Iranian

---

Delayed-type hypersensitivity reaction measured by skin testing, is an important tool for evaluation of cellular immune response in leishmaniasis and has been used recently as an indication of exposure to infection. Skin testing in leishmania infection needs a standard reagent with high specificity, sen‌sitivity, and potency. In the present work, a soluble leishmanin, and three whole parasite prepara‌tions from Leishmania major, i.e. thimerosal, phenol, and autoclaved leishmanins, were prepared un‌der standard conditions. These antigens were evaluated and compared in different foci of leishmani‌asis. The sensitivity and potency of reagents were tested in both recovered cases and patients with ac‌tive ulcer in two areas endemic to urban and rural leishmaniasis. Data obtained showed that, the sensitivity of thimerosal, phenol, and soluble leishmanins are almost equal, but are higher than auto‌claved one. Moreover, the potency of soluble leishmanin was also proved to be higher than other reagents. The purity of soluble leishmanin is higher than other three particulate preparations, and it lacks the majority of membrane antigens. As a result this reagent can be used as a standard and ideal antigen for skin testing in human leishmaniasis.

---

Partially purified antigenic fractions of Leishmania major promastigotes were obtained by sodium dodecyl sulfate (SDS-PAGE) and electro-elution. Three isolated protein fractions designated as frac‌tions (Fr.) 1, 2, and 3 correspond to 40-60, 60-80, 80-100 kilodalton (kDa) respectively. They were used for immunization of BALB/c mice against Leishmaniasis. The effects of these fractions on immune response of BALB/c mice against leishmanial infections was investigated by studying the infection course in infected mice, delayed type hypersensitivity (DTH) skin test, lymphocyte proliferation assay (LTT) in them. Subcutaneous immunization of mice with fraction 2 in conjugation with Complete Freund's Adjuvant (CFA) developed partial immunity against Leishmania major infection, and in‌duced specific DTH response. Meanwhile this fraction exhibited no exacerbating effect on leishmanial infection course. Subcutaneous immunization with fraction 1 also induced partial protection in lesser extent than fraction 2 against leishmanial infection.

---

Both urban and rural cutaneous leishmaniasis (CL) are endemic in different parts of Iran and have long been recognized in most provinces. However, there is no report of endemicity of CL in rural areas of Kashan, 200 km north of Isfahan and 260 km south of Tehran, Iran. To our knowledge, this is the first report of outbreak of cutaneous leishmaniasis in this area. To study and identify the nature of infection in this area, a survey was conducted in a borough, 30 Km south-east of Kashan on 3,530 people, using questionnaire for obtaining clinical status, and leishmanin skin test as an indicator of the prevalence of infection. In addition, 42 lesion samples from patients residing in this area were cultured, which twenty eight of them were positive. By enzyme immunoassay, using three species-specific monoclonal antibodies against common species of Leishmania in Iran, species of isolates were identified. Some of these isolates were also analyzed by PCR. The results showed the prevalence of 6% patients with lesion(s) in the population and 3.1% of people displayed lesion scar(s). Also 12% of population showed sensitivity to leishmanin. In this study the Leishmania major strain was identified in majority of cases, which showed two different patterns when analyzed by PCR. Both patterns belonged to different endemic strains of L. Major in Isfahan, which indicates the possible transmission of infection from Isfahan to this area.

---

The early cytokines production in response to live logarithmic and stationary phase promastigotes of Leishmania major was examined on the peripheral blood cells of the healthy individuals. Whole blood cultures were stimulated by either logarithmic or stationary phase promastigotes. IFN- and IL-10 productions were assessed by specific sandwich ELISA. The results showed that the logarithmic promastigotes were more potent than the stationary promastigotes in induction of IFN-. In contrast, IL-10 production was significantly higher in the supernatants of the cells stimulated by the stationary promastigotes compared to the logarithmic phase of the parasites. The effect of BCG on IL-10 and IFN- productions induced by these two types of promastigotes was also studied. BCG had augmenting effect on the cytokine production. However, the difference between the logarithmic and the stationary promastigotes was still observed, since logarithmic parasites induced higher amount of IFN- and lower amount of IL-10 compared to the stationary parasites. In parallel, intracellular expression of IL-12 and IL-10 in the CD14+ cells was studied and the same results were obtained. The logarithmic phase parasites induced significantly higher expression of IL-12 and lower expression of IL-10 compared to the stationary phase parasites which induced lower expression of IL-12 and higher expression of IL-10. These results demonstrated that logarithmic promastigotes of L. major are more potent to induce Th1 response than stationary promastigotes which might have implication in vaccine preparation.

