Avicenna Journal of Medical Biotechnology
Year:2018 | Volume:10 | Issue:2|Papers Count:11

Antibodies are proteins of the immune system that are produced by B-lymphocytes. These proteins exert their effects by recognizing and binding to their targets (antigens). A monoclonal antibody (mAb) is originally produced by a single B-cell. Production of mAbs was first introduced in 1975 using cell fusion technique and hybridoma cell production. A Hybridoma is formed by fusion of an antibody producing B-lymphocyte and a myeloma cell line....

Background: One of the most significant problems in the treatment of leukemia is the expansion of resistance to chemotherapeutic agents. Therefore, assessing the drug resistance and especially the drug resistance genes of leukemic cells is important in any treatment. The impact of Mesenchymal Stem Cells (MSCs) and hypoxic condition have been observed in the biological performance of majority of leukemic cells.Methods: MOLT-4 cells were co-cultured with MSCs in the hypoxic condition induced by Cobalt Chloride (CoCl2) for 6 and 24 hr. Then, apoptosis of cells was analyzed using annexin-V/PI staining and expression of the drug resistance genes including MDR1, MRP, and BCRP along with apoptotic and anti-apoptotic genes, including BAX and BCL2, was evaluated by real-time PCR.Results: The hypoxic condition for MOLT-4 cells co-cultured with MSCs could significantly increase the expression of MDR1 and BCRP genes (p<0.05) which are involved in drug resistance. Also, the results indicated that this condition significantly increases the expression of BCL2 (p<0.05) and reduces the apoptosis in MOLT-4 cells cocultured with MSCs in the hypoxic condition.Conclusion: These effects can demonstrate the important role of hypoxia and MSCs on the biological behavior of Acute Lymphoblastic Leukemia (ALL) cells that may lead to particular treatment outcomes.

Background: Multiple Sclerosis (MS) has been explained as an autoimmune mediated disorder in central nerve system. Since conventional therapies for MS are not able to stop or reverse the destruction of nerve tissue, stem cell-based therapy has been proposed for the treatment of MS. Astaxanthin (AST) is a red fat-soluble xanthophyll with neuroprotection activity. The aim of this study was evaluation of pre-inducer function of AST on differentiation of human Adipose- Derived Stem Cells (hADSCs) into oligodendrocyte precursor cells.Methods: After stem cell isolation, culture and characterization by flow cytometry, hanging drop technique was done for embryoid body formation. In the following, hADSCs were differentiated into oligodendrocyte cells in the presence of AST at various concentrations (1, 5, and 10ng/ml). Finally, immunocytochemistry and real-time PCR techniques were used for assessment of oligodendrocyte differentiation.Results: Flow cytometry results indicated that hADSCs were CD44, CD49-positive, but were negative for CD14, CD45 markers. In addition, immunocytochemistry results revealed that, in AST treated groups, the mean percentage of Olig 2 and A2B5 positive cells increased especially in 5ng/ml AST treated group compared to control group (p<0.001). Moreover, real-time PCR analysis confirmed the results of immunocytochemistry.Conclusion: Since hADSCs have the potential to differentiate into multi lineage cells and due to important functions of AST in regulating various cellular processes, it seems that AST can be used as a promoter for oligodendrocyte differentiation of hADSCs for being used in cell transplantation in multiple sclerosis.

Background: Cancer/Testis Antigens (CTAs) are a subgroup of tumor-associated antigens which are expressed normally in germ line cells and trophoblast, and aberrantly in a variety of malignancies. One of the most important CTAs is Developmental Pluripotency Associated-2 (DPPA2) with unknown biological function. Considering the importance of DPPA2 in developmental events and cancer, preparing a suitable platform to analyze DPPA2 roles in the cells seems to be necessary.Methods: In this study, the coding sequence of DPPA2 gene was amplified and cloned into the retroviral expression vector to produce recombinant retrovirus. The viral particles were transducted to Esophageal Squamous Cell Carcinoma (ESCC) cell line (KYS E-30 cells) and the stable transducted cells were confirmed for ectopic expression of DPPA2gene by real-time PCR.Results: According to the critical characteristics of retroviral expression system such as stable and long time expression of interested gene and also being safe due to deletion of retroviral pathogenic genes, this system was used to induce expression ofDPPA2 gene and a valuable platform to analyze its biological function was prepared. Transduction results clearly showed efficient overexpression of the gene in target cells in protein level due to high level of GFP expression.Conclusion: Such strategies can be used to produce high levels of desired protein in target cells as a therapeutic target. The produced recombinant cells may present a valuable platform to analyze the effect of DPPA2 ectopic expression in target cells.Moreover, the introduction of its potential capacity into the mouse model to evaluate the tumorigenesis of these cancer cellsin vivo leads to an understanding of the biological importance of DPPA2 in tumorigenesis. In addition, our purified protein can be used in a mouse model to produce specific antibody developing a reliable detection of DPPA2 existence in any biological fluid through ELISA system.

