Evaluation of two commercially available ELISAs for the diagnosis of Japanese encephalitis applied to field samples

Objective To compare two commercially available kits, Japanese Encephalitis‐Dengue IgM Combo ELISA (Panbio Diagnostics) and JEV‐CheX IgM capture ELISA (XCyton Diagnostics Limited), to a reference standard (Universiti Malaysia Sarawak – Venture Technologies VT ELISA). Methods Samples were obtained from 172/192 children presenting to a site in rural India with acute encephalitis syndrome. Results Using the reference VT ELISA, infection with Japanese encephalitis virus (JEV) was confirmed in 44 (26%) patients, with central nervous system infection confirmed in 27 of these; seven patients were dengue seropositive. Of the 121 remaining patients, 37 (31%) were JEV negative and 84 (69%) were JEV unknown because timing of the last sample tested was <10 day of illness or unknown. For patient classification with XCyton, using cerebrospinal fluid alone (the recommended sample), sensitivity was 77.8% (59.2–89.4) with specificity of 97.3% (90.6–99.2). For Panbio ELISA, using serum alone (the recommended sample), sensitivity was 72.5% (57.2–83.9) with specificity of 97.5% (92.8–99.1). Using all available samples for patient classification, sensitivity and specificity were 63.6% (95% CI: 48.9–76.2) and 98.4% (94.5–99.6), respectively, for XCyton ELISA and 75.0% (59.3–85.4) and 97.7% (93.3–99.2) for Panbio ELISA. Conclusion The two commercially available ELISAs had reasonable sensitivities and excellent specificities for diagnosing JEV.     

Mechanism of DNA damage by cadmium and interplay of antioxidant enzymes and agents

Cadmium is an environmental toxicant, which causes cancer in different organs. It was found that it damages DNA in the various tissues and cultured cell lines. To investigate the mechanism of DNA damage, we have studied the effect of cadmium-induced DNA damage in plasmid pBR322 DNA, and the possible ameliorative effects of antioxidative agents under in vitro conditions. It was observed that cadmium alone did not cause DNA damage. However, it caused DNA damage in the presence of hydrogen peroxide, in a dose dependent manner, because of production of hydroxyl radicals. Findings from this study show the conversion of covalently closed circular double-stranded pBR 322 DNA to the open circular and linear forms of DNA when treated with 10 muM cadmium and various concentrations of H(2)O(2). The conversion was due to nicking in DNA strands. The observed rate of DNA strand breakage was dependent on H(2)O(2) concentration, temperature, and time. Metallothionein I failed to prevent cadmium-induced DNA nicking in the presence of H(2)O(2). Of the two antioxidant enzymes (catalase and superoxide dismutase) studied, only catalase conferred significant (50-60%) protection. EDTA and DMSO exhibited protection similar to catalase, while mannitol showed only about 20% protection against DNA damage. Ethyl alcohol failed to ameliorate cadmium-induced DNA strands break. From this study, it is plausible to infer that cadmium in the presence of hydrogen peroxide causes DNA damage probably by the formation of hydroxyl ions. These results may indicate that cadmium in vivo could play a major role in the DNA damage induced by oxidative stress.     

Estradiol fatty acid esters in adipose tissue and serum of pregnant and pre- and postmenopausal women

CONTEXT: The 17beta-estradiol fatty acid esters are hormone derivatives with long-lasting estrogenic effect. They are transported in serum lipoproteins and thought to be sequestered in adipose tissue. OBJECTIVE: Our objective was to determine the 17beta-estradiol fatty acid ester concentrations in serum and adipose tissue in women of various hormonal states. DESIGN: After several chromatographic steps separating esterified from free estradiol, time-resolved fluoroimmunoassay was used as a quantifying tool. PARTICIPANTS: Samples were obtained from pregnant women undergoing cesarean section (n = 13), or premenopausal (n = 8) and postmenopausal women (n = 6) during gynecological surgery. MAIN OUTCOME MEASURES: 17beta-Estradiol and 17beta-estradiol fatty acid ester concentrations in serum, and visceral and sc adipose tissue were examined. RESULTS: The ratio of esterified to free estradiol in plasma increased with decreasing estradiol level from 0.5% in pregnant, to 15% in premenopausal and 110% in postmenopausal women. Estradiol esters constituted about 10% of the free estradiol present in adipose tissue in pregnancy. In nonpregnant women, most of the adipose tissue estradiol was in esterified form, the median ester to free ratio being elevated to 150-490%. After menopause, the overwhelming majority of estradiol in both free and esterified form was present in adipose tissue. CONCLUSIONS: The overall higher ester to free estradiol ratio in adipose tissue than in serum indicates active esterification capacity in adipose tissue. The predominance of esterified and free estradiol in postmenopausal adipose tissue compared with serum suggests in situ production and storage. Whether the estradiol esters have an independent physiological role in adipose tissue remains to be clarified.     

