Hyperhomocysteinaemia and parameters of antioxidative defence in Tunisian patients with coronary heart disease.

BACKGROUND: An imbalance between oxidative damage and antioxidative protection in association with the pathophysiology of atherosclerosis has been suggested. The aim of this study was to test the parameters of antioxidative defence and to assess their association with hyperhomocysteinaemia and the severity of coronary heart disease (CHD) in Tunisian patients. METHODS: The study population included 100 patients with CHD and 120 healthy controls. The severity of CHD was expressed as the number of affected vessels. Superoxide dismutase (SOD) activity, glutathione peroxidase (GPx) activity and total antioxidant status (TAS) concentrations were measured using commercially available methods. Plasma total homocysteine (tHcy) concentration was determined by direct chemiluminescence assay. Serum zinc (Zn) was measured by a colorimetric method. RESULTS: Compared with healthy control subjects, patients with CHD had significantly lower activities of SOD (P < 0.01), GPx (P < 0.001), and serum Zn concentrations (P < 0.001) and significantly higher tHcy concentration (P < 0.001). However TAS concentrations were not significantly different between the groups. SOD and GPx activities were negatively correlated with tHcy concentration (P < 0.05, P < 0.001, respectively). Patients with hyperhomocysteinaemia showed a lower GPx and SOD activities than patients with normohomocysteinaemia. Antioxidant enzyme activities tended to be decreased in CHD patients presenting with 0- to 3-vessel stenosis. CONCLUSIONS: This study indicates that low activity of GPx, SOD and Zn concentration are associated with CHD patients. We hypothesize that hyperhomocysteinaemia and low antioxidant enzyme activities may increase the extent of CHD.

Ex vivo lentivirus transduction and immediate transplantation of uncultured hepatocytes for treating hyperbilirubinemic Gunn rat.

BACKGROUND: Ex vivo liver gene therapy provides an attractive alternative to orthotopic liver transplantation for the treatment of liver diseases. We previously reported a protocol in which human primary hepatocytes are highly transduced in Suspension with Lentiviral vectors and Immediately Transplanted (SLIT). Here, we evaluated the SLIT approach in Gunn rats, the animal model for Crigler-Najjar syndrome type 1, a defect in bilirubin UDP-glucuronosyltransferase (BUGT). METHODS: We constructed lentiviral vectors coding for BUGT under control of an ubiquitous promoter. Control vectors contained Green Fluorescent Protein (GFP) under control of the same promoter. Hepatocytes were isolated from jaundiced Gunn rats and transduced in suspension for four hr. After washing, 2x10 hepatocytes were immediately transplanted into syngeneic rats. Bilirubinemia and bile pigments were regularly assessed after cell transplantation. The percentage and presence of transduced hepatocytes was analyzed by immunohistochemistry in GFP-transplanted animals. RESULTS: In rats receiving BUGT-transduced hepatocytes, bilirubinemia decreased by about 30%. The level of correction remained stable for up to 240 days. Bilirubin glucuronides were present in the bile of treated animals, indicating the metabolic activity of engrafted hepatocytes. In contrast, bilirubinemia in GFP-transplanted rats did not decline but rather increased. GFP-positive hepatocytes amounted to 0.5-1% of the liver, which is in agreement with the number of transplanted and genetically-modified hepatocytes (6x10). CONCLUSIONS: This work reports the first demonstration of long-term metabolic benefit after rapid transplantation of ex vivo lentivirally tranduced hepatocytes. Therefore, this study demonstrates the therapeutic proof-of-principle and potential of the SLIT approach for treating inherited metabolic liver diseases.

Hyperhomocysteinemia, paraoxonase activity and risk of coronary artery disease.

OBJECTIVES: Paraoxonase-1 (PON1) detoxifies homocysteine thiolactone (HcyT) in human blood and could thus delay the development of atherosclerosis. We investigated (a) PON1 activity and polymorphisms, and (b) the relationship between PON1 activity, homocysteine (Hcy) and the severity of CAD patients in Tunisian population. DESIGN AND METHODS: We used PCR-RFLP analysis to detect the Q192R and L55M variants of the PON1 gene in 100 patients with CAD and in 120 healthy controls. Paraoxonase activity was measured spectrophotometrically using phenylacetate as a substrate. Total plasma homocysteine concentrations were determined by direct chemiluminescence assay. RESULTS: We found an increased Hcy level in CAD patients compared to the control group (15.86+/-8.63 vs. 11.9+/-3.25 micromol/L respectively, P<0.001), and a decrease in PON1 activity in CAD patients as compared to the control group (117+/-56 vs. 181+/-73 U/mL respectively, P<0.001). PON1 Q192R and L55M polymorphisms were not associated with the presence of CAD (P=0.592, P=0.294, respectively). However, we found that PON1 activity is lower with the PON1 192RR than with PON1 192QQ genotypes in the study population. Furthermore, there were no association between PON1 L55M polymorphism and PON1 activity. We showed a significant decrease in PON1 activity in CAD patients presenting 0- to 3-vessel stenosis (155+/-39; 135+/-36; 103+/-22; 77+/-24 U/mL, respectively; P<0.001). CONCLUSION: In this study, we showed that low PON1 activity is associated with the PON1 192RR genotypes and associated with the severity of CAD in the Tunisian population. We hypothesize that high level of Hcy together with low PON1 activity results in an increased plasma HcyT plasma concentration leading to protein N-homocysteinylation and the development and progression of atherosclerosis.

