Gene regulation of pteridine reductase 1 in leishmania promasti-gotes and amastigotes using a full-length antisense construct

Pteridine metabolic pathway is unusual features of Leishmania, which is necessary for the growth of parasite. Leishmania has evolved a complex and versatile pteridine salvage network which has the ability of scavenging a wide area of the conjugated and unconjugated pteridines especially folate and biopterin. In this study, we focus on the inhibition of ptr1 gene expression. L. major ptr1 gene was cloned into pcDNA3 and digested using KpnI and BamHI. The gene was subcloned so that antisense will transcribe and called pcDNA-rPTR. Leishmania major was cultured and late logarithmic-phase promastigotes were harvested. The promastigotes were divided into two groups. One group was transfected with 50 micro g of pcDNA-rPTR, whereas the other group was transfected with pcDNA3. Transfected cells were cultured and plated onto semi-solid media. Mouse pritonean macrophages were transfected using pcDNA-rPTR-tansfected promastigotes. Western blotting was performed on mouse transfected pritonean macrophages and extracts from transfected promastigotes of L. major using a L. major ptr1 antibody raised in rabbits. The PTR1 protein was not expressed in pcDNA-rPTR- tansfected promastigotes and mouse macrophage transfected with pcDNA-rPTR- tansfected promastigotes. This approach might be used to study the pteridine salvage pathway in Leishmania or to assess the possibility of using gene expression inhibition in the treatment of leishmaniasis.

Interleukin-10 and transforming growth factor-beta in early and late lesions of patients with leishmania major induced cutaneous leishmaniasis

Cutaneous leishmaniasis is a neglected parasitic disease, which imposes massive human distress and financial costs to the endemic countries. Better understanding of host immune response to the parasite leads to helpful strategies for disease control. Interleukin [IL]-10 and transforming growth factor [TGF]-beta are important immune regulatory cytokines, which appear to develop non-healing forms of leishmaniasis. However, there is little information about the function of IL-10 and TGF-beta in old world cutaneous leismaniasis. The aim of this study was to analyze the role of IL-10 and TGF-beta in human cutaneous leishmaniasis due to Leishmania major infection. Biopsies were obtained from lesions of twenty proven cases of L. major induced cutaneous leishmaniasis. IL-10 and TGF-beta positive cells were detected by immunofluorescence staining of frozen sections and compared between two groups of patients with early and late lesions. The mean percentage of IL-10 positive cells were significantly [P= 0.035] higher in late lesions [0.51 +/- 0.24] than early ones [0.15 +/- 0.07]. Similar results were obtained for TGF-beta with mean percentages of 0.16 +/- 0.05 and 0.53 +/- 0.28 in early and late lesions respectively [P= 0.008]. IL-10 and TGF-beta are present in lesions of L. major induced cutaneous leishmaniasis and contribute to the pathogenesis of long lasting disease forms

Interleukin-10 and transforming growth factor-beta in early and late lesions of patients with leishmania major induced cutaneous Leishmaniasis

Cutaneous leishmaniasis is a neglected parasitic disease, which imposes massive human distress and financial costs to the endemic countries. Better understanding of host immune response to the parasite leads to helpful strategies for disease control. Interleukin [IL]-10 and transforming growth factor [TGF]-beta are important immune regulatory cytokines, which appear to develop non-healing forms of leishmaniasis. However, there is little information about the function of IL-10 and TGF-beta in old world cutaneous leismaniasis. The aim of this study was to analyze the role of IL-10 and TGF-beta in human cutaneous leishmaniasis due to Leishmania major infection. Biopsies were obtained from lesions of twenty proven cases of L. major induced cutaneous leishmaniasis. IL-10 and TGF-beta positive cells were detected by immunofluorescence staining of frozen sections and compared between two groups of patients with early and late lesions. The mean percentage of IL-10 positive cells were significantly [P= 0.035] higher in late lesions [0.51 +/- 0.24] than early ones [0.15 +/- 0.07]. Similar results were obtained for TGF-beta with mean percentages of 0.16 +/- 0.05 and 0.53 +/- 0.28 in early and late lesions respectively [P= 0.008]. IL-10 and TGF-beta are present in lesions of L. major induced cutaneous leishmaniasis and contribute to the pathogenesis of long lasting disease forms

