Subclinical inflammation and prothrombotic state in heart transplant recipients: impact of cyclosporin microemulsion vs. tacrolimus

BACKGROUND: Subclinical inflammation is related to adverse events in patients with coronary artery disease. In the present study, we determined the changes in hemostatic parameters and inflammatory markers in a large cohort of dyslipidemic cardiac transplant recipients compared with dyslipidemic healthy controls, and the effect of cyclosporin microemulsion (CsA) vs. tacrolimus immunoprophylaxis on these parameters. METHODS: Stable cardiac transplant recipients (n=129) aged 56.7+/-10.1 years, 79+/-42 months postcardiac transplantation, and 26 mildly dyslipidemic healthy control subjects had serum measurements for lipids and lipoproteins, hemostatic parameters, and selected inflammatory markers. Transplant recipients were randomized to either continuation of CsA maintenance or conversion to tacrolimus immunoprophylaxis and were reassessed after six months. RESULTS: CsA-maintained cardiac transplant recipients exhibited a significant elevation in Factor VIII, Von Willebrand factor, fibrinogen and PAI-I compared with healthy control subjects (all P<0.05). Similarly, cardiac transplant patients yielded a significantly elevated C-reactive protein (CRP) (4.11+/-6.25 [transplant group (TX)] vs. 2.09+/-2.21 mg/L [control group (CTL)]; P=0.0195), and homocysteine (19.2+/-8.8 [TX] vs. 9.70+/-2.45 microM [CTL]; P<0.001). VCAM, ICAM, E- and P-selectins were also significantly higher in transplant patients than in controls (all P<0.05). The conversion from CsA to tacrolimus resulted in a significant decrease in uric acid, total- and LDL-cholesterol, apolipoprotein B, creatinine, and homocysteine levels (all P<0.05). CONCLUSIONS: Stable long-term CsA-maintained cardiac transplant patients exhibit a significant and general increase in hemostatic parameters and markers for subclinical inflammation. Tacrolimus conversion improved the patient lipid profile and decreased serum creatinine, uric acid, and homocysteine without any significant effect on the other markers.     

Omega-3 fatty acids enhance tumor necrosis factor-alpha levels in heart transplant recipients.

BACKGROUND: Proinflammatory cytokines may contribute to clinical complications in heart transplant (HTx) recipients. Previous studies have shown immunomodulating effects of omega-3 fatty acids, but the results are somewhat conflicting. In this study, we examined plasma levels of tumor necrosis factor alpha (TNF-alpha), interleukin (IL) 10, and their relations to antioxidant vitamins in 45 HTx recipients before and after treatment with omega-3 fatty acids or placebo. METHODS: The patients were long-time survivors of heart transplantation, randomized in a double-blind fashion to receive omega-3 fatty acids (3.4 g/day) or placebo for 1 year. Plasma levels of cytokines were measured by enzyme immunoassays and vitamin A, vitamin E, and beta-carotene by high-performance liquid chromatography. RESULTS: In the omega-3, but not in the placebo group, there was a rise in the proinflammatory cytokine TNF-alpha (P<0.05), a decrease in the anti-inflammatory cytokine IL-10 (P=0.07), and a rise in TNF/IL-10 ratio (P<0.05) after 12 months, suggesting a proinflammatory net effect. In the omega-3 group, the increase in TNF-alpha was associated with an increase in eicosapentaenoic acid in plasma (r=0.58, P<0.02). During omega-3 fatty-acid treatment, but not during placebo, there was a decrease in vitamin E (P<0.05) and beta-carotene (P<0.05) levels, and the decrease in vitamin E was inversely correlated with the increase in TNF-alpha (r= -0.56, P<0.01). The rise in TNF-alpha levels during omega-3 fatty acids treatment was most pronounced in those patients with transplant coronary artery disease (P<0.04). CONCLUSION: Our data suggest that omega-3 fatty acids in HTx recipients may change the balance between proinflammatory and anti-inflammatory cytokines in an inflammatory direction, possibly related to prooxidative effects of these fatty acids.     