Hyperhomocysteinaemia, methylenetetrahydrofolate reductase polymorphism and risk of coronary artery disease.

BACKGROUND: Hyperhomocysteinaemia is an independent, graded risk factor for coronary artery disease (CAD). The methylenetetrahydrofolate reductase (MTHFR) polymorphism is associated with hyperhomcysteinaemia and may therefore influence individual susceptibility to CAD. We have investigated this risk factor in a Tunisian Arab population. METHODS: Polymerase chain reaction-restriction fragment length polymorphism analysis was used to detect the C677T and A1298C variants of the MTHFR gene in 100 patients with CAD and 120 healthy controls. The severity of CAD was expressed as the number of affected vessels. Plasma total homocysteine (tHcy) concentration was determined using a direct chemiluminescence assay. RESULTS: MTHFR CC, CT and TT genotype frequencies in the CAD group were significantly different from those observed in the control group (49%, 35% and 16% versus 48.3%, 45.8% and 5.8%, respectively; P = 0.031). However, MTHFR AA, AC and CC genotypes frequencies in the CAD group were not significantly different from the control group ( P = 0.568). Patients with CAD showed higher plasma tHcy concentrations than patients without CAD (15.86 +/- 8.63 micromol/L versus 11.90 +/- 3.25 micromol/L, P < 0.001). There was no association between the MTHFR polymorphisms and the number of stenosed vessels. Patients with the MTHFR TT genotype had higher plasma tHcy, serum creatinine, cholesterol and triglyceride concentrations than patients with the MTHFR CC genotype. CONCLUSIONS: The C677T polymorphism of the MTHFR gene is associated with hyperhomocysteinaemia, lipid dysregulation and the presence of CAD in this Tunisian Arab population.

Comparison of the inflammatory response between miniaturized and standard CPB circuits in aortic valve surgery.

OBJECTIVE: One of the complications of CPB is the systemic inflammatory response syndrome (SIRS). Recent developments tend to minimize the biological impact of CPB in using miniaturized closed circuit with reduced priming volume and less blood-air interface. The benefit of these miniaturized closed circuits in terms of inflammatory response has been proved in coronary surgery. However, in open heart surgery, the CPB circuit is no more closed and the benefit of the miniaturized set-up could disappear. The aim of the study is to compare the SIRS between standard and miniaturized circuits in aortic surgery. METHODS: Forty patients who underwent singular aortic valve replacement were randomly assigned either to a standard CPB (group A, n=20) or to a miniaturized CPB (group B, n=20). Pertinent clinical and surgical data were collected. Hematological parameters (leukocyte and neutrophil counts) and biochemical parameters (C-reactive protein, cytokine tests) were determined pre-, on and post-CPB. RESULTS: There were an increase in leukocyte and neutrophil counts and a decline in hematocrit in both groups. In both groups, there was a raise after CPB, in C-reactive protein, IL-6, TNF-alpha, neutrophil elastase, and IL-10. However, the raises of elastase and TNF-alpha were significantly lower after the weaning of miniaturized CPB (116+/-46 ng/ml and 10+/-4 pg/ml, respectively) compared to standard CPB (265+/-120 ng/ml, P=0.01 and 18+/-7 pg/ml, P=0.03). The raise of IL-10 is also lower with miniaturized circuit (15+/-6 pg/ml) compared to standard circuit (51+/-26, P=0.004). CONCLUSIONS: This study demonstrates in aortic surgery, the lesser inflammatory response of a miniaturized CPB compared to a standard CPB. However, there is always some inflammation after CPB and a small bio-reactive free perfusion circuit is still to be found in open heart surgery.

Effects of ursodeoxycholic acid (ursodiol) treatment on chronic viral hepatitis in heart transplant patients: results of a prospective, double-blind, placebo-randomized study.

