Carbamoylation abrogates the antioxidant potential of hydrogen sulfide.

Hydrogen sulfide (H2S) has been identified as the third gasotransmitter. Beside its role as signaling molecule in the cardiovascular and nervous system the antioxidant and cyto-protective properties of H2S have gained much attention. In the present study we show that cyanate, an uremic toxin which is found in abundant concentration in sera of patients suffering from chronic kidney disease (CKD), can abrogate the antioxidant and cytoprotective activity of H2S via S-carbamoylation reaction, a reaction that previously has only been shown to have a physiological effect on cysteine groups, but not on H2S. Carbamoylation strongly inhibited the free radical scavenging (ABTS(+·) and alkylperoxyl ROO(·)) properties of H2S. The extent of intracellular ROS formation induced by ROO(·) was diminished by H2S whereas carbamoylation counteracted the protective effect. Reagent HOCl was rapidly inactivated by H2S in contrast to the carbamoylated compound. Protein modification by HOCl was inhibited by H2S but carbamoylation significantly reduced the effect. Thus, S-carbamoylation of low molecular weight thiols by abrogating their antioxidant potential may contribute to the higher oxidative stress observed in CKD.

S-carbamoylation impairs the oxidant scavenging activity of cysteine: its possible impact on increased LDL modification in uraemia.

Carbamoylation is the non-enzymatic reaction of cyanate with amino-, hydroxy- or thiol groups. In vivo, amino group modification (N-carbamoylation) resulting in altered function of proteins/amino acids has been observed in patients suffering from uraemia due to urea-derived cyanate. Uraemia has been linked to impaired antioxidant defense. As thiol-compounds like cysteine, N-acetyl cysteine and GSH have oxidant scavenging properties one may speculate that thiol-group carbamoylation (S-carbamoylation) may impair their protective activity. Here we report on the effect of S-carbamoylation on the ABTS free radical and HOCl scavenging property of cysteine as well on its ability to protect LDL from atherogenic modification induced by AAPH generated peroxylradicals or HOCl. The results show that S-carbamoylation impaired the ABTS free radical and HOCl scavenging property of the thiol-compounds tested. The ability of the thiols to protect LDL from lipid oxidation and apolipoprotein modification was strongly diminished by S-carbamoylation. The data indicate that S-carbamoylation could impair the free radical and HOCl scavenging of thiol-amino acids reducing their protective property against LDL atherogenic modification by these oxidant species. As S-carbamoylation is most effective at pH 7 to 5 in vivo thiol-carbamoylation may especially occur at sites of acidic extracellular pH as in hypoxic/inflammatory macrophage rich areas like the atherosclerotic plaque where increased LDL oxidation has been found and may contribute to the higher oxidative stress in uraemia.

Genotypic diversity of complement component C4 does not predict kidney transplant outcome.

Gene copy number of complement component C4, which varies among individuals, may determine the intrinsic strength of the classical complement pathway. Presuming a major role of complement as an effector in transplant rejection, we hypothesized that C4 genetic diversity may partially explain the variation in allograft outcomes. This retrospective study included 1969 deceased-donor kidney transplants randomly selected from the Collaborative Transplant Study DNA bank. We determined recipient and donor gene copy number of total C4, C4 isotypes (C4A and C4B), and C4 gene length variants (C4L and C4S) by quantitative real-time PCR analysis. Groups defined according to recipient C4 gene copy number (low, intermediate, and high) had similar 10-year allograft survival. Genotypic groups showed comparable rates of graft dysfunction, treatment for rejection, immunological graft loss, hospitalization for infection, malignant disease, and death. Similarly, separate analyses of C4A, C4B, C4L, and C4S; combined evaluation of donor and recipient C4 genotype; or analysis of recipients with higher risk for rejection did not reveal considerable outcome effects. In conclusion, we did not demonstrate that C4 gene copy number associates with transplant outcome, and we found no evidence that the resulting variation in the strength of classical complement activation influences susceptibility to rejection.

