Studies on the sequential pathology of Kyasanur Forest Disease (KFD) in Mouse brain: KFD virus induces apoptosis of neurons in cerebrum and hippocampus.

The sequential pathology of Kyasanur forest disease (KFD) in mouse brain was assessed in this study. Kyasanur forest disease virus (KFDV) strain P9605 used in this study was confirmed by real-time reverse transcriptase-polymerase chain reaction targeting the NS5 gene. Mouse Lethal Dose 50 (MLD50) of the virus was determined by in-vivo mice inoculation test. One MLD50 of the KFDV was inoculated intra-cerebrally into 36 mice aged 2-3 weeks. Another group of 36 age-matched mice that served as control group were inoculated with plain media. Six mice each from infected and control groups were euthanized every 24 hrs intervals for six days. Brain tissues were collected in 10% NBF. The collected brain tissues were processed and subjected to histopathological studies by Hematoxylin and Eosin staining. Grossly, the infected mice showed symptoms of dullness, hunched back appearance, weakness, sluggish movements with indication of hind quarter paralysis on day four post-infection. These symptoms got aggravated with complete paralysis of the hind quarters, inability to move and death on 5th and 6th day post-infection. Microscopically, the brain showed apoptosis of neurons, perivascular cuffing, gliosis, congestion, neuropil vacuolation, meningitis, degeneration, and necrotic neurons. The real-time RT-PCR on hippocampus of the KFDV-infected mouse brain showed three-fold higher expression levels of Caspase 3, a crucial mediator of apoptosis. The cerebral cortex, cerebellum and hippocampus that control the motor neuron activities and muscle tone were primarily affected, possibly correlating with the gross symptoms of hind quarter paralysis, ataxia, and other motor neuron dysfunctions noticed. Taken together, these findings reveal that KFDV induces apoptosis of neurons in the cerebrum and hippocampus of KFDV infected mice. Further studies are needed to confirm if the lesions noticed in mice brain simulate the brain lesions in humans since gross motor-neuron symptoms are similar in mice as well as humans.

Evaluation of Safety and Potency of Kyasanur Forest Disease (KFD) Vaccine Inactivated with Different Concentrations of Formalin and Comparative Evaluation of In Vitro and In Vivo Methods of Virus Titration in KFD Vaccine.

We evaluated the safety and potency of the Kyasanur Forest disease (KFD) vaccine inactivated with different formalin concentrations in mice, since the side effects due to higher formalin concentrations have been a major reason for vaccine refusal. Furthermore, with an objective to reduce the use of mice in vaccine testing, we performed quantification of the KFD virus by real-time PCR and compared it with in vivo titration in mice. The KFD vaccine prepared in chicken embryo fibroblast cells was inactivated with 0.04%, 0.06%, and 0.08% concentrations of formalin. The vaccine inactivated with 0.04% and 0.06% formalin failed the safety test, whereas the KFD vaccine inactivated with 0.08% formalin was safe and potent with a log protective index of 5678 in mice. This reduced formalin content may induce no/lesser side-effects of pain/swelling which may increase the vaccine acceptance. The real-time PCR on individual KFD vaccine harvests interpreted that when the CT value of each harvest is <20, the vaccine will have sufficient viral particles to pass the potency test. Comparison of the real-time PCR on tenfold dilutions of the pooled harvests with in vivo mice inoculation test revealed that the 1MLD50 of the vaccine lies in the tenfold dilution that yields CT values between 31 and 34.

Comparative Evaluation of Intradermal vis-à-vis Intramuscular Pre-Exposure Prophylactic Vaccination against Rabies in Cattle.

Rabies is a progressively fatal viral disease affecting a wide variety of warm-blooded animals and human beings. With cattle being major part of Indian livestock population, rabies can result in significant financial losses. Immunization of livestock vulnerable to exposure is the best way to control rabies. The present study was undertaken to investigate the efficacy of a rabies pre-exposure prophylactic vaccine administered through different routes and to sequentially monitor the levels of rabies virus-neutralizing antibody (RVNA) titers in cattle. Thirty cattle were divided into five groups of six animals each. Group I and III animals were immunized with 1 mL and 0.2 mL of rabies vaccine through intramuscular (IM) and intradermal (ID) routes, respectively, on day 0, with a booster dose on day 21; Group II and IV animals were immunized with 1 mL and 0.2 mL of rabies vaccine, respectively, without the booster dose; unvaccinated animals served as a control (Group V). Serum samples were collected on days 0, 14, 28, and 90 to estimate RVNA titers using the rapid fluorescent focus inhibition test (RFFIT). The titers were above an adequate level (≥0.5 IU/mL) on day 14 and maintained up to 90 days in all animals administered the rabies vaccine through the IM and ID route with or without a booster dose. The study indicated that both routes of vaccination are safe and effective in providing protection against rabies. Hence, both routes can be considered for pre-exposure prophylaxis. However, the ID route proved to be more economical due to its dose-sparing effect.

