India-discovered levonadifloxacin & alalevonadifloxacin: A review on susceptibility testing methods, CLSI quality control and breakpoints along with a brief account of their emerging therapeutic profile as a novel standard-of-care.

BACKGROUND: Levonadifloxacin (intravenous) and alalevonadifloxacin (oral prodrug) are novel antibiotics based on benzoquinolizine subclass of fluoroquinolone, licensed for clinical use in India in 2019. The active moiety, levonadifloxacin, is a broad-spectrum antibiotic with a high potency against methicillin-resistant Staphylococcus. aureus, multi-drug resistant pneumococci and anaerobes. OBJECTIVE: This review, for the first time, critically analyses the antimicrobial susceptibility testing methods, Clinical Laboratory & Standards Institute (CLSI)-quality control of susceptibility testing and breakpoints of levonadifloxacin. Further, the genesis, discovery and developmental aspects as well as therapeutic profile of levonadifloxacin and alalevonadifloxacin are briefly described. CONTENTS: In order to aid the scientific and clinician communities with a single comprehensive overview on all the key aspects of levonadifloxacin and alalevonadifloxacin, the present article covers the reference MIC and disk diffusion methods for levonadifloxacin susceptibility testing that were approved by CLSI and the reference ranges for quality control strains published in the CLSI M100 document. The breakpoints of levonadifloxacin were derived in concordance to US FDA, European Committee on Antibiotic Susceptibility Testing (EUCAST) and CLSI approaches. Further, the article provides a brief account of challenges encountered during the discovery stages of levonadifloxacin and alalevonadifloxacin, activity spectrum and safety benefits accruing from structural novelty-linked mechanism of action. Further, the review also covers in vitro and in vivo activities, registrational clinical studies and patient-friendly features of levonadifloxacin/alalevonadifloxacin. Cumulatively, levonadifloxacin has a potential to offer a long awaited new standard-of-care treatment for the resistant Gram-positive bacterial infections.

Dynamics of clusterin protein expression in the brain and plasma following experimental traumatic brain injury.

Progress in the preclinical and clinical development of neuroprotective and antiepileptogenic treatments for traumatic brain injury (TBI) necessitates the discovery of prognostic biomarkers for post-injury outcome. Our previous mRNA-seq data revealed a 1.8-2.5 fold increase in clusterin mRNA expression in lesioned brain areas in rats with lateral fluid-percussion injury (FPI)-induced TBI. On this basis, we hypothesized that TBI leads to increases in the brain levels of clusterin protein, and consequently, increased plasma clusterin levels. For evaluation, we induced TBI in adult male Sprague-Dawley rats (n = 80) by lateral FPI. We validated our mRNA-seq findings with RT-qPCR, confirming increased clusterin mRNA levels in the perilesional cortex (FC 3.3, p < 0.01) and ipsilateral thalamus (FC 2.4, p < 0.05) at 3 months post-TBI. Immunohistochemistry revealed a marked increase in extracellular clusterin protein expression in the perilesional cortex and ipsilateral hippocampus (7d to 1 month post-TBI), and ipsilateral thalamus (14d to 12 months post-TBI). In the thalamus, punctate immunoreactivity was most intense around activated microglia and mitochondria. Enzyme-linked immunoassays indicated that an acute 15% reduction, rather than an increase in plasma clusterin levels differentiated animals with TBI from sham-operated controls (AUC 0.851, p < 0.05). Our findings suggest that plasma clusterin is a candidate biomarker for acute TBI diagnosis.

Chemoprotective effect of nanocurcumin on 5-fluorouracil-induced-toxicity toward oral cancer treatment.