Cystatin C and the risk for cardiovascular events in patients with asymptomatic carotid atherosclerosis.

BACKGROUND AND PURPOSE: Renal dysfunction is a risk factor for cardiovascular events in patients with atherosclerosis. Unlike serum creatinine or estimated glomerular filtration rate, cystatin C reflects renal dysfunction independent of factors such as sex, weight, and race. We investigated whether baseline serum levels of cystatin C predict major cardiovascular events in patients with asymptomatic carotid atherosclerosis and compared the predictive value of cystatin C to these established markers of renal function. METHODS: We prospectively studied 1004 of 1286 consecutive patients with carotid ultrasound scanning. Patients were followed for the occurrence of major cardiovascular events, a composite of myocardial infarction, percutaneous coronary intervention, coronary bypass graft, stroke, and death. RESULTS: During a median of 3 years of follow-up, we recorded 346 major cardiovascular events in 311 patients. The risk for a first major cardiovascular event increased significantly with increasing quintiles of cystatin C; hazard ratios ranged from 1.18 to 1.94 for the highest versus the lowest quintile (P<0.001 for trend). Creatinine levels showed no significant association with major cardiovascular events, and for glomerular filtration rate, only the lowest quintile was moderately associated with adverse cardiovascular outcome. CONCLUSIONS: Cystatin C was significantly and gradually associated with future cardiovascular events in patients with carotid atherosclerosis. In contrast, neither serum creatinine nor estimated glomerular filtration rate were significant predictors of adverse cardiovascular outcomes.

Hydrogen sulfide scavenges the cytotoxic lipid oxidation product 4-HNE.

Highly reactive alpha,beta-unsaturated aldehydes like 4-hydroxy-2-nonenal (4-HNE), generated from oxidation of polyunsaturated fatty acids, can bind to proteins, polynucleotides and exert cytotoxicity. 4-HNE is known to react readily with thiol and amino groups on free or bound amino acids. Recently, hydrogen sulfide (H(2)S) has been identified as an endogenous vascular gasotransmitter and neuromodulator which can reach up to 160 micromol/l in the brain. Markedly higher 4-HNE concentrations were reported in the brain of patients suffering from Alzheimer's disease. Assuming that the low molecular thiol H(2)S may react with 4-HNE, we have tested the ability of H(2)S to counteract the cytotoxic and protein-modifying activity of 4-HNE. The results show that H(2)S at physiologically relevant concentrations could effectively protect neuronal cells (SH-SY5Y) from the cytotoxic action of 4-HNE. The HNE-modification of cellular proteins was also inhibited in presence of H(2)S. These data suggest that H(2)S may be an important protective factor against carbonyl stress by inactivating/modulating the action of highly reactive alpha,beta-unsaturated aldehydes like 4-HNE in the brain.

Monocytes/macrophages in kidney allograft intimal arteritis: no association with markers of humoral rejection or with inferior outcome.

BACKGROUND: Several studies indicate that interstitial and intracapillary monocytes/macrophages (MO) represent a significant proportion of graft-infiltrating cells in renal allografts and that their presence may unfavourably affect clinical outcome. Much less is known about the role of MO in vascular rejection of transplanted kidneys. The aim of our study was to determine the cellular composition of immune cell infiltrates in intimal arteritis and to analyse whether it is associated with features of humoral immunity and impaired graft survival. METHODS: In 34 recipients with vascular rejection, we determined the proportion of intimal and interstitial MO and T-cells (expressed as ratio of CD68- and CD3-positive cells) in immunohistochemically double-labelled slides. RESULTS: Intimal arteritis is always composed of T-cells and MO with a median CD68/CD3 ratio of 1.03. In 47% of cases, however, T-cells predominate (CD68/CD3 ratio <1). The median interstitial CD68/CD3 ratio is 0.61, with T-cells dominating in 64% of cases. There is no correlation between the cellular composition of arterial and interstitial infiltrates. The proportion of interstitial and arterial MO has no impact on graft survival, and is, in contrast to previous reports on MO in allograft glomerulitis and capillaritis, not associated with C4d staining. CONCLUSIONS: Intimal arteritis in kidney allograft rejection is composed of a mixed infiltrate of MO and T-lymphocytes. In contrast to MO in PTCitis and glomerulitis, the MO in intimal arteritis are not associated with markers of humoral immune response and there are no different allograft outcomes between MO and T-lymphocyte-dominated groups.

