Association of vitamin D levels and risk of latent tuberculosis in the hemodialysis population

BackgroundVitamin D is essential in the host defense against tuberculosis (TB). Suboptimal vitamin D status is common in the hemodialysis population. Hemodialysis patients have an increased risk compared to the general population latent tuberculosis infection (LTBI). However, the association between vitamin D deficiency and LTBI in this population remains unclear.Materials and methodsWe conducted a cross-sectional study between March and May 2017. Interferon-gamma release assay (IGRA) through QuantiFERON-TB Gold In-Tube was used to assess LTBI. Plasma 25-hydroxycholecalciferol (25-OHD) levels were measured by Elecsys Vitamin D Total assay. Suboptimal vitamin D levels included vitamin D insufficiency 20–29 ng/mg and vitamin D deficiency <20 ng/mL. Predictors for LTBI were analyzed.ResultsA total of 287 participants were enrolled. The suboptimal vitamin D level was 31.4% (90/287), which including the vitamin D deficiency was 13.9% (40/287). A total of 49.1% (141/287) people received nutritional vitamin D supplementation. The prevalence of IGRA positivity in this study was 25.1% (72/287). There was no significant difference in vitamin D concentrations or the proportion of vitamin D supplementation among the IGRA-positive and IGRA-negative groups (p = 0.789 and 0.496, respectively). In multivariate analysis, age >65 years old (odds ratio (OR), 1.89; 95% CI, 1.08–3.31; p = 0.026) and TB history (OR, 3.51; 95% CI, 1.38–8.91; p = 0.008) were independent predictors of IGRA positivity.ConclusionThis is the first study to report that vitamin D deficiency was not associated with IGRA positivity in a hemodialysis population. Aging and TB history were both independent predictors for LTBI.     

Antigenic cartography using sera from sequence-confirmed SARS-CoV-2 variants of concern infections reveals antigenic divergence of Omicron

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Development of an IgG-Fc fusion COVID-19 subunit vaccine,AKS-452

AKS-452 is a biologically-engineered vaccine comprising an Fc fusion protein of the SARS-CoV-2 viral spike protein receptor binding domain antigen (Ag) and human IgG1 Fc (SP/RBD-Fc) in clinical development for the induction and augmentation of neutralizing IgG titers against SARS-CoV-2 viral infection to address the COVID-19 pandemic. The Fc moiety is designed to enhance immunogenicity by increasing uptake via Fc-receptors (FcγR) on Ag-presenting cells (APCs) and prolonging exposure due to neonatal Fc receptor (FcRn) recycling. AKS-452 induced approximately 20-fold greater neutralizing IgG titers in mice relative to those induced by SP/RBD without the Fc moiety and induced comparable long-term neutralizing titers with a single dose vs. two doses. To further enhance immunogenicity, AKS-452 was evaluated in formulations containing a panel of adjuvants in which the water-in-oil adjuvant, Montanide™ ISA 720, enhanced neutralizing IgG titers by approximately 7-fold after one and two doses in mice, including the neutralization of live SARS-CoV-2 virus infection of VERO-E6 cells. Furthermore, ISA 720-adjuvanted AKS-452 was immunogenic in rabbits and non-human primates (NHPs) and protected from infection and clinical symptoms with live SARS-CoV-2 virus in NHPs (USA-WA1/2020 viral strain) and the K18 human ACE2-trangenic (K18-huACE2-Tg) mouse (South African B.1.351 viral variant). These preclinical studies support the initiation of Phase I clinical studies with adjuvanted AKS-452 with the expectation that this room-temperature stable, Fc-fusion subunit vaccine can be rapidly and inexpensively manufactured to provide billions of doses per year especially in regions where the cold-chain is difficult to maintain.     

Redox active or thiol reactive? Optimization of rapid screens to identify less evident nuisance compounds

Compounds that exhibit assay interference or undesirable mechanisms of bioactivity are routinely encountered in assays at various stages of drug discovery. We observed that assays for the investigation of thiol-reactive and redox-active compounds have not been collected in a comprehensive review. Here, we review these assays and subject them to experimental optimization to improve their reliability. We demonstrate the usefulness of our assay cascade by assaying a library of bioactive compounds, chemical probes, and a set of approved drugs. These high-throughput assays should complement the array of wet-lab and in silico assays during the initial stages of hit discovery campaigns to pursue only hit compounds with tractable mechanisms of action.     

