Prevalence of factor VIII inhibitors among Afghan patients with hemophilia A: a first report.

: Prevalence of inhibitors in Afghan hemophilia patients has not been reported previously. Our aim was to determine the prevalence of factor VIII inhibitors among hemophilia A patients from the Kabul province of Afghanistan to identify and characterize the pattern of inhibitor formation. Clinical information and blood samples were collected from three hemophilia centers in Kabul, Afghanistan. Plasma samples were obtained from 62 patients with severe (80.5%) and 15 patients with moderate hemophilia A (19.5%) in this cross-sectional study design. All the patients were receiving on-demand treatment. The Nijmegen modification of the Bethesda assay was used to detect inhibitors. Multiplex PCR, inverse-PCR, Multiplex ligation-dependent probe amplification and direct sequencing were performed for genotyping. Inhibitor activity was detected in one out of 15 (6.7%) patients with moderate hemophilia and in six out of 62 (9.7%) with severe disease. Apart from the intron 22 inversion, five different mutations including one missense, two large and two small deletions were detected. This is the first report showing that the prevalence of inhibitors in Afghan hemophilia A patients is much lower than in other populations.

Motor Neuron Transdifferentiation of Neural Stem Cell from Adipose-Derived Stem Cell Characterized by Differential Gene Expression.

Adipose-derived stem cells (ADSC) are adult stem cells which can be induced into motor neuron-like cells (MNLC) with a preinduction-induction protocol. The purpose of this study is to generate MNLC from neural stem cells (NSC) derived from ADSC. The latter were isolated from the perinephric regions of Sprague-Dawley rats, transdifferentiated into neurospheres (NS) using B27, EGF, and bFGF. After generating NSC from the NS, they induced into MNLC by treating them with Shh and RA, then with GDNF, CNTF, BDNF, and NT-3. The ADSC lineage was evaluated by its mesodermal differentiation and was characterized by immunostaining with CD90, CD105, CD49d, CD106, CD31, CD45, and stemness genes (Oct4, Nanog, and Sox2). The NS and the NSC were evaluated by immunostaining with nestin, NF68, and Neurod1, while the MNLC were evaluated by ISLET1, Olig2, and HB9 genes. The efficiency of MNLC generation was more than 95 ± 1.4 % (mean ± SEM). The in vitro generated myotubes were innervated by the MNLC. The induced ADSC adopted multipolar motor neuron morphology, and they expressed ISLET1, Olig2, and HB9. We conclude that ADSC can be induced into motor neuron phenotype with high efficiency, associated with differential expression of the motor neuron gene. The release of MNLC synaptic vesicles was demonstrated by FM1-43, and they were immunostained with synaptophysin. This activity was correlated with the intracellular calcium ion shift and membrane depolarization upon stimulation as was demonstrated by the calcium indicator and the voltage-sensitive dye, respectively.

Decreased GFAP expression and improved functional recovery in contused spinal cord of rats following valproic acid therapy.

Many studies have illustrated that much of the post-traumatic degeneration of the spinal cord cells is caused by the secondary mechanism. The aim of this study is to evaluate the effect of the anti-inflammatory property of valproic acid (VPA) on injured spinal cords (SC). The rats with the contused SC received intraperitoneal single injection of VPA (150, 200, 300, 400 or 500 mg/kg) at 2, 6, 12 and 24 h post-injury. Basso-Beattie-Bresnahan (BBB) test and H-reflex evaluated the functional outcome for 12 weeks. The SC were investigated 3 months post-injury using morphometry and glial fibrillary acid protein (GFAP) expression. Reduction in cavitation, H/M ratio, BBB scores and GFAP expression in the treatment groups were significantly more than that of the untreated one (P < 0.05). The optimal improvement in the condition of the contused rats was in the ones treated at the acute phase of injury with 300 mg/kg of VPA at 12 h post-injury, they had the highest increase in BBB score and decrease in astrogliosis and axonal loss. We conclude that treating the contused rats with 300 mg/kg of VPA at 12 h post-injury improves the functional outcome and reduces the traumatized SC gliosis.

