A novel thermoresponsive nano carrier matrix of hyaluronic acid, methotrexate and chitosan to target the cluster of differentiation 44 receptors in tumors.

Major challenges in current cancer chemotherapy include drug resistance, low efficacy and non-selectivity, resulting in undesirable side effects. In this study, we demonstrate a solution to these challenges that involves a dual targeting approach for tumors that overexpress CD44 receptors. The approach employs a nano-formulation (tHAC-MTX nano assembly), fabricated from hyaluronic acid (HA), the natural ligand for CD44, conjugated with methotrexate (MTX) and complexed with the thermoresponsive polymer 6-O-carboxymethylchitosan (6-OCMC) graft poly(N-isopropylacrylamide) [6-OCMC-g-PNIPAAm]. The thermoresponsive component was designed to have a lower critical solution temperature of 39 °C (the temperature of tumor tissues). In-vitro drug release studies reveal faster release of the drug at the higher temperatures of the tumor tissue likely due to the conformation changes in the thermoresponsive component of the nano assembly. Drug release was also enhanced in the presence of hyaluronidase enzyme. Higher cellular uptake and greater cytotoxicity of the nanoparticles were demonstrated in cancer cells that overexpress CD44 receptors suggesting a receptor binding and cellular uptake mechanism. Such nano-assemblies which incorporate multiple targeting mechanisms have the potential to improve efficacy and decrease side effects of cancer chemotherapy.

Safety and Evaluation of the Immune Response of Coronavirus Nosode (BiosimCovex) in Healthy Volunteers: A Preliminary Study Extending the Homeopathic Pathogenetic Trial.

Objectives: Regulatory clinical Phase I studies are aimed at establishing the human safety of an active pharmaceutical agent to be later marketed as a drug. Since homeopathic medicines are prepared by a potentizing method using alcohol, past a certain dilution, their toxicity/infectivity is assumed to be unlikely. We aimed to develop a bridge study between homeopathic pathogenetic trials and clinical trials. The primary purpose was to evaluate the safety of a nosode, developed from clinical samples of a COVID-19 patient. The secondary objectives were to explore whether a nosode developed for a specific clinical purpose, such as use during an epidemic, may elicit laboratory signals worthy of further exploration. Methods: An open-label study was designed to evaluate the safety and immune response of the Coronavirus nosode BiosimCovex, given orally on three consecutive days to ten healthy volunteers. Clinical examinations, laboratory safety and immune parameters were established. Interferon-gamma, Interleukin-6, and CD 4 were measured. (CTRI registration number: CTRI/2020/05/025496). Results: No serious/fatal adverse events were reported. Laboratory tests to measure safety were unchanged. Three subjects showed elevated Interleukin-6 (IL-6) on day 17 in comparison to the baseline, and ten subjects showed elevated IL-6 on day 34. A significant difference between IL-6 observations, calculated by repeated measures ANOVA, was found to be highly significant. On day 60, the IL-6 values of nine subjects were found to return to normal. Corresponding CD4 cell elevation was observed on day 60, when compared to day 34. Conclusions: HPT may potentially extend into physiological changes with regards to immune response and should encourage future studies.

Preparation of Coronavirus nosodes sourced from a clinical sample of SARS-Cov-2 positive patient, inactivated strain, and spike glycoprotein

