Role of Hyperhomocysteinemia and Methylene Tetrahydrofolate Reductase C677T Polymorphism in Idiopathic Portal Vein Thrombosis

PURPOSE: Portal vein thrombosis (PVT) is a rare and life-threatening vascular disorder characterized by obstruction or narrowing of the portal vein. Hyperhomocysteinemia and methylenetetrahydrofolate reductase (MTHFR) C677T polymorphism has been studied in PVT patients with conflicting results. In the present study the association of hyperhomocysteinemia and MTHFR C677T polymorphism with PVT risk was investigated in Iranians. MATERIALS AND METHODS: Our study population consisted of 10 idiopathic PVT patients and 80 healthy control subjects matched for age and sex. MTHFR C677T polymorphism was genotyped by the polymerase chain reaction technique combined with restriction enzyme fragment length polymorphism (PCR-RFLP) technique and plasma total homocysteine (tHcy) levels were determined by enzyme immunoassay method. RESULTS: Mean plasma tHcy levels were significantly higher in PVT patients (20.2±6.8) than control subjects (10.9±4.7) (P=0.001). Moreover, plasma tHcy levels were significantly higher in 677T allele carriers relative to 677C allele carriers in both PVT patients (P=0.01) and control subjects (P=0.03). Neither homozygote nor heterozygote genotypes of MTHFR C677T polymorphism correlated significantly with PVT risk (P>0.05). Moreover, MTHFR C677T polymorphism didn't increase the risk of PVT under dominant (CT+TT vs. CC) or recessive (TT vs. CC+CT) genetic models analyzed (P>0.05). The difference in frequency of minor 677T allele between PVT patients and control subjects was not statistically significant (P>0.05). CONCLUSION: Based on the current study, we suggest that hyperhomocysteinemia constitutes a significant and common risk factor for PVT. Also, MTHFR C677T polymorphism is not a risk factor for PVT but is a contributing factor for elevated plasma tHcy levels.

Association of Methylenetetrahydrofolate Reductase C677T Polymorphism with Hyperhomocysteinemia and Deep Vein Thrombosis in the Iranian Population

PURPOSE: Deep venous thrombosis (DVT) is a common but elusive condition characterized by a high morbidity and mortality rate. The aim of the present study was to investigate the correlation between methylenetetrahydrofolate reductase (MTHFR) C677T polymorphism with plasma total homocysteine (tHcy) levels and DVT risk in an Iranian population. MATERIALS AND METHODS: Our study population consisted of 67 patients with a diagnosis of DVT and 67 healthy subjects as controls. Genotyping of MTHFR C677T polymorphism was performed by the polymerase chain reaction technique combined with restriction enzyme fragment length polymorphism (PCR-RFLP) and measurement of tHcy levels was done by enzyme immunoassay method. RESULTS: Plasma tHcy levels were significantly higher in DVT patients than controls (18.09+/-7.6 vs. 10.5+/-4.3, P=0.001). Also, plasma tHcy levels were significantly higher in MTHFR 677TT genotypes compared to 677CC genotypes in both DVT patients (P=0.016) and controls (P=0.03). Neither heterozygote nor homozygote genotypes of MTHFR C677T polymorphism was significantly correlated with DVT (P>0.05). The distribution of MTHFR C677T genotypes was similar between men and women in both DVT patients and controls (P>0.05). Moreover, the frequency of mutant 677T allele did not differ significantly between the two groups (28.3% vs. 21.6%, P=0.15). CONCLUSION: Based on this study, we propose that hyperhomocysteinemia but not homozygosity for MTHFR C677T polymorphism is a significant risk factor for DVT in the Iranian population. Also, MTHFR 677TT genotype is a determinant of elevated plasma tHcy levels.

Retinal pigment epithelium culture; a potential source of retinal stem cells

To establish human retinal pigment epithelial [RPE] cell culture as a source for cell replacement therapy in ocular diseases Human cadaver globes were used to isolate RPE cells. Each globe was cut into several pieces of a few millimeters in size. After removing the sclera and choroid, remaining tissues were washed in phosphate buffer saline and RPE cells were isolated using dispase enzyme solution and cultured in Dulbecco's Modified Eagle's Medium: Nutrient Mixture F-12 supplemented with 10% fetal calf serum. Primary cultures of RPE cells were established and spheroid colonies related to progenitor/stem cells developed in a number of cultures. The colonies included purely pigmented or mixed pigmented and non-pigmented cells. After multiple cellular passages, several types of photoreceptors and neural-like cells were detected morphologically. Cellular plasticity in RPE cell cultures revealed promising results in terms of generation of stem/progenitor cells from human RPE cells. Whether the spheroids and neural-like retinal cells were directly derived from retinal stem cells or offspring of trans-differentiating or de-differentiating RPE cells remains to be answered

Designing of relative quantitative Real-Time PCR for detection of CD40 gene expression in dendritic cell after suppression with antisense

