Justification of 150 mg clopidogrel in patients with high on-clopidogrel platelet reactivity.

BACKGROUND: The GRAVITAS trial showed that 150 mg clopidogrel did not improve outcome in patients with high on-clopidogrel platelet reactivity (HPR) screened by the VerifyNow assay. We aimed to determine the impact of 150 mg clopidogrel in stable angina patients with HPR identified with conventional aggregometry (LTA). MATERIALS AND METHODS: Clopidogrel-naive stable angina patients before ad hoc percutaneous coronary intervention were recruited into a randomized, double-blind, placebo-controlled trial (NCT00638326). Twelve to 24 h after the 600-mg loading dose of clopidogrel, ADP(5µM)-stimulated maximal (AGGmax), late platelet aggregation (AGGlate) and vasodilator-stimulated phosphoprotein phosphorylation (VASP-PRI) were evaluated. Patients with HPR (AGGmax ≥ 34%) were randomly allocated to 75 or 150 mg clopidogrel after 4 weeks. After control platelet function measurements at day 28, 75 mg clopidogrel was administered to all patients until 1 year. RESULTS: The study was prematurely terminated at the stage of 200 enroled patients. Administration of 150 mg clopidogrel significantly reduced platelet aggregation (AGGmax: 45·0 ± 6·8 vs. 33·8 ± 15·1, P < 0·01; AGGlate: 27·1 ± 14·7 vs. 13·8 ± 18·0, P < 0·01) and VASP-PRI (57·5 ± 15·2 vs. 37·2 ± 17·1; P < 0·01), while platelet reactivity remained unchanged in patients with HPR receiving 75 mg clopidogrel. The higher maintenance dose of clopidogrel was associated with a significant reduction in cardiovascular (CV) death and myocardial infarction (MI) (0% vs. 11·4%, P = 0·04) and in CV death, MI or target vessel revascularization (24·6% vs. 3·1%; P = 0·01) during 1 year. CONCLUSIONS: One-month administration of 150 mg maintenance dose of clopidogrel reduces platelet reactivity and might decrease the risk of thrombo-ischaemic complications in stable angina patients with HPR identified by LTA.

Prognostic significance of high on-clopidogrel platelet reactivity after percutaneous coronary intervention: systematic review and meta-analysis.

BACKGROUND: A growing number of observational studies suggest that high on-clopidogrel platelet reactivity (HPR) is associated with recurrent thrombotic events after percutaneous coronary intervention. We aimed to perform an updated systematic review and meta-analysis on the clinical relevance of HPR to summarize the available evidence and to define more precise effect estimates. METHODS: Relevant observational studies published between January 2003 and February 2010 were searched that presented intent-to-treat analyses on the clinical relevance of HPR measured with an adenosine diphosphate (ADP)-specific platelet function assay. The main outcome measures were cardiovascular (CV) death, definite/probable stent thrombosis (ST), nonfatal myocardial infarction (MI), and a composite end point of reported ischemic events. The outcome parameters were pooled with the random-effect model via generic inverse variance weighting. RESULTS: Twenty studies comprising a total number of 9,187 patients qualified. High on-clopidogrel platelet reactivity appeared to be a strong predictor of MI, ST, and the composite end point of reported ischemic events (odds ratios [95% CI]: 3.00 [2.26-3.99], 4.14 [2.74-6.25], and 4.95 [3.34-7.34], respectively; P < .00001 for all cases). According to the meta-analysis, patients with HPR had a 3.4-fold higher risk for CV death compared with patients with normal ADP reactivity (odds ratio 3.35, 95% CI 2.39-4.70, P < .00001). Although there were large differences in the methodology as well as in the definition of HPR between studies, the predicted risk for CV death, MI, or ST was not heterogeneous (I(2): 0%, 0%, and 12%, respectively; P = not significant for all cases). CONCLUSIONS: High on-clopidogrel platelet reactivity, measured by an ADP-specific platelet function assay, is a strong predictor of CV death, MI, and ST in patients after percutaneous coronary intervention.

O-GlcNAc modification of proteins affects volume regulation in Jurkat cells.