IntroductionNosodes, the homeopathicpreparationssourcedfrom biological materials including clinical samples, cultures of organisms, and diseased tissues have been in use against the source-specific infections as well as other diseases. The nosodes have demonstrated some efficacy in managing epidemics, such as influenza, dengue, and leptospirosis.This article presents the need and process of development ofnosodes from the SARS-CoV-2 to explore its prophylactic and therapeutic potentials against certain related viral diseases.Materials and methodsA clinical sample of SARS-Cov-2 positive patient,based on the cycle threshold (CT) value of the qRT-PCR, heat-inactivated SARS-CoV-2, and spike glycoprotein all were processed for making nosodesas per the method described in Homoeopathy Pharmacopoeia of India.Molecular tests, such as qRT-PCR and sterility tests were performed to establish the live organisms, RNA material, and the absence of contamination.ResultsThree variants of CoronavirusNosodewere developed using a clinical sample,heat-inactivatedSARS-CoV-2, and spike glycoprotein.In potencies 3c and above, no detectableSARS-CoV-2 RNA material was found by PCR.The analytical results for nosodes were reported as compliant for sterility testing as per the IP.ConclusionThree variants of Coronavirus nosodes were preparedwhich need to be evaluated further through pre-clinical and clinical studies.(AU)

Biosynthesis of silver nanoparticles by Pseudomonas spp. isolated from effluent of an electroplating industry.

The aim of this study was to isolate and screen bacteria from soil and effluent of electroplating industries for the synthesis of silver nanoparticles and characterize the potential isolate. Soil and effluent of electroplating industries from Mumbai were screened for bacteria capable of synthesizing silver nanoparticles. From two soils and eight effluent samples 20 bacterial isolates were obtained, of these, one was found to synthesize silver nanoparticles. Synthesis of silver nanoparticle by bacteria was confirmed by undertaking characterization studies of nanoparticles that involved spectroscopy and electron microscopic techniques. The potential bacteria was found to be Gram-negative short rods with its biochemical test indicating Pseudomonas spp. Molecular characterization of the isolate by 16S r DNA sequencing was carried out which confirmed its relation to Pseudomonas hibiscicola ATCC 19867. Stable nanoparticles synthesized were 50 nm in size and variable shapes as seen in SEM micrographs. The XRD and FTIR confirmed the crystalline structure of nanoparticles and presence of biomolecules mainly proteins as agents for reduction and capping of nanoparticles. The study demonstrates synthesis of nanoparticles by bacteria from effluent of electroplating industry. This can be used for large scale synthesis of nanoparticles by cost effective and environmentally benign mode of synthesis.

Evaluation of HiaureusTM Coagulase Confirmation Kit in Identification of Staphylococcus aureus.

INTRODUCTION: Staphylococcus aureus is a facultative anaerobic Gram positive coccal bacterium whose incidence ranges to different infections. It is a cause of various uncomplicated skin infections, abscesses, septicaemia/bacteraemia, gastroenteritis, endocarditis, toxic shock syndrome and food intoxications. Various methods with varied time, sensitivities, specificities and costs are available, but may not be used as a reliable test for the identification and differentiation of S. aureus. Therefore, there is a need to evaluate newer tests. AIM: To compare the conventional tests with a commercial available kit for reliable, cost effective identification and confirmation of S. aureus. MATERIALS AND METHODS: The current prospective study was conducted in the Department of Clinical Pathology, Haffkine Institute for a period of six months. A total of 341 clinical isolates of staphylococci isolated from pus, urine, blood culture and sterile body fluids were subjected to conventional tests like Tube Coagulase Test (TCT) using Rabbit Plasma (RP) and Human Plasma (HP), culture media such as Mannitol Salt Agar (MSA) and Deoxyribonuclease (DNase) media in parallel to HiaureusTM Coagulase Confirmation Kit (HACCK), a commercially available kit for identification of S. aureus. Amplification of the femA gene was used as a comparative reference point test to calculate the sensitivity, specificity and concordance values of the conventional tests. RESULTS: Amongst the coagulase based tests, HACCK was 100% sensitive and specific. The TCT using RP was 98.58% sensitive while TCT using HP was less sensitive (95.37%). A total of 100% specificity was observed for TCT using RP while TCT using HP was 96.68% specific. The MSA and DNase media were 97.86% vs 96.44% and 96.67% vs 91.67% sensitive and specific respectively. The combination tests had varying sensitivity and specificity ranges. The HACCK demonstrated 100% concordance with femA amplification and was labelled as an ideal perfect test (κ=1) with MSA as an alternative test for S. aureus identification. CONCLUSION: The HACCK can be used as an exclusive, reliable and cost effective test for identification of S. aureus. Alternatively, in view of the cost factor MSA either as a single test or in combination with TCT using HP could be used as screening tests and confirm discordant results with HACCK.