Dendritic cells have a critical role in control and regulation of immune responses. It is believed that these cells can be used for the treatment of many diseases. One of the methods used in immunotherapy is based on generating of tolerogenic dendritic cells through inhibition of expression costimulatory molecules. CD40 is one of the costimulatory molecules, and inhibition of expression by antisense or siRNA techniques, can generate tolerogenic dendritic cells. Generation of tolerogenic dendritic cells will be useful in the treatment of many diseases. By developing a quantitive RT-PCR for evaluation of gene expression, generation of these cells could be possible. Using proper software we designed an Antisense and transfection of dendritic cells by lipofectamine 2000 [Invitrogen] could lead us to generate tolerogenic dendritic cells. In this study dendritic cells were extracted from of Balb/c mice Spleen and the purity of this extraction was determined by flow cytometry. BCL 1 cell line as a CD40 expressing control group and Wehi-164 cell line were cultured in RPMI-1640+10%FCS. Primer design for CD40 gene and house keeping gene [GADPH] was done by bioinformatic soft wares such as Beacon designer, mfold and Blast. RNasy plus mini kit [Qiagen] was used for RNA extraction and the Purity and Integrity were determined by O.D at 260/280 and agarose gel electrophoresis. In the next step cDNA synthesized and quantitative RT-PCR for CD 40 using IQ sybergreen [Biorad] were setup. Finally, standard curve for CD40 and internal control in different RNA concentrations were performed. After transfection with lipofectamin 2000 the amount of gene suppression were quantified by qualitative RT-PCR. Using gradient real time PCR, optimum annealing temperature, C[t] and delta Rn for CD40 and GADPH were determined, annealing temperature was 59.5 degree sign c and melting temperature was 84 degree sign c. Slope of the curve and the efficacy of PCR for CD40 and GADPH genes were quantified by serial dilution method. CD 40 Standard curve efficiency is 96.5 and the slope is -3.408 and the efficacy of GADPH standard curve is 94.1 and its slope is -3.471. The amount of CD40 gene suppression by antisense in dendritic cells was 1/32 and in BCL1 cell line was 1/64. Also the transfection reagent had no effect on the gene expression. The best time for CD40 gene expression is 48h after transfection. Semi quantitative PCR, absolute quantitative PCR and relative quantitative PCR are used to evaluative the expression of different genes such as CD40. In this study we found that for making CD40 and GADPH standard curves, Biorad two steps PCR kit [IQ -sybergreen] and Oligo dt for cDNA synthesis were very suitable. In this case, GADPH is a good internal control. Also for evaluation of gene suppression relative quantitative RT-PCR was more efficient compare to other methods such as northern blotting. The CD40 gene Suppression after 48 hours in dendritic cells was 1/32 and in BCL1 cells was 1/64

Characteristics and prevalence of occult hepatitis B virus infection in patients with hepatitis C in Iran

Hepatitis B vims [HBV] infection in patients who lack detectable hepatitis B surface antigen [HBsAg] is called occult hepatitis B infection. Such infections have been frequently identified in patients with chronic hepatitis C liver disease, but their prevalence is not known.207 patients with chronic hepatitis C who were HCV-RNA and anti-HCV positive were studied for HBV-DNA by PCR, and for HBsAg and anti-HBc by ELIS A. DNA was extracted by high pure nucleic acid kit [Roche-Germany]. HB V-DNA amplification was done with a set of primer directed to the pre-S region. HBsAg and anti-HBc were evaluated by a commercially available ELIS A kit [Dade Behring].23 of 207 patients with chronic hepatitis C liver disease [11.1%] were positive for HB V-DNA [co-infection]. Among this group 17 patients [8.2%] were HBsAg negative [occult infection]. 8 of 17 patients with occult infection [47%] were anti-HBc positive and 9 were anti-HBc negative [53%]. No significant difference was found in epidemiological and biochemical parameters in patients with HCV alone in comparison with HCV co-infected with occult hepatitis B [p= 0.453 for ALT and77= 0.498 for AST]. Occult hepatitis B virus infections occur frequently in patients with chronic hepatitis C liver disease and may have clinical significance

Occult Hepatitis B as a Cause of Cryptogenic Cirrhosis

Background: occult hepatitis B virus [HBV] infection is characterized by presence of HBV infection with undetectable hepatitis B surface antigen [HBsAg]. Diagnosis of occult HBV infection requires sensitive HBV-DNA PCR assay. Recently it has been shown that occult hepatitis B may be a cause of cryptogenic liver disease. The aim of this study is the investigation of occult HBV infection among patients with cryptogenic liver disease

Methods: 65 consecutive paraffin-embedded liver tissues from cases referred to RCGLD [Research Center for Gastroenterology and Liver Diseases] and THC [Tehran Hepatitis Center] during the years 2001 and 2002 for liver biopsy because - of elevation of alanine aminotransferase [ALT] levels for more than six months were studied. Among these, 12 patients with cryptogenic liver disease were found. Human tissue DNA could be extracted in 7 of 12 patients. In these patients liver biopsies were reviewed and HBV-DNA and HBsAg and HBcAg were assayed in liver tissue by polymerase chain reaction [PCR] and immunohistochemistry [IHC], respectively

Results: histologically, chronic hepatitis, cirrhosis and nonspecific changes were reported. HBVDNA was detectable in 4 patients but IHC was negative in all. The frequency of occult HBV infection was more than 50%

Conclusions: occult HBV infection is common among patients with cryptogenic liver disease. In these patients, HBV-DNA may be detected more frequently among patients with more advanced liver pathology [cirrhosis] and more aggressive clinical course [decompensated cirrhosis]