An increasing amount of recent research has demonstrated that the hexosamine biosynthesis pathway (HBP) plays a significant role in the modulation of intracellular signaling transduction pathways, and affects cellular processes via modification of protein by O-linked beta-N-acetylglucosamine (O-GlcNAc). Besides the many known and postulated effects of protein O-GlcNAc modifications, there is little available data on the role of O-GlcNAc in cellular volume regulation. Our objective was to test the effect of increased O-GlcNAc levels on hypotonia-induced volume changes in Jurkat cells. We pretreated Jurkat cells for 1 h with glucosamine (GlcN), PUGNAc (O-(2-acetamido-2-deoxy-D-glucopyranosylidene)-amino-N-phenylcarbamate) an inhibitor of O-GlcNAcase, or a high level of glucose to induce elevated levels of O-GlcNAc. We found that the response of Jurkat cells to hypotonic stress was significantly altered. The hypotonia induced cell-swelling was augmented in both GlcN and PUGNAc-treated cells and, to a lesser extent, in high glucose concentration-treated cells. Evaluated by NMR measurements, GlcN and PUGNAc treatment also significantly reduced intracellular water diffusion. Taken together, increased cell swelling and reduced water diffusion caused by elevated O-GlcNAc show notable analogy to the regulatory volume changes seen by magnetic resonance methods in nervous and other tissues in different pathological states. In conclusion, we demonstrate for the first time that protein O-GlcNAc could modulate cell volume regulation.

Comparison of conventional aggregometry with VASP for monitoring P2Y12-specific platelet inhibition.

No consensus exists regarding the optimal estimate of light transmission aggregometry (LTA) to reflect P2Y12 ADP receptor inhibition in patients receiving thienopyridine therapy. Currently, the only completely P2Y12-receptor specific method is the measurement of vasodilator stimulated phosphoprotein (VASP) phosphorylation (PRI) with flow cytometry. In the current analysis, we aimed to test the superiority of the late platelet aggregation value over other estimates of light transmission aggregometry in determining P2Y12-receptor inhibition by comparing them to VASP-PRI. On-clopidogrel platelet reactivity was measured in 121 clopidogrel-naïve patients who underwent elective coronary stent implantation. Samples for LTA and VASP assessments were drawn at 12-18 hours after a 600-mg loading dose of clopidogrel and 25 days after the intervention. ADP 5 µM-induced maximal aggregation (AGGmax), 6-minute late aggregation (AGGlate), 6-minute disaggregation (disAGG) and area under the aggregation curve (AUC) were compared to VASP-PRI. Categorical agreement with VASP-defined normal and high platelet reactivity (HPR: PRI ≥ 50%) was calculated according to the optimal cutoff values obtained with receiver-operating characteristic (ROC) curves. The evaluation of 242 measurements showed significant, moderate-strength correlations between VASP-PRI and LTA estimates without the superiority of AGGlate over other estimates (AGGmax: ρ = 0.47; AGGlate: ρ = 0.45; disAGG: ρ = -0.44; AUC: ρ = 0.50). Notably, there were considerable intra-individual differences between VASP and LTA testing. LTA estimates were similar in classifying patients to VASP-defined normal or HPR categories (AGGmax: κ = 0.41; AGGlate: κ = 0.45; disAGG: κ = 0.44; AUC: κ = 0.44). When all estimates of LTA were compared in multivariable models, AUC proved to be the independent linear determinant of VASP-PRI and the best predictor of HPR. Estimates of LTA seem equal in determining the degree of P2Y12-receptor inhibition or in predicting VASP-defined HPR without the superiority of AGGlate over others. These results reject a commonly used hypothesis and might contribute to the standardization of the LTA assay.

Invariant Valpha7.2-Jalpha33 TCR is expressed in human kidney and brain tumors indicating infiltration by mucosal-associated invariant T (MAIT) cells.