Developmental validation of the ANDE™ rapid DNA system with FlexPlex™ assay for arrestee and reference buccal swab processing and database searching

A developmental validation was performed to demonstrate reliability, reproducibility and robustness of the ANDE System with the FlexPlex assay, including an integrated Expert System, across a number of laboratories and buccal sample variations. Previously, the related DNAscan™/ANDE 4C Rapid DNA System using the PowerPlex^®16 assay and integrated Expert System Software received NDIS approval in March 2016. The enhanced ANDE instrument, referred to as ANDE 6C, and the accompanying 6-dye, 27-locus STR assay, referred to as FlexPlex, have been developed to be compatible with all widely used global loci, including the expanded set of the CODIS core 20 loci.Six forensic and research laboratories participated in the FlexPlex Rapid DNA developmental validation experiments, testing a total of 2045 swabs, including those obtained from 1387 unique individuals. The goal of this extensive and comprehensive validation was to thoroughly evaluate and document the ANDE System and its internal Expert System to reliably genotype reference buccal swab samples in a manner compliant with the FBI’s Quality Assurance Standards and the NDIS Operational Procedures.The ANDE System, including automated Expert System analysis, generated reproducible and concordant results for buccal swabs when testing various instruments at different laboratories by a number of different operators. When testing a number of non-human DNAs, including oral bacteria, the ANDE System and FlexPlex assay demonstrated limited cross-reactivity. Potential PCR inhibitors were evaluated as part of the validation and no inhibition was detected. Reproducible and concordant profiles were generated from buccal swab samples collected with a limit of detection appropriate for buccal swab collections from arrestees. The precision and resolution of the System met industry standards for detection of microvariants and single base resolution.The integrated Expert System appropriately demonstrated the ability to correctly pass or fail profiles for CODIS upload without human review. During this comprehensive developmental validation, the ANDE System successfully interpreted over 2000 samples tested with over 99.99% concordant alleles. The data package described herein led to the ANDE System with the FlexPlex assay receiving NDIS approval in June 2018.     

Antioxidant and anti-inflammatory activities of lycopene against 5-fluorouracil-induced cytotoxicity in Caco2 cells

5-fluorouracil (5FU) is widely used to treat colorectal cancer (CC) and its main mechanisms of anticancer action are through generation of ROS which often result in inflammation. Here, we test the effect of Lycopene against 5FU in Caco2 cell line. Caco2 cells were exposed to 3 µg/ml of 5FU alone or with 60, 90, 120 µg/ml of lycopene. This was followed by assessment of cytotoxicity, oxidative stress, and gene expression of inflammatory genes. Our findings showed that Lycopene and 5FU co-exposure induced dose-dependent cytotoxic effect without compromising the membrane integrity based on the LDH assay. Lycopene also significantly enhanced 5FU-induced SOD activity and GSH level compared to control for all mixture concentrations (p < 0.01). Lycopene alone and combination with 5FU-induced expression of IL-1β, TNF-α, and IL-6. Furthermore, IFN-γ expression was significantly enhanced by only mixture of lycopene (90 µg/ml) and 5FU (p < 0.05). In conclusion, Lycopene supplementation with 5FU therapy resulted in improvement in antioxidant parameters such as catalase and GSH levels giving the cell capacity to cope with 5FU-mediated oxidative stress. Lycopene also enhanced IFN-γ expression in the presence of 5FU, which may activate antitumor effects further enhancing the cancer killing effect of 5FU.     

Genotoxicity of synthetic amorphous silica nanoparticles in rats following short‐term exposure. Part 1: Oral route

Synthetic amorphous silica (SAS) in its nanosized form is now used in food applications although the potential risks for human health have not been evaluated. In this study, genotoxicity and oxidative DNA damage of two pyrogenic (NM‐202 and 203) and two precipitated (NM‐200 and ‐201) nanosized SAS were investigated in vivo in rats following oral exposure. Male Sprague Dawley rats were exposed to 5, 10, or 20 mg/kg b.w./day for three days by gavage. DNA strand breaks and oxidative DNA damage were investigated in seven tissues (blood, bone marrow from femur, liver, spleen, kidney, duodenum, and colon) with the alkaline and the (Fpg)‐modified comet assays, respectively. Concomitantly, chromosomal damage was investigated in bone marrow and in colon with the micronucleus assay. Additionally, malondialdehyde (MDA), a lipid peroxidation marker, was measured in plasma. When required, a histopathological examination was also conducted. The results showed neither obvious DNA strand breaks nor oxidative damage with the comet assay, irrespective of the dose and the organ investigated. Similarly, no increases in chromosome damage in bone marrow or lipid peroxidation in plasma were detected. However, although the response was not dose‐dependent, a weak increase in the percentage of micronucleated cells was observed in the colon of rats treated with the two pyrogenic SAS at the lowest dose (5 mg/kg b.w./day). Additional data are required to confirm this result, considering in particular, the role of agglomeration/aggregation of SAS NMs in their uptake by intestinal cells. Environ. Mol. Mutagen. 56:218–227, 2015. © 2014 Wiley Periodicals, Inc.     