Progression of carotid stenosis detected by duplex ultrasonography predicts adverse outcomes in cardiovascular high-risk patients.

BACKGROUND AND PURPOSE: The progression of carotid stenosis reflects the activity of atherosclerotic disease and may indicate a risk for systemic atherothrombotic complications. We investigated whether progressive carotid stenosis determined by duplex ultrasonography predicts adverse outcomes in cardiovascular high-risk patients. METHODS: We prospectively studied 1065 of 1268 consecutive patients initially asymptomatic with respect to carotid disease. Carotid ultrasound investigations at baseline and after a median of 7.5 months (range, 6 to 9 months) were performed to identify patients with progressive stenosis as defined by Doppler velocity criteria. Patients were then followed up clinically for a median of 3.2 years for the occurrence of major adverse cardiovascular events (composite MACEs: myocardial infarction, percutaneous coronary or peripheral interventions, coronary or vascular surgery, amputation, stroke, and all-cause mortality). RESULTS: We found progressive carotid stenosis in 93 patients (9%) by ultrasound and thereafter recorded 495 MACEs in 421 patients (40%) during clinical follow-up. Patients with progressive carotid stenosis had a significantly increased risk for cardiovascular events compared with patients with nonprogressive disease: adjusted hazard ratios and confidence intervals were 2.01 for composite MACEs (95% CI, 1.48 to 2.67, P<0.001), 2.38 for myocardial infarction (95% CI, 1.07 to 5.35, P=0.044), 1.59 for any coronary event (95% CI, 1.10 to 2.28, P=0.011), 2.00 for stroke (95% CI, 1.02 to 4.11, P=0.035), 2.42 for any peripheral vascular event (95% CI, 1.61 to 3.62, P<0.001), and 1.75 for cardiovascular death (95% CI, 1.03 to 2.97, P=0.039). CONCLUSIONS: Progression of carotid stenosis within a 6- to 9-month interval detected by duplex ultrasound predicts midterm clinical adverse events of atherosclerosis in high-risk patients affecting the coronary, cerebrovascular, and peripheral circulations.

Single bolus injection of bilirubin improves the clinical outcome in a mouse model of endotoxemia.

Increasing serum levels of biliverdin and bilirubin was shown to be beneficial in settings of inflammation. Bilirubin was shown to be protective in LPS-induced lung injury in rats; however, the exact mechanism remains elusive. Here, we investigated whether a single bolus injection of bilirubin would exert anti-inflammatory effects in a mouse model of endotoxemia. Mice were challenged with sublethal doses (2 mg/kg body weight) of LPS, and the effects of intravenously administered bilirubin (40 mg/kg body weight) were assessed. In contrast to control animals, bilirubin-treated animals fully recovered from endotoxin shock within 24 h. Bilirubin treatment improved the clinical score significantly at all time points assessed, attenuated weight loss, and improved LPS-induced anorexia. Furthermore, bilirubin treatment inhibited LPS-induced leukocyte-endothelial interactions and leukocyte accumulation in various tissues. Expression of inflammatory genes, including endothelial adhesion molecules, but also IL-1beta and TNF-alpha, was significantly reduced in bilirubin-treated animals. Moreover, bilirubin inhibited LPS-induced expression of inflammatory genes in isolated cultured aortic endothelial cells and in bone marrow-derived macrophages. These data show that single-dose administration of bilirubin attenuates tissue injury induced by endotoxin, and that bilirubin, in addition to its antioxidant effects, also exerts potent anti-inflammatory activity.