INTRODUCTION: Cancer of oral cavity is the uncontrolled expansion of damaged cell within the mouth cavity. 5-fluorouracil (5-FU) chemotherapy was focused to kill the cancer cell, but it would affect the surrounding normal cells during oral cancer treatment. This study included the evaluation of chemoprotective effects of curcumin (CU), as an herbal remedy, on 5-FU-induced-cytotoxicity toward oral cancer treatment, loaded within a nanocarrier system. CU was combined with 5-FU chemotherapy as a combinational drug-delivery system to evaluate synergistic effects. MATERIALS AND METHODS: Nanoformulation of CU (nano-CU) and nanoformulation of 5-FU (nano-FU) were prepared by employing homogenization with high-energy sonication. The characterizations of prepared nanoformulations were evaluated on the basis of particle size, zeta potential, and polydispersity index (PDI) values. The chemopreventive effect of nano-CU on 5-FU induced-toxicity and synergistic efficacy were optimized through different in-vitro assays. RESULTS: The average particle size of nano-CU and nano-FU were up to 200 nm, negatively-charged, and shown up to 4th-day control release of the drug within the acceptable concentration. IC50 value for growth inhibition was calculated as 47.89 and 26.19 µg/ml, respectively, for nano-CU and nano-FU. OCC was pretreated with nano-CU and shown the protective effect by reducing 5-FU induced-cytotoxicity by preventing normal cells through reduced viability. The DPPH-indicated fluorescence-tagged cells had quantified for antioxidant effect as it reduces intracellular reactive oxygen species level in OCC. Along with alteration in cell protein expression, Blc2, and Bax, shows enhanced apoptosis rate in OCC. CONCLUSION: Nano-CU provides chemoprotective nature towards 5-FU induced-toxicity, along with synergistic effects in oral cancer treatment.

American Joint Replacement Registry Risk Calculator Does Not Predict 90-day Mortality in Veterans Undergoing Total Joint Replacement.

BACKGROUND: The American Joint Replacement Registry (AJRR) Total Joint Risk Calculator uses demographic and clinical parameters to provide risk estimates for 90-day mortality and 2-year periprosthetic joint infection (PJI). The tool is intended to help surgeons counsel their Medicare-eligible patients about their risk of death and PJI after total joint arthroplasty (TJA). However, for a predictive risk model to be useful, it must be accurate when applied to new patients; this has yet to be established for this calculator. QUESTIONS/PURPOSES: To produce accuracy metrics (ie, discrimination, calibration) for the AJRR mortality calculator using data from Medicare-eligible patients undergoing TJA in the Veterans Health Administration (VHA), the largest integrated healthcare system in the United States, where more than 10,000 TJAs are performed annually. METHODS: We used the AJRR calculator to predict risk of death within 90 days of surgery among 31,214 VHA patients older than 64 years of age who underwent primary TJA; data was drawn from the Veterans Affairs Surgical Quality Improvement Project (VASQIP) and VA Corporate Data Warehouse (CDW). We then used VHA mortality data to evaluate the extent to which the AJRR calculator estimates distinguished individuals who died compared with those who did not (C-statistic), and graphically depicted the relationship between estimated risk and observed mortality (calibration). As a secondary evaluation of the calculator, a sample of 39,300 patients younger than 65 years old was assigned to the youngest age group available to the user (65-69 years) as might be done in real-world practice. RESULTS: C-statistics for 90-day mortality for the older samples were 0.62 (95% CI, 0.60-0.64) and for the younger samples they were 0.46 (95% CI, 0.43-0.49), suggesting poor discrimination. Calibration analysis revealed poor correspondence between deciles of predicted risk and observed mortality rates. Poor discrimination and calibration mean that patients who died will frequently have a lower estimated risk of death than surviving patients. CONCLUSIONS: For Medicare-eligible patients receiving TJA in the VA, the AJRR risk calculator had a poor performance in the prediction of 90-day mortality. There are several possible reasons for the model's poor performance. Veterans Health Administration patients, 97% of whom were men, represent only a subset of the broader Medicare population. However, applying the calculator to a subset of the target population should not affect its accuracy. Other reasons for poor performance include a lack of an underlying statistical model in the calculator's implementation and simply the challenge of predicting rare events. External validation in a more representative sample of Medicare patients should be conducted to before assuming this tool is accurate for its intended use. LEVEL OF EVIDENCE: Level I, diagnostic study.

Best practice recommendations for the transfer of cell-based assays for the measurement of neutralizing anti-drug antibodies.

