Ahmad Gharehbaghian

Biochemical and molecular characterization of hepatocyte-like cells derived from human bone marrow mesenchymal stem cells on a novel three-dimensional biocompatible nanofibrous scaffold

Background:  There is significant interest in using nanofibers in tissue engineering from stem cells. The transdifferentiation of mesenchymal stem cells into the hepatic lineage in a nanofibrous structure has not been reported. In this study, a three dimensional nanofibrous scaffold is introduced for differentiation of human bone marrow derived mesenchymal stem cells (hBMSCs) into hepatocytes.

Nrf-2 overexpression in mesenchymal stem cells reduces oxidative stress-induced apoptosis and cytotoxicity

The most prominent capabilities of mesenchymal stem cells (MCSs) which make them promising for therapeutic applications are their capacity to endure and implant in the target tissue. However, the therapeutic applications of these cells are limited due to their early death within the first few days following transplantation. Therefore, to improve cell therapy efficacy, it is necessary to manipulate MSCs to resist severe stresses imposed by microenvironment. In this study, we manipulated MSCs to express a cytoprotective factor, nuclear factor erythroid-2 related factor 2 (Nrf2) to address this issue. Full-length human Nrf2 cDNA was isolated and TOPO cloned into TOPO cloning vector and then transferred to gateway adapted adenovirus expression vector by LR recombination reaction. Afterwards, the Nrf2 bearing recombinant virus was prepared in appropriate mammalian cell line and used to infect MSCs. The viability and apoptosis of the Nrf2 expressing MSCs were evaluated following hypoxic and oxidative stress conditions. Transient expression of Nrf2 by MSCs protected them against cell death and the apoptosis triggered by hypoxic and oxidative stress conditions. Nrf2 also enhanced the activity of SOD and HO-1. These findings could be used as a strategy for prevention of graft cell death in MSC-based cell therapy. It also indicates that management of cellular stress responses can be used for practical applications.

Efficient replacing of fetal bovine serum with human platelet releasate during propagation and differentiation of human bone marrow-derived mesenchymal stem cells to functional hepatocytes-like cells

Objectives  The aim of this study was to find out substitution effect of fetal bovine serum (FBS) with human platelet releasate (HPR) as a major growth factor source during expansion and differentiation of human bone marrow‐derived mesenchymal stem cells (hBMSC) into hepatocytes.

Low Dose Ribavirin for Treatment of Hepatitis C Virus Infected Thalassemia Major Patients; New Indications for Combination Therapy

BACKGROUND Treatment guidelines contraindicate ribavirin for treatment of hepatitis C virus (HCV) infection in thalassemia major patients. Nevertheless, the current evidence suggests that ribavirin might be tolerated by these patients. OBJECTIVES Despite this evidence, low dose ribavirin combination therapy has not been compared with peginterferon monotherapy in these patients so far. PATIENTS AND METHODS Two hundred eighty thalassemia patients with detectable HCV-RNA PCR (≥ 50 IU/mL) and liver histology consistent with chronic HCV infection were self-assigned to receive peginterferon alfa-2a (n = 81) monotherapy or its combination therapy with ribavirin, 600-800 mg QD, according to hemoglobin levels (n = 199). Treatment experienced patients were eligible for this study. RESULTS Sustained virological response (SVR) was significantly higher in patients who received ribavirin (51 % vs. 38 % P = 0.02). In multivariate regression, OR of ribavirin for prediction of SVR was 2.2 (95 % CI 1.24-3.91). The SVR was significantly higher in the ribavirin group in subgroups of patients with more than 24 years of age, elevated ALT, ferritin < 2006 ng/mL, previous treatment failure, genotype 1, positive history of splenectomy, fibrosis score of 0-4 HAI and viral load < 600,000 IU/mL. Treatment discontinuations due to the safety concerns were comparable between the treatment groups (6.5 and 8 %). Furthermore, transfusion intervals were almost halved in patients who received low dose ribavirin. CONCLUSIONS According to the present study, adult thalassemia patients with HCV infection can be treated successfully with low dose ribavirin. Hence, we strongly advise combination therapy in thalassemia patients with aforementioned clinical characteristics. Moreover, ribavirin does not seem to be beneficial in thalassemia patients below 18 years of age.

