Endothelial Nitric Oxide Synthase Gene Intron 4 (VNTR) Polymorphism and Vascular Access Graft Thrombosis

Vascular access thrombosis is a leading cause of vascular access failure in hemodialysis patients. Thrombosis is a multifactorial condition and genetic makeup can affect thrombosis risk. We conducted a study to investigate for possible associations between ecNOS gene intron 4 variable-number tandem repeat (VNTR) polymorphism and thrombosis of polytetrafluoroethylene hemodialysis arteriovenous access grafts (AVG) in Turkish patients. Fifty-five patients with end-stage renal disease who had AVGs implanted between 2000 and 2002 and 167 healthy individuals representing our healthy population were enrolled in this prospective study. Each subject provided a venous blood sample from which DNA was isolated, and polymerase chain reaction analysis was done to identify genotypes (aa, bb, ab) for ecNOS gene intron 4 VNTR polymorphism. All grafts were placed in brachioaxillary position. The subjects were divided into two groups based on duration of graft patency. The thrombosis group (Group I) comprised 26 patients who developed AVG thrombosis in the first 12 months after placement. The no-thrombosis group (Group II) comprised 29 patients whose grafts remained patient for at least 12 months. The frequency of the aa genotype in Group I was significantly higher than that in Group II (p =. 005). At 6, 12, and 24 months, the primary patency rates for the AVGs in patients with the aa genotype were significantly lower than the corresponding rates for the bb and ab genotype groupings (p =. 01, p =. 01 and p =. 04 for the three respective time points; Kaplan–Meier). ecNOS gene intron 4 VNTR polymorphism is linked with the pathogenesis of vascular access thrombosis in Turkish patients undergoing hemodialysis.     

Polymorphisms in the tumor necrosis factor-alpha gene in Turkish women with pre-eclampsia and eclampsia

The genetic background predisposing pregnant women to pre-eclampsia/eclampsia (PE/E) is still unknown. The aim of the current study was to investigate whether there is an association between the TNF-alpha-308 and 850 polymorphisms and PE or eclampsia. In this study, 40 cases of eclampsia, 113 cases of PE and 80 normotensive control cases were genotyped for the TNF-alpha-G-308A and C-850 polymorphisms. At position 308, the replacement of Guanine with Adenosine was denoted as TNF2. We found a significant difference between the TNF2 allele frequencies of the eclamptic, pre-eclamptic and normotensive controls. TNF2 (AA) polymorphism frequency was significantly higher among the eclamptics and pre-eclamptics (control : 5%, PE : 13.3%, E : 12.9%). A significantly different genotype distribution of C-850T polymorphism was observed between the PE/E and control groups, with the frequency of the variant TT genotype being significantly reduced in the preeclamptics (PE : 17% ; E : 17.5%) when compared with the control group (24.3%). We have demonstrated an association between TNF-alpha polymorphisms and pre-eclampsia susceptibility. However, it is not known whether C-850T polymorphism has a functional effect on the TNF-alpha gene. In addition, it was not possible to determine whether this polymorphism promotes the progression from PE to eclampsia because of no statistically significant difference between eclampsia and the controls.     

The effect of hepatitis C virus infection on insulin resistance in chronic haemodialysis patients

PURPOSE: To investigate the contribution of HCV infection to insulin resistance in chronic haemodialysis patients. MATERIALS AND METHODS: The study was performed with 55 patients who were on regular haemodialysis therapy three times per week. Of the 55 patients, 34 (20 females and 14 males with an average age of 40.9 years) were anti-HCV (+) and were defined as the HCV (+) group. The remaining 21 patients (8 females and 11 males with an average age of 50 years) were negative for HCV and other viral markers and were defined as the HCV (-) group. BMI of all patients were below 27. Insulin resistance (IR) was calculated according to the HOMA formula and patients were called HOMA-IR (+) if their HOMA scores were higher than 2.5. All of the HOMA-IR (+) patients in both groups were called the HOMA-IR (+) subgroup. None of the patients had a history of drug use or any diseases that were related to insulin resistance except uremia. In both groups and the healthy control group, insulin and glucose levels were studied at three different venous serum samples taken at 5- minute intervals after 12 hours of fasting. Other individual variables were studied at venous serum samples taken after 12 hours of fasting. RESULTS: HOMA scores were (3)2.5 in 22 of 34 HCV (+) patients (64.7%) and 7 of 21HCV (-) patients (33.33%) (p=0.024). Insulin levels of HCV (+) group (13.32 +/- 9.44mIU/mL) were significantly higher than HCV (-) (9.07 +/- 7.39mIU/mL) and the control groups (6.40 +/- 4.94mIU/ mL) (p=0.039 and p=0.021 respectively). HCV (+) patients were younger (40.94 +/- 17.06 and 52.62 +/- 20.64 years, respectively) and had longer dialysis duration (7.18 +/- 3.61 and 2.91 +/- 2.69 years, respectively). Significant positive correlations of HOMA score with insulin (r=0.934, p=0.000) and fasting glucose levels (r=0.379, p=0.043) were found in the HOMA- IR (+) subgroup. Also, a significant positive correlation was found between ALT and insulin levels in the HOMA IR (+) subgroup. C-peptide levels of both HCV (+) and (-) groups were significantly higher than the control group (p < 0.001). There were not any significant correlations between HOMA score and some of the other individual variables including levels of triglyceride, ferritin, ALT, iPTH and Mg in any of the groups. CONCLUSION: In chronic haemodialysis patients; HCV infection is related to a high prevalence of insulin resistance, higher insulin and glucose levels.     

