Overexpression of miR-32 in Chinese hamster ovary cells increases production of Fc-fusion protein.

The demand for industrial genetically modified host cells were increased with the growth of the biopharmaceutical market. Numerous studies on improving host cell productivity have shown that altering host cell growth and viability through genetic engineering can increase recombinant protein production. During the last decades, it was demonstrated that overexpression or downregulation of some microRNAs in Chinese Hamster Ovary (CHO) cells as the host cell in biopharmaceutical manufacturing, can improve their productivity. The selection of microRNA targets has been based on their previously identified role in human cancers. MicroRNA-32 (miR-32), which is conserved between humans and hamsters (Crisetulus griseus), was shown to play a role in the regulation of cell proliferation and apoptosis in some human cancers. In this study, we investigated the effect of miR-32 overexpression on the productivity of CHO-VEGF-trap cells. Our results indicated that stable overexpression of miR-32 could dramatically increase the productivity of CHO cells by 1.8-fold. It also significantly increases cell viability, batch culture longevity, and cell growth. To achieve these results, following the construction of a single clone producing an Fc-fusion protein, we transfected cells with a pLexJRed-miR-32 plasmid to stably produce the microRNA and evaluate the impact of mir-32 overexpression on cell productivity, growth and viability in compare with scrambled control. Our findings highlight the application of miRNAs as engineering tools and indicated that miR-32 could be a target for engineering CHO cells to increase cell productivity.

Multiple myeloma cell-derived exosomes promote favorable tumor functional performance by polarizing macrophages toward M2-like cells.

It has long been hypothesized that leukemic cells are able to modulate the fate of resident cells in the tumor microenvironment (TME) toward either supporting or immunosuppressive cells for the development of tumors. Exosomes can be a potential culprit in imposing tumor desire. There is evidence about the impact of tumor-derived exosomes on different immune cells in different malignancies. However, findings about macrophages are contradictory. Here, we evaluated the potential influence of multiple myeloma (MM)-cell-derived exosomes on the polarization of macrophages by examining hallmarks of M1 and M2 macrophages. After treatment of M0 macrophages with isolated exosomes (from U266B1), gene expression (Arg-1, IL-10, TNF-α and IL-6), immunophenotyping markers (CD206), cytokine secretion (IL-10 and IL-6), nitric oxide (NO) production, and redox potentiality of target cells were assessed. Our results revealed significantly increased expression of the genes involved in the development of M2-like cells but not M1 cells. The CD 206 marker and IL-10 protein levels were significantly increased at different time points. The expression of IL-6 mRNA and IL-6 protein secretion did not change significantly. MM-cell-derived exosomes induced significant changes in NO production and intracellular ROS levels in M0 cells.

Recent developments in miRNA based recombinant protein expression in CHO.

It is widely accepted that the growing demand for recombinant therapeutic proteins has led to the expansion of the biopharmaceutical industry and the development of strategies to increase recombinant protein production in mammalian cell lines such as SP2/0 HEK and particularly Chinese hamster ovary cells. For a long time now, most investigations have been focused on increasing host cell productivity using genetic manipulating of cellular processes like cell cycle, apoptosis, cell growth, protein secretory and other pathways. In recent decades MicroRNAs beside different genetic engineering tools (e.g., TALEN, ZFN, and Crisper/Cas) have attracted further attention as a tool in the genetic engineering of host cells to increase protein expression levels. Their ability to simultaneously target multiple mRNAs involved in one or more cellular processes made them a favorable tool in this field. Accordingly, this study aimed to review the methods of selecting target miRNA for cell line engineering, miRNA gain- or loss-of-function strategies, examples of laboratory and pilot studies in this field and discussed advantages and disadvantages of this technology.

The potential therapeutic role of PTR1 gene in non-healing anthroponotic cutaneous leishmaniasis due to Leishmania tropica.

