Effect of transgenic Leishmania major expressing mLLO-Bax-Smac fusion gene in the apoptosis of the infected macrophages.

Objectives: Leishmaniasis is a complex infection against which no confirmed vaccine has been reported so far. Transgenic expression of proteins involved in macrophage apoptosis-like BAX through the parasite itself accelerates infected macrophage apoptosis and prevents Leishmania differentiation. So, in the present research, the impact of the transgenic Leishmania major including mLLO-BAX-SMAC proapoptotic proteins was assayed in macrophage apoptosis acceleration. Materials and Methods: The coding sequence mLLO-Bax-Smac was designed and integrated into the pLexyNeo2 plasmid. The designed sequence was inserted under the 18srRNA locus into the L. major genome using homologous recombination. Then, mLLO-BAX-SMAC expression was studied using the Western blot, and the transgenic parasite pathogenesis was investigated compared with wild-type L. major in vitro and also in vivo. Results: Western blot and PCR results approved mLLO-BAX-SMAC expression and proper integration of the mLLO-Bax-Smac fragment under the 18srRNA locus of L. major, respectively. The flow cytometry results revealed faster apoptosis of transgenic Leishmania-infected macrophages compared with wild-type parasite-infected macrophages. Also, the mild lesion with the less parasitic burden of the spleen was observed only in transgenic Leishmania-infected mice. The delayed progression of leishmaniasis was obtained in transgenic strain-injected mice after challenging with wild-type Leishmania. Conclusion: This study recommended transgenic L. major including mLLO-BAX-SMAC construct as a pilot model for providing a protective vaccine against leishmaniasis.

Knockout Of BIRC5 Gene By CRISPR/Cas9 Induces Apoptosis And Inhibits Cell Proliferation In Leukemic Cell Lines, HL60 And KG1.

INTRODUCTION: Human Baculoviral inhibitor of apoptosis repeat-containing 5 (BIRC5) which encodes survivin exhibits multiple biological activities, such as cell proliferation and apoptosis. Survivin is overexpressed in numerous malignant diseases including acute myeloid leukemia (AML). Recent studies have shown that the CRISPR/Cas9 nuclease-mediated gene-editing systems are suitable approach's for editing or knocking out various genes including oncogenes. METHODS AND MATERIALS: We used CRISPR-Cas9 to knockout the BIRC5 in the human leukemic cell line, HL60, and KG1, and these cell lines were transfected with either the Cas9- and three sgRNAs expressing plasmids or negative control (scramble) using Lipofectamine 3000. The efficacy of the transfection was determined by quantitative reverse transcription-polymerase chain (RT-qPCR) and surveyor mutation assays. Cell proliferation and apoptosis were measured by MTT assay and flow cytometry, respectively. RESULTS: We have successfully knocked out the BIRC5 gene in these leukemic cells and observed that the BIRC5-knocked out cells by CRISPR/Cas9 showed a significant decrease (30 folds) of survivin at mRNA levels. Moreover, cell death and apoptosis were significantly induced in BIRC5-CRISPR/Cas9-transfected cells compared to the scramble vector. CONCLUSION: We demonstrated for the first time that targeting BIRC5 by CRISPR/Cas9 technology is a suitable approach for the induction of apoptosis in leukemic cells. However, further studies targeting this gene in primary leukemic cells are required.

BIRC5 Gene Disruption via CRISPR/Cas9n Platform Suppress Acute Myelocytic Leukemia Progression

Background: Acute myelocytic leukemia (AML) is a clonal malignancy resulting from the accumulation of genetic abnormalities in the cells. Human baculoviral inhibitor of apoptosis repeat-containing 5 (BIRC5), encodes survivin, is one of only a handful of genes that is differentially over-expressed in numerous malignant diseases including AML. Methods: The BIRC5 was silenced permanently in two AML cell lines, HL­60 and KG-1, via the CRISPR/Cas9n system. After transfection of CRISPR constructs, genomic DNA was extracted and amplified to assess mutation detection. To evaluate BIRC5 gene expression, quantitative real-time PCR was performed. Also, MTT cell viability and Annexin­V/propidium iodide flowcytometric staining were performed, and the data were analyzed using the Kolmogorov-Smirnov, Levene's, and ANOVA tests. Results: The results indicated that Cas9n and its sgRNAs successfully triggered site-specific cleavage and mutation in the BIRC5 gene locus. Moreover, suppression of BIRC5 resulted in the reduction of cell viability, and induction of apoptosis and necrosis in HL60 and KG1 suggested that the permanent suppression of BIRC5 remarkably dropped the gene expression and cells viability. Conclusion: This study reinforces the idea that BIRC5 disruption via Cas9n:sgRNAs has favorable effects on the AML clinical outcome. It thereby can be a promising candidate in a variety of leukemia treatments.

Effect of leukemia inhibitory factor on the myelinogenic ability of Schwann-like cells induced from human adipose-derived stem cells.

The Schwann cells (SCs) may be obtain from nerve biopsies for autologous transplantation. However, it is difficult to obtain sufficient amount of SCs for clinical applications. Human adipose-derived stem cells (ADSCs) can be induced to differentiate into Schwann-like cells (S-like cells) and used for autologous transplantation. However, effect of leukemia inhibitory factor (LIF) on the myelinogenic ability of SC-like cells induced from human ADSC is not investigated yet. The aim of this study was to evaluate of the effect of exogenous LIF on myelinogenic potential of differentiated cells in vitro. ADSCs were harvested from human fat tissue and characterized using flow cytometry. Human ADSCs were treated for sphere formation and LIF was added to terminal differentiation medium. GFAP/S100ß and MBP markers were used to confirm differentiation of human ADSCs, and myelinogenic ability of SC-like cells, respectively, using both immunostaining and real-time RT-PCR analysis. The analysis for GFAP(+)/S100ß(+) revealed that LIF can increase both differentiated cells rates and the percentage of myelinating SC-like cells (p < 0.05). Our data showed that SC-like cells induced from human ADSCs were able to generate myelin when exposed to LIF and these cells could be a potential source for the treatment of peripheral and central axonal injuries.