The anti-tumor response of human invariant NKT (NKT) cells is well established. A novel T cell subset, mucosal-associated invariant T (MAIT) cells, possesses similar regulatory properties to NKT cells in autoimmune models and disease. Here, we examined the clonality of four T cell subsets expressing invariant alphaTCR, including Valpha7.2-Jalpha33 of MAIT cells, in 19 kidney and brain tumors. The MAIT clonotype was identified and co-expressed with NKT clonotype in half of the tumors. In contrast, two other invariant T cell clonotypes (Valpha4 and Valpha19) were not present in tumors. Such tumors also expressed Vbeta2 and Vbeta13, the restricted TCRbeta chain of MAIT cells and the antigen-presenting molecule MR1. A high percentage of infiltrating T cells was CD8+ and expressed HLA-DR suggesting activation. Although the MAIT alphaTCR was identified in both peripheral CD56+ and CD56- subsets, infiltrating lymphocytes were CD56 negative. The clonal presence of MAIT cells in tumors correlated with the expression of pro-inflammatory cytokines but no IL-4, IL-5 and IL-10, suggesting that a pro-inflammatory subset of human MAIT cells may exist. Our data imply that a CD56- subset of MAIT cells may participate in tumor immune responses similarly to NKT cells.

Effects of PACAP on survival and renal morphology in rats subjected to renal ischemia/reperfusion.

Pituitary adenylate cyclase activating polypeptide (PACAP) occurs and exerts a variety of biological functions in the nervous system and in the peripheral organs, including the urinary system. PACAP has protective effects against myeloma kidney injury and renal ischemia. Ischemia/reperfusion injury of the kidney is a major clinical problem, and based on the protective effects of PACAP in cerebral and cardiomyocyte ischemia, the aim of the present study was to evaluate the effects of a single intravenous PACAP injection on the survival and renal morphology after varying times of ischemia. Rats were subjected to renal artery clamping for 15, 30, 45, 60, or 75 min followed by reperfusion. PACAP (100 microg) was administered intravenously before arterial clamping. We found that a 15- or 30-min renal ischemia led to no renal dysfunction, and the kidneys showed normal appearance with no difference between PACAP- and saline-treated groups. Control rats with 45 min of ischemia had increased premature death rate and showed multifocal acute tubular atrophy, while a 60-min ischemia led to death of all control animals within a few days displaying severe, multifocal Grade II tubular atrophy. In contrast, all PACAP-treated animals survived with subtle morphological changes after the 45-min ischemia. After the 60-min ischemia, death rate was significantly lower in PACAP-treated rats compared to controls, and animals showed subtle focal tubular alteration. A 75-min ischemia was not performable in controls because of deaths before the termination of ischemia. PACAP-treated rats survived longer, but they also died after 5-10 days exhibiting severe focal tubular atrophy. In summary, our results clearly show that PACAP is able to prolong the renal ischemic time, decrease mortality, and attenuate tubular degeneration after renal ischemia.

[Multidrug resistance in chronic lymphocytic leukemia]. / Multidrogrezisztencia-vizsgálatok krónikus lymphoid leukaemiában.

INTRODUCTION: New prognostic factors discovered in chronic lymphocytic leukemia have recently got into the center of clinical interest. While the predictive value of cytogenetical abnormalities, immunoglobulin heavy chain gene mutation status, CD38 and ZAP70 expression is already well known, the significance of multi-drug resistance in chronic lymphocytic leukemia is not well characterized. AIMS: The goal of this study was to characterize the multidrug resistance features in 82 patients with chronic lymphocytic leukemia at the genetical, expression- and functional level and to compare it with the patient's clinical behavior (survival and response to therapy). METHODS: Light Cycler Real Time PCR based "Single Nucleotide Polymorphism" analysis of the MDR1 gene, as a biological predictor of the expression level of P-glycoprotein was tested in 66 patients with chronic lymphocytic leukemia. P-glycoprotein expression and MDR-function was detected in 82 cases by flow cytometry (by use of anti-P-glycoprotein monoclonal antibody and calcein-verapamil functional test). Response to therapy was analyzed by statistical Fisher-test in the treated 35 patients. The survival analysis (Log-rank test) was performed on the whole population ( n = 82). RESULTS: No significant correlation was found between the three levels of multidrug resistance (genetics, phenotype, function) in our patients with chronic lymphocytic leukemia. P-glycoprotein positive cases (n = 9) were predominantly non-responders (8/9, 89%). There must be, however, other mechanisms causing non-response (total non-responders: 13/35 treated cases). Most of P-glycoprotein negative CLL patients (n = 26) responded well (21/26, 80%) to chemotherapy (responders: 22/35 treated CLL) (p < 0,001). The tendency was the same in the average expected survival rate between P-glycoprotein positive and negative patients (84 vs 203 months) but the difference was not significant (p = 0,106). CONCLUSIONS: This study proved the clinical prognostic significance of P-glycoprotein expression of leukaemic cells predicting the chemotherapy response and partially estimating the general survival of patients suffering from chronic lymphocytic leukemia.