Expression of heme oxygenase-1 in human vascular cells is regulated by peroxisome proliferator-activated receptors.

OBJECTIVE: Activation of peroxisome proliferator-activated receptors (PPARs) by lipid-lowering fibrates and insulin-sensitizing thiazolidinediones inhibits vascular inflammation, atherosclerosis, and restenosis. Here we investigate if the vasculoprotective and anti-inflammatory enzyme heme oxygenase-1 (HO-1) is regulated by PPAR ligands in vascular cells. METHODS AND RESULTS: We show that treatment of human vascular endothelial and smooth muscle cells with PPAR ligands leads to expression of HO-1. Analysis of the human HO-1 promoter in transient transfection experiments together with mutational analysis and gel shift assays revealed a direct transcriptional regulation of HO-1 by PPARalpha and PPARgamma via 2 PPAR responsive elements. We demonstrate that a clinically relevant polymorphism within the HO-1 promoter critically influences its transcriptional activation by both PPAR isoforms. Moreover, inhibition of HO-1 enzymatic activity reversed PPAR ligand-mediated inhibition of cell proliferation and expression of cyclooxygenase-2 in vascular smooth muscle cells. CONCLUSION: We demonstrate that HO-1 expression is transcriptionally regulated by PPARalpha and PPARgamma, indicating a mechanism of anti-inflammatory and antiproliferative action of PPAR ligands via upregulation of HO-1. Identification of HO-1 as a target gene for PPARs provides new strategies for therapy of cardiovascular diseases and a rationale for the use of PPAR ligands in the treatment of other chronic inflammatory diseases.

The dietary soy flavonoid genistein abrogates tissue factor induction in endothelial cells induced by the atherogenic oxidized phospholipid oxPAPC.

INTRODUCTION: Tissue factor (TF) plays a pivotal role in the generation of thrombin in atherothrombotic disease. The oxidized phospholipid 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (oxPAPC), an active compound of minimally oxidized low-density lipoprotein (MM-LDL), induces TF in endothelial cells (EC). The dietary soybean isoflavonoid genistein has been claimed to reverse several processes leading to atherosclerosis and related cardiovascular events via binding to estrogen receptors, generating nitric oxide (NO) or inhibiting tyrosine kinase-dependent pathways. METHODS AND MATERIALS: The effects and mechanisms of genistein on activity, antigen expression and mRNA levels of oxPAPC-induced TF were studied in human umbilical vein endothelial cells (HUVEC) and human aortic endothelial cells (HAEC). RESULTS AND CONCLUSIONS: Genistein abrogated oxPAPC-induced TF activity in arterial and venous human EC to basal levels, as measured by functional clotting assay, and downregulated oxPAPC-induced antigen expression measured by flow cytometry and mRNA levels quantified by real-time PCR. Western blotting and inhibitor experiments with the estrogen-receptor inhibitor ICI 182,780 and the NO-synthase inhibitor N(omega)-nitro-L-arginine methyl ester (L-NAME) showed that the effect may be mediated via inhibition of phosphorylation of ERK, but not upstream MEK1/2. The effect is not mediated by the tyrosine kinase inhibitor activity of genistein, as another tyrosine kinase inhibitor (tyrphostin 25) had no effect. Binding to the estrogen receptor or generation of NO are not involved in the action of genistein on TF. In conclusion genistein reduces oxPAPC-induced TF expression and thereby the prothrombotic phenotype of EC, further substantiating and explaining the beneficial effects of dietary genistein in preventing atherosclerosis and related cardiovascular events.

Rescue therapy with tacrolimus and mycophenolate mofetil does not prevent deterioration of graft function in C4d-positive chronic allograft nephropathy.