Background: Alzheimer’s Disease (AD) is the most prevalent cause of memory impairment in the elderly population, but the diagnosis and treatment of the disease is still challenging. Lavender aqueous extract has recently been shown to have the potential in clearing Amyloid-beta plaques from AD rat hippocampus. To elucidate the therapeutic mechanisms of lavender, serum metabolic fingerprint of Aβ-induced rat Alzheimer’s models was investigated through nuclear magnetic resonance spectrometry.Methods: For the establishment of rat Alzheimer’s models, 10 mg of Amyloid beta 1-42 was injected to male Wistar rats. The lavender aqueous extract was injected 20 days after the establishment of the models, once daily for 20 days. Serum samples were collected and metabolite fingerprints were obtained using 500MHz 1H-NMR spectrometry, following multivariate statistical analyses. The resulted metabolites were then subjected to pathway analysis tools to reveal metabolic pathways affected by the lavender extract treatment.Results: Levels of 10 metabolite markers including alanine, glutamine, serine, isoleucine, valine, carnitine, isobutyrate, pantothenate, glucose and asparagine were reversed nearly to control values after treatment with lavender extract. The results revealed that the most significantly affected pathways during treatment with lavender extract belonged to carbohydrate and amino acid metabolism, including pantothenate and CoA metabolism, glyoxilate and dicarboxylate metabolism, alanine, aspartate and glutamate metabolism, cysteine and methionine metabolism.Conclusion: As lavender extract reversed the direction of changes of some metabolites involved in AD pathogenesis, it was concluded that the extract might play a role in the disease improvement and serve as a potential therapeutic option for the treatment of AD. Moreover, the metabolites which were found in AD rats could serve as a potential marker panel for the disease; however, much further investigation and validation of the results is needed.

Background: Sheep industry has taken steps toward transforming itself into a more efficient and competitive field. There are many varieties of sheep breeds in the world that each of them serves a useful purpose in the economies of different civilizations.Ghezel sheep is one of the Iranian important breeds that are raised for meat, milk and wool. Field of spermatogonial cell technologies provides tools for genetic improvement of sheep herd and multiple opportunities for research. Spermatogonial cells are the only stem cells capable of transmitting genetic information to future generations.Methods: This study was designed to extend the technique of isolation and in vitro proliferation of spermatogonial cells in Ghezel sheep.Results: Isolated cells were characterized further by using specific markers for type A spermatogonia, including PLZF. Also, sertoli cells were characterized by vimentin which is a specific marker for sertoli cells. After 10 days of co-culture, viability rates of the cells was above 94.7%, but after the freezing process the viability rates were 74 percent.Conclusion: In this study, a standard method for isolation and in vitro proliferation of spermatogonial stem cells in Ghezel sheep was developed.

Background: The cyclin E2 (CYCE2) is an important regulator in the progression and development of NSCLC, and its ectopic expression promoted the proliferation, invasion, and migration in several tumors, including Non-Small Cell Lung Cancer (NSCLC). However, the upregulation of CYCE2 in NSCLC cells suggested that it has a key role in tumorigenicity. In addition, the RAS family proteins as oncoproteins were activated in many major tumor types and its suitability as the therapeutic target in NSCLC was proposed. Considering the crucial role of microRNAs, it was hypothesized that altered expression ofhsa-miR-30d-5p and hsa-let-7b might provide a reliable diagnostic tumor marker for diagnosis of NSCLC.Method: Real-time RT-PCR approach could evaluate the expression alteration of hsa-miR-30d-5pand hsa-let-7b and it was related to the surgically resected tissue of 24 lung cancer patients and 10 non-cancerous patients. The miRNAs expression was associated with clinicopathological features of the patients.Results: Hsa-miR-30d showed a significant downregulation (p=0.0382) in resected tissue of NSCLC patients compared with control group. Its expression level could differentiate different stages of malignancies from each other. The ROC curve analysis gave it an AUC=0.73 (p=0.037) which was a good score as a reliable biomarker. In contrast, hsa-let-7b was significantly overexpressed in tumor samples (p=0.03). Interestingly, our findings revealed a significant association ofhsa-let-7b in adenocarcinoma tumors, compared to Squamous Cell Carcinomas (SCC) (p<0.05). Also, analysis of ROC curve ofhsa-let-7b (AUC=0.74, p-value=0.042) suggests that it could be as a suitable biomarker for NSCLC.Conclusion: Together, these results suggest a possible tumor suppressor role for hsamiR-30din lung tumor progression and initiation. Moreover, upregulation of hsa-let- 7bwas associated with the tumor type.

