Impact of the NaV1.8 variant, A1073V, on post-sigmoidectomy pain and electrophysiological function in rat sympathetic neurons.

NaV1.8 channels play a crucial role in regulating the action potential in nociceptive neurons. A single nucleotide polymorphism in the human NaV1.8 gene SCN10A, A1073V (rs6795970, G>A), has been linked to the diminution of mechanical pain sensation as well as cardiac conduction abnormalities. Furthermore, studies have suggested that this polymorphism may result in a "loss-of-function" phenotype. In the present study, we performed genomic analysis of A1073V polymorphism presence in a cohort of patients undergoing sigmoid colectomy who provided information regarding perioperative pain and analgesic use. Homozygous carriers reported significantly reduced severity in postoperative abdominal pain compared with heterozygous and wild-type carriers. Homozygotes also trended toward using less analgesic/opiates during the postoperative period. We also heterologously expressed the wild-type and A1073V variant in rat superior cervical ganglion neurons. Electrophysiological testing demonstrated that the mutant NaV1.8 channels activated at more depolarized potentials compared with wild-type channels. Our study revealed that postoperative abdominal pain is diminished in homozygous carriers of A1073V and that this is likely due to reduced transmission of action potentials in nociceptive neurons. Our findings reinforce the importance of NaV1.8 and the A1073V polymorphism to pain perception. This information could be used to develop new predictive tools to optimize patient pain experience and analgesic use in the perioperative setting.NEW & NOTEWORTHY We present evidence that in a cohort of patients undergoing sigmoid colectomy, those homozygous for the NaV1.8 polymorphism (rs6795970) reported significantly lower abdominal pain scores than individuals with the homozygous wild-type or heterozygous genotype. In vitro electrophysiological recordings also suggest that the mutant NaV1.8 channel activates at more depolarizing potentials than the wild-type Na+ channel, characteristic of hypoactivity. This is the first report linking the rs6795970 mutation with postoperative abdominal pain in humans.

Perioperative thrombotic complications associated with pediatric liver transplantation: a UNOS database evaluation.

BACKGROUND: This retrospective UNOS database evaluation analyzes the prevalence of preoperative portal vein thromboses (PVT), and postoperative thromboses leading to graft failure in pediatric patients undergoing liver transplantation (LT). METHODS: The evaluation was performed in three age groups: I (0-5), II (6-11), III (12-18) years old. Factors predictive of pre- and postoperative thromboses were analyzed. RESULTS: Between 2000 and 2015, 8982 pediatric LT were performed in the US. Of those, 390 patients had preoperative PVT (4.3%), and 396 (4.4%) had postoperative thromboses. The prevalence of both types of thromboses was less in Group III than in the other two groups (3.20% vs 4.65%, p = 0.007 and 1.73% vs. 5.13%, p < 0.001, respectively). The prevalence of postoperative thromboses was significantly higher in Group I than in the other two groups (5.49% vs. 2.51%, p < 0.001). Preoperative PVT was independently associated with postoperative thromboses (OR = 1.7, p = 0.02). Children less than 5 years of age were more likely to develop postoperative thromboses leading to graft failure (OR = 2.9, p < 0.001). CONCLUSION: Younger children undergoing LT are prone to pre-and postoperative thrombotic complications. Preoperative PVT at the time of transplantation was independently associated with postoperative thromboses. Perioperative antithrombotic therapy should be considered in pediatric patients undergoing LT.

Association of frequent genetic variants in platelet activation pathway genes with large-vessel ischemic stroke in Polish population.