Experimental studies on the effects of alpha-tocopherol in small intestinal ischemia and reperfusion injury]

In the present study, we quantified the biochemical [thiobarbituric acid (TBA) reactants, superoxide dismutase (SOD) and vitamin E] and histologic changes in the small intestinal tissue after ischemia and/or reperfusion. Sixty-seven Wistar rats were divided into 7 groups; N group: control, A-I group: 30 min. ischemia, A-II group: 120 min. ischemia, B-I group: Declamp after 30 min. ischemia, B-II group: 30 min. reperfusion after 30 min. ischemia, B-III group: 30 min. reperfusion after 120 min. ischemia, E group: vitamin E administration 30 min. reperfusion after 30 min. ischemia. The levels of TBA reactants were significantly different between A-II and B-II, B-II and E (all p less than 0.01). For SOD, there were significant differences between N and B-I (p less than 0.01), N and E (p less than 0.05). For vitamin E, there were significant differences between A-I and B-I, A-I and B-I, B-II and E (all p less than 0.01). Histologic changes showed that the grade of tissue injury was more severe in B-I and B-II than in A-I, and was less in E than in B-II. It is suggested that vitamin E protected cells from injury due to oxygen free radicals.     

The effect of warm ischemic time on renal function and injury in the isolated hemoperfused kidney

BACKGROUND: The precise effect of warm ischemia on renal allograft function remains unclear and leads to variable warm ischemic time (WIT) limits advocated by transplant programs. This study aims to investigate the relationship between WIT, renal ischemia reperfusion injury, and graft function using a hemoperfused kidney model. METHODS: Porcine kidneys were perfused with normothermic blood on an isolated organ perfusion system. Kidneys were divided into four groups (n=6) and subjected to 7, 15, 25, and 40 min WIT. Physiological parameters were measured throughout the 6 hr perfusion period. Serum, tissue, and urine samples were analyzed for histological and biochemical markers of ischemia reperfusion injury. RESULTS: Creatinine clearance, urine output, renal hemodynamics, and oxygen consumption deteriorated proportionally with increasing WIT. A significant increase in plasma carbonyl levels during perfusion was seen after 25 and 40 min WIT only. Plasma 8-isoprostane levels were higher after 40 min WIT (2.5+/-1.6) vs. 7, 15, and 25 min WIT (0.65+/-0.43, 0.25+/-0.12, and 0.62+/-0.21, respectively; P<0.05). A negative correlation was shown between urine output and plasma carbonyls (r=-0.415, P<0.05) and between 8-isoprostane levels and creatinine clearance (r=-0.649, P<0.005). Caspase-3 activity was significantly higher after 7 min WIT compared with the other groups, correlating positively with creatinine clearance, urine output, and renal blood flow. CONCLUSION: The isolated organ perfusion system successfully delineated a clear WIT-dependent variation in renal function which correlated accurately with oxidative injury markers. This model may represent a clinically applicable tool for assessing graft viability.     

