Design and development of modified mRNA encoding core antigen of hepatitis C virus: a possible application in vaccine production

Introduction: Hepatitis C virus [HCV] is a ?blood-borne pathogen, resulting in liver cirrhosis and liver cancer. Despite of many efforts in development of treatments for HCV, no vaccine has been licensed yet. The purpose of this study was ?to design and prepare a specific mRNA, without 5' cap and poly [A] tail transcribed in vitro capable of coding core protein and also to determine its functionality

Methods: Candidate mRNA was prepared by in vitro transcription of the designed construct consisting of ??5' and 3' untranslated regions of heat shock proteins 70 [hsp70] mRNA, T7 promoter, internal ribosome entry site [IRES] sequences of eIF4G related to human dendritic cells [DCs], and the ?Core gene of HCV. To design the modified mRNA, the ??5' cap and poly [A] tail structures were not considered. DCs were transfected by in vitro-transcribed messenger RNA [IVT-mRNA] and the expressions of green fluorescent protein [GFP], and Coregenes were determined by microscopic examination and Western blotting assay

Results: Cell transfection results showed that despite the absence of ??5' cap and poly [A] tail, the structure of the mRNA ?was stable. Moreover, the successful expressions of GFP and Coregenes were achieved

Conclusion: Our findings indicated the effectiveness of a designed IVT-mRNA harboring the Core gene of HCV in transfecting and expressing the antigens in DCs. Considering the simple and efficient protocol for the preparation of this IVT-mRNA and its effectiveness in expressing the gene that it carries, this IVT-mRNA could be suitable for development of an RNA vaccine against HCV

Construction and production of Foxp3- Fc [IgG] DNA vaccine/fusion protein

Background: It seems that the success of vaccination for cancer immunotherapy such as Dendritic Cell [DC] based cancer vaccine is hindered through a powerful network of immune system suppressive elements in which regulatory T cell is the common factor. Foxp3 transcription factor is the most specific marker of regulatory T cells. In different studies, targeting an immune response against regulatory cells expressing Foxp3 and their removal have been assessed. As these previous studies could not efficiently conquer the suppressive effect of regulatory cells by their partial elimination, an attempt was made to search for constructing more effective vaccines against regulatory T cells by which to improve the effect of combined means of immunotherapy in cancer. In this study, a DNA vaccine and its respective protein were constructed in which Foxp3 fused to Fc[IgG] can be efficiently captured and processed by DC via receptor mediated endocytosis and presented to MHCII and I [cross priming]

Methods: DNA construct containing fragment C [Fc] portion of IgG fused to Foxp3 was designed. DNA construct was transfected into HEK cells to investigate its expression through fluorescent microscopy and flow cytometry. Its specific expression was also assessed by western blot. For producing recombinant protein, FOXP3-Fc fusion construct was inserted into pET21a vector and consequently, Escherichia coli [E. coli] strain BL21 was selected as host cells. The expression of recombinant fusion protein was assayed by western blot analysis. Afterward, fusion protein was purified by SDS PAGE reverse staining

Results: The expression analysis of DNA construct by flow cytometry and fluorescent microscopy showed that this construct was successfully expressed in eukaryotic cells. Moreover, the Foxp3-Fc expression was confirmed by SDS-PAGE followed by western blot analysis. Additionally, the presence of fusion protein was shown by specific antibody after purification

Conclusion: Due to successful expression of Foxp3-Fc [IgG], it would be expected to develop vaccines in tumor therapies for removal of regulatory cells as a strategy for increasing the efficiency of other immunotherapy means

Improvement of liver cell therapy in rats by dietary stearic acid

Background: Stearic acid is known as a potent anti-inflammatory lipid. This fatty acid has profound and diverse effects on liver metabolism. The aim of this study was to investigate the effect of stearic acid on markers of hepatocyte transplantation in rats with acetaminophen [APAP]-induced liver damage

Methods: Wistar rats were randomly assigned to 10-day treatment. Stearic acid was administered to the rats with APAP-induced liver damage. The isolated liver cells were infused intraperitoneally into rats. Blood samples were obtained to evaluate the changes in the serum liver enzymes, including activities of aspartate aminotransferase [AST], alanine aminotransferase [ALT] and alkaline phosphatase [ALP] and the level of serum albumin. To assess the engraftment of infused hepatocytes, rats were euthanized, and the liver DNA was used for PCR using sex-determining region Y [SRY] primers