Functional hepatocyte-like cells derived from human bone marrow mesenchymal stem cells on a novel 3-dimensional biocompatible nanofibrous scaffold.

Aim To supporting growth and functional differentiation of adult stem cells into hepatocytes in a well-controlled manner, we performed differentiation of human bone marrow mesenchymal stem cells (hBMSCs) to hepatocytes-like cells on a constructed 3-dimensional (3D) nanofibrous biocompatible scaffold. Methods After characterization of the hBMSCs isolated from human bone marrow, the performance of the cells seeded and their proliferation on the scaffold was evaluated by scanning electron microscopy (SEM) and 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Different approaches such as immunocytochemistry, reverse transcriptase polymerase chain reaction (RT-PCR), and biochemical assays were used to estimate the ability of hBMSC-derived cells to express hepatocyte-specific markers. Results Scanning electron micrographs and MTT analysis revealed the cells were able to expand and remained biologically and metabolically active for 21 days. Immunocytochemical analysis of albumin and α-fetoprotein showing the accumulation of these markers in differentiated cells was confirmed by RT-PCR. Additional markers such as cytochrome P450 3A4, cytokeratin-18, and cytokeratin-19 detected by RT-PCR showed progressive expression during 3 weeks of differentiation on 3D scaffold. The hepatocyte-like cells displayed several characteristics of metabolic functions as judged by production of albumin, urea, transferrin, serum glutamic pyruvic transaminase (SGPT), and serum oxaloacetate aminotransferase (SGOT). Levels of above-mentioned markers, except SGOT in differentiated cells on scaffold, were found to be significantly greater than in the 2D culture system (p<0.05). Conclusion Overall data suggest that the engineered nanofibrous scaffold is a conductive matrix for functional hBMSC-derived hepatocyte-like cells and is promising for maintenance of hepatocytes suitable for implantation.

An Estimate of Transfusion-Transmitted Infection Prevalence in General Populations

Dear Editor, Blood donations saves the lives of millions of people worldwide, and blood transfusion is essential to the effectiveness of the health care system by supporting modern medicine as its pivotal role in patient interventions [1][2]. New, more sophisticated medical and surgical procedures such as transplant, heart surgery, and trauma or cancer treatment depend highly on blood transfusions in each country. Moreover, blood samples improve the quality of life of multitransfused patients. In developing countries transfusion-transmitted infections (TTIs) often threaten the safety of patients requiring blood transfusion, and medical providers face serious challenges with blood availability, safety, and affordability. It is estimated that 45% of the 80 million blood donations across the globe are collected each year in developing countries that comprised roughly 80% of the world’s population [3][4]. On a national level, countries should secure the safety of patients requiring blood transfusion by creating standard blood-transfusion establishments with solid support from policy makers and adequate and consistent government funding. One of the most prominent factors in ensuring safe blood samples is to have a national program for donor selection, recruitment, retention, and education to minimize donations from donors who might transmit diseases to recipients [2][4]. In addition, to ensure blood safety it is important to monitor and evaluate TTIs, including hepatitis B virus (HBV) and hepatitis C virus (HCV), among blood donors and in the general population. The prevalence of transfusion transmitted infections among blood donors in well-structured health care system with a well-organized blood establishment can be used as a reliable tool for statistical estimations of those infectious agent that can be transmitted through blood products including Hepatitis B and C. Indeed, the prevalence size of HCV or HBV among blood donors in such countries can contribute to statistical estimation of those viruses in the general population[5]. In Petrovic’s study, the prevalence rates of chronic HBV and HCV in the general populations of the Tuzla Canton in Bosnia and Herzegovina have been estimated by the adjusted prevalence of HBs-Ag and anti-HCV seropositive samples among first-time blood donors. In the current study the statistical analysis of prevalence rates has been performed based on incidence calculation of the estimated ratio between the two populations (the first-time blood donors and general population) using Poisson distribution method through reviewing published papers[6]. However, epidemiological studies of HBV and HCV in general populations have faced difficulties in obtaining sufficient sample sizes, gender and age distributions, and financial support. Nevertheless, the process of blood-donor selection aims to identify and recruit nonremunerated, apparently healthy people willing to donate blood. The donated blood should be free of bloodborne infectious agents that can be transmitted to recipients, and this depends highly on the structure of the country’s health care system [6][7]. In addition, blood-transfusion establishments should meet the international requirements for standard operating procedures that play a pivotal role in protecting the safety of both donors and recipients. Especially after the AIDS epidemic, the concern about blood safety in multitransfused patients has increased greatly. As the authors have explained, recruited blood donors are usually healthier because they are from people in the community who tend to exhibit healthier behaviors and safer life styles[8]. In conclusion, the accuracy of estimating TTI prevalence in the general population by surveying first-time blood donors depends on how similar the demographic characteristics of the two populations are. What we perceive from this study are significant differences in TTI prevalence rates between first-time and regular blood donors (data not shown here but available upon request), particularly in terms of demographic characteristics (e.g., cultural-economic issues), the structure of regional or country health care systems, blood transfusion service structures, blood donation incentives, blood type of donors (paid-donors, family replacement donors, or volunteer nonremunerated blood donors). Indeed, it seems that the populations of the countries or regions in which the reviewed studies were conducted are not demographically compatible or similar enough for valid inferences to be made.