BACKGROUND: Chronic viral hepatitis averages 15% to 20% in heart transplant patients. Several studies have shown that ursodiol may improve liver biochemistry in patients with chronic hepatitis. We used a double-blind randomized controlled trial to evaluate the effect of ursodiol in heart transplant patients with chronic viral hepatitis. METHODS: Thirty heart patients with chronic viral hepatitis B, C, or non-A-G received ursodiol, 800 mg per day (group 1), and 30 received placebo (group 2) for 12 months. Endpoints were improvement in liver biochemical tests and in total Knodell score. Intent-to-treat and per-protocol analyses were performed. RESULTS: At entry, both groups were comparable for all of the studied parameters. During the study period, serum alanine aminotransferase, aspartate aminotransferase, and gamma-glutamyl transpeptidase variations were not different between group 1 and group 2 patients. Knodell score improved in 20% of group 1 patients and in 43% of group 2 patients (NS). Adverse events or mortality were not different in the two groups during the study period. Similar results were observed by intent-to-treat and per-protocol analyses. CONCLUSIONS: A 12-month course of ursodiol therapy had no effect on liver enzymes or liver histology in heart transplant patients with chronic hepatitis.

Hyperhomocysteinemia, paraoxonase concentration and cardiovascular complications in Tunisian patients with nondiabetic renal disease.

OBJECTIVES: Hyperhomocysteinemia is associated with an increased risk of cardiovascular diseases. We determine homocysteine levels (Hcy), paraoxonase (PON1) concentration and their relationship on cardiovascular complications in patients with chronic renal disease (CRD). DESIGN AND METHODS: The study population included 100 CRD patients and 120 healthy controls. Renal function was assessed using the eGFR by the MDRD study equation. Patients were considered to have CRD when the eGFR was <60 mL/min/1.73 m(2). Hcy concentrations were determined by direct chemiluminescence assay. PON1 concentration was measured spectrophotometrically using phenylacetate as a substrate. RESULTS: We found an increased Hcy levels and a decreased eGFR and PON1 concentration in CRD patients compared to the control group (P<0.001, P<0.001, P<0.01 respectively). Patients with cardiovascular complications showed an increased Hcy levels and a lower PON1 concentration than patients without cardiovascular complications (P<0.001, P<0.01 respectively). CONCLUSION: We showed that hyperhomocysteinemia and low PON1 concentration are associated with CRD and markedly associated in patients with cardiovascular complications. Additional effects contribute to the severity of renal disease and increase the incidence of cardiovascular disease.

Endothelial nitric oxide synthetase, methylenetetrahydrofolate reductase polymorphisms, and cardiovascular complications in Tunisian patients with nondiabetic renal disease.

OBJECTIVES: The relationship between endothelial nitric oxide synthase (eNOS), methylenetetrahydrofolate reductase (MTHFR) gene polymorphisms and cardiovascular disease (CVD) in Tunisian patients with chronic renal disease (CRD) has not been examined. We investigated (a) the relationship of these gene polymorphisms with the presence and the severity of renal disease, and (b) their relationships with CVD in these patients. DESIGN AND METHODS: We used PCR-RFLP analysis to detect the eNOS G894T, MTHFR C677T and A1298C variants in 100 patients with CRD and in 120 healthy controls. RESULTS: MTHFR C677T and A1298C polymorphisms were not associated with the presence of renal disease. However, we found that eNOS G894T polymorphism was associated with the presence and severity of renal disease and with CVD in CRD patients (P=0.028, P=0.018, P=0.016 respectively). We showed that 894T allele was an independent risk factor of severity of renal disease and the incidence of CVD (P=0.01 and P<0.01 respectively). CONCLUSION: The G894T polymorphism of the eNOS gene is associated with severity of renal disease. Presence of the 894T allele aggravated renal damage and increased the incidence of CVD in Tunisian CRD patients.

Comparative study on in vitro effects of homocysteine thiolactone and homocysteine on HUVEC cells: evidence for a stronger proapoptotic and proinflammative homocysteine thiolactone.

Hyperhomocysteinemia is an independent risk factor for the development of atherosclerosis. However the underlying mechanisms responsible for endothelial cell injury with increased plasma concentration of homocysteine or homocysteine derivatives remains still incompletely elucidated. In this study, we investigated the ability of homocysteine (Hcy) and homocysteine thiolactone (HcyT) to induce cell death and IL-8 secretion in primary human umbilical vein endothelial cells (HUVEC). Hcy and HcyT were both cytotoxic and capable of promoting cell death, as measured by caspase-3 activation and DNA fragmentation. ELISA assays clearly demonstrated that Hcy and HcyT strongly activated IL-8 release. Furthermore, our results showed that HcyT was much more efficient than Hcy in activating caspase-3 or in inducing IL-8 secretion. The use of antioxidants such as vitamin C and vitamin E strongly but not completely reduced programmed cell death and chemokine release suggesting that other pathways different than reactive oxygen species are also involved. This study suggests that Homocysteine derivatives like HcyT might possess stronger cytotoxicity and pro-inflammatory properties and that Hcy derivatives levels should therefore be more taken into account during diagnostics.

Hyperhomocysteinemia, endothelial nitric oxide synthase polymorphism, and risk of coronary artery disease.