Multicentric Analytical and Inter-observer Comparability of Four Clinically Developed Programmed Death-ligand 1 Immunohistochemistry Assays in Advanced Clear-cell Renal Cell Carcinoma

BackgroundPrevious studies have suggested increased clinical benefit with inhibition of programmed death-ligand 1 (PD-L1)/programmed death-1 in patients with PD-L1–positive locally advanced/metastatic renal cell carcinoma (RCC). We examined the analytical and inter-observer comparability of PD-L1–positivity across 4 clinically developed immunohistochemistry assays in clear-cell RCC (CCRCC).Materials and MethodsRandomly selected archived, formalin-fixed, paraffin-embedded nephrectomy specimens from 201 patients with locally advanced CCRCC were screened using VENTANA SP142. From these, 30 cases were selected based on their tumor-infiltrating immune cell (IC) PD-L1 status (PD-L1-IC–positivity of < 1%, 1%-5%, or > 5%; 10 cases each). These cases were stained for PD-L1 using VENTANA SP142 and SP263, and DAKO 22C3 and 28-8, and scored for PD-L1 expression on IC and tumor cells (TC) by trained readers at 5 sites.ResultsAdjusted mean percentages of PD-L1-IC–positivity and PD-L1-TC–positivity varied from 4.0% to 4.9% and from 1.3% to 10.7%, respectively, between assays. Inter-assay differences in PD-L1-IC–positivity were small and non-significant (P = .1938 to .9963); for PD-L1-TC–positivity, significant differences were observed between VENTANA SP142 and the other assays (P ≤ .0001) and between VENTANA SP263 and DAKO 28-8 (P = .0248). Intra-class correlation values showed moderate-to-high inter-reader agreement for each assay for PD-L1-IC–positivity and for 3 assays for PD-L1-TC–positivity.ConclusionsIn this first multicenter analytical comparison study of PD-L1 assays in CCRCC, PD-L1–positivity could be assessed reproducibly using all 4 assays for IC and for 3 of the 4 assays for TC.     

Effectiveness of various methods of formaldehyde neutralization using monoethanolamine

Formaldehyde is the most commonly used fixative chemical for the preservation of human cadavers used for educational purposes in the United States. Formaldehyde is also a known carcinogenic agent whose exposure level is regulated by guidelines of the Occupational Safety and Health Administration. Various methods for formaldehyde neutralization exist, yet many donations programs do not take any steps to neutralize the formaldehyde in embalmed donor bodies. The effectiveness of monoethanolamine (MEA) in neutralizing formaldehyde is well documented when used as a final injection during embalming. The purpose of this study is to report the effectiveness of several post‐embalming techniques of formaldehyde neutralization. Twenty‐four donor bodies were assigned to four experimental groups of six. For the three experimental groups, the techniques tested involve delivery of a 20:1 dilution of deionized water:MEA via recannulization and gravity flow infusion, compartment injection, and alternate wetting solution containing four percent MEA. Our results indicated that spray bottle delivery was not effective in neutralization of formaldehyde compared to the control group, but that formaldehyde levels decreased when recannulization or compartment injection were used. The most effective method of formaldehyde neutralization was compartment injection of MEA solution (P < 0.01). The results of this study indicate that, in situations where MEA is not used as a final infusion during embalming, compartment injection of MEA solution is an effective method of formaldehyde neutralization. Clin. Anat. 28:449–454, 2015. © 2015 Wiley Periodicals, Inc.     

Genetic toxicity assessment using liver cell models: past,present, and future

ABSTRACT

Genotoxic compounds may be detoxified to non-genotoxic metabolites while many pro-carcinogens require metabolic activation to exert their genotoxicity in vivo. Standard genotoxicity assays were developed and utilized for risk assessment for over 40 years. Most of these assays are conducted in metabolically incompetent rodent or human cell lines. Deficient in normal metabolism and relying on exogenous metabolic activation systems, the current in vitro genotoxicity assays often have yielded high false positive rates, which trigger unnecessary and costly in vivo studies. Metabolically active cells such as hepatocytes have been recognized as a promising cell model in predicting genotoxicity of carcinogens in vivo. In recent years, significant advances in tissue culture and biological technologies provided new opportunities for using hepatocytes in genetic toxicology. This review encompasses published studies (both in vitro and in vivo) using hepatocytes for genotoxicity assessment. Findings from both standard and newly developed genotoxicity assays are summarized. Various liver cell models used for genotoxicity assessment are described, including the potential application of advanced liver cell models such as 3D spheroids, organoids, and engineered hepatocytes. An integrated strategy, that includes the use of human-based cells with enhanced biological relevance and throughput, and applying the quantitative analysis of data, may provide an approach for future genotoxicity risk assessment.