Neutralizing Antibody Response and Efficacy of Novel Recombinant Tetravalent Dengue DNA Vaccine Comprising Envelope Domain III in Mice.

BACKGROUND: Dengue is a global arboviral threat to humans; causing 390 million infections per year. The availability of safe and effective tetravalent dengue vaccine is a global requirement to prevent epidemics, morbidity, and mortality associated with it. METHODS: Five experimental groups (6 mice per group) each of 5-week-old BALB/c mice were immunized with vaccine and placebo (empty plasmid) (100 µg, i.m.) on days 0, 14 and 28. Among these, four groups (one group per serotype) of each were subsequently challenged 3 weeks after the last boost with dengue virus (DENV) serotypes 1-4 (100 LD50, 20 µl intracerebrally) to determine vaccine efficacy. The fifth group of each was used as a control. The PBS immunized group was used as mock control. Serum samples were collected before and after subsequent immunizations. EDIII fusion protein expression was determined by Western blot. Total protein concentration was measured by Bradford assay. Neutralizing antibodies were assessed by TCID50-CPE inhibition assay. Statistical analysis was performed using Stata/IC 10.1 software for Windows. One-way repeated measures ANOVA and Mann-Whitney test were used for neutralizing antibody analysis and vaccine efficacy, respectively. RESULTS: The recombinant EDIII fusion protein was expressed adequately in transfected 293T cells. Total protein concentration was almost 3 times more than the control. Vaccine candidate induced neutralizing antibodies against all four DENV serotypes with a notable increase after subsequent boosters. Vaccine efficacy was 83.3% (DENV-1, -3, -4) and 50% (DENV-2). CONCLUSION: Our results suggest that vaccine is immunogenic and protective; however, further studies are required to improve the immunogenicity particularly against DENV-2.

Preparation, standardization and in vitro safety testing of Mycobacterium nosodes (Emtact- polyvalent nosode).

BACKGROUND: Most of the nosodes in the homeopathic pharmacopeia have been sourced from obscure pathological material over a century ago; of which no scientific documentation is available. METHOD: A method for preparation and standardization of univalent and polyvalent Mycobacterium nosodes (labeled as Emtact), using different strains of Mycobacterium tuberculosis was developed. The committee comprising microbiologists, scientist, pharmacist, homeopaths and clinicians had reviewed and approved the method of preparation of nosode. Preparation of the nosode was based on the reference in the Homeopathy Pharmacopoeia of India (HPI), group N-IV. Strains of M. tuberculosis viz. Standard strain H37Rv, multi-drug resistant (MDR) M. tuberculosis, Mycobacterium bovis (BCG vaccine) and Mycobacterium avium were identified, procured and documented. Twenty billion viable cells for each strain were taken for Original Stock Nosode (OSN). The original stock was prepared by suspending the microbial cells into water for injection (WFI) (1 ml). As per the Indian Pharmacopoeia (IP) monograph, sterility testing was done for different potencies. Polymerase Chain Reaction (PCR) was performed for 30c potency for detection of any DNA material of the source organisms. RESULT: A polyvalent (multi-strain) and univalent M. tuberculosis nosodes were prepared for research and clinical use. No growth of Mycobacterium was observed in any of the samples above 5c potency. The in-vitro testing for nosode (30c) was found to be free from any organism and DNA material. CONCLUSION: Mycobacterium nosodes sourced from individual strain and polyvalent Emtact nosode in vitro testing results found to be satisfactory for its handling and utilization. The nosode seems to be safe and may be tested further in vivo to explore its therapeutic application.