Selegiline is an efficient and potent inducer for bone marrow stromal cell differentiation into neuronal phenotype.

OBJECTIVE: Bone marrow stromal cells (BMSCs) were documented as a feasible candidate for cell therapy. Several protocols with one or more steps, using different chemicals, were used for inducing BMSCs differentiation into neuronal phenotype. Many of these chemicals were reported to be of mutagenic, teratogenic or carcinogenic properties. The purpose of this work was to evaluate the neuronal inductivity of selegiline to BMSCs. METHODS: Selegiline was used to induce BMSCs following pre-induction with beta-mercaptoethanol and retinoic acid. The neuronal phenotype was evaluated using antineurofilament 200, neurofilament 68, anti-Map-2 and synapsin I antibodies. In addition, reverse transcription polymerase chain reaction was used for the expression of brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), neurotrophin 3 (NT-3), neuroligin 1 and post-synaptic density protein 95 (PSD-95). RESULTS: The result of the work showed that the induced BMSCs were immunoreactive for neurofilaments 200 and 68. Moreover, anti-Map-2 and synapsin I antibodies showed a structure morphologically consistent with synapses. Reverse transcription polymerase chain reaction showed that the induced BMSCs could express BDNF, NGF, NT-3, neuroligin 1 and PSD-95. The time course used was 1, 3, 6, 12, 24, 36 and 48 hours, and the percentages of neurofilament 200 immunoreactive cells were analysed using the logistic growth curve. DISCUSSION: The results showed that selegiline was efficient and a potent inducer for BMSCs into neuronal phenotype, which can be used in the cell therapy in neurodegeneration and traumatic nervous tissue injuries.

Downregulation of IGF-IR expression by RNAi inhibits proliferation and enhances chemosensitization of human colon cancer cells.

PURPOSE: Colon cancer is the second leading cause of cancer death worldwide. Elevated expression of insulin-like growth factor-I receptor (IGF-IR) is a frequent genetic abnormality seen in this malignancy. For a better understanding of its role in maintaining the malignant phenotype, we used RNA interference (RNAi) directed against IGF-IR in our study. The aim of this study was to examine the anti-proliferation and chemosensitization effects elicited by a decrease in the transcription and protein levels of IGF-IR by RNAi in SW480 colon cancer cells. METHODS: A plasmid-based polymerase III promoter system was used to deliver and express short interfering RNA (siRNA) targeting IGF-IR to reduce its expression in SW480 cells. Western blot analysis was used to measure the protein level of IGF-IR. We assessed the effects of IGF-IR silencing on cancer cell growth by a cell growth curve. The effect of the 5-fluorouracil (5-FU)-induced cell death by knockdown of IGF-IR was also investigated by methyl thiazolyl tetrazolium assay. RESULTS: Transfection of siRNA targeting IGF-IR was shown to reduce IGF-IR messenger RNA levels by 95%. Western blotting detected a similar inhibition of IGF-IR protein levels in those cells. The cells transfected with PKD-short hairpin RNA-IGF-IR-V2 significantly decreased cell growth and rendered cells more sensitive to chemotherapy. The highest proliferation inhibitory and chemosensitization ratios were 53 +/- 2% and 1.78, respectively. CONCLUSION: This study indicates that downregulation of IGF-IR results in significant inhibition of tumor growth in vitro. It also provides a promising strategy to chemotherapy efficacy in human tumors and forming a basis for future in vivo trials.

SiRNA-mediated IGF-1R inhibition sensitizes human colon cancer SW480 cells to radiation.