BACKGROUND: Humoral alloresponses may contribute to chronic allograft nephropathy (CAN) in a subset of kidney transplant recipients. For chronic humoral rejection, the efficacy of rescue therapy with tacrolimus and mycophenolate mofetil has been suggested. METHODS: Eleven recipients with C4d-positive CAN (index biopsy performed after a median of 3 years posttransplantation), who had been on cyclosporine A-based immunosuppression, were converted to tacrolimus, and if not part of basal therapy, to mycophenolate mofetil. We evaluated the effect of this tacrolimus/mycophenolate mofetil rescue therapy on clinical outcomes and on alloantibody formation detected with flow cytometric testing of panel-reactive antibody. RESULTS: Tacrolimus/mycophenolate mofetil rescue therapy (plus anti-rejection treatment in six recipients with additional signs of acute cellular rejection) failed to prevent progressive deterioration of graft function. Four patients returned to dialysis after 4 to 18 months. Serial post-transplant serology detected HLA class I and/or II reactivity in seven recipients. Tacrolimus/mycophenolate mofetil therapy did not affect the time course of alloantibody levels. One patient with C4d-positive transplant glomerulopathy, who did not respond to tacrolimus/mycophenolate mofetil rescue therapy, developed nephrotic-range proteinuria associated with a rapid decline of allograft function. Despite considerable reduction in alloantibody levels and nearly complete clearance of C4d deposits, immunoadsorption failed to prevent graft failure in this patient. CONCLUSION: Our data argue against the efficacy of tacrolimus/mycophenolate mofetil rescue therapy in established C4d-positive chronic allograft dysfunction. Prospective trials are needed to evaluate whether early initiation of this or other antihumoral strategies are capable of effectively preventing alloantibody-mediated chronic graft injury.

C4d in pediatric renal allograft biopsies: a marker for negative outcome in steroid-resistant rejection.

Recently, deposition of C4d, reflecting complement activation via the classical pathway, has been established as marker of antibody-mediated rejection. As C4d can be detected in paraffin sections, it allows for retrospective analysis in populations with low case loads, such as in pediatric transplantation. In this study we re-evaluated consecutive renal transplant biopsies obtained since 1990 in 36 children (18 boys, 18 girls) who had received their allograft (nine living, 27 cadaveric) at an age of 10.12+/-4.4 yr. Clinical indications for biopsy were 16 acute steroid resistant rejections (ASRs), 11 chronic rejections and nine other diagnoses. Overall, C4d deposition was found in nine cases (25%), eight of them with diagnosed ASR. Six out of these eight allografts were lost during 36 months of clinical follow-up, a significantly higher rate than in C4d-negative biopsies (p<0.05). C4d status therefore turned out to be an excellent predictor for inferior graft survival following ASR. None of the other histopathologic markers were sensitive for humoral rejections. In conclusion, the high prevalence of C4d-positive staining in ASR demonstrates the importance of the humoral part of the immune system in pediatric transplantation. The worse outcome of C4d-positive rejections despite massive immunosuppressive therapy clearly indicates the need for innovative therapies in this high-risk population.

A microsatellite polymorphism in the heme oxygenase-1 gene promoter is associated with risk for melanoma.

Heme oxygenase-1 (HO-1) has been demonstrated to play an important role in the regulation of signaling systems, which are involved in the control of cell cycle progression and apoptosis. Recently, a (GT)n dinucleotide repeat polymorphism in the HO-1 promoter was shown to modulate HO-1 gene expression. Short (<25 GT) repeats are associated with an increased HO-1 upregulation after stimulation than are longer repeats. Malignant melanoma (MM) is the most serious cutaneous malignancy with high tendency to aggressive growth and resistance to apoptosis. Therefore, we sought to study the influence of this polymorphism on the progression of MM. We determined the HO-1 promoter genotype in 152 patients with MM and 398 healthy controls and studied their association in regard to susceptibility to MM, Breslow thickness and disease-free survival. In our study, the homozygous short allele with <25 (GT)n repeats (S/S) was found more frequently in the melanoma group compared to the healthy control population (21 and 12%, respectively). The calculated risk for acquiring primary MM in S/S carriers was 2-fold higher compared to those with L-allele types (95% confidence interval: 1.2-2.4, p = 0.03). Additionally, the S/S genotype was significantly associated with primary tumors with deeper Breslow thickness compared to L-allele (>25 repeats) carriers (mean Breslow thickness: 4.0 +/- 2.9 mm versus 3.1 +/- 1.7 mm, p = 0.03). These data suggest that HO-1 might render a higher risk for MM in S/S genotype individuals and could represent an important candidate gene in the pathogenesis and growth of malignant melanoma.