Association between carcinosarcoma and the transplanted kidney.

BACKGROUND: Renal carcinosarcoma is a rare tumor with 12 reported cases in the world literature. To our knowledge, carcinosarcoma of a renal allograft has not been reported to date. CASE REPORT: A multifocal urothelial carcinosarcoma of a transplanted kidney in a 49-year-old woman is described. Genomic analysis of the extracted nuclei of all the neoplastic cells showed uniformly XY genotype proving the transplant origin of the tumor. RESULTS: The carcinogenic role of immunosuppressive medications in kidney-transplanted patients is reported in the literature. In this case, immunosuppression may have promoted the carcinosarcoma. CONCLUSION: Renal transplant patients should be monitored for the development of malignancy in the allograft and elsewhere.

[Multidrug resistance: diagnostic approaches and difficulties]. / Többszörös gyógyszer-rezisztencia: diagnosztikai megközelítések es nehézségek.

During the mid sixties scientists recognized that tumour cells can be resistant to a variety of chemotherapeutical drugs of different chemical structure simultaneously. They named this phenomenon multidrug resistance (MDR). Following this observation, number of in vitro and in vivo experiments proved that transmembrane proteins of the cell membrane are responsible for the mechanism. Many details of the underlying biochemical mechanisms were explored during the past decade. Nowadays the importance of MDR is well appreciated in different walks of medical science. MDR is an important problem during the treatment of many haematological conditions and solid organ tumors. Also, MDR is an important factor during immunosuppressant therapy of the transplanted patients. In spite of extensive research there are many uncertainties around MDR. This brief review describes the present options in the investigation of MDR. Based upon the MDR genotyping and expression level the likelihood of drug resistance may be predicted with reasonable accuracy. Additional information may be obtained by measuring the P-glycoprotein expression on the cell surface and the outward transport of test molecules from the cells. Although the tests described above provide significant help in predicting MDR or in the confirmation of existing MDR there is no consensus about the laboratory diagnosis.

Activated platelet-derived microparticle numbers are elevated in patients with severe fungal (Candida albicans) sepsis.

BACKGROUND: The treatment of severe sepsis highly depends on the identification of bacteria or fungi from blood and/or other body materials. Although widely available blood culturing and risk assessment scores are not completely reliable, current guidelines do not recommend the wide empirical use of antifungal medications based on questionable benefit or possible side-effects. We aimed to test whether platelet-derived microparticle (MP) measurements can improve the early detection of the infective agent behind sepsis. METHODS: Thirty-three consecutive severe septic patients from our university intensive care unit were included in our prospective study. MP number and surface antigen characteristics were followed by flow cytometry on days 1 (admission), 3 and 5. For microbiological identification, various specimens were collected on admission and in case of overall status deterioration. RESULTS: On admission, septic patients showed elevated annexin V and constitutive platelet marker (CD41)-positive MP numbers compared with volunteers. Mixed fungal septic patients showed significantly elevated annexin V and CD41-positive particle numbers on day 1 (P < 0.05) compared with the non-fungal septic group. Adhesive platelet marker (CD42a) harbouring vesicles were negligible in the non-fungal group, while fungal septic patients showed significantly elevated numbers in all measurements (P < 0.01). Particles from activated platelets (PAC1) had elevated numbers in the first and fifth study days compared with non-fungal septic patients (P < 0.05). CONCLUSIONS: The measurement of CD42a- and PAC1-positive microparticles may provide important additional information which can help to improve the early instalment of antifungal therapy of severe septic patients.

