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Nanosilver in the treatment of localized cutaneous leishmaniasis caused by Leishmania major (MRHO/IR/75/ER): an in vitro and in vivo study
M Mohebali,M.M Rezayat,K Gilani,S Sarkar
DARU : Journal of Pharmaceutical Sciences , 2009,
Abstract: "n "n "n "n "nBackground and the purpose of the study: "n This study was designed to evaluate the effectiveness of different concentrations of Nanosilver solution against Iranian strain of Leishmania major (MRHO/IR/75/ER) both in vitro and in vivo "n "non BALB/c mice model for the first time. "nMethods: "n this is an interventional study which was conducted on the infected macrophages by L. major amastigotes in vitro. In order to confirm the in vitro results, various concentration of Nanosilver solution were administered topically on skin lesions caused by L.major "n "nin 78 inbred BALB/c mice as test or interventional and 52 mice as control groups once or twice daily for 14 days. Results and major conclusion : Results of this study showed that different concentration of Nanosilver reduced proliferation of L. major amastigotes compared with the control wells but the differences were not statistically significant. Also, different concentrations of Nanosilver did not decrease the lesion sizes and amastigote counts in the BALB/c mice significantly. Secondary infection was significantly decreased in Nanosilver- treated groups compared with control groups (p<0.01). In conclusion, Nanosilver seems to be effective for control of secondary infection of localized cutaneous leishmaniasis.
Cloning and Expression of TRYP6 Gene from Leishmania major (MRHO/IR/75/ER)
G Eslami,R Salehi,H Hejazi,A Khamesipour
Iranian Journal of Arthropod-Borne Diseases , 2008,
Abstract: Background: Leishmania, needs to detoxify the macrophage derived potent peroxides (H2O2). Tryparedoxin path-way contains tryparedoxin peroxidase (TSA or TRYP). The aim of the study was to detect the full-length gene se-quence and its encoded protein of the LmTRYP6 gene (EU251502), and comparison the gene sequence with LmTRYP6 (LmjF15.1140), another previously reported member of this gene family. Methods: L.major (MRHO/IR/75/ER) promastigotes were cultured, DNA and RNA were extracted and the inter-ested gene was amplified using PCR and RT-PCR methods. PCR/ RT-PCR fragments were purified and cloned first in pTZ57R/T and then in pET15b expression vector. The expressed protein was verified using western blot method. Char-acterization of the expressed protein was performed bioinformatically. Results: Molecular evaluation revealed that the cloned LmTRYP6 gene (EU251502) encoded a predicted 184 amino acid long protein with a theoretical isoelectric point of 6.1101. Alignment showed a number of changes in amino acid composition including the replacement of highly conserved Trp177 by Cys in LmTRYP6 (ABX26130). Conclusion: So far no study has been done on this group, i.e. TRYP6 gene, from tryparedoxin peroxidase family. The low homology with LmTRYP6 (LmjF15.1140) and vast array of differences observed in the gene under study (LmTRYP6; EU251502) could open new windows in the field of anti-Leishmania combat. Based on its important role in the viability and successful establishment of the parasite in the host organism it looks to be very good candi-date for vaccine development and any other sort of novel drug development.
Pathogenicity Variations of Susceptibility and Resistance to Leishmania major MRHO/IR/75/ER Strain in BALB/c and C57BL/6 mice
Marzieh Amini,Hossein Nahrevanian,Mahin Farahmand
Iranian Journal of Parasitology , 2008,
Abstract: Background: To compare the pathogenicity differences in two susceptible Balb/c and resistant C57bl/6 mice infected with Leishmania major MRHO/IR/75/ER as a prevalent strain of zoonotic cutaneous leishmaniasis in Iran. Methods: Mice were assigned into four groups as control and infected BALB/c and C57BL/6 mice. Experimental leishma-niasis was initiated by (s. c) injection of the 2×106 L. major promastigotes into the basal tail of infected groups. The devel-opment of lesions was determined weekly by measuring the two diameters. After 10 weeks, all mice were killed humanly, target tissues including lymph node, spleen and liver from each mouse were removed, weighted, and their impression smears were prepared. Results: Proliferation of amastigotes inside macrophages, pathogenicity signs in two susceptible, resistant hosts was varied, and these variations were depended on mice strain. Conclusion: Host immunity may modify clinical signs and could affect the proliferation of amastigotes inside macro-phages, the size of lesions, the survival rates, the degree of hepatomegaly and splenomegaly and the percentage of amasti-gotes in lesion, liver, spleen, lymph node and brain smears.