Background: Proinflammatory cytokines have been known to be elevated in patients with Chronic Heart Failure (CHF). Given the importance of proinflammatory cytokines in the context of the failing heart, the prevalence of Tumor Necrosis Factor-a (TNF-a), Interleukin (IL) -6 polymorphisms in patients with CHF was studied due to ischemic heart disease.Methods: Forty three patients with ischemic heart failure were enrolled in this study and compared with 140 healthy individuals. The allele and genotype frequency of four Single Nucleotide Polymorphisms (SNPs) within the IL-6 (-174, nt565) and TNF-a (-308, -238) genes were determined, using Polymerase Chain Reaction with Sequence-Specific Primers (PCR-SSP) assay.Results: The frequency of the TNF-a (-238) A/A genotype was significantly higher in patients comparing to controls (p=0.043), while TNF-a G/A genotype at the same position decreased significantly, in comparison with controls (p=0.018). The most frequent haplotype for TNF-a was A/A in the patient group in comparison with controls (p=0.003). There was no significant difference in allele and genotype frequencies of IL-6 at positions -174 and nt565, and TNF-a at position -308.Conclusion: Certain alleles, genotypes, and haplotypes in TNF-a, but not IL-6, gene were overrepresented in patients with ischemic heart failure, which may, in turn, predispose individuals to this disease.

Background: Multiple Sclerosis (MS) is the most common cause of neurologic disability in young adults. Recently, theAIRE gene was identified as a genetic risk factor for several autoimmune diseases in genome wide association studies. The aim of this study was to further investigate the possible role of theAIRE gene in susceptibility to MS in Iranian population.Methods: A total of 112 MS patients and 94 ethnically matched controls were included in the study. The Single-Nucleotide Polymorphism (SNP) (rs1800520, C>G) with a global MAF=0.2282/1143 was selected and genotyped using HRM real-time PCR method.Results: Results showed that AIRE SNP rs1800520 was significantly less common in the MS patients than in healthy controls (17.8vs.28.7%, pc=0.032, OR=0.54, 95% CI 0.279, 1.042). Also, the frequency of allele G was significantly higher among the control group than in the case group (37.77vs.25%, pc=0.014). Interestingly, mRNA transcribed on the rs1800520 SNP showed decreased free energy than the wild type suggesting that its increased stability may be responsible for the different activities of the polymorphic AIRE molecule.Conclusions: This is the first study investigating the relationship between AIRE gene and the susceptibility to MS. These results indicated that the rs1800520 SNP is not a susceptibility gene variant for the development of MS in Iranian population.

Background: Klebsiella pneumoniae (K. pneumoniae) is an opportunistic pathogen that could be resistant to many antimicrobial agents. Resistance genes can be carried among gram-negative bacteria by integrons. Enzymatic inactivation is the most important mechanism of resistance to aminoglycosides. In this study, the frequencies of two important resistance geneaac (6’) -IIa and ant (2’’) -I, and genes coding integrase I and II, inK. pneumoniae isolates resistant to aminoglycosides were evaluated.Methods: In this cross-sectional study, an attempt was made to assess the antibiotic susceptibility of 130K. pneumoniae isolates obtained from different samples of patients hospitalized in training hospitals of Yazd evaluated by disk diffusion method.The frequencies ofaac (6’) -IIa, ant (2’’) -I, intl1, and intl2 genes were determined by PCR method. Data were analyzed by chi-square method using SPSS software (Ver.16).Results: our results showed that resistance to gentamicin, tobramycin, kanamycin, and amikacin were 34.6, 33.8, 43.8, and 14.6%, respectively. The frequencies ofaac (6’) -IIa, ant (2’’) -I, intl1, and intl2 genes were 44.6, 27.7, 90, and 0%, respectively.Conclusion: This study showed there are high frequencies of genes coding aminoglycosides resistance inK. pneumoniae isolates. Hence, it is very important to monitor and inhibit the spread of antibiotic resistance genes.

Background: Exact mechanisms of fetal harm following vitrification are still unknown.This study was conducted to evaluate the cryopreservation impact on the expression of EpidermalGrowth Factor Receptor (EGFR) gene in mouse 2-cell and blastocysts.Methods: To stimulate ovulation in mice, hCG was injected, followed by collecting 2-cells and blastocysts after 44-46 and 88-89hr, respectively. These embryos were divided into two case and control groups. The fresh case group was cryopreserved using cryotop and warmed after 4 mounts. Normal 2-cells were selected based on their morphology and their RNA was extracted. Quantitative expression ofEGFR gene in both groups was investigated by applying real time-PCR.Results: The statistical Real-Time (RT) -PCR analyses performed using SPSS revealed that the expression level ofEGFR gene was diminished in the case group compared to the control group.Conclusion: The current study indicated the negative effect of cryopreservation on expression amount ofEGFR gene in 2-cell and blastocyst mouse embryos.