Expression and purification of recombinant human coagulation factor VII fused to a histidine tag using Gateway technology

BACKGROUND Factor VII (FVII) is a plasma glycoprotein that participates in the coagulation process leading to the generation of fibrin. The aim of this study was to construct, express and purify recombinant FVII fused to a polyhistidine (his) tag using Gateway technology. METHODS To construct the entry clone, blunt-end FVII cDNA and subsequent polymerase chain reaction (PCR) product isolated from a HepG2 cell line was TOPO-cloned into a pENTR TOPO vector. To construct the expression clone, a LR recombination reaction was carried out between the entry clone and destination vector, pDEST26. Chinese hamster ovary (CHO) cells were transfected with 1 microg of DNA of PDEST26-FVII using the FuGENE HD transfection reagent. Two cell lines that permanently expressed recombinant FVII were established. The expression of recombinant FVII was confirmed by reverse transcriptase PCR and enzyme-linked immunosorbent assay. Culture medium containing his-tagged FVII was added to the nickel-nitrilotriacetic acid resin column and bound protein was eluted. The purified protein was detected by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and western blot analysis. The biological activity of the recombinant FVII was determined by a prothrombin time assay using FVII-depleted plasma. RESULTS The results showed that human recombinant FVII was successfully cloned and the accuracy of the nucleotide sequence of the gene and its frame in the vector were confirmed by DNA sequencing. Stable clones transfected with the construct expressed FVII mRNA and related protein but no expression was detected in the CHO cells containing an empty vector. A protein of about 52 KDa was detected in SDS-PAGE and was further confirmed by western blot analysis. A three-fold decrease in clotting time was observed using this recombinant FVII. CONCLUSION As far as we are aware, this is the first report of expression of recombinant FVII fused with a his-tag through Gateway technology. The next steps, including large scale expression, purification, activation and stabilisation, are underway.

Emerging Infectious Threats to the Blood Supply: Seroepidemiological Studies in Iran – a Review

The risk of transfusion-transmitted infections has been greatly reduced by improvements in donor screening and testing. However, newly recognized blood-borne infectious agents can be threats to blood safety. In order to evaluate the prevalence some of these agents in blood donors, a systematic review was conducted. Data were obtained from published papers related to HGV, Torque Teno virus (TTV), HTLV, West Nile virus (WNV) and SEN virus (SEN-V). Based on these studies, the prevalence of HGV varied from 1 to 8.6% for anti-E2 and from 0 to 4.8% for HGV RNA. The prevalence of TTV DNA and HTLV-I varied from 2.7 to 79.5% and from 0.013 to 2.3%, respectively. The WNV-specific IgM antibody and WNV RNA are negative in blood donors. Prevalence rates of SEN-V in Iranian blood donors range from 23 to 90.8%. Consequences of these infectious agents for blood safety are different. Thus, the need to perform laboratory screening as well as effectiveness and efficiency of laboratory tests depend on pathogenicity level and epidemiological conditions of emerging infections. However, being prepared based on the current level of risk and interventions to reduce the risk can be effective in reducing the potential threat for blood supply.