BACKGROUND: Drug resistance is a common phenomenon frequently observed in countries where leishmaniasis is endemic. Due to the production of the pteridine reductase enzyme (PTR1), drugs lose their efficacy, and consequently, the patient becomes unresponsive to treatment. This study aimed to compare the in vitro effect of meglumine antimoniate (MA) on non- healing Leishmania tropica isolates and on MA transfected non-healing one to PTR1. METHODS: Two non-healing and one healing isolates of L. tropica were collected from patients who received two courses or one cycle of intralesional MA along with biweekly liquid nitrogen cryotherapy or systemic treatment alone, respectively. After confirmation of L. tropica isolates by polymerase chain reaction (PCR), the recombinant plasmid pcDNA-rPTR (antisense) was transfected via electroporation and cultured on M199. Isolates in form of promastigotes were treated with different concentrations of MA and read using an enzyme-linked immunosorbent assay (ELISA) reader and the half inhibitory concentration (IC50 ) value was calculated. The amastigotes were grown in mouse macrophages and were similarly treated with various concentrations of MA. The culture glass slides were stained, and the mean number of intramacrophage amastigotes and infected macrophages were assessed in triplicate for both stages. RESULTS: All three transfected isolates displayed a reduction in optical density compared with the promastigotes in respective isolates, although there was no significant difference between non-healing and healing isolates. In contrast, in the clinical form (amastigotes), there was a significant difference between non-healing and healing isolates (p < 0.05). CONCLUSION: The results indicated that the PTR1 gene reduced the efficacy of the drug, and its inhibition by antisense and could improve the treatment of non-healing cases. These findings have future implications in the prophylactic and therapeutic modality of non- healing Leishmania isolates to drug.

Enhancement of Expression Level of Modified t-PA (TNKase) in Leishmania tarentolae by Induction System

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.

Monoclonal antibodies expression improvement in CHO cells by PiggyBac transposition regarding vectors ratios and design.

Establishing stable Chinese Hamster Ovary (CHO) cells producing monoclonal antibodies (mAbs) usually pass through the random integration of vectors to the cell genome, which is sensitive to gene silencing. One approach to overcome this issue is to target a highly transcribed region in the genome. Transposons are useful devices to target active parts of genomes, and PiggyBac (PB) transposon can be considered as a good option. In the present study, three PB transposon donor vectors containing both heavy and light chains were constructed, one contained independent expression cassettes while the others utilized either an Internal Ribosome Entry Site (IRES) or 2A element to express mAb. Conventional cell pools were created by transferring donor vectors into the CHO cells, whereas transposon-based cells were generated by transfecting the cells with donor vectors with a companion of a transposase-encoding helper vector, with 1:2.5 helper/donor vectors ratio. To evaluate the influence of helper/donor vectors ratio on expression, the second transposon-based cell pools were generated with 1:5 helper/donor ratio. Expression levels in the transposon-based cells were two to five -folds more than those created by conventional method except for the IRES-mediated ones, in which the observed difference increased more than 100-fold. The results were dependent on both donor vector design and vectors ratios.

Comparison of HCV Core and CoreE1E2 Virus-Like Particles Generated by Stably Transfected Leishmania tarentolae for the Stimulation of Th1 Immune Responses in Mice.

BACKGROUND: Virus-like particles (VLPs) could be improved into successful immunogens as well as a potent delivery vehicle, but however, the current expression systems for VLPs production have some limitations. METHOD: Recently, we developed a novel strategy to produce two HCV VLPs containing core or coreE1E2 proteins using stably transfected Leishmania tarentolae promastigotes. Then, BALB/c mice were injected by both viral like particles in different immunization strategies such as homologous DNA-, homologous VLP-, and heterologous DNA/ VLP-based immunizations. RESULTS: TEM microscopy indicated HCV core and HCV coreE1E2 VLP assembly with average size of 30-40 and 40-60 nm after purification, respectively. Our results showed that homologous immunizations with both HCV core or coreE1E2 VLPs significantly induced anti-core or anti- coreE1E2 antibody responses, respectively as well as secretion of IFN-γ cytokine as compared to other strategies. Moreover, DNA-prime/VLP-boost regimens significantly elicited higher levels of IFN-γ and antibody responses in comparison with homologous DNA/DNA regimens. The groups immunized with homologous or heterologous coreE1E2 VLPs showed markedly higher immune responses as compared to groups immunized with core VLP regimens against coreE1E2 protein. CONCLUSION: The crude HCV VLPs generated by Leishmania expression system could elicit a Th1- type response as a promising vaccine candidate against HCV infections.

