Live attenuated Leishmania major p27 gene knockout as a novel vaccine candidate: A study on safety, protective immunity and efficacy against canine leishmaniasis caused by Leishmania infantum.

Canine leishmaniasis (CanL) is an important parasitic e disease caused by Leishmania infantum and is transmitted by female phlebotomine sand flies primarily between canines and secondarily to humans. Recently, we showed that immunization with Leishmania major p27 gene knockout (Lmp27-/-) as a live attenuated vaccine was safe, induced immunogenicity, and protected against the development cutaneous and visceral leishmaniasis in mice. The p27 protein is a component of the COX protein complex which is responsible for ATP production. In this study, we analyzed the Lmp27-/- candidate vaccine potential with this regard to the safety and induction of immunogenicity and protection against CanL. Variables such a clinical manifestation, anti-Leishmania antibodies using direct agglutination test (DAT), lymphocyte proliferation, delayed-type hypersensitivity (DTH), bone marrow aspiration (BMA) and parasite burden using parasitological and molecular examinations were measured. The results demonstrated that the Lmp27-/- vaccinated group showed no clinical signs after inoculation with Lmp27-/- mutant during a 12-month follow-up, and had significantly higher T-cell responses (Lymphocyte proliferation and DTH), lower seroconversion and parasite burdens following a challenge inoculation with L. infantum after 6-mounth. In conclusion, vaccination with Lmp27-/- parasites would be safe and provide significant immunoprotectivity and efficacy against infection with wild type (WT) L. infantum.

Hepatoprotective and antioxidant effects of Hedera helix extract on acetaminophen induced oxidative stress and hepatotoxicity in mice.

Exposure to high doses of acetaminophen is the most common cause of drug induced liver injury. We investigated the protective effects of Hedera helix extract against acetaminophen induced oxidative stress and hepatotoxicity using a mouse model. We used two control groups: group A was given 0.9% NaCl, group B was an acetaminophen control that was given a single injection of 600 mg/kg acetaminophen. T1-T4 groups were pretreated orally with different doses of H. helix extract. The mice were sacrificed and blood samples were collected to estimate the levels of glutathione peroxidase (GPx), malondialdehyde (MDA), superoxide dismutase (SOD) and total bilirubin. Liver samples also were used for histopathological studies. We found that acetaminophen significantly increased the levels of serum ALP, ALT, AST and MDA, and also significantly reduced the antioxidant factors, CAT, GPX and SOD. H. helix extract treatment produced a significant reduction in the levels of ALP, ALT, AST and MDA in serum and restored the levels of CAT, GPX and SOD to control levels. The histopathological findings were consistent with the biochemical findings. H. helix extract exhibited antioxidant and hepatoprotective effects against acetaminophen induced liver damage.

Evaluation of Th17 associated antigen in Old World Cutaneous Leishmaniasis: A comparative study in acute versus chronic human cutaneous Leishmaniasis using immunohistochemistry.

There are little information about Th17 cells and cutaneous Leishmaniasis (CL), due to an important effect of Th17 cells on immune response, it is worth to explore the role of Th17 on CL. The purpose of this study was to assess Th17 population in patients with acute vs. chronic CL lesions in comparison with skin samples collected from healthy volunteers in an endemic region of Old World CL. A total of 49 patients with clinical manifestations of chronic (n=16) and acute (n=33) CL lesions were recruited. The clinical diagnosis of CL was confirmed by direct smear or PCR. Biopsy specimens from prelesional skin of non-infectious lesions of 30 healthy individuals were used as control. Tissue sections of 3µm thickness were prepared and used for immunohistochemistry (IHC) analysis with primary antibody specific for Th17 associated antigen (CD161). For IHC, Envision+ (DakoCytomation) system was used and developed by using diaminobenzidine (DakoCytomation). The mean age of 33 patients with acute CL and the mean age of 16 patients with chronic CL were accordingly 45.24±16.43 and 33.56±15.87. In acute and chronic CL the mean (±standard deviation) and median (±interquartile range) were accordingly 2.92±2.21, 2.56±2.9 and 2.1±1.99, 1.54±2.81. In healthy controls the mean (±standard deviation) and median (±interquartile range) were 0.72±0.41 and 0.61±0.58 respectively. With pairwise comparison of acute, chronic and control groups, there were significant difference between acute and control (P value < 0.001), chronic and control (P value = 0.043). The results showed that there was an increasing cellular response of Th17 in both acute and chronic CL patients. Th17 was significantly higher in patients with acute and chronic CL lesions in comparison with healthy control group. However, there was no significant difference between acute and chronic infection concerning to Th17 cells.