Preoperative antithrombin III activity predicts outcome after surgical repair of acute type A aortic dissection.

BACKGROUND: Acute Stanford type A aortic dissection is associated with substantial perioperative morbidity and mortality. A sepsis-like state may lead to antithrombin (AT) III consumption and deficiency. The impact of preoperative AT III activity on outcome in patients undergoing emergency surgery is yet unknown. METHODS: We measured preoperative AT III activity in 99 consecutive patients undergoing emergency aortic surgery for Stanford type A aortic dissection during a 4-year period in a retrospective study. Cardiovascular co-morbidities, risk factors and surgical data were recorded and patients were followed for 30-day mortality, and occurrence of multiple organ failure (MOF). RESULTS: During the first 30 days, 15 patients (15%) died, and 8 patients (8%) had MOF. Median AT III levels (IQR) in 30-day non-survivors versus survivors were 64% (52-72) versus 90% (75-97) (p<0.001), and in patients with versus without MOF were 66% (52.3-77.3) versus 88% (72-96) (p=0.018), respectively. Adjusted odds ratios for 30-day mortality and MOF for AT III activity (per % increments) were 0.92 (p=0.007), and 0.96 (p=0.012), respectively, indicating a significant inverse relationship between AT III activity and outcome. CONCLUSION: There is a strong inverse association between preoperative AT III activity and adverse outcome in patients undergoing surgical repair of acute Stanford type A aortic dissection. Larger studies are necessary to determine a cut-off value for AT III and to assess whether patients with low AT III levels benefit targeted therapeutic interventions.

Aluminum ions stimulate the oxidizability of low density lipoprotein by Fe2+: implication in hemodialysis mediated atherogenic LDL modification.

OBJECTIVE: Al(3+) stimulates Fe(2+) induced lipid oxidation in liposomal and cellular systems. Low-density lipoprotein (LDL) oxidation may render the particle atherogenic. As elevated levels of Al(3+) and increased lipid oxidation of LDL are found in sera of hemodialysis patients, we investigated the influence of Al(3+) on LDL oxidation. MATERIALS AND METHODS: Using different LDL modifying systems (Fe(2+), Cu(2+), free radical generating compounds, human endothelial cells, hemin/H(2)O(2) and HOCl), the influence of Al(3+) on LDL lipid and apoprotein alteration was investigated by altered electrophoretic mobility, lipid hydroperoxide-, conjugated diene- and TBARS formation. RESULTS: Al(3+) could stimulate the oxidizability of LDL by Fe(2+), but not in the other systems tested. Al(3+) and Fe(2+) were found to bind to LDL and Al(3+)could compete with Fe(2+) binding to the lipoprotein. Fluorescence polarization data indicated that Al(3+) does not affect the phospholipid compartment of LDL. CONCLUSIONS: The results indicate that increased LDL oxidation by Fe(2+) in presence of Al(3+) might be due to blockage of Fe(2+) binding sites on LDL making more free Fe(2+) available for lipid oxidation.

Microsatellite polymorphism in the heme oxygenase-1 gene promoter and cardiac allograft vasculopathy.