Efficacy of microwave and infrared radiation in the treatment of the skin lesions caused by leishmania major in an animal model

Cutaneous leishmaniasis caused by L. major is an important public health problem in endemic areas. The aim of this study was to explore the therapeutic effect of microwave and or infrared radiation in the treatment of lesion induced in BALB/c mice by L. major inoculation. The footpad lesion was induced in BALB/c mice by inoculation of L. major promastigotes subcutaneously. The lesion was treated with 600 watts power, 2.450 GHz frequency and/or infrared device with 150 watts and a wave length of 890 nanometres. The size of the lesion was recorded by footpad swelling measurement ever 10 days. The lesion growth was significantly hampered in treated mice compared with the untreated control group [P<0.05]. Infrared radiation was more effective than microwave in inhibiting ulcer enlargement. Infrared radiation and microwave significantly hampered L. major lesion growth in BALB/c mice. This therapeutic effect was more in infrared radiation treated mice than microwave treated mice

Cutaneous leishmaniasis in Bam: a comparative evaluation of pre- and post- earthquake years [1999-2008]

The recent devastating earthquake of December 26 in Bam, 2003 created various risk factors; caused a sharp increase in incidence of anthroponotic cutaneous leishmaniasis [ACL] cases and reached to an epidemic proportion. The objective of this study was to evaluate the status of ACL cases five years before the earthquake compared to the cases occurred five years after the earthquake [1999-2008]. Status of disease was assessed retrospectively for the five years before the earthquake and prospectively for the five years after the earthquake. Identification was confirmed by smear and polymerase chain reaction [PCR]. The mean annual incidence of ACL for the period from 1999 to 2003 was 1.9 per 1000 comparing to post earthquake period, which was 7.6 per 1000. Most of the infection was in individuals of <20 years, more frequently in females before the earthquake, whilst in contrast, there was a progressive rise in the number of cases, significantly in male individuals of >20 years [P< 0.0001] in post earthquake era. The anatomical distribution of lesions considerably changed during the two periods. Most of the cases were limited to three zones within the city prior to the earthquake, whereas it was spread throughout different zones after the earthquake. PCR indicated that the CL was due to Leishmania tropica in the city. The results strongly suggest that in natural disasters such as earthquakes various precipitating factors in favor of disease will be created, which in turn provide a suitable condition for propagation of the vector and the transmission of the parasite

Leishmanicidal activity of films containing paromomycin and gentamicin sulfate both in vitro and in vivo

Based on the efficacy of paromomycin ointment and recent ongoing clinical trials of combination of paromomycin and gentamicin, a new physical form of films of the paromomycin and gentamicin was prepared and anti-Leishmania activities of the prepared films were assessed in vitro and in vivo. Paromomycin 15% and gentamicin 0.5% was incorporated in a film using ethyl cellulose and HPMC [Hydroxyl Propyl Methyl Cellulose]. In order to assess the drug release and anti-Leishmania activities of the preparation, a clone L. major parasite was established using a set of modified NNN medium without overaly liquid layer. Therapeutic effects of the films were evaluated using Balb/c mice model. The mice were inoculated with 2x10[6] L. major promastigotes [MRHO/IR/75/ER] and then when the lesions developed the mice were randomly divided in 3 groups, 10 mice per group, and treated with either perpetrated films or placebo for 28 days or left untreated. Growth inhibition of cloned promastigotes showed that the films have enough releasing capacity and in vivo system, the films containing paromomycin and gentamicin was able to reduce the lesion size and induced complete cure in 80% of the mice but relapse was seen in 60% of the cured mice and overall 50% cure rate was seen during 20 weeks period of the study. It seems that the prepared films might be further used in human clinical trials

IL-23 and IL-27 levels in macrophages collected from peripheral blood of patients with healing vs non-healing form of cutaneous leishmaniasis