Bacterial superinfection in zoonotic cutaneous leishmaniasis.

BACKGROUND: Zoonotic Cutaneous Leishmaniasis (ZCL) is a polymorphic disease. It is generally accepted that bacterial superinfection may play a role in the clinical appearance of the lesions and may delay or prevent the healing process. However, the pattern of bacterial pathogens involved has rarely been investigated. MATERIAL/METHODS: The aim of this study was to identify the bacterial species contaminating the suspected ZCL and their susceptibility to commonly used antibiotics. Microscopic examination of stained smears and cultures were used to differentiate ZCL from non-ZCL lesions in a rural area north of Isfahan, Iran from July to December 2009. Bacteria were isolated from the lesions and identified and antibiotic susceptibility was determined by standard microbiological techniques. RESULTS: The results show that 602 (68%) of 855 patients were positive for ZCL, of which 83.4% with volcano-shape, 8.8% psoriasiform, 6.6% popular form and 1.2% with other atypical forms of ZCL. The bacteria were isolated from 66.8% of ZCL (70% of volcano-shape, 60% of psoriasiform and 25% of popular form) and 64.7% of non-ZCL lesions. The most common species were Staphylococcus aureus (41.7%) and S. epidermidis (28%) followed by Bacillus sp. Streptococcus pyogenes, Escherichia coli, Klebsiella sp., Proteus sp., Enterobacter sp. and Pseudomonas aeroginosa. Ciprofloxacin, Erythromycin, Cefazolin and Clindamycin were the most effective antibiotics. CONCLUSIONS: Bacterial superinfection appears to be very common in ZCL, but its prevalence is not different from that of non-ZCL lesions and it has little effect on the clinical appearance of anthroponotic cutaneous Leishmaniasis (ACL). Local lesion care and management of bacterial superinfection must be considered in the treatment of ZCL.

Comparative molecular epidemiology of Leishmania major and Leishmania tropica by PCR-RFLP technique in hyper endemic cities of Isfahan and Bam, Iran.

BACKGROUND: Leishmania is an obligate intracellular protozoa, and the sandfly, as a vector, transmits infectious forms of the parasite to the vertebrate host. The etiologic agents of cutaneous leishmaniasis (CL), Leishmania major and Leishmania tropica, are the most prevalent factor in Iran, especially in the Isfahan and Bam regions. Because of the importance of CL in endemic regions and the interaction of species diversity factors in developing control strategies, several isolated Leishmania species from 2 hyperendemic regions of Iran, Isfahan and Bam cities, were examined in this study by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). MATERIAL/METHODS: In this study, 340 samples were taken from clinically suspected CL patients to prepare slides for direct microscopy and cultures for promastigotes by PCR-RFLP. The internal transcribed spacer 1 (ITS1) region of genomic DNA was extracted and amplified with LITSr and L5.8s primers. Amplification by PCR-RFLP was performed to determine 4 kinds of genotype pattern of the species in the 2 main cities of Isfahan and Bam. Some of the product samples were sequenced and analyzed. RESULTS: Two genotypic groups were detected from L. major isolates, LmA and LmB; also L. tropica showed 2 patterns, LtA and LtB, in comparison with standard species. The most prevalent genotypes related to isolates of Isfahan were LmA and of Bam were LtA. These 2 genotypes were recorded as major etiologic factors of CL in these 2 regions. CONCLUSIONS: Leishmania major and L. tropica, the causative agents of zoonotic CL and anthroponotic CL, respectively, in Isfahan and Bam, are genetically highly polymorphic species, and a correlation may exist between genetic heterogeneous and clinical manifestation and geographic regions of the disease in humans.

Phenotypic characterizations and comparison of adult dental stem cells with adipose-derived stem cells.

OBJECTIVES: Mesenchymal stem cells or "multipotent stromal cells" are heterogeneous cell population with self-renewal and multilinage differentiation. The aim of this study was to examine and compare the expression of important stem cell surface markers on two populations of mesenchymal stem cells, one derived from human exfoliated deciduous teeth and the other derived from human adipose tissue. These new stem cells will offer a promising avenue for prevention and reversal of many human diseases such as type 1 diabetes and prevention of liver fibrotic process. METHODS: Mesenchymal stem cells were isolated and cultured from human adipose tissue and dental pulp of human exfoliated deciduous teeth. The cultured cells then were harvested and stained by different fluorescent labeled monoclonal antibodies against surface markers and were analyzed using flow cytometry. RESULTS: Both different cell populations expressed CD44, CD90 and CD13 (stem cell markers) with similar intensity. They did not express hematopoietic markers (CD11b, CD19 and CD34), and lymphocyte or leukocyte antigens CD3, CD7, CD20, CD14, CD45, CCR5 (CD195), CD11b and CD10 on their surfaces. Two different cell types demonstrated different levels of expression in CD56 and CD146. Mesenchymal stem cells from human exfoliated deciduous teeth were positive for CD105 and were negative for CCR3 and CCR4 expression. CONCLUSIONS: Both cell populations derived from adipose tissue and dental pulp showed common phenotypic markers of mesenchymal stem cells. In conclusion, mesenchymal stem cells could be isolated and cultured successfully from dental pulp of human exfoliated deciduous teeth, they are very good candidates for treatment and prevention of human diseases.