Status of blood transfusion in World Health Organization-Eastern Mediterranean Region (WHO-EMR): Successes and challenges

BACKGROUND Blood products are used for patient treatment and survival in the cases of major surgery, hematological disorders or cancer therapy. Presently the main blood components are not yet replaceable by artificial products and all activities related to blood transfusion is highly dependent on the healthcare development of each country. The World Health Organization Eastern Mediterranean Region (WHO-EMR) comprises of 21 member states with variable socio-economic status effective on blood transfusion activities. The fundamental motivation behind this research was to accumulate some data of blood practices in this region and to have an appropriate image of the WHO-EMR region. MATERIAL AND METHODS The data were collected through the published papers or data, blood transfusion services websites, and the other health official websites like WHO. RESULTS Among WHO-EMR countries there are some with a nationally organized blood transfusion establishment such as Egypt, Iran, Iraq, Kuwait, Morocco, Oman, Pakistan, and Syria. In a few, blood transfusion administrations are hospital-based like Saudi Arabia. The others are run by Red Crescent such as Bahrain, Tunisia and UEA or by Red Cross like Lebanon. Only Iran and UAE succeed to have 100% voluntary non-remunerated blood donors; however, most of them are still under the weight of family/replacement blood donation such as Afghanistan, Egypt, Iraq, Lebanon, Morocco, Saudi Arabia and Sudan or even paid donors like Pakistan and Yemen. The haemovigilance and training programs have been implemented in some countries including Bahrain, Iran, Jordan, Kuwait, Oman, Qatar, Saudi Arabia, Tunisia and UAE. Unfortunately, there are rare and inaccessible information about some EMR states like Djibouti, Palestine and Somalia so that little data can be independently discovered. CONCLUSION In these countries different measures ought to be additionally designated to ensure blood products adequacy and safety such as the development of well-coordinated national blood transfusion centers with the increased government commitment, the establishment of a well-organized system for voluntary non-remunerated blood donor recruitment, the establishment of well-equipped laboratories for screening donor samples for transfusion transmitted infections (TTIs) for at least mandatory tests recommended by WHO, the implementation of an educational/training program for professionals working in the blood transfusion establishments or hospitals, the regular audit on the quality and the integrity of the blood transfusion chain, and the development of a regional network of collaboration to bolster the neighboring nations in the EMR through effective communication tools.

Identification of potential predictive markers of dexamethasone resistance in childhood acute lymphoblastic leukemia

Response to dexamethasone (DEXA), as a hallmark drug in the treatment of childhood acute lymphoblastic leukemia (ALL), is one of the pivotal prognostic factors in the prediction of outcome in ALL. Identification of predictive markers of chemoresistance is beneficial to selecting of the best therapeutic protocol with the lowest effect adverse. Hence, we aimed to find drug targets using the 2DE/MS proteomics study of a DEXA-resistant cell line (REH) as a model for poor DEXA responding patients before and after drug treatment. Using the proteomic methods, three differentially expressed proteins were detected, including voltage dependent anion channel 1 (VDAC1), sorting Nexin 3 (SNX3), and prefoldin subunit 6 (PFDN6). We observed low expression of three proteins after DEXA treatment in REH cells. We subsequently verified low expression of resulted proteins at the mRNA level using the quantitative PCR method. These proteins are promising proteins because of their important roles in drug resistance and regulation of apoptosis (VDAC1), protein trafficking (SNX3), and protein folding (PFDN6). Additionally, mRNA expression level of these proteins was assessed in 17 bone marrow samples from children with newly diagnosed ALL and 7 non-cancerous samples as controls. The results indicated that independent of the molecular subtypes of leukemia, mRNA expression of VDAC1, SNX3, and PFDN6 decreased in ALL samples compared with non-cancerous samples particularly in VDAC1 (p < 0.001). Additionally, mRNA expression of three proteins was also declined in high-risk samples compared with standard risk cases. These results demonstrated diagnostic and prognostic value of these proteins in childhood ALL. Furthermore, investigation of protein-protein interaction using STRING database indicated that these proteins involved in the signaling pathway of NR3C1 as dexamethasone target. In conclusion, our proteomic study in DEXA resistant leukemic cells revealed VDAC1, SNX3, and PFDN6 are promising proteins that might serve as potential biomarkers of prognosis and chemotherapy in childhood ALL.