We recommend the application of a strategically designed step-wise approach to transfer cell-based assays that includes assessing analytical performance (through a fit for purpose validation and/or design of experiment robustness characterization), clinical performance (i.e., concordance) and performance or proficiency testing for long-term method monitoring. Here we focus on the application of this strategy to cell-based assays for the measurement of neutralizing anti-drug antibodies. This application is unique in that it requires a custom cell-based assay to be used over a long period of time (potentially phase 1a through the life of a marketed product) with the confidence of consistent method performance and result reporting. But, the process is adaptable to a variety of assay types and applications. We present lessons learned from two cell-based assay transfers that met relevant challenges while implementing alternative permutations of the recommended method transfer process.

A signaling-enhanced chimeric receptor to activate the ICOS pathway in T cells.

Activation of the inducible costimulator (ICOS) signaling pathway in T cells is difficult to assess with bioassays, because most T cell lines do not constitutively express ICOS. Additionally, engagement of ICOS by its natural ligand B7 related protein 1 (B7RP1) is insufficient to elicit ICOS signaling, but requires simultaneous costimulation of the T cell receptor (TCR) to be effective. Here we describe a genetically engineered human T cell line that expresses a chimeric receptor (ICOS-CD3) consisting of full-length human ICOS fused at its C-terminal end to the cytoplasmic domain of human CD3 zeta. When engaged by B7RP1, ICOS-CD3 initiated signaling independently of TCR costimulation and induced substantially more IL-2 secretion in Jurkat T cells compared to wildtype ICOS. We demonstrate that this signaling-enhanced chimeric receptor can be used in simple and sensitive bioassays to detect bioactive B7RP1, anti-B7RP1 drugs, and the presence of corresponding neutralizing anti-drug antibodies.

Preparation of (+)-trans-isoalliin and its isomers by chemical synthesis and RP-HPLC resolution.

Naturally occurring (+)-trans-isoalliin, (R(C)R(S))-(+)-trans-S-1-propenyl-L-cysteine sulfoxide, is a major cysteine sulfoxide in onion. The importance of producing it synthetically to support further research is very well recognized. The (+)-trans-isoalliin is prepared by chemical synthesis and reversed-phase (RP)-HPLC. First, S-2-propenyl-L-cysteine (deoxyalliin) is formed from L-cysteine and allyl bromide, which is then isomerized to S-1-propenyl-L-cysteine (deoxyisoalliin) by a base-catalyzed reaction. A mixture of cis and trans forms of deoxyisoalliin is formed and separated by RP-HPLC. Oxidation of the trans form of deoxyisoalliin by H2O2 produces a mixture of (-)- and (+)-trans-isoalliin. Finally, RP-HPLC is used successfully in separating (-)- and (+)-trans-isoalliin, and hence, (+)-trans-isoalliin is synthesized for the first time in this study. In addition, the (±) diastereomers of cis-isoalliin are also separated and purified by RP-HPLC.

HPLC-MTT assay: anticancer activity of aqueous garlic extract is from allicin.

A strategy using reversed-phase high-performance liquid chromatography (HPLC), thin layer chromatography (TLC), mass spectrometry (MS), nuclear magnetic resonance (NMR), chemical synthesis, and MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) cell viability assay to identify allicin as the active anticancer compound in aqueous garlic extract (AGE) is described. Changing the pH of AGE from 7.0 to 5.0 eliminated interfering molecules and enabled a clean HPLC separation of the constituents in AGE. MTT assay of the HPLC fractions identified an active fraction. Further analysis by TLC, MS, and NMR verified the active HPLC fraction as allicin. Chemically synthesized allicin was used to provide further confirmation. The results clearly identify the active compound in AGE as allicin.

Turmeric - A new treatment option for lichen planus: A pilot study.

Turmeric is dried rhizome of the perennial herbs curcumalonga. It is called Haldi in Hindi, turmeric in English, ukon in Japanese. It has been used in Asian Medicine since the second millennium BC. It's utility is referred to in the ancient Hindu script the Ayurveda. Pathogenesis of the OLP should be taken in consideration for the treatment point of view. The Cell mediated immunity to secondary antigenic change in oral mucous membrane is thought to play a major role in its pathogenesis modified keratocyte surface antigens are the primary target for cytotoxic cellular response. Curcumin also been shown to have immune modulatory effect involving activation of host macrophages and natural killer cells and modulation of lymphocytes mediated function.