Results: The levels of AST, ALT and ALP in the serum of rats with APAP-induced liver INJURY were significantly increased and returned to the levels in control group by day six. The APAP-induced decrease in albumin was significantly improved in rats through cell therapy, when compared with that in the APAP-alone treated rats. SRY PCR analysis showed the presence of the transplanted cells in the liver of transplanted rats

Conclusion: Stearic acid-rich diet in combination with cell therapy accelerates the recovering of hepatic dysfunction in a rat model of liver injury

L-carnitine Effectively Induces hTERT Gene Expression of Human Adipose Tissue-derived Mesenchymal Stem Cells Obtained from the Aged Subjects

BACKGROUND AND OBJECTIVES: Human mesenchymal stem cells (hMSCs) are attractive candidates for cell therapy and regenerative medicine due to their multipotency and ready availability, but their application can be complicated by the factors such as age of the donors and senescence-associated growth arrest during culture conditions. The latter most likely reflects the fact that aging of hMSCs is associated with a rise in intracellular reactive oxygen species, loss of telomerase activity, decrease in human telomerase reverse transcriptase (hTERT) expression and finally eroded telomere ends. Over-expression of telomerase in hMSCs leads to telomere elongation and may help to maintain replicative life-span of these cells. The aim of this study was to evaluate of the effect of L-carnitine (LC) as an antioxidant on the telomerase gene expression and telomere length in aged adipose tissue-derived hMSCs. METHODS: For this purpose, cells were isolated from healthy aged volunteers and their viabilities were assessed by MTT assay. Quantitative gene expression of hTERT and absolute telomere length measurement were also performed by real- time PCR in the absence and presence of different doses of LC (0.1, 0.2 and 0.4 mM). RESULTS: The results indicated that LC could significantly increase the hTERT gene expression and telomere length, especially in dose of 0.2 mM of LC and in 48 h treatment for the aged adipose tissue-derived hMSCs samples. CONCLUSION: It seems that LC would be a good candidate to improve the lifespan of the aged adipose tissue-derived hMSCs due to over-expression of telomerase and lengthening of the telomeres.

Comparative effects of Nucleostemin silencing in human Molt-4 and Jurkat leukemia T-ALL cells

Nucleostemin [NS], a stem cell-abundant nucleolar protein, is critical for maintaining the self-renewal and proliferative properties of normal and cancerous stem cells. Recent data suggests that NS signaling is important for proliferation of T-cells and leukemia cells. This study was conducted to verify the role of NS in pathogenesis and treatment of T-cell acute lymphocytic leukemia [T-ALL]. Our results revealed that RNA interference-mediated NS silencing primarily affected clonogenicproperty of T-ALL cells by limiting their self-renewal potential in vitro.These effects were accompanied with inhibition of proliferation and early apoptosis in Jurkat cells [p53-null] while late apoptosis in Molt-4 [p53 functional] T-ALL cells. Collectively, our results suggest that NS is a critical regulator in self-renewal and apoptosis of different T-ALL cells. This suggests therapeutic potential of this gene in leukemia

Altered of microRNA expression level in oligospermic patients

MicroRNA [miRNA] is small endogenous, single strand RNA molecules that regulate gene expression at post-transeriptional level through several mechanisms to affect key cellular event including male germ cells differentiation, proliferation, development and apoptosis. Mutation and/or aberrant expression of miRNAs have been associated with progression of various disorders, including infertility. The purpose of this research was to study the estrogen receptor beta [ERbeta], hsa-mir-21 and, hsa-mir-22 expression level in oligospermic infertile and control fertile men and correlation between them. In this study, the change in mir-21, mir-22 expression and their common target gene [ERbeta] expression levels were evaluated in oligospermic infertile men [n= 43] compared with 43 age matched healthy control by Real-Time PCR methods. Expression analysis by qRT-PCR test on miRNA have identified that mir-21, mir-22 levels were significantly higher than those in normal controls [p<0.0001] and ER beta expression level significantly decreased in comparison with the normal group [p<0.0001]. Our study showed that mir-21 and mir-22 are indirectly involved in spermatogenesis by regulating of the estrogen receptor and might have a diagnostic and prognostic value in men infertility