---

Leishmaniasis is a major infectious disease of considerable public health in more than 86 countries around the world. Several approaches toward vaccine development against this disease have been taken. Glycoprotein (gp63) is conserved among diverse species of Leishmania and has induced immunological responses in murine models. Therefore, this glycoprotein has been considered as a second generation vaccine for Leishmaniasis and for potential diagnostic antigen. Recombinant vaccine using gp63 in cocktail form is one of the candidates. Since, Pichiapastoris expression system is similar to that of the eukaryotic genes, refolding and glycosylation aspects of the expressed protein, gp63 gene from NIH strain of L. major cloned into BamHI site of pHIL-S1 as yeast expression vector (shuttle vector). This vector carries sequences of acid phosphatase (PHO1) signal peptide from yeast. The construction transfected into the P. pastoris using lithium chloride method. Recombinant clones were screened on histidin minus media. The expression of rgp63 was studied by methanol after induction. The expressed recombinant protein was confirmed by Western blotting and electron microscopy. The expression level of rgp63 was more than 30%. Since the rgp63 expression in P. pastoris was active in SDS-PAGE gelatin gel, therefore it should be very similar to the native form.

---

Both zoonotic and anthroponotic cutaneous leishmaniasis (CL) caused by L. major and L. tropica, respectively, are endemic in different parts of Iran. This study was performed to investigate the new changes in epidemiological pattern of CL, and to identify the species of Leishmania and the strains of L. major isolated from different endemic areas of Iran. Seventy-two isolates from 245 samples collected from different endemic areas of Iran were characterized by monoclonal antibodies (mAb) specific for L. major, L. tropica, and L. infantum. Flow cytometry, isoenzyme analysis and PCR amplification were used for identification. Isoenzyme analysis was carried out by PAGE and cellulose acetate. Eight enzymes including malate dehydrogenase (MDH), malic enzyme (ME), glucose 6-phosphate dehydrogenase (G6PD), glucose phosphate isomerase (GPI), nucleoside hydrolase inosine (NHi), nucleoside hydrolase deoxy inosine (NHd), superoxide dismutase (SOD) and 6-phosphogluconate dehydrogenase (6PGD) were used for isoenzyme analysis. PCR assay was developed using specific primers against kinetoplast DNA. The isolates were compared with reference strains (RS). Data obtained from different methods showed 45 out of 72 isolates were similar to RS of L. major and 22 similar to L. tropica, and also five samples were identified as Crithidia. Isoenzyme migration pattern of L. major isolates was indistinguishable from each other in six enzymatic systems but differences were observed in profile of two enzymes of SOD and MDH. The data indicate that L. major species are dominant in the studied endemic areas, and different strains of L. major are present in different geographic areas of Iran. Moreover, it seems that enzymatic system is more useful approach than others for characterization of Leishmania species and L. major strains

---

The onset of infections by Leishmania parasites mainly caused by amastigote growth inside the macrophages. One of the important properties of lesion-derived amastigote is thought to be the attachment of various host proteins including immunoglobulins on the surface of amastigote. In this study, the presence of immunoglobulins on the surface of lesion-derived amastigotes was detected by Western blotting using three different peroxidase conjugated anti-heavy chain antibodies and peroxidase conjugated anti-mouse IgG antibody. Then, a new technique was developed for isolation of lesion-derived amastigotes. This technique simply consists of a microbiological plate covered by rabbit anti-mouse immunoglobulins. Overnight incubation of the infected cell suspension isolated from mice lesion on such plate at 4°C, was followed by gentle washing and isolation of the amastigotes. The results showed that the surface of amastigote has covered with different amount of immunoglobulins such as IgG, IgM, and IgA detected by pixel analysis software. With this technique, which was comparable with other techniques, pure amastigote was isolated. This is a simple and reliable method for isolation of real amastigotes from lesion of the infected BALB/c mice