INOS and IFN? Gene Expression in Leishmania major-Infected J774 Cells Treated With Miltefosine
Shahram Khademvatan,Mohammad Javad Gharavi,Elham Yousefi,Jasem Saki
International Journal of Pharmacology , 2011,
Abstract: Miltefosine is the new drug of choice for the treatment of leishmaniasis. The aim of this work was to study the molecular mechanisms and immunomudolatory properties of miltefosine in J774 cell line infected with the Leishmania major (MRHO/IR/75/ER) parasite. In this experimental study infected J774 cell line by L. major, treated by miltefosine and incubated for 72 h. Total RNA was extracted and cDNA was synthesis. RT- PCR was used for study of IFN? and iNOS gene expression. IFN? and iNOS proteins were analyzed by Western blotting. Cell culture supernatant was examined by ELISA for of IL12 and IL10 concentration. After 4 h incubation, miltefosine increased iNOS and IFN? gene expression in L. major infected J774 cell line. Western blot analysis of extracted cell proteins showed 130 and 17 kDa of bonds related to iNOS and IFN?, respectively. After 48 h treatment with miltefosine, analysis of cell supernatant with ELISA showed a significant increase of IL12 but no change in IL10 Cytokine. Study showed that miltefosine in addition to its direct effect can improve cellular immunity with rising of IFN? and of iNOS genes expression that are able to activate macrophages.
The Effect of Alkanna tincturia and Peganum harmala Extracts on Leishmania major (MRHO/IR/75/ER) in vitro
Ruhallah Yousefi,Fatemeh Ghaffarifar,Abdolhosein Dalimi Asl
Iranian Journal of Parasitology , 2009,
Abstract: "nBackground: Cutaneous leishmaniasis is an important health problem caused by Leishmania spp. As there is no vaccine, drug treatment is the only way to tackle leishmaniasis. In the present study, inhibitory and kill-ing effects of Peganum harmala and Alkana tinctoria extracts on amastigotes and promastigotes forms of Leishmania were evaluated in-vitro."nMethods: The seeds of Peganum harmala, Stems and roots of Alkanna tictoria were collected and crude extrac-tion carried out. In this experimental study, Leishmania major promastigotes were cultured in RPMI-1640 with 10% FBS at 22-26°C, and infected macrophages with amastigotes were cultured in RPMI-1640 with 10% FBS at 37°C in 5% CO2. Then the extracts of each plant were added to cultivated parasites and incubated for 3 days. Promastigote and amastigote assay was carried out using counting assay based on growth inhibition."nResults: The results indicated that both extractions can inhibit the growth of promastigotes, and in concentra-tions of 40μg/ml of P. harmala , 200μg/ml of A. tincturia, and 20 μg/ml of equal combination of P. hamala and A. tincturia are Inhibitory Concentration (IC50) for parasites growth. By adding these concentra-tions of the extracts to the infected macrophages in the culture, their effects were separately eva-lu-ated. The mean of amastigotes number in macrophages in the culture with P. harmala, A. ticturia, combina-tion and control groups were 0.7, 0.7, 0.6, 2.3 amastigotes per macrophage, respectively."nConclusion: By this method, inhibition of intracellular and extracellular growth of L. major was demon-strated suggesting that, plant drugs with efficacy and safe products can be applied as new treatment for cutane-ous leishmaniasis.
THE EFFECT OF STACHYS LAVANDULIFOLIA VAHL. AND MESPILUS GERMANICA L. LEAVES HYDROALCOHOLIC EXTRACTS ON LEISHMANIA MAJOR (MRHO/IR/75/ER) IN VITRO
M Asadi,S Bahrami,R Ansari Samani
Jundishapur Journal of Natural Pharmaceutical Products , 2010,
Abstract: Cutaneous leishmaniasis is endemic in 88 different countries. There are an estimated 1.5 million new cases each year, with over 90% occurring in Afghanistan, Algeria, Iran, Iraq, Saudi Arabia, and Syria and in Brazil and Peru. This study is the first one for evaluating the effect of hydroalcoholic extracts of Stachys lavandulifolia Vahl. and Mespilus germanica L. leaves against L. major (MRHO/IR/75/ER) promastigotes. The results of this study showed that, extracts of S. Lavandulifolia and M. germanica leaves are effective on activity against the proliferation of promastigotes of L. major suggest that these extracts might be a promising approach for developing new anti-leishmanial drugs.