Evaluation of Th17 associated antigen in Old World Cutaneous Leishmaniasis: A comparative study in acute versus chronic human cutaneous Leishmaniasis using immunohistochemistry

@#There are little information about Th17 cells and cutaneous Leishmaniasis (CL), due to an important effect of Th17 cells on immune response, it is worth to explore the role of Th17 on CL. The purpose of this study was to assess Th17 population in patients with acute vs. chronic CL lesions in comparison with skin samples collected from healthy volunteers in an endemic region of Old World CL. A total of 49 patients with clinical manifestations of chronic (n=16) and acute (n=33) CL lesions were recruited. The clinical diagnosis of CL was confirmed by direct smear or PCR. Biopsy specimens from prelesional skin of non-infectious lesions of 30 healthy individuals were used as control. Tissue sections of 3μm thickness were prepared and used for immunohistochemistry (IHC) analysis with primary antibody specific for Th17 associated antigen (CD161). For IHC, Envision+ (DakoCytomation) system was used and developed by using diaminobenzidine (DakoCytomation). The mean age of 33 patients with acute CL and the mean age of 16 patients with chronic CL were accordingly 45.24±16.43 and 33.56±15.87. In acute and chronic CL the mean (±standard deviation) and median (±interquartile range) were accordingly 2.92±2.21, 2.56±2.9 and 2.1±1.99, 1.54±2.81. In healthy controls the mean (±standard deviation) and median (±interquartile range) were 0.72±0.41 and 0.61±0.58 respectively. With pairwise comparison of acute, chronic and control groups, there were significant difference between acute and control (P value < 0.001), chronic and control (P value = 0.043). The results showed that there was an increasing cellular response of Th17 in both acute and chronic CL patients. Th17 was significantly higher in patients with acute and chronic CL lesions in comparison with healthy control group. However, there was no significant difference between acute and chronic infection concerning to Th17 cells.

PEGylation of cationic liposomes encapsulating soluble Leishmania antigens reduces the adjuvant efficacy of liposomes in murine model.

Although there have been several attempts to develop a vaccine against leishmaniasis, no vaccine in human has been developed yet. Liposomes consisting of 1, 2-dioleoyl-3-trimethylammonium-propane (DOTAP) encapsulating soluble Leishmania antigens (SLA) enhance protective immunity of SLA against Leishmania major infection in mice. However, they immobilized at the injection site because of their positive charge. To overcome the problem, shielding the surface charge with polyethylene glycol (PEGylation) was chosen in this study. Liposomal SLA consisting different concentrations of PEG (1.9%-15% mol) were prepared. BALB/c mice were immunized three times in 3 weeks intervals with different formulations. Lesion development and parasite burden in footpad and spleen were evaluated to specify the type of generated immune response and extent of protection. Th1/Th2 cytokine profiles and IgG isotypes were also analysed. The maximum protection was observed in mice immunized with Lip-SLA or pLip-SLA (1.9%) due to smaller footpad swelling, reduction in parasite load, an increase in IgG2a and IFN-γ production. Our results showed that immunization of mice with a high level of PEG (>7.5%) did not improve protective immunity of liposomal SLA. The presence of PEG, particularly more than 3.75%, is not recommended for protection against leishmaniasis.

The importance of the patient voice in vaccination and vaccine safety-are we listening?

Much has been written about the patient-physician relationship over the years. This relationship is essential in maintaining trust in the complex arena of modern diagnostic techniques, treatment and prevention, including vaccines and vaccine safety. However, a great deal of this material was written from the viewpoint of clinicians and academics. The patient voice may be positive or negative, fragmented or complex. Information sources are weighed and treated differently, according to the value system and risk perceptions of the individual. In post-trust societies, when people have less confidence in health authorities, communication needs to be more than a paternalistic top-down process. Notions of empowerment and individual patient choice are becoming crucial in medical care. The 'voice of the patient', which includes healthy individuals receiving vaccines, needs to be heard, considered and addressed. With respect to childhood immunizations, this will be the voice of the parent or caregiver. The key to addressing any concerns could be to listen more and to develop a communication style that is trust-based and science-informed. Regulatory agencies are encouraging clinical and patient-reported outcomes research under the umbrella of personalized medicine, and this is an important step forward. This paper attempts to reflect the paradigm shift towards increasing attention to the patient voice in vaccination and vaccine safety.

Tumour Necrosis Factor-alpha (TNF-α) and its soluble receptor type 1 (sTNFR I) in human active and healed leishmaniases.