---

Recent scientific evidence indicates that distinct patterns of susceptibility in BALB/c mice to Leishmania major infection are attributable to the differential expansion of distinct CD4+ T-cell subsets and their cytokines production. Production of the Th1 cytokine IFN-g is associated with resistance, whereas production of the Th2 cytokines IL-4 and IL-10 are associated with extreme susceptibility. The major host immune defense mechanism against Leishmania is activation of macrophages by INF-γ derived from T cells. The inability of susceptible hosts to mount the immune response necessary to activate macrophage and destroy the parasites may be due to the parasite-specific proteins that are able to suppress the immune system. In the present study, we have semi-purified the excreted antigens of Leishmania major promastigote and amastigote by column chromatography. The isolated fraction showed a potent immunosuppressive activity on normal BALB/c mice lymphocytes stimulated with mitogens. Fifteen microgram of the isolated fraction caused 81% suppression of lymphocyte proliferation. These data may suggest that the parasite by secreting immunosuppressive factor down regulate the immune system and as a result survive in the body

---

capture ELISA system was developed for diagnosis of visceral leishmaniasis (VL) using a monoclonal antibody raised against an antigen previously detected in the urine of VL patients. Urine samples from confirmed VL cases from Yemen, Nepal, Spain, Sudan and Brazil were tested in the capture ELISA in comparison with urine samples from endemic and non-endemic areas along with urine samples from patients with malaria, brucellosis, schistosomiasis and patients with non-infectious diseases. All of VL patient samples from different geographical areas (apart from 2 samples from Brazil) gave a positive result, while no cross-reaction was found with the control samples. The results obtained with the capture-ELISA were compared to those obtained with KAtex, a previously described latex agglutination test, showed that the KAtex and the new ELISA are comparable in terms of specificity (100%) but a better sensitivity (94.1%) was found for the capture-ELISA. Moreover, the capture-ELISA adds a useful quantitative dimension to antigen detection. In addition, the boiling of urine samples, which is necessary for KAtex, was not required in the capture-ELISA. These results suggest that the antigen detection in urine by the new capture ELISA system provides a useful method for diagnosis of VL and fulfils the requirements of a non-invasive method for diagnosis of VL

---

It is now well documented that interferon gamma (IFN-γ) is the indispensable cytokine for inducing protective immunity against experimental and human cutaneous leishmaniaisis. The importance of IFN-γ receptor (IFN-γR) has also been studied. In the present study, we made attempts to find out whether L. major infection is able to alter the expression of IFN-γR in vivo. In addition, we studied the responsiveness to IFN-γ ex vivo. To do that, we assessed the expression of CD119 (IFN-γRα ) on CD45+ cells isolated from draining lymph nodes of infected and uninfected BALB/c and C57BL/6 mice by flow cytometry. The MFI (mean fluorescence intensities) of CD119 on uninfected BALB/c mice were 192.8 ± 18.4 but the CD119 MFI of infected BALB/c mice were remarkably decreased (107.9±40.8). CD119 MFI of uninfected and infected C57BL/6 mice were 276.2 ± 17.1 and 140.4±43.0 respectively Moreover, we measured the production of nitric oxide (NO) by these cells in the presence of IFN-γ in order to study the function of IFN-γR. NO production by draining lymph nodes cells of infected C57BL/6 mice in response to recombinant murine IFN-γ was significantly higher than the cells of infected BALB/c mice (37.5 ± 0.6 and 11.6 ± 0.5 µM respectively, p<0.05). Therefore, our results confirm the in vitro reports regarding the impairment of IFN-γ responsiveness due to Leishmania infection.