Platelets are critically involved in the development of cerebral ischemia. Our study aimed to establish an association between frequent (minor allele frequency (MAF) > 5%) genetic polymorphisms in 84 candidate genetic loci previously linked to platelet reactivity by the use of next-generation sequencing of exons from pooled DNA samples in Polish patients with a history of large-vessel ischemic stroke. Genetic analysis was performed on blood samples obtained from 500 patients (diagnosed with acute non-cardioembolic ischemic stroke with coexisting large-artery atherosclerosis) and age/sex/history of smoking matching 500 controls of Polish origin with high risk of cardiovascular disease. Sequencing of 10 pools (five for each ischemic and control groups) was performed on the Ilumina HiSeq2500 sequencer which generated an average of 36.1 (22.7-45.9 range) million pair-end 101 bp reads and 5.3 (3-7 range) Gbp per pooled sample consisting of 100 subjects. In total, we observed 789 frequent polymorphisms in the sequenced 84 genes (703 of single-nucleotide polymorphism (SNP) type and 86 indels). When the MAF between control and stroke groups was compared, only two intronic polymorphisms (1 SNP and 1 indel) in RGS7 (rs127445 36) and ANKS1B (rs398098426) genes, respectively, show statistically significant differences, which persisted after individual genotyping of the variants and adjustment for potential confounding factors. From the remaining variants, 35 polymorphisms displayed various degrees of nominal significance (from 0.6.3 × 10-5 to 5 × 10-2) and 754 polymorphisms did not show any statistical significance when comparison was evaluated for differences in MAF between the study groups. In conclusion, the results of the study demonstrate statistically significant differences in two frequent intronic genetic variants (in RGS7 and ANKS1B) that could be associated with the platelet function between ischemic stroke patients with coexisting large-vessel atherosclerosis and control patients having high vascular risk.

Intraoperative auditory evoked potential recordings are more reliable at signal detection from different sensor sites on the forehead compared to bispectral index.

Bispectral index (BIS) and auditory evoked potential (AEP) monitoring require the attachment of forehead sensors, posing difficulties when the surgical field involves the forehead. This study analyzed the relationship between BIS values and AEP indices from different sites on the head to establish alternative sensor locations for AEP recording. Thirty patients scheduled for elective surgery under sevoflurane anesthesia were randomly assigned to the forehead, nose or mandible groups (n = 10 patients per group). AEP sensors were placed at the assigned position for each group and BIS sensors were placed on the forehead. BIS value and AEP index were simultaneously recorded from induction until emergence from general anesthesia. Relationships between BIS values and AEP indices were analyzed using a regression method and compared between groups using Pearson's correlation coefficients. Square regression models better expressed the relationships than linear models in all groups. The z-transformed coefficient in the forehead group was the same as the nose group (p = 0.24) and significantly different in the mandible group (p = 0.0046). These findings suggest that AEPs can be accurately recorded from sensors placed on the nose. Nasal AEP might be useful for monitoring electrical activity in the brain during surgeries involving the forehead.

Population-Specific Associations of Deleterious Rare Variants in Coding Region of P2RY1-P2RY12 Purinergic Receptor Genes in Large-Vessel Ischemic Stroke Patients.

The contribution of low-frequency and damaging genetic variants associated with platelet function to ischemic stroke (IS) susceptibility remains unknown. We employed a deep re-sequencing approach in Polish patients in order to investigate the contribution of rare variants (minor allele frequency, MAF < 1%) to the IS genetic susceptibility in this population. The genes selected for re-sequencing consisted of 26 genes coding for proteins associated with the surface membrane of platelets. Targeted pooled re-sequencing (Illumina HiSeq 2500) was performed on genomic DNA of 500 cases (patients with history of clinically proven diagnosis of large-vessel IS) and 500 controls. After quality control and prioritization based on allele frequency and damaging probability, follow-up individual genotyping of deleterious rare variants was performed in patients from the original cohort. Gene-based analyses identified an association between IS and 6 rare functional and damaging variants in the purinergic genes (P2RY1 and P2RY12 locus). The predicted properties of the most damaging rare variants in P2RY1 and P2RY12 were confirmed by using mouse fibroblast cell cultures transfected with plasmid constructs containing cDNA of mutated variants (FLIPR on FlexStation3). This study identified a putative role for rare variants in P2RY1 and P2RY12 genes involved in platelet reactivity on large-vessel IS susceptibility in a Polish population.