VLP production in Leishmania tarentolae: A novel expression system for purification and assembly of HPV16 L1.

Viral like particles (VLPs) have been used as immunogen for improvement of preventive vaccines against several viral infections in preclinical and clinical trials. These constructs can stimulate both cellular and humoral immunity. Two prophylactic HPV L1 VLP vaccines known as Gardasil and Cervarix were commercialized worldwide. However, there are main problems for expression and purification of VLPs in eukaryotic expression systems such as baculovirus and yeast leading to high cost of these vaccines. A novel Leishmania protozoan system has been applied to produce different recombinant proteins due to unique properties including generation of similar proteins with mammalian, easy handling, and large-scale culture. In the current study, we developed a novel strategy to produce HPV L1 VLP using stably transfected Leishmania cells. The positive transfectants were analyzed by SDS-PAGE and Western blot analysis. The assembly of purified L1 protein was detected by TEM microscopy. Finally, C57BL/6 mice were immunized by crude VLPs and antibody responses were assessed. The results of electronic microscopy revealed average 55-60 nm for L1 VLP. Furthermore, high IgG1 and IgG2a antibody responses were generated by L1 VLPs in mice similar to L1 VLPs produced in baculovirus-infected insect cells. Regarding the results, the amount of recombinant protein generated by Leishmania was 2-3mg/500 ml media, suggesting further optimization of this system for using in large animals and human.

Expression and Purification of HCV Core and Core-E1E2 Proteins in Different Bacterial Strains.

BACKGROUND: Hepatitis C virus (HCV) is a main public health problem causing chronic liver infection and subsequently liver cirrhosis and lethal hepatocellular carcinoma (HCC). Vaccination based on HCV capsid proteins has attracted a special interest for prevention of viral infections. The core protein is a basic and evolutionary most conserved protein, which regulates the cellular processes related to viral replication and pathogenesis. The envelope E1 and E2 proteins involve in generation of the infectious particles, viral entry by binding to a host cell receptor, and modulation of the immune responses. OBJECTIVES: In current study, the efficient generation of recombinant core and core-E1E2 proteins was developed in bacterial expression systems. MATERIALS AND METHODS: The expression of HCV core and core-E1E2 proteins was performed using prokaryotic pET-28a and pQE-30 expression systems in BL21/ Rosetta, and M15 strains, respectively. The recombinant proteins were purified using affinity chromatography under native conditions and also reverse staining method. Finally, the levels of recombinant proteins were assessed by BCA kit and spectrophotometer. RESULTS: The data showed a clear band of ~573 bp for HCV core and ~2238 bp for core-E1E2 genes in agarose gel. Moreover, a ~21 kDa band of core protein and a ~83 kDa band of core-E1E2 protein were revealed in SDS-PAGE. The affinity chromatography could not purify the core and core-E1E2 proteins completely, because of low affinity to Ni-NTA bead in comparison with reverse staining method. CONCLUSIONS: This study is the first report for purification of HCV core and core-E1E2 proteins using the reverse staining procedure with no need of any chromatography columns. The BL21 strain was more potent than Rosetta strain for HCV core protein in pET 28a expression system. Furthermore, M15 strain was suitable for expression of coreE1E2 in pQE-30 bacterial system.

A dual drug sensitive L. major induces protection without lesion in C57BL/6 mice.