Classification of carotid artery Doppler signals in the early phase of atherosclerosis using complex-valued artificial neural network

In this study, carotid arterial Doppler ultrasound signals were acquired from left carotid arteries of 38 patients and 40 healthy volunteers. The patient group had an established diagnosis of the early phase of atherosclerosis through coronary or aortofemoropopliteal angiographies. Results were classified using complex-valued artificial neural network (CVANN). Principal component analysis (PCA) and fuzzy c-means clustering (FCM) algorithm were used to make a CVANN system more effective. For this aim, before classifying with CVANN, PCA method was used for feature extraction in PCA-CVANN architecture and FCM algorithm was used for data set reduction in FCM-CVANN architecture. Training and test data were selected randomly using 10-fold cross validation. PCA-CVANN and FCM-CVANN architectures classified healthy and unhealthy subjects for training and test data with about 100% correct classification rate. These results shown that PCA-CVANN and FCM-CVANN classified Doppler signals successfully.     

Fetuin-A levels are increased in the adipose tissue of diabetic obese humans but not in circulation

Background

The hepatokine fetuin-A is linked to obesity and type 2 diabetes, but its presence and expression in adipose tissue remain unclear. In this study, we aimed to assess the circulating levels of fetuin-A and its expression in subcutaneous adipose tissue (SAT) from diabetic and non-diabetic obese subjects and their modulation by exercise.

Methods

SAT and blood were obtained from adults obese (diabetic, n=118 and non-diabetic, n=166) before and after a 3-month exercise program (diabetic, n=40 and non-diabetic, n=36, respectively). Plasma fetuin-A was assayed using ELISA. The presence and expression of fetuin-A in SAT, peripheral blood mononuclear cells (PBMCs) and cell lines (3T3-L1, THP-1, HepG2, RAW 264.7) were analysed using confocal microscopy, immunoblotting and qRT-PCR.

Results

Plasma fetuin-A level did not significantly differ between diabetic and non-diabetic obese subjects. However, when the non-diabetic group was divided into metabolically healthy and unhealthy phenotypes, significantly higher fetuin-A level was observed in the unhealthy sub-group. Circulating fetuin-A was mainly associated with glycaemic markers. In SAT, fetuin-A protein level was significantly higher in the diabetic obese subjects but its mRNA was not detected. Similarly, fetuin-A protein was detected in PBMCs, but its mRNA was not. In line with this, the use of various cell lines and culture media indicated that the presence of fetuin-A in SAT and PBMCs was due to its uptake from circulation rather than its endogenous expression. Finally, physical exercise decreased fetuin-A levels in both plasma and SAT in both groups.

Conclusions

Fetuin-A levels increased in association with diabetes in SAT but not in circulation in the obese subjects. Moreover, physical exercise decreased fetuin-A level. Fetuin-A potentially acts as a hepatokine taken up by other tissues, such as adipose tissue.

