Lentiviral mediating genetic engineered mesenchymal stem cells for releasing IL-27 as a gene therapy approach for autoimmune diseases

Autoimmune diseases precede a complex dysregulation of the immune system. T helper17 [Th17] and interleukin [IL]-17 have central roles in initiation of inflammation and subsequent autoimmune diseases. IL-27 significantly controls autoimmune diseases by Th17 and IL-17 suppression. In the present study we have created genetic engineered mesenchymal stem cells [MSCs] that mediate with lentiviral vectors to release IL-27 as an adequate vehicle for ex vivo gene therapy in the reduction of inflammation and autoimmune diseases. In this experimental study, we isolated adipose-derived MSCs [AD-MSCs] from lipoaspirate and subsequently characterized them by differentiation. Two subunits of IL-27 [p28 and EBI3] were cloned in a pCDH-513B-1 lentiviral vector. Expressions of p28 and EBI3 [Epstein-Barr virus induced gene 3] were determined by real time polymerase chain reaction [PCR]. MSCs were transduced by a pCDH-CMV-p28-IRESEBI3- EF-copGFP-Pur lentiviral vector and the bioassay of IL-27 was evaluated by IL-10 expression. Cell differentiation confirmed true isolation of MSCs from lipoaspirate. Restriction enzyme digestion and sequencing verified successful cloning of both p28 and EBI3 in the pCDH-513B-1 lentiviral vector. Real time PCR showed high expressions level of IL-27 and IL-10 as well as accurate activity of IL-27. The results showed transduction of functional IL-27 to AD-MSCs by means of a lentiviral vector. The lentiviral vector did not impact MSC characteristics

α:Non-α and Gγ:Aγ globin chain ratios in thalassemia intermedia patients treated with hydroxyurea

<p><b>OBJECTIVES</b>To elucidate the possible ways by which hydroxyurea molecules affect globin chain (α or β-like) synthesis.</p><p><b>METHODS</b>A total of 23 thalassemia intermedia patients (13 male and 10 female) aged between 5 and 26 years were treated for five months with 15 mg/(kg·day) of hydroxyurea. Hemoglobins electrophoresis and globin chain electrophoresis was performed on each sample at different time points before and during the treatment.</p><p><b>RESULTS</b>Fetal hemoglobin increased significantly in most patients and average episodes of transfusion decreased. Both Gγ and Aγ-globin chains increased significantly and α-globin:Nonα-globin chain as well as Gγ-globin:Aγ globin chains ratios decreased.</p><p><b>CONCLUSIONS</b>Improvement in α:non-α ratio and consequent decrease of free α-globin chain might be the cause of beneficial effects of hydroxyurea therapy. Two patients who felt better didn't show significant increase in their fetal hemoglobin level, and this is in contradiction with the hypothesis claiming that the HbF level increase is the cause of such therapeutic effect. In spite of the unclear mechanism of action of this drug, hydroxyurea therapy had noticeable impacts on thalassemia intermedia and also sickle cell disease and even patients suffering from thalassemia major.</p>

Beta globin frameworks in thalassemia major patients from North Iran

Four combinations of five neutral sequence changes at rs713040, rs10768683, rs7480526, rs7946748, and rs1609812 occurring in the human beta globin gene defined as frameworks have been reported in beta globin gene. Here we report for the frequency of these frameworks in thalassemia major patients of North Iran beta globin gene framwork of 46 thalassemia major patients of north Iran were determined using Denaturing Gradient Gel Electrophoresis. All these frameworks called framework 1, 2, 3, 3a were present at the frequency of 23.9%, 45.7%, 6.5% and 23.9% respectively. These frameworks may be used for tracking mutant alleles in prenatal diagnosis programs

[IVS8 polyT and M470V polymorphisms in healthy individuals and cystic fibrosis patients in Mazandaran province, Iran]