BACKGROUND: The heme oxygenase-1 (HO-1) isoenzyme has recently been suggested to protect transplants from ischemia-reperfusion and immunologic injury. Inducibility of this enzyme is modulated by a (GT)n dinucleotide length polymorphism in the HO-1 gene promoter. Short (class S) repeats are associated with greater up-regulation of HO-1 than are long repeats. In the present study, we investigated the impact of the promoter polymorphism on the development of cardiac allograft vasculopathy (CAV) in human heart transplants. METHODS: We enrolled 152 recipients of a heart allograft with at least 1 year survival post-transplantation in this retrospective study. The HO-1 genotype was assessed using genomic DNA isolated from paraffin-embedded allograft biopsy specimens. Patients were followed angiographically for CAV. Angiographic vessel-wall abnormalities were defined as CAV, and a stenosis of more than 50% in at least 1 vessel area was defined as severe CAV. RESULTS: Eighty-seven patients (57%) had received a heart from a donor with at least 1 class S allele. Within the mean follow-up period of 9 years, 95 patients (63%) showed signs of CAV, among which 60 patients (40%) developed severe CAV. The frequency of CAV and severe CAV was not significantly different between class S allele recipients and non-recipients (CAV, 57/87 vs 38/65, p = 0.12; severe CAV, 35/87 vs 25/65, p = 0.30). CONCLUSION: In contrast to recent findings in renal allografts and vascular injury, the HO-1 gene promoter polymorphism does not show an association with the development of CAV in heart transplants.

Association of neutrophils and future cardiovascular events in patients with peripheral artery disease.

OBJECTIVE: We hypothesized that higher neutrophil counts are associated with an increased incidence of major adverse cardiovascular events (MACE) in patients with clinically advanced atherosclerosis. METHODS: We prospectively studied 398 patients (233 men; median age, 69 years) with symptomatic peripheral artery disease who were admitted to the inpatient ward of the angiology department of a tertiary care university hospital in a cohort study. Total and differential white blood cell (WBC) counts were obtained, and patients were followed for MACE, defined as myocardial infarction, percutaneous coronary interventions, coronary artery bypass grafting, stroke, carotid revascularization, and death. RESULTS: During a median follow-up of 20 months, 140 MACE occurred in 105 patients (26%). Multivariate Cox proportional hazards analysis was used to assess the association of differential WBC count parameters (in tertiles) with MACE and their interrelation with traditional cardiovascular risk factors and other parameters of inflammation. Patients with neutrophil counts >5.8 G/L (upper tertile) exhibited an increased adjusted risk for all MACE (hazards ratio [HR], 1.83; P = .017), death (HR, 3.39; P = .010), and the composite of myocardial infarction, stroke, and death (HR, 2.20; P = . 012) compared with patients in the lower tertile (<4.4 G/L), independently of traditional cardiovascular risk factors and levels of high-sensitivity C-reactive protein. Only neutrophils, but not eosinophils, basophils, monocytes, lymphocytes, or the total WBC count showed a significant association with cardiovascular outcome. CONCLUSION: In patients with peripheral artery disease, neutrophil counts in the upper tertile (>5.8 G/L) indicate a substantially increased risk for major adverse cardiovascular events, adding to the prognostic information of traditional atherothrombotic risk factors and other parameters of inflammation.

Expression of kidney-specific cadherin distinguishes chromophobe renal cell carcinoma from renal oncocytoma.

Distinguishing renal oncocytoma from chromophobe and other renal carcinomas is essential, considering their differing biological potentials. Although renal oncocytoma is considered a benign tumor, chromophobe renal cell carcinoma has potentially malignant biological behavior. The numerous reported studies on distinguishing these 2 entities have been based on morphological, histochemical, immunohistochemical, ultrastructural, and cytogenetic features. But none of these features has proven to be reliably specific, especially in tumors with overlapping phenotypes. We report a novel immunohistochemical approach based on the expression of a recently described kidney-specific cadherin (Ksp-cadherin) for the differential diagnosis of these 2 tumors. We compared Ksp-cadherin expression in 212 renal tumors, including 102 clear cell renal carcinomas, 46 papillary renal cell carcinomas, 30 chromophobe carcinomas, 3 collecting duct carcinomas, and 31 oncocytomas. In addition, we examined the expression of epithelial membrane antigen, vimentin, CK7, and Hale's colloidal iron staining. We found that chromophobe renal cell carcinomas consistently (96.7% of cases) demonstrated a distinctive membrane pattern of Ksp-cadherin expression, whereas renal oncocytomas (3.2%), clear cell renal cell carcinomas (0%), papillary renal cell carcinomas (2.2%), and collecting duct carcinomas (0%) usually did not express Ksp-cadherin. CK7 expression was found in 90.0%, 6.5%, 7.8%, 76.1%, and 33.3% of these tumor cases, respectively. Whereas CK7 was detected in different types of renal cell carcinomas, Ksp-cadherin was expressed almost exclusively in chromophobe renal cell carcinomas. Immunohistochemical analysis of Ksp-cadherin offers a fast, reliable approach for the distinguishing between renal oncocytoma and chromophobe renal cell carcinoma that is applicable for routine pathology laboratory studies without the need for time-consuming and costly ancillary studies.