HIV epidemiology in the Northwestern Federal District of Russia: dominance of HIV type 1 subtype A

A rapidly advancing epidemic of HIV-1 infection has been documented among injecting drug users (IDUs) in Russia. The Northwestern Federal District was the first of the seven Russian Federal Districts involved in a drug-related HIV epidemic through an outbreak in Kaliningrad in 1996. The Northwestern Federal District has a high HIV prevalence rate having reached 252 per 100,000 by the end of 2003, exceeding the Russian average (180) by 1.4 times. The epidemic peaked in 2001. Since then the annual number of new cases has decreased, probably reflecting saturation among at least some IDU populations. However, at the same time, the heterosexual spread of HIV has become more prominent. To study the genetic epidemiology of HIV-1, samples were collected from 150 individuals covering a wide geographical area and different transmission groups in the Northwestern Federal District. Phylogenetic analysis revealed that an Eastern European subtype A HIV-1 strain similar to those reported earlier among IDUs in other regions of Russia accounted for 80% of HIV-1 infections and was the predominant subtype in six out of seven administrative territories studied both among IDUs and heterosexually infected persons. As an exception to the dominant role of the Eastern European subtype A strain, the CRF03-AB strain was found to be dominant in the city of Cherepovets located in the north central European Russian territory of Vologda Oblast. This is the first report of the CRF03-AB strain causing an outbreak outside the Kaliningrad region.     

Fabrication and in vivo evaluation of Nelfinavir loaded PLGA nanoparticles for enhancing oral bioavailability and therapeutic effect

Nelfinavir mesylate (NFV) is an anti-viral drug, used in the treatment of Acquired Immunodeficiency Syndrome (AIDS). Poor oral bioavailability and shorter half-life (3.5–5 h) remain a major clinical limitation of NFV leading to unpredictable drug bioavailability and frequent dosing. In this context, the objective of the present study was to formulate NFV loaded poly (lactic-co-glycolic acid) (PLGA) nanoparticles (NPs), which can increase the solubility and oral bioavailability along with sustained release of the drug. NFV loaded PLGA-NPs were prepared by nanoprecipitation method using PLGA and Poloxomer 407. The prepared NPs were evaluated for particle size, zeta potential, morphology, drug content, entrapment efficiency (EE) and in vitro dissolution studies. Oral bioavailability studies were carried out in New Zealand rabbits by administering developed NFV PLGA-NPs and pure drug suspension. PLGA-NPs prepared by using 1:4 ratio of drug and PLGA, with a stirring rate of 1500 rpm for 4 h. The prepared NPs were in the size of 185 ± 0.83 nm with a zeta potential of 28.7 ± 0.09 mV. The developed NPs were found to be spherical with uniform size distribution. The drug content and EE of the optimized formulation were found to be 36 ± 0.19% and 72 ± 0.47% respectively. After oral administration of NFV PLGA-NPs, the relative bioavailability was enhanced about 4.94 fold compared to NFV suspension as a control. The results describe an effective strategy for oral delivery of NFV loaded PLGA NPs that helps in enhancing bioavailability and reduce the frequency of dosing.     

Chemoprotective effect of monoisoamyl 2, 3‐dimercaptosuccinate (MiADMS) on cytokines expression in cadmium chloride treated human lung cells

Cadmium is commercially profitable element, but it causes toxicity in humans and animals leading to diseases in various organs. The main route of cadmium exposure to humans is through inhalation. Lungs respond to insult through secretion of cytokines. In this study, the chemoprotective effect of monoisoamyl 2, 3‐dimercaptosuccinate (MiADMS) was evaluated on viability and cytokines expression in CdCl₂ treated human lung A549 cells by cytokine array. Cells were treated with 0, 50, 75, and 100 µM CdCl₂ alone, 300 µM MiADMS alone, and co‐treated with 300 µM MiADMS and 75 µM CdCl₂ for 24 h. The viability was measured by crystal violet dye. The level of cytokines in the cells' lysate and cell culture medium was measured using Ray Biotech's Human Cytokine Array 6 in control cells, 75 µM CdCl₂ alone and MiADMS co‐treated cells. Array results were validated by ELISA kit. The CdCl₂ caused a dose dependent decrease in cell viability, while MiADMS co‐treatment resulted in a significant increase in viability of CdCl₂ treated cells. Morphology of the cells treated with CdCl₂ was destroyed, while MiADMS restored the lost shape in CdCl₂ treated cells. In addition, the cells co‐treated with MiADMS and CdCl₂ showed modulation of cytokines expression in comparison to the CdCl₂ alone treated cells. The ELISA results showed the similar pattern of cytokine expression as Human Cytokine Array and validated the array results. These results clearly show the chemoprotective effect of MiADMS and suggest that MiADMS can be used as antidote at moderate dose against CdCl₂ toxicity. © 2014 Wiley Periodicals, Inc. Environ Toxicol 30: 704–711, 2015.     

PEG-SO(3)H catalyzed synthesis and cytotoxicity of α-aminophosphonates

One pot three-component PEG-SO(3)H catalyzed reaction of 4-(Pyridin-4-yl)benzaldehyde and triethyl phosphite with various primary amines afforded α-aminophosphonates with high yields by the Kabachnik-Field's reaction. These new structurally diversified set of α-aminophosphonates (4a-j) were evaluated for their anti-tumor activity on human chronic myeloid leukemia cells (K 562), human colon carcinoma cells (Colo 205) along with non-cancerous human embryonic kidney cells (HEK 293). They showed moderate activity on both cancerous cells and non-cancerous cells.