PURPOSE: Insulin like growth factor receptor 1 (IGF-1R) is well-documented to play a key role in radiation response and tumor radiosensitivity, thus offering an attractive clinic drug target to enhance tumor sensitivity to anti-cancer radiotherapy. MATERIAL AND METHODS: Human colon carcinoma SW480 cells were transfected with the specific small interference RNA (siRNA) expression vector (pkD-shRNA-IGF-1R-V2) designed to target IGF-1R mRNA. The expression of IGF-1R mRNA and its protein among the transfected and untransfected cells were detected by semi-quantitative RT-PCR and ELISA assay. The changes in cell radiosensitivity were examined by MTT assay. RESULTS: Transfection of mammalian expression vector pkD containing IGF-1R siRNA was shown to reduce IGF-1R mRNA levels by up to 95%. ELISA assay detected a similar inhibition of IGF-1R protein levels in cells transfected with IGF-1R siRNA. SW480 cells transfected with the expression vector for siRNA significantly rendered cells more sensitive to radiation and the highest radiation enhancement ratio was 2.02 +/- 0.08. CONCLUSION: These data provide the first evidence that specific siRNA fragment (pkD-shRNA-IGF-1R-V2) targeting human IGF-1R mRNA is able to enhance colon cancer radiosensitivity. Also results indicated that, combining IGF-1R siRNA and radiation significantly enhances antitumor efficacy compared with either modality alone.

Knockdown of IGF-IR by RNAi inhibits SW480 colon cancer cells growth in vitro.

BACKGROUND AND AIMS: Colon cancer is the second leading cause of death due to cancer worldwide. Elevated expression of IGF-IR is a frequent genetic abnormality seen in this malignancy. The aim of the study was to examine the anti-growth effects elicited by a decrease in the protein level of IGF-IR by RNA interference (RNAi) in SW480 cells. METHODS: A plasmid-based polymerase III promoter system was used to deliver and express short interfering RNA (siRNA) targeting IGF-IR to reduce its expression in SW480 cells. The expression of IGF-1R protein was detected by Western blot. We assessed the effects of IGF-IR silencing on cancer cell growth by a growth curve. RESULTS: We prepared a type of IGF-IR short hairpin RNA (shRNA) expression vector that could efficiently inhibit the expression of IGF-IR in SW480 cells. At 48 h after transfection, the expression inhibition rate was 92 +/- 2% at mRNA level detected by RT-PCR analysis. Western blotting detected a similar inhibition of IGF-IR protein levels in cells transfected with pkD-shRNA-IGF-IR-V2. Downregulation of IGF-IR resulted in significant inhibition of cancer cell growth in vitro. The cell growth inhibition rates at 24, 48, and 72 h after pkD-shRNA-IGF-IR-V2 transfection were 32.06, 47.61, and 35.36%, respectively. CONCLUSIONS: Our data show that decreasing the IGF-IR protein level in SW480 cells by RNAi could significantly inhibit tumor growth in vitro, implying the therapeutic potential of RNAi on the treatment of colon cancer by targeting overexpression oncogenes such as IGF-IR. IGF-IR may be a potential therapeutic target for human colon cancer.

Transdifferentiation of bone marrow stromal cells into Schwann cell phenotype using progesterone as inducer.

Bone marrow stromal cells (BMSCs) were reported to transdifferentiate into Schwann cells by a two-stage protocol, using beta-mercaptoethanol and retinoic acid (BME-RA) as preinducers (preinduction stage: PS) and platelet derived growth factor (PDGF), basic fibroblast growth factor (bFGF), forskolin (FSK) and heregulin (HRG) as inducers (induction stage: IS). In this study, six groups were used, group one was used as control (PS: BME-RA; IS: PDGF, bFGF, FSK and HRG). In group 2, the preinducer was similar to group 1, and in the induction stage, progesterone replaced HRG. In groups 3 and 4, the preinducer was progesterone; and at the induction stage, the inducer was similar to groups 1 and 2. Accordingly, in groups 5 and 6, the preinducer was FSK. The immunohistochemical differentiation markers were S-100 and P0, and RT-PCR markers were OCT-4 and P0 at the preinduction stage, while at the induction stage P0 and NeuroD were used. The results of the study showed that S-100 was expressed in the groups after the induction stage, however, P0 was not expressed in any group. There was not any significant difference between the percentage of S100 positive cells in the 1st and 2nd groups. P0 was expressed at the mRNA level in the undifferentiated BMSCs and in the 3rd and 4th groups after the preinduction and the induction stages. The conclusion of this study is that progesterone can induce BMSCs into Schwann cell phenotype.