Effects of curcuma longa extract on telomerase activity in lung and breast cancer cells

The purpose of this study is to evaluate the effect of Curcuma longa extract on the telomerase gene expression in QU-DB lung cancer and T47D breast cancer cell lines. The present study is an experimental research. Using 3 different phases n-hexane, dichloromethane and methanol, total extract of Curcuma longa in a serial dilution was prepared and three phases was analyzed for determining which phase has more curcuminoids. Then the extract cytotoxicity effect was tested on breast cancer cell line [T47D], and lung cancer cell line [QU-DB] by 24, 48 and 72 h MTT [Dimethyl thiazolyl diphenyl tetrazolium] assay. Then, the cells were treated with serial concentrations of the extract. Finally, total protein was extracted from the control and test groups, its quantity was determined and telomeric repeat amplification protocol [TRAP] assay was performed for measurement of possible inhibition of the telomerase activity. Cell viability and MTT-based cytotoxicity assay show that the total extract of Curcuma longa has cytotoxic effect with different IC50s in breast and lung cancer cell lines. Analysis of TRAP assay also shows a significant reduction in telomerase activity on both cancer cells with different levels. Curcuma longa extract has anti-proliferation and telomerase inhibitory effects on QU-DB lung cancer and T47D breast cancer cells with differences in levels of telomerase inhibition

Adipocyte derived hormones gene expression, resistin and visfatin, in AGS gastric cancer cell line

Adipose tissue has characteristics of an endocrine organ which releases a number of adipocyte-specific factors, known as adipocytokines. It has recently suggested that adipocytokines might play a role in pathogenesis and progression of certain cancers, especially in gastric cancer. This study has managed to investigate endogenous and/or exogenous expression of Visfatin and Resistin in gastric cancer cell line. Cell culture and semi-quantitative reverse transcription polymerase chain reaction has performed to measure mRNA and protein expression of Resistin and Visfatin in gastric cancer cell lines. ELISA test has performed for cell lysate and supernatant of cell culture to measure Resistin and Visfatin protein expression and secretion. Human gastric cancer cell line [AGS cell line] has found to express Visfatin mRNA and protein but Resistin mRNA and protein has not expressed. Visfatin has expressed endogenously in AGS human gastric cancer cells. Conversely Resistin has no expression. The results of this study has suggested that expression of adipocytokine proteins in real samples, could be a biomarker for gastric cancer

Evaluation of hs-CRP, antioxidant markers and MDA in patients of coronary artery disease [CAD] containing non-smokers and non-diabetics

Background: The oxidative stress and inflammation are cooperative events involved in atherosclerosis development. In the present study we assessed the association of MDA, antioxidant markers, high sensitive C - reactive protein [hs-CRP] and lipid status parameters in the patients with coronary artery disease [CAD]. Significant risk factors such as cigarette and diabet were excluded from the study. Methods: Oxidative stress parameters for example Malondialdehyde [MDA], antioxidant markers including: erythrocyte superoxide dismutase[SOD], Glutathion peroxidase [GPX], Total antioxidant capacity[TAC], The inflammation marker and serum lipid status parameters were measured in 120 subjects including 60 CAD patients with angiographically diagnosed CAD and 60 CAD-free subjects as a control group, also diabetics, smoking patients, patients with malignancy, renal and liver disease, and other disease were excluded from the study. Results: The serum MDA and hs-CRP levels were increased significantly as compared to controls. However, erythrocyte SOD, GPX activities and TAC level were reduced significantly in patients [P‹0.05 in all cases]. The levels of total cholesterol, Triglyceride, LDL-C were significantly higher and that of HDL-C was meaningfully lower than those of control [P‹0.05 in all cases]. Conclusion: The association between oxidative stress parameters, antioxidant markers, the inflammation index and lipid status parameters suggest their involvement in atherosclerosis development that may lead to CAD progression