     

Effects of Flutamide on [Methyl-H]-Choline Uptake in Human Prostate Cancer-3 Cells: A Pilot Study

Background: Positron emission tomography using [methyl-¹¹C]-choline is effective in imaging many types of cancer, especially prostate cancer (PC). The antiandrogen flutamide is often used as part of the initial treatment of PC. Data on the effect of flutamide on and methylcholine incorporation into PC-3 cells are lacking in the experimental and literature work.Objectives: The aims of this study were to assess whether human PC-3 cells are susceptible to flutamide and whether the drug modulates the uptake of [methyl-³H]-choline into these cells.Methods: PC-3 cells were treated for 3 days with flutamide (≤100 nmol/L), inhibiting growth by 20% to 70% with control cells included. Two viability tests (cytotoxic analyses), the thiazole blue assay and the trypan blue exclusion method, were used to determine the median inhibitory concentration for flutamide (10 nmol/L). Control and flutamide-treated cells were incubated with [methyl-³H]-choline for 10 minutes and then in nonradioactive medium for 10 minutes to simulate the rapid blood clearance of [methyl-¹¹C]-choline tracer that occurs within 5 to 20 minutes, and then extracted using organic and aqueous solvents to determine the intracellular distribution of the tracer. Protein assay and flow-cytometry analysis were used to determine protein content and DNA synthesis in both control and treated cells. The uptake of [methyl-³H]-choline was normalized to protein content and expressed as mean (SD) dpm/1Jg protein (n = 6).Results: PC-3 cell proliferation was inhibited with flutamide treatment. After treatment of PC-3 cells with flutamide 10 nmol/L for 3 days, cells accumulated DNA during the S phase. Mean (SD) [methyl-³H]-choline uptake was found to be significantly lower with flutamide 10-nmol/L-treated cells compared with control cells (65.95 [0.72] vs 114.21 [0.57] dpm/1Jg protein; P < 0.001); the difference between the 5-nmol/L-treated cells and controls was nonsignificant.Conclusions: In this pilot study, flutamide inhibited tumor cell growth and proliferation and decreased (modulated) the uptake of [methyl-³H]-choline into androgen receptor-negative PC-3 cells. These results suggest that flutamide might inhibit proliferation by an androgen-independent mechanism.     

Erroneous introduction of femoral venous catheter into a broad ligament varicose vein causing hemoperitoneum in a woman with postpartum thrombotic microangiopathy

Femoral venous catheterization is a common procedure in critical care patients. Pregnant women and those in the postpartum period are at risk of various complications such as shock, acute kidney injury, and thrombotic microangiopathic syndromes requiring hemodialysis and plasma exchange, which may necessitate central venous catheterization. Femoral vein catheters may also sometimes be needed. These women may have underlying pelvic congestion and varicosities. Here we present a 24-year-old female patient, who has been treated for postpartum thrombotic microangiopathy with initial clinical improvements, became hemodynamically unstable with diffuse abdominal tenderness and a significant drop in the hemoglobin/hematocrit. Her abdominal ultrasound showed fluid in the peritoneal cavity with hemorrhagic diagnostic tap. The patient underwent exploratory laparotomy which unexpectedly revealed an erroneously introduced femoral vein catheter into a broad ligament varicose vein causing hemoperitoneum and evident ovarian injury. Puncturing of broad ligament varicosities causing hemoperitoneum in peripartum women has not been previously reported as a complication of femoral vein catheterization. This indicates that femoral catheterization in pregnant and peripartum women should be cautiously done and that development of acute abdominal issues, following insertion of femoral vein catheter should raise clinical suspicion and warrant evaluation of catheter misplacement.     

Synthesis and pharmacological evaluation of pyrazolopyrimidopyrimidine derivatives: anti-inflammatory agents with gastroprotective effect in rats

We report the synthesis of new anti-inflammatory 1,7-dihydropyrazolo[3′,4′:4,5]pyrimido[1,6-a]pyrimidine 5 from aminocyanopyrazole. All compounds were characterized by physical, chemical and spectral studies. Preliminary pharmacological evaluation of the resulting products showed that compounds 5a, b, f (50–100 mg/kg, i.p) are active anti-inflammatory agents in carrageenan-induced rat paw oedema assay, and their effects are comparable to that of acetylsalicylic–lysine (300 mg/kg, i.p.), used as a reference drug. The nature of substituent (Y, R₃) had a pronounced effect on the anti-inflammatory activity. Studies of structure–activity relationships have led to selection of compound ethyl-3,5-dimethyl-7-imino-N ¹-phenyl-1,7-dihydropyrazolo[3′,4′:4,5]pyrimido[1,6-a]pyrimidine-6-carboxylate, 5f which exhibited the most potent anti-inflammatory activity. In addition, the compounds 5a, b, f showed a significant gastroprotective effect against HCl/EtOH-induced gastric ulcer.