Impact of genetic variants on post-clopidogrel platelet reactivity in patients after elective percutaneous coronary intervention.

AIM: To determine the effect of various SNPs on post-clopidogrel platelet reactivity and clinical outcome. MATERIALS & METHODS: Cytochrome 2C19 (CYP2C19) loss-of-function (LOF; *2, *3) and gain-of-function (GOF; *17) allelic variants, together with ABCB1 (3435 C→T and 2677 G→T/A) and paraoxonase-1 (PON-1; 192 Q→R) SNPs were analyzed in 189 patients after elective stent implantation who participated in a randomized, placebo-controlled trial (NCT00638326). Platelet reactivity was determined with light transmission aggregometry and vasodilator stimulated phosphoprotein phosphorylation (VASP-PRI) 12-24 h after 600 mg clopidogrel. High on-treatment platelet reactivity (HTPR) was defined according to the consensus definition (ADP 5 µM >46%; VASP-PRI>50%). RESULTS: In the case of CYP2C19 genotypes, a gene-dose effect was observed in ADP reactivity with the lowest values in GOF homozygotes and the highest degree in patients carrying two LOF alleles. The odds for HTPR also increased with the number of LOF alleles. There were no significant differences in platelet reactivity according to PON-1 or ABCB1 genotypes. In multivariate analysis, the presence of a CYP2C19 LOF allele turned out to be the independent determinant of HTPR. Although the study was not powered to clinical outcome (not LOF heterozygotes), only patients with two LOF alleles had a significantly higher risk for cardiovascular death, myocardial infarction or unplanned target vessel revascularization at 1 year compared with non-LOF carriers. CONCLUSION: Genetic variants in CYP2C19 have a gene-dose effect on post-clopidogrel platelet reactivity, with homozygote LOF carriers having the highest risk for HTPR and for adverse ischemic events. Neither ABCB1 nor PON-1 genotypes significantly influenced platelet reactivity or outcome.

Mice deficient in pituitary adenylate cyclase activating polypeptide (PACAP) show increased susceptibility to in vivo renal ischemia/reperfusion injury.

Pituitary adenylate cyclase activating polypeptide (PACAP) is a neuropeptide with well-known cytoprotective effects. We have reported earlier that PACAP decreases mortality and the degree of tubular atrophy in a rat model of renal ischemia/reperfusion injury. Recently, we have shown that kidney cultures isolated from PACAP deficient mice show increased susceptibility to renal oxidative stress. Based on these previous studies, we raised the question whether PACAP deficient mice display increased sensitivity to in vivo kidney ischemia/reperfusion. PACAP⁻/⁻ mice underwent 45 or 60 min of renal ischemia followed by 2 weeks reperfusion. Kidneys were processed for histological analysis. Sections stained with PAS-haematoxylin were graded for the following parameters: degree of tubular dilation, Bowmann's capsule dilation, lymphocyte and macrophage infiltration, thyroidization and the disappearance of the PAS-positive glycocalyx from under the brush border. In other sets of experiments, tissue cytokine expression and the level of the endogenous antioxidant superoxide dismutase (SOD) were also determined after 60 min ischemia/reperfusion. Our results show that while intact kidneys were not different between wild-type and PACAP deficient mice, marked differences were observed in the histological structures in groups that underwent ischemia/reperfusion. PACAP deficient mice had a worse histological outcome, with significantly higher histological scores for all tested parameters. Cytokine expression was also markedly different between wild-type and PACAP deficient mice. In addition, the level of SOD was significantly lower in PACAP⁻/⁻ animals after ischemia/reperfusion. In conclusion, the lack of endogenous PACAP leads to higher susceptibility to in vivo renal ischemia/reperfusion, suggesting that PACAP has an endogenous renoprotective effect.

Effects of PACAP on mitochondrial apoptotic pathways and cytokine expression in rats subjected to renal ischemia/reperfusion.