Simultaneous determination of three steroidal glycoalkaloids in Solanum xanthocarpum by high performance thin layer chromatography.

A new high-performance thin-layer chromatographic (HPTLC) method has been developed for the simultaneous quantitation of three bioactive steroidal glycoalkaloid (SGA) markers, solasonine (SN), solamargine (SM) and khasianine (KN) in the plant Solanum xanthocarpum. Extraction efficiency of targeted SGAs from plant matrix using methanol and acidified methanol were studied using percolation, ultrasonication and microwave techniques. The separation was achieved on silica gel 60F(254) TLC plates using chloroform-methanol-water as mobile phase. The quantitation of SGAs was carried out using the densitometric reflection/absorption mode at 520 nm after post chromatographic derivatization using Dragendorff's reagent. The method was validated for peak purity, precision, accuracy, robustness, limit of detection (LOD) and quantitation (LOQ). Method specificity was confirmed using retention factor (R(f)), Vis spectral correlation and electrospray ionization mass spectra (ESI-MS) of marker compounds in the sample track.

Multiplexed evaluation of a cell-based assay for the detection of antidrug neutralizing antibodies to panitumumab in human serum using automated fluorescent microscopy.

The method described here employs a high-content cell-based assay format for the detection of neutralizing antibodies (NAbs) to panitumumab, a fully human monoclonal antagonistic antibody to the human epidermal growth factor (EGF) receptor in human serum (screening assay). A specificity assay was also developed and qualified to confirm that the neutralizing activity was attributable to the presence of NAbs and not due to serum interference (serum interference assay). The ArrayScan IV HCS reader was used for the measurement of tyrosine phosphorylation of the epidermal growth factor receptor (EGFR) and STAT-1 redistribution between the cytoplasm and nucleus in the human epidermoid carcinoma cell line A431. Assay conditions were developed by (1) optimizing the response of the A431 cells to recombinant human EGF in pooled human serum, (2) evaluating the ability of panitumumab to inhibit the EGF response, and (3) assessing the assay's sensitivity for detecting a positive control affinity purified rabbit polyclonal anti-panitumumab antibody. Panitumumab dose-dependently inhibited 4 ng/mL EGF, and the positive control antibody showed a dose-dependent neutralization of 50 ng/mL panitumumab. The experiments indicated that in comparison to STAT-1 translocation, EGFR phosphorylation was the optimal endpoint for the screening and serum interference assays. Assay cut points were derived for the screening and serum interference assays by obtaining normalized ratios of mean fluorescence intensity values obtained with EGFR phosphorylation by testing sera from healthy human donor sera. The assay sensitivity was determined to be 0.125 microg/mL for the positive control antibody.

Arthritic pain among Latinos: results from a community-based survey.

OBJECTIVE: To examine factors associated with pain among Latinos with arthritis, identify common coping strategies and potentially effective interventions, and determine whether pain levels affect the level of interest in potentially useful programs. METHODS: Using a convenience sampling approach and a combination of face-to-face and telephone surveys, 588 Latino adults in Oregon with arthritis were interviewed. The intensity of pain during a typical day was assessed using a scale ranging from 0 (no pain) to 10 (worst pain). A score of >or=7 was defined as severe pain. RESULTS: More than 60% of Latinos reported severe pain. Results from an ordinary least square regression indicated that among Latinos with arthritis, women, those with lower levels of education, and those reporting poor or fair self-rated health and functional limitations had higher levels of pain, after controlling for confounders. Those with severe pain were more likely than those with lower levels of pain to use over the counter medicine and home remedies to manage their arthritis. In addition, Latinos with greater pain were more likely to be interested in arthritis management programs. CONCLUSION: These findings have important implications for public health policy. The strong interest of Latinos in various arthritis and joint pain management programs could prove to be an important avenue for supporting a population with high levels of arthritic pain and lack of health insurance. These pain management programs are all the more appealing, given the availability of a number of evidence-based, low-cost interventions.