Cystic fibrosis [CF] is a multiorgan autosomal recessive disorder. As CF is highly heterogeneous in Iran and many mutations have a low frequency, routine molecular diagnostic methods are not very efficient. The use of highly polymorphic intragenic markers not only can facilitate phenotype prediction in prenatal diagnosis by gene tracking, but also can lead to the demonstration of possible associations between haplotypes and specific mutations. We determined IVS8 polyT and M470V polymorphisms in exon 10 of CFTR gene in this case-control study. Polymorphisms of IVS8 polyT in 53 patients with CF were referred to Amirkola children's Hospital of Babol University of Medical Sciences, 2007 to 2011 and 49 fertile healthy individuals were determined by reverse dot blot method. M470V polymorphism was analyzed by PCR-RFLP. In IVS8 polyT study, T7 was the most frequent allele in healthy individuals than patients with CF [respectively, 82.8% Vs. 77.2%]. T9 was more abundant in patients with CF than normal individuals [respectively, 21.7% Vs. 7.4%, P=0.005]. T9/T9 genotype was more frequent in patients than healthy individuals [respectively, 15.1% and 2%, P=0.032]. Study for M470V polymorphism showed that M/V was the most common genotype in normal individuals and patients with CF [respectively, 49% and 40.4%]. M-T9 haplotype was highly associated with the disease in both patients with CF and normal individuals [respectively, 19.1% and 2.4%, [P<0.001]. The allelic distribution and heterozygosity results suggest that both M470V and IVS8 polyT can be helpful in the prenatal diagnosis of CF in Northern Iranians with a positive family history of the disease

Detecting common CFTR mutations by reverse dot blot hybridization method in cystic fibrosis first report from northern Iran

Cystic fibrosis and its distribution vary widely in different countries and/or ethnic groups. Common CFTR mutations were reported from Iran, but the northern population was not or underrepresented in those studies. The aim of this study was to determine the frequency of common CFTR mutations in children from northern Iran. Thirty unrelated Iranian cystic fibrosis patients aged less than 11 years and living in Mazandaran province were screened for 5 common CFTR gene mutations. deltaF508, N1303K, G542X, R347H and W1282X using Reverse Dot Blot method. Only one mutation, DeltaF508, was found in 7 patients accounting for 21.7% [13/60] of alleles. These findings can be used for planning future screening and appropriate genetic counseling programs in Iranian CF families

[Frequency of beta-globin mutations in beta-thalassemia patients from east of Mazandaran]

Beta-thalassemia is the most common inherited disorder in the world, especially in Iran. According to Iranian thalassemia society registry, 18616 thalassemia patients now living in Iran, which Mazandaran and Fars provinces have the most patients. Previous reports have shown that the frequency of b-thalassemia carriers is more than 10% in Mazandaran province. Although b-thalassemia is very heterogenous in the molecular level, but in each population, 5 to 10 mutations are more common. In this research common mutation in eastern area of Mazadaran province was investigated. 5 to 10 ml peripheral blood samples were collected from volunteer patients who were referred to Boali Sina Hospital in Sari. DNA was extracted from blood, then 20 different mutations were screened and detected using two different methods, ARMS-PCR and Reverse-Dot Blot in Thalassemia Research Center in Sari and Amir Kola Thalassemia Center. From 240 chromosomes investigated in 120 b-thalassemia patients in total, 96.25% mutations were identified. 13 different mutations were identified from 231 chromosomes. Among different mutations investigated, IVSII-1G>A was detected as the most common with frequency of 68/3%, which was homozygous in 64 individuals [53/3%] and compound heterozygous with other mutations in 34 individuals [28/3%] respectively. Mutations C8[-AA], codon22[G>A]/ FSC 22/23/24[-7bp], codon 30[G>A], and IVSII-1G>A were identified in 83% of chromosomes which were studied [200 chromosomes from 240]. Mutation IVSII-1G>A is the most common mutation in northern provinces [Gilan, mazandaran, Golestan] in recent study. Also, comparison of these results with the similar finding from other provinces showed that the distributions of mutations in the northern area are different with northwest, south or southeast of the country

[Common CFTR gene mutations in cystic fibrosis patients in Mazandaran province - Iran]

Cystic fibrosis [CF] is the most common inherited disorder in Caucasian populations caused by mutation in cystic fibrosis transmembrane conductance regulator [CFTR]. The type of mutations and their distributions varies widely between different countries and/or ethnic groups. The aim of this study was to characterize mutations involved in this disease in Mazandaran province, Iran. In this descriptive study thirty unrelated Iranian cystic fibrosis patients were screened for deltaF508, N1303K, G542X, R347H and W1282X mutations in the CFTR gene using Reverse Dot Blot method during 2004-06. This technique uses biotinilated PCR products for simultaneous hybridization with several normal and mutant probes specific to known mutations fixed on Biodyne C membranes. DeltaF508 mutation was found in 13 [21.66%] alleles. 6 patients were homozygote and one was compound heterozygote for this mutation. These findings reveal an important heterogeneity of CFTR gene mutations in Mazandaran Province. Thus regarding the relative low rate of detectable mutations, it is necessary to undertake larger studies for molecular diagnosis of cystic fibrosis in this province