African Americans have a lower prevalence of portal vein thrombosis at the time of liver transplantation.

BACKGROUND: Perioperative vascular thrombotic events in patients undergoing liver transplantation (LT) are associated with significant morbidity and mortality. METHODS: In this retrospective UNOS database analysis, we evaluated the prevalence of portal vein thrombosis (PVT) and factors contributing to PVT development in different ethnic groups. RESULTS: Of the 47 953 LT performed between 2002 and 2015, we identified 3642 cases of PVT. African Americans (AA) had a significantly lower prevalence of PVT compared to other ethnic groups (p = 0.0001). Multivariable regression analysis confirmed that AA were less likely than other ethnicities to have PVT (OR = 0.6). AA cohort was more likely to have infectious or autoimmune causes of liver failure (OR = 1.6, 1.7 respectively) as well as higher creatinine and INR compared to other groups (OR = 1.6, 1.3 respectively). AA's were less likely to have encephalopathy, ascites, or variceal bleeding, which might indicate lower portal pressures. AA's were listed for LT later than other ethnicities and had both a lower functional status and higher MELD score at the time of registration. DISCUSSION: AA's had a significantly lower prevalence of preoperative PVT despite having a greater number of factors predisposing to thrombosis. This predisposition should be considered before instituting perioperative antithrombotic therapy.

Analysis of Common Single Nucleotide Polymorphisms in Complex Regional Pain Syndrome: Genome Wide Association Study Approach and Pooled DNA Strategy.

OBJECTIVE: The objective of this study was to use a genome-wide association (GWAS) approach and pooled DNA strategy to search for new genomic loci associated with complex regional pain syndrome (CRPS). DESIGN: The study cohort consisted of 230 patients with established diagnosis of CRPS. The control group consisted of 230 age- and gender-matched subjects without chronic pain. We tested the association of common single nucleotide polymorphisms (SNPs), genotyped using a high-density microarray platform, with CRPS phenotype. This was followed by individual genotyping of the most significant SNPs identified in the microarray genomic scan, in both original discovery (N = 115) and independent verification (N = 115) groups of patients with CRPS, as well as in the appropriate matched control subjects. RESULTS: The results of our study provide no support for the initial hypothesis of the existence of an association between any investigated genomic targets (including GWAS for all genomic loci available on the microarray, and focused scan of the HLA locus on chromosome 6) and CRPS phenotype. CONCLUSIONS: Despite the fact that we interrogated about 83% of all of common SNPs in the human genome, we did not find evidence that any of the investigated common SNPs may be associated with CRPS phenotype.

Next-generation re-sequencing of genes involved in increased platelet reactivity in diabetic patients on acetylsalicylic acid.

The objective of this study was to investigate whether rare missense genetic variants in several genes related to platelet functions and acetylsalicylic acid (ASA) response are associated with the platelet reactivity in patients with diabetes type 2 (T2D) on ASA therapy. Fifty eight exons and corresponding introns of eight selected genes, including PTGS1, PTGS2, TXBAS1, PTGIS, ADRA2A, ADRA2B, TXBA2R, and P2RY1 were re-sequenced in 230 DNA samples from T2D patients by using a pooled PCR amplification and next-generation sequencing by Illumina HiSeq2000. The observed non-synonymous variants were confirmed by individual genotyping of 384 DNA samples comprising of the individuals from the original discovery pools and additional verification cohort of 154 ASA-treated T2DM patients. The association between investigated phenotypes (ASA induced changes in platelets reactivity by PFA-100, VerifyNow and serum thromboxane B2 level [sTxB2]), and accumulation of rare missense variants (genetic burden) in investigated genes was tested using statistical collapsing tests. We identified a total of 35 exonic variants, including 3 common missense variants, 15 rare missense variants, and 17 synonymous variants in 8 investigated genes. The rare missense variants exhibited statistically significant difference in the accumulation pattern between a group of patients with increased and normal platelet reactivity based on PFA-100 assay. Our study suggests that genetic burden of the rare functional variants in eight genes may contribute to differences in the platelet reactivity measured with the PFA-100 assay in the T2DM patients treated with ASA.