Chitosan Encapsulated Meloxicam Nanoparticles for Sustained Drug Delivery Applications: Preparation, Characterization, and Pharmacokinetics in Wistar Rats.

Meloxicam (MLX) is currently used in the therapeutic management of both acute and chronic inflammatory disorders such as pain, injuries, osteoarthritis, and rheumatoid arthritis in both humans and animals. Gastrointestinal toxicity and occasional renal toxicity were observed in patients taking it for a long-term period. Meloxicam's late attainment of peak plasma concentration results in a slow onset of action. The goal of the current study was to prepare and characterize chitosan encapsulated meloxicam nanoparticles (CEMNPs) with high bioavailability and less gastro intestinal toxicity in order to prevent such issues. The size of the prepared CEMNPs was approximately 110-220 nm with a zetapotential of +39.9 mV and polydispersity index of 0.268, suggesting that they were uniformly dispersed nanoparticles. The FTIR and UV-Vis spectroscopy have confirmed the presence of MLX in the prepared CEMNPs. The pharmacokinetics have been studied with three groups of male Wistar rats receiving either of the treatments, viz., 4 mg·kg-1 of MLX and 1 or 4 mg·kg-1 of CEMNPs. Plasma samples were collected until 48 h post administration, and concentrations of MLX were quantified by using reverse (C18) phase HPLC. Non-compartmental analysis was applied to determine pharmacokinetic variables. Upon oral administration, the maximum concentration (Cmax) was reached in 4 h for CEMNPs and 6 h for MLX. The mean area under the plasma MLX concentration-time curve from 'zero' to infinity (AUC0-∞), half-life (t1/2ß), and mean resident time (MRT) of 1 mg·kg-1 of CEMNPs was 1.4-, 2-, and 1.8-fold greater than 4 mg·kg-1 of MLX. The prepared CEMNPs demonstrated quicker absorption and prolonged release along with a significant improvement in the bioavailability of MLX, paving a prospective path for the development of drugs with enhanced bioavailability with less side effects.

Effect of Telmisartan on Arsenic-Induced (Sub-chronic) Perturbations in Redox Homeostasis, Pro-inflammatory Cascade and Aortic Dysfunction in Wistar Rats.

An experimental study was conducted in male Wistar rats to explore the antioxidant potential of telmisartan (an AT1 receptor blocker) to overcome arsenic ('As')-induced perturbations in redox homeostasis pro-inflammatory cytokines, prostaglandin-E2 levels and aortic dysfunction in Wistar rats. Wistar rats were randomly divided into four groups of six each. Group-I served as untreated control, while group-II received sodium (meta) arsenite (NaAsO2) (10 mg/kg b.wt. p.o) for a period of 60 days. Experimental rats in group-III received treatment similar to group-II, but in addition received telmisartan (with 1% aqueous solution of Tween 80) @ 10 mg/kg b.wt. (p.o) for a similar duration, while rats in group-IV received telmisartan alone. Arsenic exposure resulted in significant (p < 0.05) elevation in the levels of superoxide anion ([Formula: see text]) radicals (control: 768.20 ± 126.77 vs group-II: 1232.75 ± 97.85 pmol of NBT reduced/min/mg protein). Telmisartan administration showed significant (p < 0.05) reduction in [Formula: see text] generation (815.34 ± 43.41 pmol of NBT reduced/min/mg protein). Sub-chronic exposure to 'As' significantly (p < 0.05) decreased the activities of SOD, CAT, GPx and GR activity and GSH levels in the aorta, thus induced lipid peroxidation (LPO) measured as measured in terms of thiobarbituric acid reactive substances (TBARS) called as malondialdehyde (MDA). However, the administration of telmisartan effectively countered the LPO (24.03 ± 1.18 nmol of MDA/g) on account of restoring the levels of aforesaid antioxidant defense system. Telmisartan administration effectively attenuated the 'As'-induced surge in pro-inflammatory cytokines (viz., IL-1ß, IL-6 and TNF-α) levels, as well as countered the activity of cyclooxygenase (COX2) as indicated by a significant (p < 0.05) decrease in PGE2 level in the aorta. In addition to it, there was a significant (p < 0.05) decrease in plasma angiotensin II (Ang-II) levels in experimental rats receiving telmisartan. Quantitative RT-PCR studies revealed that sub-chronic exposure to 'As' upregulated the Nox2 mRNA expression, but there was a 1.2-fold reduction in expression level upon co-administration of telmisartan. Histopathological examination revealed marked recovery from 'As'-induced disruption of tunica adventitia and loss of connective tissue in experimental rats receiving telmisartan. The study concludes that telmisartan can overcome aortic dysfunction induced by sub-chronic exposure to arsenic through drinking water in experimental rats through restoration of redox balance, attenuation of pro-inflammatory cytokines and mediators and downregulation of Nox2 mRNA expression.