[Serum levels of adiponectin in non-diabetic and diabetic obese individuals]

Adiponectin is an adipose tissue-derived hormone that low levels of this hormone are associated with obesity, insulin resistance, and type 2 diabetes. The aim of this study was to compare the serum levels of adiponectin in diabetic and non-diabetic obese individuals. As a cross-sectional study 35 obese individuals with type 2 diabetes mellitus and 35 non-diabetic obese subjects were enrolled. Two groups were matched for age, gender and body mass index. Fasting lipid profile was measured via the enzymatic methods. The Nyco Card HbA1c Kit was used to measure HbA1c. The Serum Adiponectin, insulin and glucose levels were measured via an enzyme immunoassay, using a commercially available kit and glucose oxidase methods, respectively. The HOMA and QUICKI indices were used to determine insulin resistance and insulin sensitivity, respectively. The mean of insulin resistance index [HOMA-IR], HbA1c, diastolic blood pressure, triglyceride and fasting glucose in diabetes were significantly higher than non-diabetics [P<0.05]. The serum Adiponectin levels was significantly lower in diabetes than non-diabetics [15.74 +/- 6.70 vs. 21.52 +/- 9.35] and was significantly higher in women than men [19.38 +/- 7.33 vs. 12.68 +/- 4.28] among diabetic and [24.63 +/- 10.52 vs. 17.83 +/- . 6.21] among non-diabetics groups. Type 2 diabetes mellitus is associated with low serum adiponectin concentrations and probably adiponectin involved in the pathophysiology linking obesity to type 2 diabetes

Study of leptin gene expression changes in streptozotocin-induced diabetic rats

Leptin, a peptide hormone, is the product of [ob] Gene. Leptin regulate body weight and composition through reducing appetite and energy expenditure in rodents and humans. The aim of this study was to evaluate differences in expression of Leptin Gene in different tissues of streptozotocin induced diabetic rats. 40 Sprague Dawely rat were selected. Intra peritoneal injection was carried out in 20 rats and another 20 rats were used as control. After injection of 60mg/kg Streptozotocin, animals were transformed into diabetic. Glucose was measured by glucose oxidase method. Leptin and insulin were measure by commercially available immunoassay kits. After one week treatment, different tissues including adipose tissues, Spleen, epidydimis, and Liver of both control and experimental animals were dissected. For investigation of any changes of the Leptin gene expression in different tissues, RNA was extracted using Trizol method. By using RT-PCR technique, Leptin cDNA and beta-actin cDNA as internal control were constructed and PCR was carried out. The RT-PCR products were detected on 2% agarose gel using electrophoresis. Mean serum levels of Leptin was 5.23 +/- 0.45 ng/ml before injection of streptozotocin and markedly decreased in STZ induced diabetic rats to 0.79 +/- 0.25 ng/ml. This decrease was statistically significant [P<0.05]. There was a direct and significant correlation between leptin and insulin in streptozotocin-induced diabetic rats [r=0.37, P<0.05] while, this was reverse in control rats [r= -0.28, P<0.05]. Using RT-PCR method, Leptin gene expression in different tissues including fat epidydimis, liver, and spleen showed that the intensity of leptin band with 452 bp was decreased in diabetic rats in comparison to normal rats. Actin Gene expression was identified in PCR products having 403 bp and the intensity was constant in both groups. The reduction rates of [ob] mRNA in fat epidydimis tissue in STZ diabetic rats was remarkable in comparison to Spleen and Liver. It is speculated that Leptin gene could be under regulation of insulin dependent mechanism in diabetic rats and by modulating Leptin gene expression in diabetic patients, it may be useful in clinical practices

Study of serum levels of stem cell factor in patients with bladder cancer

Stem cell factor [SCF] has recently been identified as a multi-potential growth factor that acts on early different progenitor cells of various lineages and triggers its biologic effects by binding to its receptor, c-Kit. The main aim of this study was to evaluate serum levels of SCF in bladder cancer patients, and its possible relation with clinical course of disease. The serum SCF level was determined in bladder cancer patients, as our subject cases. The study group consisted of 35 bladder cancer patients and 35 healthy individuals as controls. The samples were prepared in two separated times, before operation and 2