The role of tumour necrosis factor-alpha (TNF-α) is not fully understood in human leishmaniasis. We analysed the alterations in the levels of TNF-α, soluble TNF receptor type 1 (sTNFR I), IL-17 and IL-22 productions in active and healed leishmaniases. Blood samples were collected from volunteers with active cutaneous leishmaniasis (ACL), the same subjects after lesion healing (healed CL = HCL), volunteers with active visceral leishmaniasis (AVL), healed VL (HVL) and healthy controls. Levels of cytokines were titrated on Leishmania Ag-stimulated PBMC culture. The mean level of TNF-α production from stimulated cells was significantly higher in ACL than controls (P < 0·001) and significantly reduced after treatment in HCL volunteers (P < 0·05). The mean level of sTNFR I production was significantly higher in ACL than controls (P < 0·001) and significantly reduced after treatment in HCL volunteers (P < 0·05). The mean level of IL-22 production in AVL was significantly higher than controls (P < 0·05) and was significantly lower in HVL compared with AVL (P < 0·001) and controls (P < 0·05). The levels of TNF-α (P = 0·0025) and sTNFR I (P < 0·01) productions from PBMCs showed significant decreasing trend after treatment in each CL volunteer. Reduction in TNF-α is associated with clinical response to treatment and healing of CL lesions due to L. major.

Plasma levels of tumor necrosis factor-alpha (TNF-α), TNF-α soluble receptor type 1 (sTNFR I) and IL-22 in human leishmaniasis.

The role of pro-inammatory cytokine tumor necrosis factor-alpha (TNF-α) in human leishmaniasis is not fully understood. We analyzed the alterations in the plasma levels of TNF- α, soluble TNF receptor type 1 (sTNFR I), IL-17 and IL-22 in the volunteers with leishmaniasis. Blood samples were collected from patients with active cutaneous leishmaniasis (CL), the same CL patients after standard antimonial therapy as healed CL, active visceral leishmaniasis (VL) and healed VL volunteers. Levels of the cytokines were titrated on plasma samples by sandwich ELISA method. The mean level of TNF-α was significantly higher in active CL patients than healthy controls (P<0.001) and significantly reduced after treatment in the same volunteers (P<0.001). The mean level of sTNFR I was significantly higher in active CL patients than healthy controls (P<0.05). The mean level of IL-22 in plasma of the AVL patients was significantly higher than that of healthy control group (P<0.05). There is a negative correlation between the levels of TNF-α and sTNFR I and healing of CL. Measurement of cytokines in plasma samples is more feasible than cell culture in evaluation of immune response in human leishmaniasis.

Effects of combined therapy with silymarin and glucantime on leishmaniasis induced by Leishmania major in BALB/c mice.

Leishmania major is resistant to the traditional treatments in many parts of the world. PgpA, a member of (ABC) transporter superfamily, has been identified in Leishmania involved in antimony resistance. Silymarin can inhibit PgpA. The aim of this study was to determine the effect of combined therapy with glucantime and silymarin on Cutaneous Leishmaniasis. The effects of silymarin on response of L. major to glucantime were evaluated with amastigote macrophage and mice model of leishmaniasis. Immediately after injection in mice inoculated into footpads with L. major amastigote, systemic treatment was performed and the size of footpad swelling was measured twice a week. 4 and 8 weeks after the beginning of the treatment, splenic parasite burden was done. Silymarin showed no significant effect on the response of L. major promastigotes to glucantime. 2 formulations (glucantime 25 µm with silymarin 25 µm or 12.5 µm) reduced cell death in amastigote assays. The effect of silymarin on footpad swelling was detected when the combination of low-dose glucantime (20 mg/kg) with 25-50 mg/kg silymarin (especially 50 mg/kg) were used at day 22 of post infection (P<0.05). According to the parasite burden data, use of silymarin in the presence of different doses of glucantime, did not show significant effect compared to glucantime alone. The results of this study suggest that silymarin in conjunction with glucantime may have benefit effects in murine model of cutaneous leishmaniasis.

Plasma levels of tumor necrosis factor-alpha (TNF-α), TNF-α soluble receptor type 1 (sTNFR I) and IL-22 in human leishmaniasis

The role of pro-inammatory cytokine tumor necrosis factor-alpha (TNF-α) in human leishmaniasis is not fully understood. We analyzed the alterations in the plasma levels of TNF- α, soluble TNF receptor type 1 (sTNFR I), IL-17 and IL-22 in the volunteers with leishmaniasis. Blood samples were collected from patients with active cutaneous leishmaniasis (CL), the same CL patients after standard antimonial therapy as healed CL, active visceral leishmaniasis (VL) and healed VL volunteers. Levels of the cytokines were titrated on plasma samples by sandwich ELISA method. The mean level of TNF-α was significantly higher in active CL patients than healthy controls (P<0.001) and significantly reduced after treatment in the same volunteers (P<0.001). The mean level of sTNFR I was significantly higher in active CL patients than healthy controls (P<0.05). The mean level of IL-22 in plasma of the AVL patients was significantly higher than that of healthy control group (P<0.05). There is a negative correlation between the levels of TNF-α and sTNFR I and healing of CL. Measurement of cytokines in plasma samples is more feasible than cell culture in evaluation of immune response in human leishmaniasis.