---

Background: The therapy of leishmania infection is difficult and each year 1.5 million new cases of cutaneous leishmaniasis and 500,000 new cases of visceral leishmaniasis are estimated, therefore, there is a need for an effective vaccine. Monoclonal antibody (mAb) is one of the suitable methods for isolation and purification of leishmania antigens. In this report, we produced several mAb against leishmania infantum antigens for antigen purification to be used as candidate vaccine. Methods: BALB/c mice were injected with freeze-thawed promastigote twice together with Freund adjuvant. Three days before fusion, antigen in saline was injected into the tail vain and then mice were killed and the spleen lymphocytes were fused with myeloma SP2/0. Results: Five mAb against promastigote form of Leishmania infantum parasite were obtained. Western-blot analysis showed that these mAb recognize a band of 57- kDa protein either in parasite lysate or on whole L. infantum, L. tropica, L. major and L. donovani. It seems that the 57 kDa-protein is the major surface leishmania antigen (gp63) that is neither stage-specific nor differentially regulated. These mAb do not recognize the recombinant gp63 antigen and seems recognizing only the native form of a gp63 isoform. The IgG1 mAb was purified by affinity column and was used to purify 57 kDa antigens from Leishmania lysate. Conclusion: Since these antibodies recognizing one specific protein band in 4 different strains of leishmania, they could be used for leishmania diagnostic kits and also for purification of antigen to be tested for its protective effect against leishmania infection.

---

Background: Currently, there are no effective vaccines against leishmaniasis, and treatment using pentavalent antimonial drugs is occasionally effective and often toxic for patients. The PTR1 enzyme, which causes antifolate drug resistance in Leishmania parasites encoded by gene pteridine reductase 1 (ptr1). Since Leishmania lacks pteridine and folate metabolism, it cannot synthesize the pteridine moiety from guanine triphosphate. Therefore, it must produce pteridine using PTR1, an essential part of the salvage pathway that reduces oxidized pteridines. Thus, PTR1 is a good drug-target candidate for anti-Leishmania chemotherapy. The aim of this study was the cloning, expression, and enzymatic assay of the ptr1 gene from Iranian lizard Leishmania as a model for further studies on Leishmania. Methods: Promastigote DNA was extracted from the Iranian lizard Leishmania, and the ptr1 gene was amplified using specific primers. The PCR product was cloned, transformed into Escherichia coli strain JM109, and expressed. The recombinant protein (PTR1 enzyme) was then purified and assayed. Results: ptr1 gene was successfully amplified and cloned into expression vector. Recombinant protein (PTR1 enzyme) was purified using affinity chromatography and confirmed by Western-blot and dot blot using anti-Leishmania major PTR1 antibody and anti-T7 tag monoclonal antibody, respectively. The enzymatic assay was confirmed as PTR1 witch performed using 6-biopterin as a substrate and nicotinamide adenine dinucleotide phosphate as a coenzyme. Conclusion: Iranian lizard Leishmania ptr1 was expressed and enzymatic assay was performed successfully.

---

Background: Zoonotic cutaneous leishmaniasis (ZCL) due to Leishmania major is increasing in many parts of Iran. This disease originally is a disease found in gerbils. Leishmania parasites are transmitted by sandflies that live and breed in gerbil burrows. Nested PCR amplified Leishmania ITS1-5.8S rRNA gene in both main reservoir host “Rhombomys opimus” and in the “Phlebotomus papatasi” main vector of ZCL, in Iran. Population differentiation and seasonal variation of sandflies were analyzed at a microgeographical level in order to identify any isolation by distance, habitat or seasons. Methods: Populations of sandflies were sampled from the edges of villages in Natanz, Isfahan province, Iran, using the Centers for Disease Control traps and sticky papers. Individual sandflies were identified based on external and internal morphological characters. Nested PCR protocols were used to amplify Leishmania ITS1-5.8S rRNA gene, which were shown to be species-specific via DNA sequence. Results: A total of 4500 sandflies were collected and identified. P. papatasi, Phlebotomus sergenti and Phlebotomus jacusieli from genus Phlebotomus and Sergentomyia sintoni and Sergentomyia clydei from genus Sergentomyia were identified in this region. P. papatasi was the most abundant sandfly in the collections. Ten out of 549 female P. papatasi and four out of 19 R. opimus were found to be infected with L. major. Conclusion: Seasonal activity of sandflies starts in June and ends in November. Abundance of P. papatasi was in September. Finding and molecular typing of L. major in P. papatasi and R. opimus confirmed the main vector and reservoir in this region.

