Antileishmanial effect of silver nanoparticles and their enhanced antiparasitic activity under ultraviolet light

Leishmaniasis is a protozoan vector-borne disease and is one of the biggest health problems of the world. Antileishmanial drugs have disadvantages such as toxicity and the recent development of resistance. One of the best-known mechanisms of the antibacterial effects of silver nanoparticles (Ag-NPs) is the production of reactive oxygen species to which Leishmania parasites are very sensitive. So far no information about the effects of Ag-NPs on Leishmania tropica parasites, the causative agent of leishmaniasis, exists in the literature. The aim of this study was to investigate the effects of Ag-NPs on biological parameters of L. tropica such as morphology, metabolic activity, proliferation, infectivity, and survival in host cells, in vitro. Consequently, parasite morphology and infectivity were impaired in comparison with the control. Also, enhanced effects of Ag-NPs were demonstrated on the morphology and infectivity of parasites under ultraviolet (UV) light. Ag-NPs demonstrated significant antileishmanial effects by inhibiting the proliferation and metabolic activity of promastigotes by 1.5- to threefold, respectively, in the dark, and 2- to 6.5-fold, respectively, under UV light. Of note, Ag-NPs inhibited the survival of amastigotes in host cells, and this effect was more significant in the presence of UV light. Thus, for the first time the antileishmanial effects of Ag-NPs on L. tropica parasites were demonstrated along with the enhanced antimicrobial activity of Ag-NPs under UV light. Determination of the antileishmanial effects of Ag-NPs is very important for the further development of new compounds containing nanoparticles in leishmaniasis treatment

The effect of the various freezing methods on the cell viability of cryopreserved HEp-2 and rabbit kidney cells

Bu çalışmada bovin serum albumininin, primer tavşan böbrek hücre kültürü ile HEp-2 devamlı hücre kültürlerinin kriyoprezervasyonunda hücre canlılığı üzerindeki rolü araştırıldı. Öncelikle hücrelerin yavaş, orta hızlı ve hızlı dondurma metodlarıyla kriyoprezervasyonu yapıldı. Kriyoprotektan madde olarak dimetil sülfoksit, gliserol ve Rowe karışımı kullanıldı. Hücreler %5 ve %10'luk kriyoprotektan madde ve 0, 5, 10, 20 ve 30g/mL bovin serum albumin içeren dondurma solüsyonları ile donduruldu. Deneylerde en yüksek hücre canlılığı yavaş dondurma metodunda elde edilirken, bu metotda dondurma solüsyonuna katılan bovin serum albuminin konsantrasyonuyla doğru orantılı olarak hem primer tavşan böbrek hem de HEp-2 devamlı hücrelerinin canlılığı üzerinde istatiksel olarak anlamlı bir artış meydana getirdiği tespit edildi (p0.05).In this study, the role of bovine serum albumin on the viability of cryo-preserved primary rabbit kidney cell- and HEp-2 continuous cell cultures was studied. Firstly, the cryopreservations of cells were done via slow, intermediate and rapid freezing methods. Dimethyl sulfoxide, glycerol and Rowe mix were used as cryoprotectant agents. The cells were frozen with the solutions containing 5% and 10% cryoprotectant agents and 0, 5, 10, 20, 30g/mL bovine serum albumin, separately. While the highest cell viability was obtained with slow freezing method, in this method, it has been determined that the concentration of bovine serum albumin in the freezing solution proportionally increased the cell viability both on primary rabbit kidney cells and HEp-2 continuous cells (0.05)