Serum Brain-Derived Neurotrophic Factor is Related to Platelet Reactivity but not to Genetic Polymorphisms within BDNF Encoding Gene in Patients with Type 2 Diabetes.

BACKGROUND: The aim of this study was to investigate the association between serum concentrations of the brain-derived neurotrophic factor (BDNF), platelet reactivity and inflammatory markers, as well as its association with BDNF encoding gene variants in type 2 diabetic patients (T2DM) during acetylsalicylic acid (ASA) therapy. MATERIAL/METHODS: This retrospective, open-label study enrolled 91 patients. Serum BDNF, genotype variants, hematological, biochemical, and inflammatory markers were measured. Blood samples were taken in the morning 2-3 h after the last ASA dose. The BDNF genotypes for selected variants were analyzed by use of the iPLEX Sequenom assay. RESULTS: In multivariate linear regression analysis, CADP-CT >74 sec (p<0.001) and sP-selectin concentration (p=0.03) were predictive of high serum BDNF. In multivariate logistic regression analysis, CADP-CT >74 sec (p=0.02) and IL-6 concentration (p=0.03) were risk factors for serum BDNF above the median. Non-significant differences were observed between intronic SNP rs925946, missense SNP rs6265, and intronic SNP rs4923463 allelic groups and BDNF concentrations in the investigated cohort. CONCLUSIONS: Chronic inflammatory condition and enhanced immune system are associated with the production of BDNF, which may be why the serum BDNF level in T2DM patients with high platelet reactivity was higher compared to subjects with normal platelet reactivity in this study.

Autoimmune conditions are associated with perioperative thrombotic complications in liver transplant recipients: A UNOS database analysis.

BACKGROUND: End stage liver disease (ESLD) is associated with significant thrombotic complications. In this study, we attempted to determine if patients with ESLD, due to oncologic or autoimmune diseases, are susceptible to thrombosis to a greater extent than patients with ESLD due to other causes. METHODS: In this retrospective study, we analyzed the UNOS database to determine the incidence of thrombotic complications in orthotopic liver transplant (OLT) recipients with autoimmune and oncologic conditions. Between 2000 and 2012, 65,646 OLTs were performed. We found 4,247 cases of preoperative portal vein thrombosis (PVT) and 1,233 cases of postoperative vascular thrombosis (VT) leading to graft failure. RESULTS: Statistical evaluation demonstrated that patients with either hepatocellular carcinoma (HCC) or autoimmune hepatitis (AIC) had a higher incidence of PVT (p = 0.05 and 0.03 respectively). Patients with primary biliary cirrhosis (PBC), primary sclerosing cholangitis (PSC) and AIC had a higher incidence of postoperative VT associated with graft failure (p < 0.0001, p < 0.0001, p = 0.05 respectively). Patients with preoperative PVT had a higher incidence of postoperative VT (p < 0.0001). Multivariable logistic regression demonstrated that patients with AIC, and BMI ≥40, having had a transjugular intrahepatic portosystemic shunt, and those with diabetes mellitus were more likely to have preoperative PVT: odds ratio (OR)(1.36, 1.19, 1.78, 1.22 respectively). Patients with PSC, PBC, AIC, BMI ≤18, or with a preoperative PVT were more likely to have a postoperative VT: OR (1.93, 2.09, 1.64, 1.60, and 2.01, respectively). CONCLUSION: Despite the limited number of variables available in the UNOS database potentially related to thrombotic complications, this analysis demonstrates a clear association between autoimmune causes of ESLD and perioperative thrombotic complications. Perioperative management of patients at risk should include strategies to reduce the potential for these complications.