BACKGROUND: Hyperhomocysteinemia is an independent, graded risk factor for coronary artery disease (CAD). The G894T variant of endothelial nitric oxide synthase (eNOS) was postulated to be associated with hyperhomocysteinemia and could influence individual susceptibility to CAD. The aims of this study were to investigate (a) the relationship of the eNOS G894T polymorphism with the presence and the severity of CAD and (b) the possible relationship between hyperhomocysteinemia and the eNOS G894T variant for the risk of CAD severity in a Tunisian population. METHODS: We used PCR with restriction fragment length polymorphism analysis to detect the G894T variant of the eNOS gene in 100 patients with CAD and 120 healthy controls. The severity of CAD was expressed by the number of affected vessels. Total plasma homocysteine concentrations were determined by direct chemiluminescence assay. RESULTS: The frequencies of the eNOS GG, GT, and TT genotypes in the CAD group were significantly different from those in the control group (45%, 44%, and 11% vs 60%, 35.8% and 4.2%, respectively; P = 0.035). There was no association between the eNOS G894T genotype frequencies and the number of stenosed vessels (P = 0.149). In the CAD group, the coexistence of the 894 GT or TT genotypes and hyperhomocysteinemia led to an increased risk of CAD severity. CONCLUSION: The G894T polymorphism of the eNOS gene is associated with the presence of CAD, and in conjunction with hyperhomocysteinemia, increased the risk of CAD severity in a Tunisian population.

Taurine status and response to intravenous taurine supplementation in adults with short-bowel syndrome undergoing long-term parenteral nutrition: a pilot study.

Taurine deficiency in patients on long-term parenteral nutrition may be involved in cholestasis. We aimed to assess plasma taurine and tauro-conjugated bile acids in adults with short-bowel syndrome and their response to intravenous taurine. Thirty-two adult patients, who had been on taurine-free parenteral nutrition for a mean of 59 (SE 14) months for short-bowel syndrome, were studied retrospectively. In a second study, a subgroup of ten patients with chronic cholestasis received taurine-enriched (6.0 (SE 0.6) mg/kg per d) parenteral nutrition for 55 (SE 13) months. Post-absorptive plasma taurine and bile acid concentrations were measured and liver function tests routinely sampled. At baseline, plasma taurine was lower in patients with a jejunal length of less than 35 cm (group A, n 16) than in those with a jejunal length of 35 cm or more (group B, n 16): 43 (SE 3) v. 58 (SE 4) micromol/l (P=0.01). The groups were no different in terms of chronic cholestasis (12/16 v.13/16 patients), total bile acids (26 (SE 13) v.14 (SE 5) micromol/l) or the ratio of tauro-conjugated:glyco-conjugated bile acids (5 (SE 2) v.8(SE 4)%, usual range 30-60%). After supplementation, there was an increase in plasma taurine level (63 (SE 8) v. 43 (SE 4), P=0.007) but was no change in either total bile acids or the ratio of tauro-conjugated: glyco-conjugated bile acids. There was a significant decrease in aspartate aminotransferase level. Long-term parenteral nutrition for short-bowel syndrome is associated with an impaired tauro-conjugation of bile acids (enterohepatic pool), irrespective of plasma taurine level (systemic pool) and despite long-term taurine intravenous supplementation.

Successful gene therapy of the Gunn rat by in vivo neonatal hepatic gene transfer using murine oncoretroviral vectors.

Crigler-Najjar type 1 disease (CN1) is a rare inherited metabolic disease characterized by complete absence of hepatic UDP-glucuronosyl transferase (UGT1), resulting in high levels of unconjugated bilirubin. CN1 is an attractive candidate disease for gene therapy. Here we show that in vivo neonatal hepatocyte transduction using recombinant oncoretroviral vectors results in long-term and complete phenotype correction in Gunn rats, a model for CN1. Two-day-old newborn Gunn rats were injected via the temporal vein with 200 microL UGT1 or control beta-galactosidase retroviral vectors. In UGT1-injected animals, bilirubinemia was normal at 6 weeks (3 micromol/L) and remained in the normal range (i.e., <10 micromol/L) for more than 34 weeks. In contrast, in beta-galactosidase-injected animals as well as in noninjected controls, bilirubinemia remained at a high level (i.e., >100 micromol/L) during the whole experimental follow-up. Large amounts of bilirubin monoglucuronides and diglucuronides were present in the bile of treated animals. Finally, polymerase chain reaction and reverse transcription polymerase chain reaction analysis as well as Western blot confirmed the presence and expression of UGT1 almost exclusively in the liver. The estimated proportion of transduced hepatocytes was in the range of 5% to 10%. In conclusion, complete and permanent correction of hyperbilirubinemia in newborn Gunn rats using retroviral vectors can be obtained, paving the way for future gene therapy for CN1.