 3CL^pro. Of these, only five exhibited significant inhibition of 3CL^pro in cells: GC376, boceprevir, Z-FA-FMK, calpain inhibitor XII, and GRL-0496. This assay should greatly facilitate efforts to identify more potent inhibitors of SARS-CoV-2 3CL^pro.     

Relationship between diabetic retinopathy microalbuminuria and other modifiable risk factors

BackgroundDiabetic Retinopathy (DR) is an important microvascular complication of diabetes that can lead to irreversible blindness. Microalbuminuria is strongly associated with diabetic retinopathy and can be used as a reliable marker of diabetic retinopathy.AimTo assess the association between DR, microalbuminuria, and other modifiable risk factors in patients with type 2 diabetes.Methodology3090 patients with T2DM visiting North Delhi Diabetes Centre, New Delhi between July 2016 to October 2019 were evaluated for the clinical and biochemical parameters that included urinary albumin, HbA1C, lipid profiles, serum creatinine estimation and underwent biothesiometry.Results3090 patients (1350 females and 1740 males), with mean age of 52.7 ± 9.2 years and diabetes duration ranging from 1 to 19 years (mean 9.4 ± 6), duration of less than 5 years, 6–10 years and more than 10 years in 52%, 26% and in 22%, respectively. Duration of diabetes was strong predictor of retinopathy (p = 0.001). The HbA1c and BMI in patients with DR was significantly higher than in those without DR. 18.2% patients were diagnosed to have retinopathy. Peripheral neuropathy was observed in 24.2% and was positively associated with DR (p = 0.002). 33.9% and 4.5% patients had microalbuminuria macroalbuminuria, respectively and 9.7% patients had creatinine >1.3 mg/dL. There was significant positive relationship between different grades of retinopathy and albuminuria.ConclusionsOur study is a large real-world study that demonstrates that HbA1c, BMI, duration of diabetes, microalbuminuria and peripheral neuropathy are relatively, yet cohesively contributing factors towards varying grade of retinopathy.     

Restoration of spermatogenesis by lentiviral gene transfer: offspring from infertile mice

Disruption of spermatogenesis found in azoospermia and oligozoospermia is thought to be of primarily genetic origin. Sl/Sl(d) mutant mice offer a model system in which lack of transmembrane type c-kit ligand (KL2) expression on the somatic Sertoli cell surface results in disruption of spermatogenesis. We investigated the ability of adeno-, adeno-associated-, retro-, and lentiviral vectors to transduce Sertoli cells and found that transduction with either adeno- or lentiviral vectors led to reporter gene expression for more than 2 mo after testicular tubule injection. Because adenoviral vectors showed toxicity, lentiviral vectors were used to express the c-kit ligand in Sl/Sl(d) Sertoli cells. Restoration of spermatogenesis was observed in all recipient testes. Furthermore, the sperm collected from recipient testes were able to generate normal pups after intracytoplasmic sperm injection. None of the offspring carried the transgene, suggesting the inability of lentiviral vectors to infect spermatogenic cells in vivo. We propose that lentiviral vectors can be used for gene therapy of male infertility without the risk of germ-line transmission.     

Detoxifying enzymes in human ovarian tissues: comparison of normal and tumor tissues and effects of chemotherapy

Summary Many anticancer drugs exert their cytotoxic effects via formation of oxygen free radicals. Cellular thiols, glutathione (GSH)-dependent enzymes and other redox enzymes are involved in the metabolism of these anticancer drugs and of the oxygen free radicals that may be generated during their metabolism. We quantified these biochemical parameters in cytosol from human ovarian tissues. We compared non-protein thiol levels, GSH transferase, GSH peroxidase, Superoxide dismutase, catalase, DT diaphorase and aldehyde dehydrogenase activity in serous ovarian tumors (n=15), other malignant ovarian tumors (n=12), benign ovarian tissue (n=10) and histologically normal ovarian tissue (n=12). Mean GSH transferase and DT diaphorase activitie were similar in serous and other malignant ovarian tumors. GSH transferase activity was decreased in malignant tissues relative to normal and benign tissues. Mean DT diaphorase and Superoxide dismutase activities were increased in the malignant tissues, although this was not statistically significant. The mean levels of all anzymes except Superoxide dismutase and aldehyde dehydrogenase in benign tissues were fairly similar to the mean levels found in normal tissue samples. Tissues from patients with serous ovarian tumors, who had received cyclophosphamide and cisplatin prior to surgery, also were analyzed (n=7). Except for aldehyde dehydrogenase, all the parameters measured were decreased in these samples relative to serous tissue from untreated patients. These biochemical analyses may be useful in understanding the mechanisms involved in the response to chemotherapy.Abbreviations ANOVA analysis of variance - GSH glutathione Partial support for this study was obtained from the Comprehensive Cancer Center of Metropolitan Detroit, core grant CA-22453, National Institutes of Health.     

Circulating T-Cell Subsets,Monocytes, and Natural Killer Cells in Peripartum Cardiomyopathy: Results From the Multicenter IPAC Study

Objective

The aim of this work was to evaluate the hypothesis that the distribution of circulating immune cell subsets, or their activation state, is significantly different between peripartum cardiomyopathy (PPCM) and healthy postpartum (HP) women.

Unusual immunophenotype of CD8+ T cells in familial hemophagocytic lymphohistiocytosis

Familial hemophagocytic lymphohistiocytosis (FHL) is an inherited, fatal disorder of infancy. We report here a 17-day-old female infant who presented with high fever, hepatosplenomegaly, hypertriglyceridemia, hypofibrinogenemia, thrombocytopenia, and liver failure. Leukocytosis was detected with circulating "atypical" lymphoid cells. Flow cytometric studies revealed expanded subpopulations of CD8+ T cells with unusual immunophenotypic features, including a subset that lacked CD5 expression. A liver biopsy showed hemophagocytic lymphohistiocytosis with exuberant infiltrates of CD8+ T cells that lacked perforin. Mutational studies revealed a 666C-->A (H222Q) missense mutation in the perforin gene. T-cell receptor studies on flow-sorted T-cell subpopulations revealed no evidence of monoclonality. Analysis of T-cell receptor excision circle levels indicated long proliferative history in the aberrant CD8+ T-cell subsets. This case provides an instructive example of uncontrolled reactive proliferation of CD8+ T cells in FHL, resulting in atypical morphology and unusual immunophenotypic features that might suggest malignancy in other clinical settings.