pH-Zone-refining centrifugal partition chromatography for preparative isolation and purification of steroidal glycoalkaloids from Solanum xanthocarpum.

pH-Zone-refining centrifugal-partition chromatography (CPC) was successfully applied in the separation of complex polar steroidal glycoalkaloids of close Rf values, directly from a crude extract of Solanum xanthocarpum. The experiment was performed with a two phase solvent system composed of ethyl acetate/butanol/water (1:4:5 by volume) where triethylamine (5 mM) was added to the upper organic mobile phase as an eluter and TFA (10 mM) to the aqueous stationary phase as a retainer. Separation of 1 g of crude extract over CPC resulted in two distinct pH-zones. The fractions collected in pH-zone i afforded 72 mg of solasonine while the fractions collected in pH-zone ii were slightly impure, hence were purified over medium pressure LC, which afforded 30 mg of solasonine and further 15 mg of solamargine (SM). The steroidal glycoalkaloids, SM and solasonine were isolated in 93.3 and 91.6% purity, respectively. The isolated alkaloids were characterized on the basis of their (1)H, (13)C-NMR, and ESI-MS data.

Immunogenicity of biologically-derived therapeutics: assessment and interpretation of nonclinical safety studies.

An evaluation of potential antibody formation to biologic therapeutics during the course of nonclinical safety studies and its impact on the toxicity profile is expected under current regulatory guidance and is accepted standard practice. However, approaches for incorporating this information in the interpretation of nonclinical safety studies are not clearly established. Described here are the immunological basis of anti-drug antibody formation to biopharmaceuticals (immunogenicity) in laboratory animals, and approaches for generating and interpreting immunogenicity data from nonclinical safety studies of biotechnology-derived therapeutics to support their progression to clinical evaluation. We subscribe that immunogenicity testing strategies should be adapted to the specific needs of each therapeutic development program, and data generated from such analyses should be integrated with available clinical and anatomic pathology, pharmacokinetic, and pharmacodynamic data to properly interpret nonclinical studies.

Development and validation of a quantitative cell-based bioassay for comparing the pharmacokinetic profiles of two recombinant erythropoietic proteins in serum.

An in vitro cell-based bioassay was developed and validated to assess the pharmacokinetic profiles of two novel therapeutic recombinant proteins (EP1 and EP2) with erythropoiesis stimulating properties in Sprague-Dawley rats. While immunoassays are the standard choice for evaluating the pharmacokinetic parameters of drugs, no immunoassay was available for EP2, necessitating the need for a quantitative bioassay capable of measuring both EP1 and EP2 separately so that appropriate comparisons could be made. The bioassay described here utilizes a sub clone of the murine 32D cell line transfected with the gene encoding for the human erythopoietin (HuEPO) receptor. Erythropoietin (EPO), EP1 and EP2 exert their proliferative effect on the cell line by signaling through the HuEPO receptor. The proliferation induced by the erythropoietic proteins was measured by [methyl-(3)H]thymidine incorporation into the cellular DNA. The assay was conducted in 96-well microtiter plates and had relatively high throughput. The Guidelines of the International Conference on Harmonization (ICH) were followed for the validation of the different assay parameters including robustness, linearity, accuracy, precision, limit of quantitation (LOQ) and specificity. The robustness of the bioassay is demonstrated by the lack of an effect of age of the 32D cell culture on the performance of the EP2 bioassay. The bioassay demonstrated good linearity, yielding a coefficient of determination of 0.99 or higher for both EP1 and EP2. The assay showed reproducible dose-response curves for EP1 in the range of 0.039-2.5 ng/mL and for EP2 in the range of 0.125-8 ng/mL. The accuracy estimates ranged between 98% and 108% for EP1 and between 90% and 110% for EP2 in the reproducible range mentioned above. Intermediate precision (within-plate R.S.D.) in the same range was within 26% and 17% for the EP1 and EP2 bioassays, respectively. The validated bioassays for EP1 and EP2 were utilized to quantitatively analyze serum samples from a pharmacokinetic study conducted to compare the profiles of the two compounds in Sprague-Dawley rats.