Leishmaniasis is a major health problem in some endemic areas and yet, no vaccine is available against any form of the disease. Historically, leishmanization (LZ) which is an inoculation of individual with live Leishmania, is the most effective control measure at least against cutaneous leishmaniasis (CL). Due to various reasons, LZ is not used today. Several live attenuated Leishmania have been developed but their use is limited. Previously, we developed a transgenic strain of L. major that harbors two suicide genes tk and cd genes (lmtkcd+/+) for use as a challenge strain in vaccine studies. These genes render the parasite susceptible to Ganciclovir (GCV) and 5-flurocytosine (5-FC). The dual drug sensitive strain of L. major was developed using gene targeting technology using a modified Herpes Simplex Virus thymidine kinase gene (hsv-tk) sensitive to Ganciclovir antibiotic and Saccharomyces cerevisae cytosine deaminase gene (cd sensitive to 5-flurocytosine) that were stably introduced into L. major chromosome. BALB/c mice inoculated with lmtkcd+/+ developed lesions which upon treatment with GCV and 5-FC completely healed. In the current study, the transgenic lmtkcd+/+strain was assessed as a live vaccine model to determine the time necessary to develop a protective immune response. C57BL/6 mice were inoculated with the transgenic lmtkcd+/+strain, and treated at the time of inoculation (day 0) or at day 8 after inoculation. Immunized animals were challenged with wild-type L. major, and complete protection was induced in mice that were treated at day 8. The results show that in contrast to leishmanization, in group of mice inoculated with a dual sensitive L. major development and persistence of lesion is not necessary to induce Th1 response and protection.

Identification of the serotypes of bacterial meningitis agents; implication for vaccine usage.

BACKGROUND AND OBJECTIVES: Bacterial meningitis is one of the most serious infections and should be treated as emergency. As it has significant morbidity and mortality throughout the world, every country should have precise information regarding the etiological agents of disease and populations at risk to design public health prevention strategy. In the present study in addition of evaluation of common etiological agents (Haemophilus influenzae, Neisseria meningitidis, and Streptococcus pneumoniae) in bacterial meningitis cases, we sero-grouped or serotyped the obtained agents in order to predict the usefulness of existing vaccines against bacterial meningitis. MATERIALS AND METHODS: Cerebrospinal fluid of 182 suspected meningitis patients were collected, from which 114 cases were approved by biochemical, microbiological and molecular tests as bacterial meningitis. The isolated bacteria were serogrouped or serotyped to determine the dominant serotypes. RESULTS: Streptococcus pneumoniae accounted for 36%, Haemophilus influenza for 26% and Neisseria meningitidis for 14% of cases. From 13 serogroups of N. meningitides the most frequent serogroups, were meningococcus group B (51%), C(24%) A (18%), Z(2%), W135 (1%) and 3% was not identified. In H. influenzae group only serotype b (100%) have been identified and in pneumococcal meningitis the most common serotype among our cases were 18C (44%) followed by14 (17%), 19A (13%), 6A (9%), 7F (4%), 4(3%), 3 (3%), 9V (2%), 8 (2%), 23f (2%), 5 (1%). CONCLUSION: Since there is no nationwide mass immunization program for common agents of bacterial meningitis in Iran, the result of this study can be used to improve the existing vaccines to cover the detected serotypes and consequently reduce the incidence of bacterial meningitis.

Anti-leishmanial and toxicity activities of some selected Iranian medicinal plants.

Leishmaniasis is caused by protozoan parasites belonging to the genus Leishmania. Cutaneous leishmaniasis is the most common form of leishmaniasis in Iran. As there is not any vaccine for leishmaniasis, treatment is important to prevent the spreading of parasites. There is, therefore, a need to develop newer drugs from different sources. The aim of this study was to assess anti-leishmanial activity of the ethanolic extracts of 17 different medicinal plants against Leishmania major promastigotes and macrophage cell line J774. The selection of the hereby studied 17 plants was based on the existing information on their local ethnobotanic history. Plants were dried, powdered, and macerated in a hydroalcoholic solution. Resulting extracts have been assessed for in vitro anti-leishmanial and brine shrimp toxicity activities. Four plants, Caesalpinia gilliesii, Satureia hortensis, Carum copticum heirm, and Thymus migricus, displayed high anti-leishmanial activity (IC50, 9.76 ± 1.27, 15.625 ± 3.76, 15.625 ± 5.46, and 31.25 ± 15.44 µM, respectively) and were toxic against the J774 macrophage cell line at higher concentrations than those needed to inhibit the parasite cell growth (IC50, 45.13 ± 3.17, 100.44 ± 17.48, 43.76 ± 0.78, and 39.67 ± 3.29 µM, respectively). Glucantime as positive control inhibited the growth of L. major promastigotes with IC50 = 254 µg/ml on promastigotes (1 × 10(6)/100 µ/well) of a log phase culture, without affecting the growth of J774 macrophages. These data revealed that C. gilliesii, S. hortensis, C. copticum heirm, and T. migricus extracts contain active compounds, which could serve as alternative agents in the control of cutaneous leishmaniasis. The activity of these herbs against L. major promastigotes and macrophage cell line J774 was reported for the first time in our study.