Investigation of the antiviral effect of vepesid on HSV type 2

Objective: Vepesid is a semisynthetic derivative of phodophylotoxin extracted from Phodophylum peltatum, which is in the group of plant alkaloids. In this study, the antiviral effect of Vepesid against herpes simplex virus type 2 (HSV-2) in in vitro conditions was investigated. Materials and Methods: In this investigation, a HEp-2 continuous cell line that was derived from human larynx cancer cells was used. The experiments were done in culture plates with smooth bases consisting of 96 wells. Cultivation of cells was realized in EMEM medium with 10% fetal bovine serum at an atmosphere of 37°C with 5% CO2. The toxicity of investigated agents and sensitivity of HEp-2 cells were evaluated according to the Reed and Muench method. The 50% effective dose (ED50) of the antiviral agent was expressed as the concentration that inhibits the cytopathological effect in half of the quadruplicate test cultures. Acyclovir was included as a positive control drug for HSV. The experiments were done in two stages. In the first stage, the concentration of Vepesid that did not affect proliferation was determined by means of morphological and biochemical parameters (pH, pCO2, pO2, Na+). In the second stage, the antiviral effect of that dose was examined on 100 TCID{50} of HSV-2. Results: The concentrations of Acyclovir and Vepesid (3.12 mg/ml, 6.28 mg/ml) were toxic and 1.56 mg/ml of both agents did not affect cell proliferation. These amounts of Acyclovir and Vepesid inhibited viral reproduction and had no cytopathological effect on cells. Na+ content of the culture medium was 138 ± 2.54 mEq/L for the control group, 126 ± 1.65 mEq/L for the virus control, 142 ± 0.3 for the Acyclovir, 142 ± 0.4 for the Acyclovir + virus, 143 ± 1.1 for Vepesid, 141 ± 0.2 for the virus + Vepesid. LD50 of Acyclovir and Vepesid was found to be 71% for a virus titer of 100 TCID50. Conclusion: We found that Vepesid prevented the viral replication of herpes simplex virus type 2. We think that the study of Vepesid as the treatment for patients with herpes simplex virus (HSV) is very important in terms of its clinical and epidemiological nature.Objective: Vepesid is a semisynthetic derivative of phodophylotoxin extracted from Phodophylum peltatum, which is in the group of plant alkaloids. In this study, the antiviral effect of Vepesid against herpes simplex virus type 2 (HSV-2) in in vitro conditions was investigated. Materials and Methods: In this investigation, a HEp-2 continuous cell line that was derived from human larynx cancer cells was used. The experiments were done in culture plates with smooth bases consisting of 96 wells. Cultivation of cells was realized in EMEM medium with 10% fetal bovine serum at an atmosphere of 37°C with 5% CO2. The toxicity of investigated agents and sensitivity of HEp-2 cells were evaluated according to the Reed and Muench method. The 50% effective dose (ED50) of the antiviral agent was expressed as the concentration that inhibits the cytopathological effect in half of the quadruplicate test cultures. Acyclovir was included as a positive control drug for HSV. The experiments were done in two stages. In the first stage, the concentration of Vepesid that did not affect proliferation was determined by means of morphological and biochemical parameters (pH, pCO2, pO2, Na+). In the second stage, the antiviral effect of that dose was examined on 100 TCID{50} of HSV-2. Results: The concentrations of Acyclovir and Vepesid (3.12 mg/ml, 6.28 mg/ml) were toxic and 1.56 mg/ml of both agents did not affect cell proliferation. These amounts of Acyclovir and Vepesid inhibited viral reproduction and had no cytopathological effect on cells. Na+ content of the culture medium was 138 ± 2.54 mEq/L for the control group, 126 ± 1.65 mEq/L for the virus control, 142 ± 0.3 for the Acyclovir, 142 ± 0.4 for the Acyclovir + virus, 143 ± 1.1 for Vepesid, 141 ± 0.2 for the virus + Vepesid. LD50 of Acyclovir and Vepesid was found to be 71% for a virus titer of 100 TCID50. Conclusion: We found that Vepesid prevented the viral replication of herpes simplex virus type 2. We think that the study of Vepesid as the treatment for patients with herpes simplex virus (HSV) is very important in terms of its clinical and epidemiological nature

Flow cytometric analysis of the effects of methotrexate and vepesid on the HEp-2 cell cycle