In this study the level of IL-23 and IL-27 produced by macrophages derived from peripheral blood mononuclear cell culture collected from patients with healing or non-healing form of cutaneous leishmaniasis lesion were compared before and after treatment with live Leishmania to explore whether IL-23 or IL-27 plays any role in healing process of cutaneous lesions induced by L. major. Twenty patients resident in Isfahan Province, with healing or non-healing form of cutaneous leishmaniasis lesion caused by Leishmania major participated in this study. In vitro productions of IL-23 and IL-27 by peripheral blood derived macrophages, before and after stimulation with live L. major [MRHO/IR/75/ER] promastigotes were evaluated using ELISA method. Patient with healing form of lesion received no treatment and patient with non-healing form of lesion received at least 2 courses of glucantime. The mean production of IL-23 and IL-27 from macrophages of patients with healing form of lesion was significantly higher than patients with non-healing form of lesion. The levels of IL-23 and IL-27 in culture supernatants before and after stimulation in healing form of CL was significantly higher than non- healing form of CL [P < 0.001]. IL-23 and IL-27 might play a role in human leishmaniasis and further studies are needed to understand the role of IL-23 and IL-27 in leishmaniasis

Production and purification of anti-rhombomys opimus immunoglobulins

Zoonotic cutaneous leishmaniasis [ZCL] is an increasing public health problem in some endemic regions. Horseradish peroxidase [HRP] conjugated rabbit anti-Rhombomys opimus [R. opimus] Ig is needed for immunoblotting and ELISA tests used to explore the immune response of the rodents against the sand fly saliva. In this study, the production of HRP conjugated rabbit anti-R. opimus Ig was conducted for the first time. Rhombomys opimus Ig was purified from serum by protein G affinity chromatography column and injected into rabbit to produce anti-R. opimus Ig antibody. The titration of antibody against R. opimus Ig in rabbit serum was checked using indirect ELISA. Rabbit anti-R. opimus Ig was purified by Sepharose-4B-R. opimus Ig column. Reactivity of this antibody was assessed by indirect ELISA and was conjugated to HRP by periodate method. Approximately 3.5 mg Ig was purified from 1 ml R. opimus serum using protein G affinity chromatography column. The molecular weight of purified R. opimus Ig was estimated about 150 kDa by SDS-PAGE. Nearly 2.3 mg rabbit anti-R. opimus Ig was purified from 1 ml immunized rabbit serum. The purified antibody was conjugated to HRP and the optimum titer of HRP conjugated rabbit anti-R. opimus Ig was determined as 1:8000 using direct ELISA. HRP conjugated rabbit anti-Gerbil IgG has been produced by a few companies, but to our knowledge HRP conjugated rabbit anti-R. opimus Ig is not commercially available. Production of HRP conjugated rabbit anti-R. opimus Ig is considerably helpful for immunological studies of R. opimus, the main reservoir host of ZCL in Iran as well as some other countries

Molecular epizootiology of rodent leishmaniasis in a hyperendemic area of Iran

Zoonotic cutaneous leishmaniasis [ZCL] is an expanding disease and public health problem in Iran. In the current study, natural Leishmania infection rate and seasonal fluctuation of the infection in Rhombomys opimus population of a hyperendemic focus of ZCL in Iran was investigated. The study was conducted from October 2006 to October 2008 in Esfahan Province, central part of Iran. An extensive sampling of rodents using Sherman traps was done in different seasons. Nested PCR assay was used for detection and identification of Leishmania species and the results were confirmed using PCR-RFLP. Leishmania infection rate was 58.6% [34 of 58] using nested PCR. 44.8% of the gerbils were infected only with L. turanica and 1.7% with L. gerbilli alone. A mixed natural infection with L. major and L. turanica was seen in 12.1% of the rodents. L. major infection alone was not seen in R. opimus population in the study area. The highest and lowest Leishmania infection rates were observed in fall and spring respectively. L. turanica infection was observed throughout the year whereas mixed infections with L. major and L. turanica was not seen in spring. It is concluded that in the study area, L. major, L. gerbilli and L. turanica circulate in the population of R. opimus. Leishmania major infection usually accompanied by L. turanica in naturally infected gerbils with the highest rate in fall. It is recommended that the role of L. turanica in the epidemiology and transmission of ZCL be revisited