Pituitary adenylate cyclase activating polypeptide (PACAP) is a neuropeptide with highly efficient cytoprotective actions. Its neuroprotective effects are well-known, but PACAP is able to exert similar actions in non-neuronal cells. Recently, we have shown that PACAP prolongs renal ischemic time, decreases mortality, and attenuates tubular degeneration in a rat model of renal ischemia/reperfusion, but the mechanism of renoprotection is not yet known. Therefore, the aim of the present study was to obtain further insight into the renoprotective effects of PACAP by examining its direct effects of PACAP on mitochondrial permeability transition in vitro and on the expression of the anti-apoptotic Bcl-2 and cytokines/chemokines in kidney tissues following 45 and 60 min renal ischemia and reperfusion in vivo. We found that PACAP did not have any direct effect on mitochondrial permeability transition. Cytokine array revealed that the expression of a few cytokines/chemokines was strongly increased after ischemia/reperfusion, which was ameliorated by PACAP treatment. Furthermore, in rats subjected to renal ischemia, PACAP treatment counteracted the ischemia/reperfusion-induced decrease of the anti-apoptotic Bcl-2, both after 45 and 60 min ischemia, as analyzed by Western blot. In summary, we showed that PACAP could attenuate tissue injury involving both anti-inflammatory and anti-apoptotic effects, but not directly acting on mitochondrial permeability transition.

Histologic findings after gastrocystoplasty in rabbits.

PURPOSE: The aim of this study was to investigate the long-term histologic changes after bladder augmentation with gastric segment in an animal subject. MATERIALS AND METHODS: Gastrocystoplasty was performed in 13 young, 3-month-old male rabbits. Open biopsies were taken from the native bladder and the gastric segment preoperatively and at 3, 6, and 12 months postoperatively. Sections were examined with H&E and periodic acid-Schiff (PAS) staining. Indirect immune peroxidase method was additionally applied to detect the carcinoembrionic antigen, the proliferative activity, and the gene for the tumor protein p53 in the epithelium. RESULTS: On the native bladder, at the 3-month follow-up, polyps, mucosal edema, submucosal fibrosis, and squamous cell metaplasia were detected, which did not change during the follow-up. On the gastric segment, at the 3-month follow-up, parietal cell hyperplasia and inflammatory mucosal overgrowth were detected; at the 6-month follow-up, inflammation or atrophy of the gastric mucosa and colonic-type metaplasia was found. These alterations remained unchanged during later course of follow-up. Neither dysplasia nor malignancy was observed during the 12-month follow-up. CONCLUSIONS: The present study supports the clinical observations of low cancer risk after gastrocystoplasty and may indicate different effect of gastric secretion on uroepithelium and that of urine on gastric mucosa.

Simultaneous presence of neutrophil alveolitis and Ki-67 positivity of alveolar macrophages in dermato-/polymyositis and systemic sclerosis.

OBJECTIVE: The proportion of cell proliferation-associated nuclear Ki-67 antigen positive alveolar macrophages was compared in the bronchoalveolar lavage fluids (BAL) of patients with dermato-/polymyositis (DM-PM), scleroderma-myositis overlap syndrome (Scl-M), systemic sclerosis (SSc), and undifferentiated connective tissue disease (UCTD). PATIENTS AND METHODS: We examined 8 patients with DM-PM, 3 with Scl-M, 11 with SSc, and 10 with UCTD. The patients were selected on the basis of their interstitial lung involvement diagnosed by non-invasive methods. RESULTS: Increased Ki-67 expression in alveolar macrophages was observed in three cases of SSc (27%) and in 4 of myositis (36%). In UCTD and in healthy volunteers a low level of Ki-67 expression was detected (in both groups 5%). A positive correlation was found between the percentage of the Ki-67 positive alveolar macrophages and the percentage of granulocytes in BAL fluid of the investigated patients with different systemic autoimmune diseases and healthy controls, indicating that increased local proliferation of differentiated large alveolar macrophages can be detected in cases with neutrophil alveolitis. There was also a correlation between the extent of ground glass opacity on HRCT and elevated proportion of proliferating alveolar macrophages in BAL fluid.