The Effect of Aqueous Garlic Extract on Interleukin-12 and 10 Levels in Leishmania Major (MRHO/IR/75/ER) Infected Macrophages
MJ Gharavi,M Nobakht,S Khademvatan,F Fani
Iranian Journal of Public Health , 2011,
Abstract: Background: The aim of the present study was to investigate the immunomodulation effects of aqueous garlic extract (AGE) in the cultured macrophages infected by Leishmania major.Methods: After J774 macrophages proliferation in RPMI1640 and incubation with Leishmania for 72 hours, AGE was added in doses of 9.25, 18.5, 37, 74 and 148 mg/ml for 18, 24 and 48 hours and cell culture supernatants were harvested. The Leishmania infected J774 cells to assess the cell viability was examined using trypan blue and methylthiazol tetrazolium assay (MTT). An enzyme-linked immunosorbent assay (ELISA) was performed on cell culture supernatants for measurement of interleukin IL-10 and IL-12.Results: Dose of 37 mg/ml for 48 hours of garlic extract was the most potent dose for activation of amastigotes infected macrophages. In addition, AGE increased the level of IL-12 in Leishmania infected cell lines significantly.Conclusions: AGE treated cell is effective against parasitic pathogens, and AGE induced IL-12 differentially affected the immune response to invading Leishmania parasites.
Miltefosine induces metacaspase and PARP genes expression in Leishmania infantum
Khademvatan, Shahram;Gharavi, Mohammad Javad;Saki, Jasem;
Brazilian Journal of Infectious Diseases , 2011, DOI: 10.1590/S1413-86702011000500005
Abstract: objectives: apoptosis is the process of programmed cell death (pcd) that occurs in both animal and plant cells. protozoan parasites possess metacaspase and these caspase-related proteases could be involved in the pcd pathways in these organisms. therefore we analyzed the activities of metacaspase and parp genes in leishmania infantum (mcan/ir/96/lon49) treated with miltefosine. materials and methods: anti-leishmania activity of miltefosine was studied by treatment of cultured promastigotes with various concentration of miltefosine. mtt assay and annexin-v fluos staining by using facs flow cytometry methods were used. cytotoxic potential of hepc on the amastigots of l.infantum was evaluated in j774 cell line. in addition, metacaspase and parp genes expression of treated l. infantum were studied. results: miltefosine led to dose-dependent death of l. infantumwith features compatible with apoptosis. over expression of metacaspase and parp was seen 6 hr after treatment. conclusions: our study showed that miltefosine exerts cytotoxic effect on l. infantum via an apoptotic-related mechanism.
Cloning and Sequencing of Leishmania major Thiol-Specific-Antioxidant Antigen (TSA) Gene
F Tabatabaie,F Ghaffari far,A Dalimi,Z Sharifi
Iranian Journal of Parasitology , 2007,
Abstract: Background: Leishmaniasis is caused by parasitic protozoa of the genus Leishmania which, in the infected host are obli-gate intracellular parasite. TSA is the immuno-dominant antigen of Leishmania major which is considered as the most promising molecule for a recombinant or DNA vaccine against leishmaniasis. Methods: Genomic DNA of TSA protein was extracted and amplificated as a template. Then the PCR product was cloned into pTZ57R/T vector. Finally, the recombinant plasmid was extracted from transformed Escherichia coli (TG1 strain) and sequenced. Results: MRHO/IR/75/ER (an Iranian strain) of L. major and TSA gene (Accession number LmjF15.1080) were used. Se-quence analysis of cloned TSA gene into pTZ57R/T vector showed high homology of 90% with LmjF15.1080 (TSA gene) and strain "LV39" (Accession no. AF069386) and strain "Friedlin" (Accession no.AF044679). Conclusion: We cloned TSA gene of L. major successfully. Recombinant plasmid was confirmed. It is ready to express recombinant protein for further studies.
The Effect of Garlic Extract on Expression of INFγ And Inos Genes in Macrophages Infected with Leishmania Major  [PDF]
MJ Gharavi,M Nobakht,SH Khademvatan,E Bandani
Iranian Journal of Parasitology , 2011,
Abstract: Background: The study was aimed to show the effect of molecular mechanism of Aqueous Garlic Extract (AGE) on expression of IFNγ and iNOS genes in Leishmania major.Methods: Leishmania major promastigotes (MRHO/IR/75/ER) were added to the in-vitro cultured J774 cell line, the cells were incubated for 72 hours. Various concentrations of garlic extract (9.25, 18.5, 37, 74, 148 mg/ml) were added to the infected cells. MTT assay was applied for cellular proliferation. After 72 hours of incubation, supernatants were collected and total RNA was extracted from the infected cells. The express of IFNγ and iNOS genes were studied by RT-PCR method.Results: The colorimetric MTT assay after 3 days of incubation showed cytotoxic effect of garlic extract with an IC50 of 37 mg/ml. In addition, IFNγ and iNOS genes expression by RT-PCR indicated that garlic extract lead to over expression of these genes in J774 cell line infected with L. major.Conclusion: Garlic extract exerts cytotoxic effect on infected J774 cell line. In addition, the hypothesis that garlic can improve cellular immunity with raising the expression of IFNγ and of iNOS genes con-firmed.
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