Whatman paper [FTA Cards] for storing and transferring Leishmania DNA for PCR examination

Diagnosis of cutaneous leishmaniasis [CL] is often made based on clinical manifestation. Correct diagnosis and identification of the parasite are crucial for choosing the effective treatment and for epidemiological studies. On the other hand, determination of Leishmania species is necessary for designing appropriate control programs. Diagnosis by PCR is becoming a [gold standard]. For PCR preparation, storage and shipments of specimens are necessary. In this study, Whatman filter paper [FTA Card] was used to store and transfer samples for Leishmania identification using PCR. Among the patients who had CL lesion and referred to Parasitology Laboratory of Emam Reza Hospital, Mashhad, Iran, 44 consented cases with positive results in their direct smear were selected. An informed consent form and a questionnaire were completed and three different types of samples [direct smear, NNN culture, and spot on FTA card] were collected. DNA extraction and PCR were carried out on three different samples from each patient. PCR results using Whatman paper samples revealed a significant difference [P<0.0001] compared to the culture method but no significant difference was seen between PCR results using samples stored on Whatman paper and direct smears. The use of FTA cards is simple, rapid, and cost-effective, and can be readily employed for large-scale population screening, especially for regions where the specimens are to be transported from distant places to the laboratory

Gene cloning of Iranian Leishmania major mannose-1-phosphate guanyltransferase

Leishmania is an obligatory intracellular protozoan parasite, which infects human beings when infected sand fly vector takes a blood meal. Most efforts are towards designing an effective vaccine to prevent leishmaniasis. In this way, development of candidate antigen for vaccine has special important. In this study, we cloned mannose-1-phosphate guanyltransferase gene of Iranian L .major in pET32a expression vector. Primers based on L. major mannose-1-phosphate guanyltransferase sequence gene was designed and synthesized. DNA of Leishmania promastigotes was extracted and PCR reaction was done. PCR product was cloned into pTZ57R and sub cloned into pET32a expression vector. Recombinant plasmid containing 1140 bp as L. major mannose-1-phosphate guanyltransferase gene was extracted and confirmed by restriction analysis. PCR product was sequenced and deposited to GenBank. There were some differences in amino acid sequences between Iranian L. major mannose-1-phosphate guanyltransferase and others previously accepted in GenBank. We amplified and cloned Iranian L. major mannose-1-phosphate guanyltransferase successfully

Flow cytometric analysis of leishmania reactive CD4[+]/CD8[+] lymphocyte proliferation in cutaneous leishmaniasis

Determination of the division histoty of T cells in vitro is helpful in the study of effector mechanisms against infections. Technique described here uses the intracellular fluorescent label carboxyfluorescein diacetate succinimidyl ester [CFSE] to monitor the proliferation. In a cross sectional study, blood samples were collected from 7 volunteers with history of cutaneous leishmaniasis [CL] and one healthy control from endemic areas in Isfahan province who referred to the Center for Research and Training in Skin Diseases and Leprosy [CRTSDL], then CD4[+]/CD8[+] lymphocytes and CD14[+] monocytes were isolated from peripheral blood mononuclear cells [PBMC] using mAbs and magnetic nanoparticles. CFSE labeled CD4[+] or CD8[+] lymphocytes cultured with autologous monocytes in the presence of PHA, SLA, live Leishmania major or as control without stimulation. Cells were harvested after 7 days and were analyzed using flow cytometry. Five consecutive divisions were monitored separately. Stimulation of CD4[+] or CD8[+] lymphocytes from CL subjects with SLA showed a significant difference in proliferation comparing with unstimulated cells [P< 0.05]. The significant difference in the percentages of CD4[+] cells stimulated with SLA was revealed at different divisions for each subject. In CD8[+] lymphocyte, significant stronger stimulation of SLA was evident later in the proliferation process. The mean number of divisions in both CD4[+]/CD8[+] lymphocytes stimulated with SLA was significantly greater than when stimulated with live L. major [P=0.007 / P=0.012, respectively]. The percentage of divided cells might be calculated separately in each division. The cells remained active following CFSE staining and there is possibility of functional analysis simultaneously