Apoptotic Markers in Donor Hearts After Brain Death vs Circulatory Death

BackgroundUse of donation after circulatory death (DCD) hearts is becoming more prevalent in cardiac transplantation. However, there is no standardized approach to myocardial preservation, and little data exists on ultrastructural changes in DCD hearts. We have previously identified increased proapoptotic and proinflammatory activity in brain dead donor (BDD) hearts that subsequently exhibit primary graft failure and lower levels in DCD left atrial tissue. This study further investigates these markers and correlates them with cardiac function in DCD hearts.MethodsThis prospective study used donor hearts deemed unsuitable for transplant after gaining institutional ethics approval; 11 human hearts were obtained from 5 DCD donors and 6 BDDs. All hearts were preserved by continuous microperfusion for 4 hours with a cold crystalloid solution and then were evaluated on a blood perfusion bench rig. After 4 hours perfusion and working assessment, tissues from all cardiac chambers were stored for later messenger RNA (mRNA) analysis for proapoptotic and proinflammatory markers.ResultsSignificantly raised levels of caspase-1, BNIP3, and NADPH oxidase mRNA expression were identified in cardiac chambers from BDD hearts compared to DCD hearts, and these differences were exaggerated in older donors. In the pooled analysis, lower expression of caspase-1, NF-κB1, and BNIP3 mRNA correlated with developed pressure at 1 hour after reperfusion in the right ventricle, but not the left.ConclusionCompared to BDD hearts, DCD hearts exhibit less stimulation of proapoptotic cascades and reactive oxygen species, potentially reducing their susceptibility to ischemic reperfusion injury.     

Bax ablation protects against hepatic ischemia/reperfusion injury in transgenic mice.

Apoptosis appears to be a central mechanism of cell death following reperfusion of the ischemic liver. The aim of this study was to determine the effect of decreased expression of the proapoptotic Bax gene on hepatic apoptotic warm ischemia/reperfusion (I/R) injury. Three groups of mice were studied: homozygotic knockout mice (Bax-/-); heterozygotic (Bax+/-); and wild type (Bax+/+). Isolated mouse livers were subjected to 90 minutes of ischemia (37 degrees C) followed by 15 minutes of reperfusion. Bax and Bcl-2 expression in liver tissue homogenates was measured by Western blot. Serum liver enzyme levels were measured and intrahepatic caspase-3 activity was determined by fluorimetric assay. Oil red O (ORO) staining was performed for fat detection. Apoptotic cells were identified by morphological criteria, immunohistochemistry for caspase-3, and terminal deoxynucleotidyl transferase-mediated 2'-deoxyuridine 5'-triphosphate nick-end labeling (TUNEL) assay. At 1 minute of reperfusion, the ischemic (Bax-/-) livers were characterized by statistically significantly lower liver enzyme levels and lower caspase-3 activity than the ischemic (Bax+/+) livers (P<0.05 for both). The reduction in postischemic apoptotic hepatic injury in the ischemic Bax-/- livers group was confirmed morphologically, by the significantly reduced microvesicular steatosis as determined by ORO staining, fewer apoptotic hepatocyte cells detected (P<0.05); immunohistochemically, by the significantly weaker activation of caspase-3 compared to the ischemic group (P<0.05); and by TUNEL assay (P<0.05). Similar levels of antiapoptotic Bcl-2 protein expression were detected in all 3 groups of ischemic livers on Western blots. Bax protein was not expressed in Bax-deficient livers and was detected in Bax+/+ normal livers. In the Bax+/- livers, levels of the damage markers were moderate. In conclusion, The better tolerance of Bax knockout livers to I/R injury suggests that the Bax gene may serve as a potential target for therapeutic intervention in hepatic I/R injury.     

Moderate hyperoxia (40%) increases antioxidant levels in mouse tissue

BACKGROUND: Oxygen is routinely administered to patients to improve clinical outcome. Since studies have shown that administering 100% oxygen can cause unwanted side effects, intermediate concentrations of 40% oxygen are used in clinical practice. In this study, we examined whether the breathing of 40% oxygen causes beneficial effects upon tissue levels of antioxidants such as vitamin E, vitamin C, and glutathione. METHODS: Four-month-old mice were separated into two groups: control (n = 11) and experimental (n = 11). The treatment group was administered 40% oxygen for 10 days. Brain, heart, lung, liver, testes, and skeletal muscle were harvested and tissue antioxidant levels were determined by HPLC. RESULTS: Vitamin E concentrations were higher in brain, heart, lung, liver, and testes of the treatment group (P < 0.05). Glutathione concentrations were higher in the lung tissue only (P < 0.05). No differences were found in vitamin C levels. CONCLUSIONS: The data suggest that mice respond to oxidative stress by increasing tissue vitamin E incorporation and cellular synthesis of glutathione in the lung when exposed to moderate levels (40%) of hyperoxia.     