Cloning and expression of truncated form of tissue plasminogen activator in Leishmania tarentolae.

An expression cassette containing kringle 2 and serine protease domains (K2S), tissue plasminogen activator (tPA), together with a signal sequence derived from Leishmania tarentolae and two fragments of the small subunit ribosomal RNA locus, was introduced into L. tarentolae. The transfected cells produced recombinant K2S (rK2S) protein extracellularly with serine protease activity. Expression and enzyme activity of rK2S in the supernatant was 930 i.u./ml. The specific activity of purified rK2S was 7.4 U/mg of protein. Replacement of the human signal sequence tPA with the signal sequence derived from Leishmania increased the secretion of recombinant protein up to 30 times.

Increased expression of recombinant human tissue plasminogen activator in Leishmania tarentolae.

Recombinant tissue plasminogen activator (rt-PA) is one of the most important thrombolytic agents for treating cardiovascular obstructions such as stroke. Glycoprotein rt-PA is a serine protease, consisting of 527 amino acids of which 35 are cysteine residues. A variety of recombinant protein expression systems have been developed for heterologous gene expression in prokaryotic and eukaryotic hosts. In recent years, Leishmania tarentolae has been considered because of its safety aspects and special attributes in expression of complex proteins. In this study, two expression cassettes, each one including two copies of t-PA cDNA, were used for integration into the L. tarentolae genome by electroporation. Transformed clones were selected in the presence of appropriate antibiotics. Expression of active rt-PA was confirmed by Western blot and Zymography tests. Real-time PCR analysis was applied to investigate the presence of multiple t-PA gene copies in the parasite genome. Correlation of t-PA gene dosage and production rate was confirmed with real-time PCR. It was shown that the expression level of rt-PA in L. tarentolae is at least 480 IU/mL of culture media. This concentration of rt-PA is seven times higher than what was reported in previous studies in L. tarentolae and some other eukaryotic systems.

Expression of a novel chimeric truncated t-PA in CHO cells based on in silico experiments.

Tissue plasminogen activator (t-PA) is one of the fibrin-specific serine proteases that play a crucial role in the fibrinolytic system. The rapid clearance of the drug from the circulation, caused by its active uptake in the liver, has lead to complicated clinical applications. Different forms of plasminogen activators have been developed to treat thrombotic disease. Deletion of the first three domains of t-PA by gene manipulation techniques has shown a significant increase in its plasma half life. In order to compensate the disadvantage of higher bleeding risk, a novel chimeric truncated form of t-PA with 394 amino acids and more fibrin affinity compared to the truncated form was designed to be expressed in Chinese Hamster Ovarian (CHO) cells. The recombinant chimeric plasminogen activator consists of kringle 2 and serine protease (K2S) domains of t-PA, namely GHRP-SYQ-K2S. The level of expression was found to be 752 IU/ml with 566,917 IU/mg specific activity, based on amidolytic activity. The fibrin binding of this novel chimeric truncated t-PA was 86% of the full length t-PA at a fibrinogen concentration of 0.2 mg/ml. This could be a promising approach with more desirable pharmacodynamic properties compared to existing commercial forms.

Human Tissue Plasminogen Activator Expression in Escherichia coli using Cytoplasmic and Periplasmic Cumulative Power.

Tissue plasminogen activator (tPA) is a serine protease, which is composed of five distinct structural domains with 17 disulfide bonds, representing a model of high-disulfide proteins in human body. One of the most important limitations for high yield heterologous protein production in Escherichia coli (E. coli) is the expression of complex proteins with multiple disulfide bridges. In this study the combination of two distinct strategies, manipulated cytoplasm and native periplasm, was applied to produce the functional full length tPA enzyme in E. coli. Using a PelB signal peptide sequence at 5' site of tPA gene, the expression cassette was prepared and subsequently was transformed into a strain with manipulated oxidizing cytoplasm. Then the induction was made to express the protein of interest. The SDS-PAGE analysis and gelatin hydrolysis confirmed the successful expression of functional tPA. The results of this study showed that complex proteins can be produced in E. coli using the cumulative power of both cytoplasm and periplasm.