Objective: To determine the mechanism of action of Methotrexate and Vepesid on the HEp-2 cells isolated from human laryngeal cancer cells morphologically and flow cytometrically (G1, G2, S and PI). Materials and Methods: The HEp-2 continuous cell line was used. Cultivation of the cells was realized in EMEM medium with 10% fetal bovine serum at an atmosphere of 37şC with 5% $CO_2$. Six different concentrations of Vepesid and Methotrexate were prepared by diluting with deionized water (5 $\mu$g/ml, 50 mg/ml, 500 $\mu$g/ml). The morphological and cell cycle parameters of HEp-2 cells were determined by inverted microscope and flow cytometer respectively. Results: In the morphological examination, Vepesid was found to have a more significant cytopathologic effect on the cells than Methotrexate, whereas in the flow cytometric examination, it was found that whilst Methotrexate stopped the cells at the S and G2 phases, Vepesid did that only at the G1 phase. Conclusion: Both the flow cytometric and cell morphological analysis showed Vepesid to be more effective than Methotrexate on HEp-2 cells. Results of studies conducted show the mechanism of action of these drugs to be dependent on the origin of the cell and on the drug type.Objective: To determine the mechanism of action of Methotrexate and Vepesid on the HEp-2 cells isolated from human laryngeal cancer cells morphologically and flow cytometrically (G1, G2, S and PI). Materials and Methods: The HEp-2 continuous cell line was used. Cultivation of the cells was realized in EMEM medium with 10% fetal bovine serum at an atmosphere of 37şC with 5% $CO_2$. Six different concentrations of Vepesid and Methotrexate were prepared by diluting with deionized water (5 $\mu$g/ml, 50 mg/ml, 500 $\mu$g/ml). The morphological and cell cycle parameters of HEp-2 cells were determined by inverted microscope and flow cytometer respectively. Results: In the morphological examination, Vepesid was found to have a more significant cytopathologic effect on the cells than Methotrexate, whereas in the flow cytometric examination, it was found that whilst Methotrexate stopped the cells at the S and G2 phases, Vepesid did that only at the G1 phase. Conclusion: Both the flow cytometric and cell morphological analysis showed Vepesid to be more effective than Methotrexate on HEp-2 cells. Results of studies conducted show the mechanism of action of these drugs to be dependent on the origin of the cell and on the drug type

Utility of the microculture method for Leishmania detection in non-invasive samples obtained from a blood bank

In recent years, the role of donor blood has taken an important place in epidemiology of Leishmaniasis. According to the WHO, the numbers of patients considered as symptomatic are only 5-20% of individuals with asymptomatic leishmaniasis. In this study for detection of Leishmania infection in donor blood samples, 343 samples from the Capa Red Crescent Blood Center were obtained and primarily analyzed by microscopic and serological methods. Subsequently, the traditional culture (NNN), Immunochromatographic test (ICT) and Polymerase Chain Reaction (PCR) methods were applied to 21 samples which of them were found positive with at least one method. Buffy coat (BC) samples from 343 blood donors were analyzed: 15 (4.3%) were positive by a microculture method (MCM); and 4 (1.1%) by smear. The sera of these 343 samples included 9 (2.6%) determined positive by ELISA and 7 (2%) positive by IFAT. Thus, 21 of (6.1%) the 343 subjects studied by smear, MCM, IFAT and ELISA techniques were identified as positive for leishmaniasis at least one of the techniques and the sensitivity assessed. According to our data, the sensitivity of the methods are identified as MCM (71%), smear (19%), IFAT (33%), ELISA (42%), NNN (4%), PCR (14%) and IC T (4%). Thus, with this study for the first time, the sensitivity of a MCM was examined in blood donors by comparing MCM with the methods used in the diagnosis of leishmaniasis. As a result, MCM was found the most sensitive method for detection of Leishmania parasites in samples obtained from a blood bank. In addition, the presence of Leishmania parasites was detected in donor bloods in Istanbul, a non-endemic region of Turkey, and these results is a vital importance for the health of blood recipients. (C) 2013 Elsevier B.V. All rights reserved