Effect of cold perfusion and perfluorocarbons on liver graft ischemia in a donation after cardiac death model.

BACKGROUND: Effects of two perfluorocarbon (PFC) formulations (perfluorodecalin emulsion and perfluorodecalin liquid) on the quality of liver graft preservation, in a donation after cardiac death (DCD) rat model, were investigated. The significance of continuous graft perfusion during cold preservation was also explored. MATERIALS AND METHODS: DCD model: 30 min after cardiopulmonary arrest was initiated, livers were excised and flushed with cold University of Wisconsin (UW) solution (± PFC) and preserved in the same solution for 8 h. The study groups were preserved as follows: group 1: no perfusion; group 2: perfusion with UW; group 3: PFC was administered before cardiac arrest and the liver was perfused with UW alone; and groups 4 and 5: perfused with UW + 1 of two PFCs. In a baseline group used only for comparison of gene expression, livers were quick-frozen after cardiac arrest. Microarrays were used to analyze liver messenger RNA transcripts. Histopathologic, immunohistochemical, and ADP/ATP ratio evaluations were performed to assess the quality of graft preservation. RESULTS: Significant decreases in downregulation and increases in upregulation of hepatic genes (relative to baseline) were demonstrated in all perfusion groups. This trend was most pronounced in the PFC groups. Lower fat content and ADP/ATP ratio and a reduction in Caspase 3 activation were found in all perfusion groups. CONCLUSION: Hypothermic perfusion of rat DCD liver grafts with oxygenated UW solution (± PFC) produced superior preservation compared with nonperfusion storage. The observed changes in expression of hepatic genes may represent a protective effect in the DCD model.

Genetic factors associated with pharmacotherapy and background sensitivity to postoperative and chemotherapy-induced nausea and vomiting.

Postoperative nausea and vomiting (PONV) continues to be a most common complication of surgery and anesthesia. It has been suggested that the inherited factors may play a significant role in the background sensitivity to both PONV and also chemotherapy-induced nausea and vomiting (CINV), including resistance to antiemetic prophylaxis and/or therapy. This notion could be best exemplified by occurrence of PONV in several generations of families and concordance of PONV in monozygotic twins. The most frequently addressed issue in the research on genomic background of PONV/CINV relates to the inherited resistance to the antiemetic treatment (pharmacogenomics), and in lesser degree to their genomic background. The most common group of antiemetics consists of 5HT3 receptor antagonists, and this group was an initial target of pharmacogenomic research. Most research approaches have been based on the investigation of polymorphic variations in the target for the antiemetic 5HT3 receptor antagonists, i.e., serotonin receptor subunits A and B (HTR3A and HTR3B). The other area of pharmacogenomic investigations includes metabolic pathways of 5HT3 antagonists, in particular polymorphic variants of the CYP450 2D6 isoform (CYP2D6) because most of them are metabolized in various degrees by the CYP2D6 system. The results of targeted genomic association studies indicate that other genes are also associated with PONV and CINV, including OPRM1, and ABCB1. In addition, genes such as DRD2 and CHRM3 genes have recently been associated with PONV. The new genome-wide association studies seem also to indicate that the background genomic sensitivity to PONV and CINV might be multifactorial and include several genomic pathways.

Association of µ-opioid receptor gene (OPRM1) haplotypes with postoperative nausea and vomiting.