Expression of human tissue plasminogen activator in the trypanosomatid protozoan Leishmania tarentolae.

A variety of recombinant protein expression systems have been developed for heterologous genes in both prokaryotic and eukaryotic systems such as bacteria, yeast, mammals, insects, transgenic animals and transgenic plants. Also, it has been reported that Leishmania tarentolae, a trypanosomatid protozoan parasite of the white-spotted wall gecko (Tarentola annularis), has the capability of expressing heterologous genes. Trypanosomatidae are rich in glycoproteins, which can account for more than 10% of total protein. The oligosaccharide structures of their glycoproteins are similar to those of mammals with N-linked galactose, and sialic acid residues. For a variety of reasons, including the glycosylation patterns and the secondary structures of some of these proteins, synthesis in eukaryotic system is highly preferable. In addition, formation of native disulfide bonds in complex eukaryotic proteins is tremendously important. In the present study, we tried to express the tPA (tissue plasminogen activator) gene in L. tarentolae. This protein is a thrombolytic agent with 527 amino acid residues. tPA possesses serine-protease activity, with 35 cysteine residues that participate in the formation of 17 disulfide bonds. We have used an expression cassette, including the alpha intergenic regions of Leishmania major and two sites at the 3'- and 5'-ends, for homologous recombination in L. tarentolae, in addition to antibiotic-resistant genes. Southern-blot analysis showed that the human tPA gene had been inserted into the genome of the parasite. The expression of the tPA at the mRNA and protein levels was confirmed. It was shown that the expressed tPA in this system was 70 i.u. (international units)/ml of culture media, which is much higher than levels reported previously in other systems.

Leishmaniasis vaccine candidates for development: a global overview.

A vaccine against different forms of leishmaniasis should be feasible considering the wealth of information on genetics and biology of the parasite, clinical and experimental immunology of leishmaniasis, and the availability of vaccines that can protect experimental animals against challenge with different Leishmania species. However, there is no vaccine against any form of leishmaniasis for general human use. One major factor is the lack of a conceived market for human leishmaniasis vaccines. Hence pharmaceutical industries involved in vaccine development are not interested in investing millions of dollars and a decade that is required for developing a new vaccine. Besides, leishmaniasis is a local/regional problem and not a global one. According to the estimates of the World Health Organization, 90 per cent of visceral leishmaniasis occurs in five countries (Bangladesh, Brazil, India, Nepal and Sudan). Those in need are amongst the poorest people in these countries. It should therefore be the objectives of these countries to develop a vaccine. Fortunately, both Brazil and India have designated the control of visceral leishmaniasis as a top priority for their respective Ministries of Health. The purpose of this review is to present only the vaccines in use and those in development for use in dogs or humans. This is not an exhaustive review of vaccine discovery or the principles of clinical immunology underlying vaccine development.

Development of a recombinant Leishmania major strain sensitive to ganciclovir and 5-fluorocytosine for use as a live vaccine challenge in clinical trials.

To provide a safer live challenge strain for use in clinical vaccine trials, a double drug sensitive strain of Leishmania major was derived using advances in gene targeting technology by stably introducing into the chromosome a modified HSV-1 thymidine kinase gene (tk), conferring increased sensitivity to ganciclovir (GCV), and a Saccharomyces cerevisiae cytosine deaminase gene (cd), conferring sensitivity to 5-fluorocytosine (5-FC). In vitro studies showed that the homozygous L. major (tk-cd+/+) promastigotes were killed by either drug alone, and together the drugs acted synergistically. In vivo infection studies showed that progressively growing lesions in BALB/c mice, caused by L. major (tk-cd+/+), were completely cured by 2 weeks of treatment with either drug alone or in combination. Treated animals showed no signs of reoccurrence of infection for at least 4 months when the experiments were terminated.