---

Background: Heat shock proteins (HSP) are highly conserved molecules with many immunological functions. They are highly immunogenic with important role in cancer immunotherapy and in vaccine development against infectious diseases. As adjuvant, HSP can augment the immunogenicity of weak antigens and can stimulate antigen presenting cells. Although vaccines have been successful for many infectious diseases, progress in leishmaniasis has not been achieved. In this report, the protective effect of HSP-enriched soluble leishmania antigen (SLA) was determined. Methods: BALB/c mice were immunized 3× with HSP-enriched SLA and SLA alone and 10 days after final boost. They were infected with 106 stationary phase promastigote of Leishmania major and immunological responses were followed until nine weeks. Results: No significant differences were observed in lymphocyte proliferation, footpad swelling, parasite burden, nitric oxide or IL-12 cytokine between HSP-enriched or SLA groups. Although the levels of IFN-γ, IL-4, TGF-β, IgG1 and IgG2b were increased in both groups, IFN-γ was significantly higher in SLA group and IgG2a in HSP-enriched SLA. Conclusion: These results indicate that HSP direct the immune system towards Th2 pattern and does not have protective role in L. major infection.

---

Background: Molecular diversity of Leishmania major and its morphological changes have become a controversial issue among researchers. Some aspects of polymorphic shapes of amastigotes in clinical manifestations along with molecular variation were evaluated among suspected patients of some exceptional zoonotic cutaneous leishmaniasis locations in Northern Khuzestan, Southwestern Iran. Methods: Suspected patients (n = 165) were sampled in zoonotic cutaneous leishmaniasis foci over two consecutive years during 2012-2014. Prepared smears were stained, scaled and measured by ocular micrometer. DNA was extracted from smears ITS-rDNA and Cytochrome b (Cyt b) markers were amplified, and PCR products were digested by BsuR1 restriction enzyme. Then the RFLP and sequencing were employed. Results: Only L. major was identified in patients containing regular amastigotes' shapes (oval or round) with a size of 2-4 µm in each of classical wet, dry, mixed lesions. Meanwhile, irregular shapes (spindle, pear, or cigarette) were observed separately in non-classical wet lesions with more than 4 µm. Interestingly, a few amastigotes with an external flagellum were observed in some lesions. All sequenced ITS-rDNA and Cyt b genes of L. major did not show any molecular variation (&chi 2 P > 0.05), including only one common haplotype (GenBank access no. EF413075). Conclusion: Findings proved that unlike other endemic foci, there is not a meaningful correlation between phenotypic and genotypic features of L. major isolates. This study is considered as the first comprehensive report to incriminate morphometric shapes of L. major amastigotes, which enhances our knowledge concerning their relevance with various clinical appearances and genotypic traits.

---

Background: Cutaneous leishmaniasis is one of the most important parasitic diseases in humans. In this disease, one of the responsible organisms is Leishmania major, which is transmitted by sandfly vector. There are specific differences in biochemical profiles and metabolite pathways in logarithmic and stationary phases of Leishmania parasites. In the present study, ¹H NMR spectroscopy was used to examine the metabolites outliers in the logarithmic and stationary phases of promastigotes in L. major to enlighten more about the transmission mechanism in metacyclogenesis of L. major. Methods: Promastigote was cultured, logarithmic and stationary phases were separated by the peanut agglutinin, and cell metabolites were extracted. ¹H NMR spectroscopy was applied, and outliers were analyzed using principal component analysis. Results: The most altered metabolites in stationary and logarithmic phases were limited to citraconic acid, isopropylmalic acid, L-leucine, ornithine, caprylic acid, capric acid, and acetic acid. Conclusion: ¹H NMR spectroscopy could play an important role in the characterization of metabolites in biochemical pathways during a metacyclogenesis process. These metabolites and their pathways can help in exploiting a transmission mechanism in metacyclogenesis, and outcoming data might be used in the metabolic network reconstruction of L. major modeling.