Use of topical liposomes containing meglumine antimoniate (Glucantime) for the treatment of L. major lesion in BALB/c mice.

Treatment of cutaneous leishmaniasis (CL) is a dream for the patients, health care authorities and scientists. The aim of this study was to develop a topical liposomal meglumine antimoniate (MA, Glucantime™) (Lip-MA) formulation and evaluate the therapeutic effects of the preparation on lesion induced by Leishmania major in BALB/c mice. Liposomes containing 22.5% MA (6.4% Sb(+5)) with and without oleic acid (LMA-OA and LMA) were formulated using fusion method plus homogenization and characterized for the size and encapsulation efficiency. The penetration of MA from the LMA-OA and LMA formulations through and into the skin was checked in vitro using Franz diffusion cells fitted with mouse skin at 37°C for 8h. The in vitro permeation data showed that almost 1.5% of formulations applied in the mouse skin were penetrated and the amount retained in the skin was about 65%. The 50% effective dose of LMA and LMA-OA against amastigotes of L. major was 46.36 and 41.01 µg/ml, respectively. LMA or LMA-OA was used topically twice a day for 4 weeks to treat the lesion induced by L. major in susceptible BALB/c mice. The results showed a significantly (P<0.001) smaller lesion size in the treated groups of mice compared to the control groups which received either empty liposomes or phosphate-buffered saline (PBS). The spleen parasite burden was significantly (P<0.001) lower in the treated groups compared to the control groups receiving either empty liposomes or PBS at the end of the treatment period. However, when the treatment was stopped, the lesion size progressed and spleen parasite burden increased in LMA and LMA-OA groups, but still was significantly less than the control groups (P<0.05). There was no significant difference between the two formulations of LMA and LMA-OA. The results suggested that topical liposomes containing MA might be an appropriate choice for clinical trials for the treatment of CL.

Interleukin 4 (IL-4) gene promoter polymorphisms in Rhombomys opimus, the main reservoir of zoonotic cutaneous leishmaniasis.

Great gerbils (Rhombomys opimus) are the most common gerbils in center to northeast of Iran as well as central Asia and serve as reservoirs for the zoonotic agents, including Leishmania major, the principal etiologic agent of zoonotic cutaneous leishmaniasis (ZCL). The outcome of L. major infection in gerbils is not uniform. Among several immune-related factors including cytokine genes, the polymorphism in interleukin 4 (IL-4) promoter gene showed a great impact on outcome and pathological symptoms of L. major infection at least in mouse model. In this study gerbils' IL-4 promoter gene polymorphism is assessed. Specific primers were designed to develop a PCR-based assay to amplify IL-4 promoter gene to possibly define IL-4 promoter gene polymorphism in great gerbil populations with a range of Leishmania infection and symptoms collected from different foci of the central, north and northeast regions of Iran. The results showed that the designed primers amplify 689bp of the promoter gene. Sequence analysis of the promoter gene revealed five polymorphic sites assembly six haplotypes among the gerbil populations. Further studies are needed to assess whether or not the five polymorphisms cause different outcome phenotypes following infection with L. major in great gerbils. The data might be used to characterize the immune responses of R. opimus against L. major infection.

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

BACKGROUND: 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. METHODS: 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 µg 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. RESULTS: The PTR1 protein was not expressed in pcDNA-rPTR- tansfected promastigotes and mouse macrophage transfected with pcDNA-rPTR- tansfected promastigotes. CONCLUSION: 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.

TLR2 and TLR4 in cutaneous leishmaniasis caused by Leishmania major.