Evaluation of DNA damage in leukocytes of G6PD-deficient Iranian newborns (Mediterranean variant) using comet assay.

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is the most common inherited disease, which causes neonatal hemolytic anemia and jaundice. Recent studies of our group showed that the Mediterranean variant of this enzyme (Gd-Md) is the predominant G6PD in Iranian male infants suffering from jaundice; this variant is classified as severe G6PD deficiency. Considering the importance of G6PD reaction and its products NADPH and glutathione (GSH) against oxidative stress, we hypothesized the failure of detoxification of H(2)O(2) in G6PD-deficient white blood cells that could probably induce primary DNA damage. For the evaluation of DNA damage, we analyzed mononuclear leukocytes of 36 males suffering from the Gd-Md deficiency using alkaline single cell gel electrophoresis (SCGE) or comet assay. The level of DNA damage was compared with the level of basal DNA damage in control group represented by healthy male infant donors (of the same age group). Visual scoring was used for the evaluation of DNA damages. The results showed that the mean level of the DNA strand breakage in mononuclear leukocytes of 36 male G6PD-deficient (Gd-Md) infants was significantly higher (P < 0.001) than those observed in the normal lymphocytes. In conclusion, this investigation indicates that the mononuclear leukocytes of the Gd-Md samples may be exposed to DNA damage due to oxidative stress. This is the first report using comet assay for evaluation of DNA damage in severe G6PD deficiency samples.

Combined glucose-6-phosphate dehydrogenase and glucosephosphate isomerase deficiency can alter clinical outcome.

Glucosephosphate isomerase (GPI) deficiency in humans is an autosomal recessive disorder, which results in nonspherocytic hemolytic anemia of variable clinical expression. A 4-year-old female with severe congenital hemolytic anemia had low red cell GPI activity of 15.5 IU/g Hb (50% of normal mean) indicating GPI deficiency. Subsequent DNA sequence analysis revealed a novel homozygous 921C to G mutation in the GPI gene sequence, predicting a Phe307 to Leu replacement. Strikingly, the red cell GPI activity in this patient was higher than that found in a second patient expressing the same GPI variant, with a more severe clinical phenotype. We propose that the hemolysis in the first patient may be modified by an accompanying deficiency of glucose-6-phosphate dehydrogenase (G6PD). The proband's red cell G6PD activity was reduced at 4.5 IU/g Hb (50% of normal mean) and molecular studies revealed heterozygosity for the G6PD Viangchan mutation and a skewed pattern of X-chromosome inactivation, producing almost exclusive expression of the mutated allele. The G6PD Viangchan variant is characterised by severe enzyme deficiency, but not chronic hemolysis. This study suggests that the metabolic consequences of a combined deficiency of GPI and G6PD might be responsible for a different clinical outcome than predicted for either defect in isolation.

Three major glucose-6-phosphate dehydrogenase-deficient polymorphic variants identified in Mazandaran state of Iran.

We report the first investigation of glucose- 6-phosphate dehydrogenase (G6PD) deficiency among the Mazandaranians in the north of Iran. We analysed the G6PD gene in 74 unrelated G6PD-deficient men with a history of favism. Molecular analysis revealed three major different polymorphic variants: G6PD Mediterranean 66.2% (49 out of 74), G6PD Chatham 27% (20 out of 74), G6PD Cosenza 6.75% (5 out of 74). These findings indicated a higher prevalence of G6PD Chatham in this Iranian population than anywhere else in the world. In addition, the distribution of these G6PD variants is more similar to that found in an Italian population than in other Middle Eastern countries.