Effects of antioxidant supplementation on blood cyclosporin A and glomerular filtration rate in renal transplant recipients.

BACKGROUND: Transplant recipients have elevated oxidative stress, which has prompted suggestions that supplementary antioxidants may be beneficial. However, only a small number of clinical trials have investigated antioxidant supplementation in transplant recipients, with very few data on their effects on patients' immunosuppressive therapy. METHODS: A randomized placebo-controlled single-blind crossover trial was conducted in 10 renal transplant recipients (RTRs) taking cyclosporin A (CsA) as part of their immunosuppressive therapy. Each phase of the trial lasted 6 months, with a 6 month wash-out period in between. During one of the phases, patients consumed a tablet twice per day which delivered 400 IU/day of vitamin E, 500 mg/day of vitamin C and 6 mg/day of beta-carotene. RESULTS: During antioxidant supplementation, there was no change in CsA dose. Antioxidant supplementation resulted in a significant decrease (P<0.05) in blood trough CsA by 24% (mean+/-SD, pre- 127.3+/-38.9, post- 97.2+/-30.7 microg/ml) compared with no change while taking the placebo (pre- 132.2+/-50.6, post- 138.6+/-56.0 microg/ml). The glomerular filtration rate was significantly (P<0.05) improved by 12% during antioxidant supplementation (pre- 66.9+/-20.7, post- 75.0+/-20.1 ml/min/1.72 m2), with no change during the placebo phase (pre- 66.8+/-11.8, post- 66.7+/-16.1 ml/min/1.72 m2). There were no significant differences (P>0.05) in markers of oxidative stress (malondialdehyde, susceptibility of plasma to oxidation) or plasma antioxidant enzymes. CONCLUSION: In CsA-treated RTRs, antioxidant supplementation decreased blood CsA, which may affect adequacy of immunosuppression.     

膀胱癌患者手术前后机体氧化应激改变及意义

目的探讨膀胱癌患者手术前后机体氧化应激变化及临床意义。方法 173例膀胱癌患者入选研究,其中移行上皮癌163例,腺癌5例,鳞癌4例,肉瘤1例;非肌层浸润性膀胱癌121例,肌层浸润性膀胱癌52例。分别检测手术前后患者血清VitC、VitE、超氧化物歧化酶（SOD）、谷胱苷肽过氧化物酶（GPx）、丙二醛（MDA）、总抗氧化活性（AOA）等。结果非肌层浸润性膀胱癌患者血清VitC、VitE、SOD、GPx均明显低于肌层浸润性膀胱癌,P〈0.05,MDA含量高于肌层浸润性膀胱癌,P〈0.05。手术后患者血清VitC、VitE、SOD、GPx、AOA均明显高于手术前（P〈0.05）,但MDA含量明显低于手术前（P〈0.05）。结论膀胱癌患者机体氧化应激随其恶性程度升高而升高,手术治疗后机体氧化应激水平降低。     

维生素E治疗对老年雌性大鼠骨量、骨代谢以及自噬水平影响

目的 观察维生素E对老年雌性大鼠骨量、骨代谢作用及自噬水平的影响.方法 10只年轻雌性大鼠和20只老年雌性Sprague-Dawley大鼠被随机分为三组:对照组(10只年轻大鼠)、模型组(10只老年大鼠)和治疗组(10只老年大鼠).在实验过程中,治疗组接受维生素E治疗.治疗12周后,通过Micro-CT和HE切片染色检...     