PAI-1 4G/5G insertion/deletion promoter polymorphism and microvascular complications in type 2 diabetes mellitus.

BACKGROUND: Plasminogen activator inhibitor-1 (PAI-1) plays an important role in the regulation of fibrinolysis and extracellular matrix turnover. PAI-1 4G/5G insertion/deletion polymorphism in the PAI-1 promoter region has been shown to modulate PAI-1 plasma levels. We investigated the relationship between this polymorphism and the prevalence of diabetic nephropathy and retinopathy in patients with type 2 diabetes in the Austrian population. PATIENTS AND METHODS: 147 consecutive patients with type 2 diabetes mellitus (96 men, 51 women; median age, 65 years; IQR, 59-71) were analyzed for the PAI-1 4G/5G genotype. RESULTS: The genotype distribution in the individuals tested was as follows: 17% (n = 25) 5G/5G, 54% (n = 80) 4G/5G, and 29% (n = 42) 4G/4G. Patients homozygous for allele 4G had a significantly higher risk of diabetic proliferative retinopathy than patients without signs of diabetic retinopathy or nonproliferative retinopathy (OR, 7.3; 95% CI, 1.4-38.8; P = 0.02). No significant associations were observed between the PAI-1 genotype and the presence of albuminuria. CONCLUSION: According to our results, diabetic proliferative retinopathy might be associated with the prevalence of PAI-1 genotype 4G/4G.

Risk factors for capillary C4d deposition in kidney allografts: evaluation of a large study cohort.

BACKGROUND: Capillary deposition of the complement split product C4d has turned out to be a valuable marker of antibody-mediated rejection. The impact of pre- and posttransplant variables including particular immunosuppressive regimens on the frequency of C4d deposition has not yet been systematically investigated in a large multivariate analysis. METHODS: In this retrospective study, the authors evaluated the incidence of C4d deposition in 388 kidney transplant recipients subjected to diagnostic biopsy within the first 6 months and analyzed the influence of potential confounders on the rate of C4d-positive graft dysfunction by applying multivariate logistic regression. RESULTS: Sixty-six recipients (17%) developed linear C4d deposits in at least a quarter of peritubular capillaries, a finding associated with inferior 1-year allograft survival (73% vs. 88% in C4d-negative patients, P=0.0003). A 50% reduction in the odds of C4d-positive graft dysfunction was found if calcineurin inhibitor or mycophenolate mofetil (MMF) therapy was started 2 to 4 hr before transplantation when compared with initiation after surgery (adjusted odds ratio [OR], 0.5; P=0.03). No differences with respect to C4d staining results were found for the use of tacrolimus, MMF, or sirolimus, or for cyclosporine C2 monitoring. Retransplantation (OR, 3.6; P<0.001) and presensitization (OR, 3.1; P=0.002) turned out to be strong independent risk factors for C4d deposition. CONCLUSIONS: The authors' results suggest a reduced risk of C4d-positive graft dysfunction for patients receiving immunosuppression before transplantation. Apart from first dose timing, no influence of particular immunosuppressive strategies on C4d staining results was found.