Anticancer efficacy of 6-thioguanine loaded chitosan nanoparticles with or without curcumin.

6-Thioguanine encapsulated chitosan nanoparticles (6-TG-CNPs) has formulated by the ionic-gelation method. Morphologically, the 6-TG-CNPs were spherical and showed mean size, PDI, zeta potential, and entrapment efficiency of 261.63 ± 6.01 nm, 0.34 ± 0.10, +15.97 ± 0.46 mV and 44.27%, respectively. The IR spectra confirmed the 6-TG complex with chitosan. The in vitro drug release profile of 6-TG-CNPs revealed an increase in sustained-release (91.40 ± 1.08% at 48 h) at pH 4.8 compared to less sustained-release (73.96 ± 1.12% at 48 h) at pH 7.4. The MTT assay was conducted on MCF-7 and PA-1 cell lines at 48 h incubation to determine % cell viability. The IC50 values of 6-TG, 6-TG-CNPs, and curcumin for MCF-7 were 23.09, 17.82, and 15.73 µM, respectively. Likewise, IC50 values of 6-TG, 6-TG-CNPs, and curcumin for PA-1 were 5.81, 3.92, and 12.89 µM, respectively. A combination of 6-TG-CNPs (IC25) with curcumin (IC25) on PA-1 and MCF-7 showed % cell viability of 43.67 ± 0.02 and 49.77 ± 0.05, respectively. The in vitro cytotoxicity potential in terms of % cell viability, early apoptosis, G2/M phase arrest, and DNA demethylating activity of 6-TG-CNPs alone and combination with curcumin proved to be more effective than that of 6-TG on PA-1 cells.

Antiproliferative and tumor inhibitory studies of 2,3 disubstituted 4-thiazolidinone derivatives.

4-Thiazolidinone derivatives were synthesized using T3P®-DMSO media as a cyclodehydrating agent. All the molecules were tested for their cytotoxicity against leukemic cell lines. The compound 3-(4-bromophenyl)-2-(4-(dimethylamino)phenyl)thiazolidin-4-one (4e) with electron donating substituent at para position of phenyl ring displayed considerable cytotoxicity against Reh and Nalm6 cells with an IC50 value of 11.9 and 13.5 µM, respectively. Furthermore, the compound 4e tested for tumor regression studies induced by EAC in Swiss albino mouse. Both in vitro and in vivo results suggested significant antiproliferative activity of compound 4e in Reh cells and mouse tumor tissue treated with compound 4e showed multifocal areas of necrosis and numerous number of apoptotic cells.

Amelioration of hepatotoxicity induced by aflatoxin using citrus fruit oil in broilers (Gallus domesticus).

This study was conducted to evaluate the efficacy of citrus fruit oil (CFO; 2.5 g kg(-1)) on the clinicopathological changes in broilers fed with diets containing 1 ppm of aflatoxin (AF). A total of 160 Ross 308 broiler chicks of 1-day-old were procured from a commercial hatchery, divided randomly on 7th day of age into four groups with two replicates of 20 birds each and fed with basal diet (group A), basal diet + CFO (group B), basal diet + AF (group C) and CFO + basal diet + AF (group D). The gross and histopathological changes in the liver, kidney, spleen, thymus and bursa of Fabricius were investigated and relative organ weights were calculated. Slight to moderate hydropic degeneration, fatty change with the formation of cyst in some cases, periportal necrosis, infiltration of heterophils and mononuclear cells and bile duct hyperplasia were observed in chicks fed with 1 ppm AF-containing diet. The addition of CFO to AF-containing diet moderately decreased the magnitude and severity of lesions (hydropic degeneration and bile duct hyperplasia) in the liver. The supplementation of CFO to the basal diet did not produce any adverse effects in birds.

Evaluation of immune response and resistance to diseases in tiger shrimp, Penaeus monodon fed with biofilm of Vibrio alginolyticus.

Immune response in juvenile tiger shrimp, Penaeus monodon fed with biofilm (BF) and free cells (FC) of Vibrio alginolyticus was studied by evaluating the hemocyte count, phenoloxidase activity and antibacterial activity. The above immune responses were higher in BF fed shrimp than that in FC fed or control shrimp. Among the different doses of BF of V. alginolyticus tested, 10(9) cfu g(-1) shrimp day(-1) for two weeks could evoke higher immune response. BF fed shrimp were more resistant to injection challenge with V. alginolyticus and whitespot syndrome virus (WSSV) with significantly higher RPS compared to that with FC fed and control shrimp. Better resistance was also reflected by rapid clearance of V. alginolyticus and WSSV from the hemolymph as confirmed by immunodot and histopathology.