Vitamin E Succinate Enhances Steatotic Liver Energy Status and Prevents Oxidative Damage Following Ischemia/Reperfusion

We have previously shown that treatment of steatotic livers with vitamin E succinate decreases liver injury and increases survival after ischemia/reperfusion (I/R). It is now understood that compromised energy status is associated with increased injury following liver ischemia in the setting of hepatic steatosis at least partially as a result of increased reactive oxygen species (ROS) and induction of mitochondrial uncoupling protein-2 (UCP2). Given the association between ROS, mitochondrial function, and UCP2, it was our goal to determine whether the protective effects of vitamin E succinate were associated with decreased ROS injury, down-regulation of UCP2, or improvement of ATP levels following I/R. To test this, leptin deficient (ob/ob) mice with steatotic livers that had received other 50 IU of vitamin E succinate supplement per day or control chow for 7 days were subjected to total hepatic ischemia (15 minutes) followed by reperfusion. We measured liver expressions of ATP, glutathione (GSH), and UCP2 as well as mitochondrial DNA damage. Vitamin E treatment decreased hepatic UCP2 expression and increased ATP and GSH levels prior to I/R. These levels were maintained at 1 hour after I/R. At 24 hours, while hepatic UCP2 expression, ATP, and GSH levels were similar to those of mice not receiving vitamin E, mitochondrial DNA damage was blocked. These results revealed that vitamin E succinate decreased hepatic UCP2 expression, reduced oxidative stress, and improved mitochondrial function in mice with steatotic livers before and after I/R, identifying mechanisms of protection in this setting.     

N-acetylcysteine Improves Early Cardiac Isograft Function in a Rat Heterotopic Transplantation Model

Reactive oxygen species play an important role in the early phase of ischemia-reperfusion injury. N-acetylcysteine (NAC), an antioxidant, has shown protective effects in ischemia-reperfusion injuries in some organ transplantations; however, its roles in cardiac transplantation have not been thoroughly evaluated. Lewis rats were divided into three groups (n = 8 in each group): sham, control, and NAC group. They were subjected to abdominal heterotopic cardiac transplantation and followed for 24 hours postoperation. For the NAC group, a bolus of NAC (150 mg/kg) was infused at 30 minutes before reperfusion, followed by 20 mg/kg/h for 1 hour; another 150 mg/kg NAC intraperitoneally administered 12 hours after reperfusion. Cardiac function and blood inflammation markers were measured at both 2 and 24 hours after reperfusion. Oxidative stress markers and neutrophil infiltration were evaluated in heart tissue at 24 hours postoperatively. Echocardiography showed that NAC significantly improved the postoperative fractional shortening of isografts (P < .01), although NAC had no effect on the heart rate. Serum lactate dehydrogenase also indicated a significant decrease in the NAC group at 24 hours posttransplantation (P = .02) Serum tumor necrosis factor-α and interleukin-1 concentrations which rapidly increased postoperatively were reduced by administration of NAC. In transplanted hearts, the increased malondialdehyde level and myeloperoxidase activity were partially reversed by NAC (both P < .01); NAC also replenished endogenous glutathione. Finally, neutrophil infiltration in isografts was reduced in the NAC group. Recipient treatment with continuous intravenous NAC protected cardiac isografts against posttransplantation ischemia-reperfusion injury, probably through its antioxidant and anti-inflammatory properties.     

Expression of IL-8 during reperfusion of renal allografts is dependent on ischemic time