Genetic variants, such as single-nucleotide polymorphisms (SNPs), of the µ-opioid receptor gene (OPRM1) might be associated with individual differences in opioid sensitivity, as well as with the incidence and severity of postoperative nausea and vomiting (PONV). The goal of the present study was to determine, in a cohort of Japanese surgical patients, genotypes and haplotypes of several SNPs in the OPRM1 gene, and their association with PONV during the early (first 24 h) postoperative period. We examined the incidence and severity of PONV, during the first 24 h after surgery, in 85 Japanese patients receiving intravenous patient-controlled analgesia fentanyl analgesia for postoperative pain control. Eight tag SNPs of the OPRM1 gene (rs1799971, A/G; rs510769, G/A; rs4870266, G/A; rs3798683, G/A; rs1323042, A/C; rs609623, C/T; rs9397685, A/G; and rs644261, C/G) were selected based on their minor allele frequency (>10%) and linkage disequilibrium strength (<80%), and genotyped for haplotype analysis and determination of associations with PONV. Only one out of eight investigated SNPs, rs9397685, in the intronic part of the OPRM1 gene was associated with differences in the occurrence and severity of PONV. We also found four common haplotypes with a frequency of >10% in the investigated patients, including GGGAACAC (33%), AGGGACAC (19%), GGGAACGC (12%), and AGAGACAC (10%). The severity of PONV in carriers of the GGGAACGC haplotype was significantly lower than in the carriers of the other haplotypes (P < 0.05). One intronic SNP, rs9397685, and haplotypes constructed from eight SNPs within the OPRM1 gene locus might be involved in the severity of PONV associated with general anesthesia and opioid administration. This novel finding, if validated and verified in larger and additional ethnic cohorts, might contribute to better knowledge of the contribution of the OPRM1 gene to PONV.

Heterologous expression of the human wild-type and variant NaV 1.8 (A1073V) in rat sensory neurons.

BACKGROUND: Silent inflammatory bowel disease (IBD) is a condition in which individuals with the active disease experience minor to no pain. Voltage-gated Na+ (NaV ) channels expressed in sensory neurons play a major role in pain perception. Previously, we reported that a NaV 1.8 genetic polymorphism (A1073V, rs6795970) was more common in a cohort of silent IBD patients. The expression of this variant (1073V) in rat sympathetic neurons activated at more depolarized potentials when compared to the more common variant (1073A). In this study, we investigated whether expression of either NaV 1.8 variant in rat sensory neurons would exhibit different biophysical characteristics than previously observed in sympathetic neurons. METHODS: Endogenous NaV 1.8 channels were first silenced in DRG neurons and then either 1073A or 1073V human NaV 1.8 cDNA constructs were transfected. NaV 1.8 currents were recorded with the whole-cell patch-clamp technique. KEY RESULTS: The results indicate that 1073A and 1073V NaV 1.8 channels exhibited similar activation values. However, the slope factor (k) for activation determined for this same group of neurons decreased by 5 mV, suggesting an increase in voltage sensitivity. Comparison of inactivation parameters indicated that 1073V channels were shifted to more depolarized potentials than 1073A-expressing neurons, imparting a proexcitatory characteristic. CONCLUSIONS AND INFERENCES: These findings differ from previous observations in other expression models and underscore the challenges with heterologous expression systems. Therefore, the use of human sensory neurons derived from induced pluripotent stem cells may help address these inconsistencies and better determine the effect of the polymorphism present in IBD patients.

Association between plasma cyclic guanosine monophosphate levels and hemodynamic instability during liver transplantation.