Cutaneous leishmaniasis (CL) is a self-healing skin disease which rarely for unknown reason(s) the lesion develops to a non-healing form. It seems that the initial contact of Leishmania parasites with the host innate immune system is an important step in the outcome of the disease. Recent studies suggested that toll-like receptors (TLRs) play a role in Leishmania recognition. In this study, the level of TLR2 and TLR4 was checked in patients with healing form of lesion and compared with that of patients with non-healing form of lesion caused by Leishmania major. Gene expression of TLR2 and TLR4 in peripheral blood-derived macrophages, before and after stimulation with live L. major promastigotes, was evaluated using quantitative real-time reverse transcription PCR and flow cytometry. The results showed that the mean relative gene expression and difference membrane expression of TLR2 in macrophages of patients with healing form of lesion were significantly higher than patients with non-healing form of lesion (P < 0.0001 and P = 0.0034), respectively, and the mean relative gene expression and difference in protein expression of TLR4 in macrophages of patients with healing form of lesion were significantly higher than that of patients with non-healing form of lesion (P = 0.021 and P = 0.002), respectively. The data suggested a possible role for TLR2 and TLR4 in the outcome of CL lesion. Further studies are needed to understand more about the detail role of the immune factors in leishmaniasis.

Efficacy of Microwave and Infrared Radiation in the Treatment of the Skin Lesions Caused by Leishmania major in an Animal Model.

BACKGROUND: 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. METHODS: 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 every 10 days. RESULTS: 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. CONCLUSION: 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.

IL-23 and IL-27 Levels in Macrophages Collected from Peripheral Blood of Patients with Healing Vs Non-Healing Form of Cutaneous Leishmaniasis.

BACKGROUND: 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. METHODS: 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. RESULTS: 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). CONCLUSION: 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.

Interleukin-10 and Transforming Growth Factor-ß in Early and Late Lesions of Patients with Leishmania major induced Cutaneous Leishmaniasis.

BACKGROUND: 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)-ß 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-ß in old world cutaneous leismaniasis. The aim of this study was to analyze the role of IL-10 and TGF-ß in human cutaneous leishmaniasis due to Leishmania major infection. METHODS: Biopsies were obtained from lesions of twenty proven cases of L. major induced cutaneous leishmaniasis. IL-10 and TGF-ß positive cells were detected by immunofluorescence staining of frozen sections and compared between two groups of patients with early and late lesions. RESULTS: 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-ß with mean percentages of 0.16±0.05 and 0.53±0.28 in early and late lesions respectively (P= 0.008). CONCLUSION: IL-10 and TGF-ß 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-ß in Early and Late Lesions of Patients with Leishmania major Induced Cutaneous Leishmaniasis.

BACKGROUND: 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)-ß 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-ß in old world cutaneous leismaniasis. The aim of this study was to analyze the role of IL-10 and TGF-ß in human cutaneous leishmaniasis due to Leishmania major infection. METHODS: Biopsies were obtained from lesions of twenty proven cases of L. major induced cutaneous leishmaniasis. IL-10 and TGF-ß positive cells were detected by immunofluorescence staining of frozen sections and compared between two groups of patients with early and late lesions. RESULTS: 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-ß with mean percentages of 0.16±0.05 and 0.53±0.28 in early and late lesions respectively (P= 0.008). CONCLUSION: IL-10 and TGF-ß are present in lesions of L. major induced cutaneous leishmaniasis and contribute to the pathogenesis of long lasting disease forms.

Cloning, expression and dynamic simulation of TRYP6 from Leishmania major (MRHO/IR/75/ER).

Leishmania, a digenetic protozoan parasite causes severe diseases in human and animals. Efficient evasion of toxic microbicidal molecules, such as reactive oxygen species and reactive nitrogen species is crucial for Leishmania to survive and replicate in the host cells. Tryparedoxin peroxidase, a member of peroxiredoxins family, is vital for parasite survival in the presence of antioxidant, hence it is one of the most important molecules in Leishmania viability and then, it may be an appropriate goal for challenging against leishmaniasis. After cloning and sub-cloning of TRYP6 from Leishmania major (MRHO/IR/75/ER), homology modeling of the LmTRYP6 was proposed to predict some functional property of this protein. The refined model showed that the core structure consists of a seven ß stranded ß-sheet and five α helices which are organized as a central 7-stranded ß2-ß1-ß5-ß4-ß3-ß6-ß7 surrounded by 2-stranded ß-hairpin, α helices A and D on one side, and α helices B, C and E on the other side. The peroxidatic active site is located in a pocket formed by the residue Pro45, Met46, Thr49, Val51, Cys52, Arg128, Met147 and Pro 148. The catalytic Cys52, located in the first turn of helix αB, is in van der Waals with a Pro45, a Thr49 and an Arg128 that are absolutely conserved in all known Prx sequences. In this study, an attractive molecular target was studied. These results might be used in designing of drugs to fight an important human pathogen.

Production and Purification of Anti-Rhombomys opimus Immunoglobulins.

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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.