Cloning and expression of TRYP6 gene from ieishmania major [MRHO/IR/75/ER]

Leishmania, needs to detoxify the macrophage derived potent peroxides [H2O2]. Tryparedoxin pathway contains tryparedoxin peroxidase [TSA or TRYP]. The aim of the study was to detect the full-length gene sequence 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. L.major [MRHO/IR/75/ER] promastigotes were cultured, DNA and RNA were extracted and the interested 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. Characterization of the expressed protein was performed bioinformatically. 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]. 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 candidate for vaccine development and any other sort of novel drug development

[Immune response evaluation against autoclaved leishmania major ncapsulated in liposome with different Tm in murine model]

Leishmaniasis is a parasitic disease which is caused by different species of Leishmania. Protozoa of the Leishmania species cause leishmaniasis. The protective immunity against leishmaniasis is the cell-mediated immunity [CMI]. Autoclaved Leishmania major [ALM] has been used as vaccine in clinical trial; however, its efficacy has been low. Liposomes are microscopic vesicles consisting of phospholipid bilayers which enclose aqueous compartments and are used as an immunoadjuvant. The aim of this study was to evaluate the immune response of ALM entrapped in liposome with different phase transition temperature [Tm] in BALB/c mice. Liposomes containing ALM were prepared as dehydration-rehydration vesicles [DRV] and composed of dipalmitoylphosphatidylcholine [DPPC, Tm 41°C] and cholesterol or phosphatidylcholine [PC, Tm -10°C] and cholesterol in a molar ratio of 7:2 DRV-DPPC-Chol- ALM [180 microg], DRV-PC-Chol-ALM [180 microg], ALM alone [180 microg], PBS, and a control empty liposome were injected separately subcutaneously [SC] in female BALB/c mice [10 per group], 3 times in three weeks interval. The mice were tested for DTH with freeze-thawed L. major promastigotes [1.5 × 10[7]] SC to the left footpad and PBS to the right footpad for control at 3 weeks after the last booster. The footpad swellings were measured after 24, 48 and 72 hours. Blood samples were collected before second and third vaccination and also before DTH test to titrate the anti-Leishmania antibodies [IgG total, IgG1 and IgG2a] by ELISA method. The sizes of liposome were between 0.5-3 microm. Percent encapsulation of ALM in DRV-DPPC- Chol-ALM and DRV-PC-Chol-ALM was 46 and 43 respectively. The results of DTH showed that footpad swelling in DRV-DPPC-Chol-ALM and DRV-PC-Chol-ALM is significantly more than control groups. The results of ELISA showed that the titer of IgG total in PBS, control empty liposomes and ALM alone is low and it is significantly less than DRV-DPPC- Chol-ALM and DRV-PC-Chol-ALM. There were no differences in the titer of IgG1 in the different groups; however, the titer of IgG2a was significantly higher in DRV-DPPC-Chol- ALM compared to DRV-PC-Chol-ALM and control groups. The results indicated that liposome prepared by higher Tm phospholipid seems to be a suitable immunoadjuvant for ALM in to improve the CMI

[Soluble CD26 and CD30 concentration in cutaneous leishmaniasis]

Introduction: Cutaneous leishmaniasis [CL] is the most common form of leishmaniasis that usually heals spontaneously with unsightly scar but rarely non-healing lesion of CL develops which is refractory to all types of therapy. It is shown that Th1 and Th2 response is associated with healing and non-healing form of the diseases, respectively. On the other hand, it is reported that CD26 and CD30 are associated with Th1 and Th2 types of response, respectively. In some diseases, there is a relationship found between level of CD26 and CD30 and Th1 and Th2 responses

Objective: The goal of this study was to determine the concentration of soluble CD26 and soluble CD30 [sCD26 and sCD30] as a possible marker for Th1/Th2 response in CL

Materials and Methods: The blood samples were taken from 36 patients with healing form of the lesion and 10 patients with non-healing form of CL who were referred to Razi hospital or Center for Research and Training in Skin Diseases and Leprosy in Tehran during 2003-2004. As a control blood samples were taken from 23 volunteers with no history of CL. In this study, the concentration of sCD26 and sCD30 were measured in plasma by ELISA method