BACKGROUND: Ischemia/reperfusion injury is an inherent consequence of solid organ transplantation that increases tissue inflammation and negatively impacts organ transplant function and survival. This study investigated the expression levels of chemokine and chemokine receptor genes in living versus cadaver donor renal allografts before and after reperfusion. METHODS: This study involved 39 renal transplant patients (19 cadaveric and 20 living donor). The ischemia biopsy was taken just before graft declamping and the reperfusion biopsy 30 min after declamping. Whole-cell RNA was isolated and chemokine (IL-8, CCL2/MCP-1, CXCL10/IP-10 and CCL5/RANTES) and chemokine receptor (CCR2 and CCR5) expression was tested by quantitative PCR. RESULTS: Just prior to declamping, ischemic cadaveric donor grafts had higher expression of CXCL10/IP-10 but not IL-8 or CCL2/MCP-1 than living donor grafts. IL-8 expression increased 50% from ischemia to reperfusion in living donor grafts but increased more than 13-fold during reperfusion of cadaver donor grafts. Increased total ischemia time induced greater IL-8 expression during reperfusion. MCP-1 expression also increased during reperfusion of living and cadaver donor grafts but differences were not observed between the two groups of grafts. RANTES, CCR2, and CCR5 expression did not change in ischemic vs. reperfusion biopsies. CONCLUSIONS: The expression of chemokines directing neutrophil and macrophage recruitment increases during reperfusion of living and cadaveric donor renal allografts. Expression levels of IL-8 correlate with the ischemic time imposed on the renal graft. Early tissue injury may be attenuated by strategies antagonizing chemokines directing the recruitment of neutrophils and macrophages into kidney grafts.     

Effects of testosterone and vitamin E on the antioxidant system in rabbit testis

The aim of the study was to investigate the effects of testosterone propionate and vitamin E on the antioxidant system in the testis. Thirty-two male New Zealand White rabbits were randomly divided into four groups. The first group was used as control. The second group was injected with testosterone propionate, the third group vitamin E and the fourth group vitamin E and testosterone propionate combination. All treatments were carried out during 6 weeks and oxidative parameters were evaluated in homogenized testicular tissue. The levels of vitamin E and the activity of glutathione peroxidase were lower (P < 0.05) in the testosterone group than in controls. However, vitamin C and malondialdehyde levels were higher (P < 0.05) in this group than in controls. The levels of reduced glutathione, beta-carotene, vitamin C and E increased, but malondialdehyde levels decreased in the vitamin E group, when compared with controls (P < 0.05). Vitamin E and beta-carotene levels were significantly higher (P < 0.05) in the combination group than in testosterone group. However, MDA levels were lower (P < 0.05) in combination group than in the testosterone group. In conclusion, administration of testosterone propionate led to a significant elevation of oxidative stress. Vitamin E is quite an effective antioxidant which protects rabbit testis against lipid peroxidation, and, testosterone-induced lipid peroxidation could be improved by additional vitamin E treatment.     

电针足三里穴对依托咪酯麻醉大鼠急性应激反应的影响

目的探讨电针足三里穴对依托咪酯麻醉大鼠应激反应的影响。方法选择雄性SD大鼠60只,体重280~310g,随机分为六组:空白对照组(C组)、模型组(M组)、依托咪酯60mg/kg组(E1组)、依托咪酯30 mg/kg组(E2组)、依托咪酯60 mg/kg联合电针组(EA1组)和依托咪酯30mg/kg复合电针组(EA2组),每组10只。C组大鼠不进行任何麻醉手术操作,其余五组均建立低位尾干切断模型。建模后,E1组和EA1组大鼠腹腔注射依托咪酯60mg/kg,E2组和EA2组大鼠腹腔注射依托咪酯30mg/kg。EA1组和EA2组大鼠于麻醉状态行双侧足三里穴治疗30min(频率2/100Hz,疏密波,电流强度1mA)。采用ELISA法检测血清ACTH和Cor的浓度,Western blot法检测下丘脑室旁核组织c-fos蛋白的含量。结果 M组ACTH、Cor浓度和c-fos蛋白含量明显高于C组(P0.05);E1组、E2组、EA1组、EA2组ACTH、Cor浓度和c-fos蛋白含量明显低于M组(P0.05);E2组、EA1组ACTH、Cor浓度和c-fos蛋白含量明显高于E1组(P0.05);EA2组ACTH浓度和c-fos蛋白含量明显低于E2组(P0.05)。结论电针大鼠足三里穴可双向调节依托咪酯对急性应激反应的影响,其机制可能与其调节下丘脑c-fos蛋白水平有关。