---

Visceral leishmaniasis (VL) is a zoonotic disease caused by leishmania species. Dogs are considered to be the main reservoir of VL. A number of methods and antigen-based assays are used for the diagnosis of leishmaniasis. However, currently available methods are mainly based on direct examination of tissues for the presence of parasites, which is highly invasive. A variety of serological tests are commonly applied for VL diagnosis, including indirect fluorescence antibody test, enzyme-linked immunosorbent assay (ELISA), dot-ELISA, direct agglutination test, Western-blotting, and immunochromatographic test. However, when soluble antigens are used, serological tests are less specific due to cross-reactivity with other parasitic diseases. Several studies have attempted to replace soluble antigens with recombinant proteins to improve the sensitivity and the specificity of the immunodiagnostic tests. Major technological advances in recombinant antigens as reagents for the serological diagnosis of VL have led to high sensitivity and specificity of these serological tests. A great number of recombinant proteins have been shown to be effective for the diagnosis of leishmania infection in dogs, the major reservoir of L. infantum. Although few recombinant proteins with high efficacy provide reasonable results for the diagnosis of human and canine VL, more optimization is still needed for the appropriate antigens to provide high-throughput performance. This review aims to explore the application of different recombinant proteins for the serodiagnosis of VL in humans and dogs.

---

Background: The expression of bio-therapeutic proteins in mammalian cells, such as CHO, attains high homogeneity related to post-translational modifications. Although CHO remains the most popular cell line for bestselling biotherapeutic proteins on the market, there are still drawbacks such as expensive culture media, long time line, and high drug cost. Recently, researches on a novel Leishmania protozoan system have confirmed that this low level eukaryote could represent a competitive alternative to the mammalian cell lines. Methods: The full length of coding sequence of modified tPA TNKase tenecteplase) was synthesized and cloned into an inducible expression vector of L. tarentolae T7-TR cells. Results: The expression of the construct was driven by a Tet-inducible promoter. A Leishmania secretory signal sequence was also added to the expression cassette to facilitate the release of the recombinant protein into the medium. The secretory recombinant protein was analyzed and confirmed by SDS-PAGE and Western blot analyses. The expression level of TNKase in this novel system of L. tarentolae was 810 IU/mL after induction, which means that the percentage of expression increases two times compared to previous models in L. tarentolae. The TNKase activity was comparable with Activase. Conclusion: Our results suggested that expressed TNK (modified tPA) is functionally compatible with Activase regarding their effect on fibrinolysis. Given the post-translational modification similarities between mammalian and L. tarentolae, it is speculated that this system is capable of producing complex proteins such as tPA similar to mammalian system, with easier manipulation and non-expensive method.

---

Background: Leishmania tropica is the cause of more than one form of leishmaniasis and lacks a known reservoir animal. This study compares the potential infectivity of recombinant and wild-type L. tropica in BALB/c mice. Methods: The potential infectivity of recombinant L. tropica^EGFP or L. tropica^EGFP-LUC by two different, the subcutaneous and intradermal, routes was compared using a range of classical detection methods and bioluminescence imaging (BLI). Results: In addition to the results obtained from classical diagnostic approaches, the BLI signals were detected in footpads and ears of L. tropica-infected animals. The BLI revealed that a bioluminescence signal can be observed at the inoculation site. The stability of the BLI remained constant in the footpad, but the signal was detectable for only three months in the pinna due to the decline in infection over time. Conclusion: The presented data are a precise verification of the assumption that BALB/c mice could be used as an experimental model for L. tropica infectivity. 

---

Leishmaniasis is caused by protozoan Leishmania parasites that are transmitted through female sandfly bites. The disease is predominantly endemic to the tropics and semi-tropics and has been reported in more than 98 countries. Due to the side effects of anti-Leishmania drugs and the emergence of drug-resistant isolates, there is currently no encouraging prospect of introducing an effective therapy for the disease. Hence, it seems that the key to disease control management is the introduction of an effective vaccine, particularly against its cutaneous form. Advances in understanding underlying immune mechanisms are feasibale using a variety of candidate antigens, including attenuated live parasites, crude antigens, pure or recombinant Leishmania proteins, Leishmania genes encoding protective proteins, as well as immune system activators from the saliva of parasite vectors. However, there is still no vaccine against different types of human leishmaniasis. In this study, we review the works conducted or being performed in this field.