Results: The results showed that the plasma levels of sCD26 and sCD30 were significantly higher in non-healing form of the disease than healing form of CL or control group [p<0.05]. The level of sCD30 was more prominent than sCD26. There was no significant difference in the level of sCD26 or sCD30 markers in healing form of CL compared to normal control group

Conclusion: Overall it seems that the level of sCD30 might be a useful marker to study the immune response in CL, which needs to be studied further

Safety, immunogenicity and possible efficacy of immunochemotherapy of persistent post Kala-azaz dermal leishmaniasis

Post kala-azar dermal leishmaniasis [PKDL] is a common skin condition that follows successful treatment of visceral leishmaniasis [VL] in Sudanese patients. Lesions persist for years in 15% of patients and are viewed as reservoirs for the disease. Drug treatment is protracted, toxic and costly. Cure is strongly correlated with conversion in the leishmanin skin test. To determine safety, immunogenecity and possible efficacy of Alum-precipitated autoclaved L. major + BCG VL candidate vaccine combined with sodium stibogluconate [SSG] in patients with persistent PKDL. Following informed consent, the vaccine mixture was administered in a stepwise manner as follows: 5 patients received a single intradermal injection of 10 microg, 5 patients received a single dose of 100 microg and 2 patients received 4 doses of 100 microg at weekly intervals. Subsequently, the three groups of patients received means of 63.0 +/- 8.0, 53.0 +/- 5.0 and 40 days courses of SSG treatment respectively and were cured. Side effects were minimal and were confined to the vaccine injection site. Following completion of the safety study, eight patients were injected with 4-6 vaccine doses of 100 microg/dose at weekly intervals in combination with SSG. Patients were closely followed up in hospital, with minimal side effects and complete clearance of the skin rash in forty days. Alum/ALM + BCG vaccine mixture plus SSG was safe and was apparently effective in healing persistent PKDL lesions. SSG treatment duration could be shortened with the SSG/vaccine combination

Immune response evaluation against leishmaniasis using liposomes containing rLmSTI1 in murine model

Leishmaniasis is a parasitic disease spread by the bite of infected sand flies. Protozoa of the Leishmania species cause leishmaniasis. The protective immunity against leishmaniasis is the cell-mediated immunity [CMI]. LmSTI1 is a candidate for the development of vaccine against cutaneous leishmaniasis [CL]. Liposomes are microscopic vesicles consisting of phospholipid bilayers which enclose aqueous compartments and are used as an immunoadjuvant. The aim of this study was to formulate liposome preparations containing recombinant rLmSTI1 to induce Th1 response in BALB/c mice against infection with L. major. Liposomes containing rLmSTI1 were prepared as dehydration-rehydration vesicles [DRV] and composed of distearoylphosphatidylcholine [DSPC] and cholesterol [CHOL] in a molar ratio of 2:1 [DSPC/CHOL-rLmSTI1]. The average size of liposome formulations was 1.1micro m checked by light microscope and particle size analyzer. DSPC/CHOL-rLmSTI1 [2micro g]; soluble rLmSTI1 [2micro g]; PBS, and a control empty liposome were injected separately subcutaneously [SC] in female BALB/c mice [10 per group], 3 times in three week intervals. The mice were challenged with L. major promastigotes [1.5 X 10[6]] SC to the left footpad and PBS to the right footpad for control at 3 weeks after the last booster. The footpad swellings were measured weekly for 12 weeks. Blood samples were collected on the day before and 12 weeks after challenge to titrate the anti-Leishmania antibodies [IgG total, IgG1 and IgG2a] by ELISA method. The parasite burden in spleen was determined at 15weeks after challenge. The results showed that in the group that received DSPC/CHOL-rLmSTI1, footpad thickness was significantly less; IgG2a titer was higher with very few parasites in the spleen compared to the other groups. The results indicated that encapsulation of rLmSTI1 in liposome seems to be a suitable tool to improve the CMI and rate of protection in murine model of leishmaniasis