The activation of cyclic guanosine monophosphate (cGMP) production in patients with end-stage liver disease (ESLD) has been associated with hemodynamic instability during orthotopic liver transplantation (OLT). The aim of this prospective, observational study was to investigate the involvement of cGMP in the mediation of profound hypotension during liver graft reperfusion. An additional objective was to determine whether preoperative cGMP levels are associated with intraoperative hemodynamic instability. Forty-four consecutive patients undergoing OLT were included in the study. Blood samples for cGMP analysis were obtained from (1) the radial artery before the surgical incision; (2) the radial artery, portal vein, and flush blood during the anhepatic phase; and (3) the radial artery 20 minutes after liver graft reperfusion. On the basis of a statistical analysis, the patients were divided into 2 groups: group 1 (preoperative cGMP level ≥ 0.05 µmol/L) and group 2 (preoperative cGMP level < 0.05 µmol/L). We demonstrated a significant correlation between the preoperative levels of cGMP and the amount of catecholamine required to maintain hemodynamic stability during reperfusion (r = 0.52, P < 0.001), the length of the hospital stay (r = 0.38, P = 0.01), and the length of the intensive care unit (ICU) stay (r = 0.44, P = 0.004). We also demonstrated a significantly higher intraoperative catecholamine requirement (P < 0.001) and a prolonged postoperative ICU stay (P = 0.02) in group 1 patients versus group 2 patients. In conclusion, this study demonstrates increased baseline cGMP production in patients with ESLD, which is significantly associated with severe hypotension during OLT. We suggest that preoperative levels of cGMP correlate with hemodynamic instability during liver graft reperfusion.

New single nucleotide polymorphisms associated with differences in platelets reactivity in patients with type 2 diabetes treated with acetylsalicylic acid: genome-wide association approach and pooled DNA strategy.

The objective of this study was to use genome-wide association approach and pooled DNA strategy to search for new genomic loci associated with inter-individual differences in platelet reactivity in the diabetic patients during acetylsalicylic acid (ASA) treatment. Study cohort consisted of 297 diabetic patients who had been taking ASA (75 mg daily) for at least 3 months. We tested association of single nucleotide polymorphisms (SNPs) genotyped using high density microarray platform with several platelet reactivity assays, followed by individual genotyping of most significant SNPs identified in the microarray genomic scan. The highest statistical significance (p value of 0.0001-0.008 in individual genotyping) was observed for SNP located within the regulatory G-protein signaling (RGS) 7 gene (rs2502448) using recessive genetic model. The diabetic patients on ASA treatment and homozygotes for its minor allele were characterized by increased odds ratio of at 3.45 (confidence interval: 1.82-6.53) for high on ASA platelet reactivity (i.e. impaired ASA response) when compared with homozygotes for wild-type allele. The genome-wide approach might provide an opportunity to identify novel candidate genes and pathways related to platelet activation in diabetic patients.

Effect of common single-nucleotide polymorphisms in acetylsalicylic acid metabolic pathway genes on platelet reactivity in patients with diabetes.

BACKGROUND: Platelet reactivity in patients on acetylsalicylic acid (ASA) therapy can be influenced by physiological or pathological conditions affecting ASA pharmacokinetics or pharmacodynamics. The mechanism of such variability in the therapeutic response to ASA, particularly in diabetic patients, is poorly understood. The rate of elimination of ASA and its metabolite, salicylic acid (SA), is likely a major factor determining drug efficacy. The objective of this study was to investigate the effect of genetic polymorphisms in the selected candidate genes within the ASA metabolic pathway on the platelet reactivity and concentration of ASA and thromboxane A(2) (TxA(2)) metabolites in a population of patients with type 2 diabetes mellitus (T2DM). MATERIAL AND METHODS: The study cohort consisted of 287 Caucasians with T2DM who had been taking ASA tablets at the dose of 75 mg per day for at least 3 months. Platelet reactivity analyses were performed using VerifyNow Aspirin and PFA-100 assays. The measured ASA metabolite included salicylic acid (ASA), and TxA(2) metabolites included serum TxB(2) and urinary 11-dh-TxB(2). Genotyping for the selected 18 single-nucleotide polymorphisms (SNPs) within 5 genes of the ASA metabolic pathway was performed using a Sequenom iPLEX platform. RESULTS: No statistically significant association was observed between the investigated SNPs genotypes, platelet reactivity, and measured metabolites in the investigated cohort of patients. CONCLUSIONS: The results of our study failed to confirm that the selected variants in the genes within the ASA metabolic pathway might contribute to platelet reactivity in a diabetic population treated with ASA.

Dissimilar effects of stereoisomers and racemic hydroxychloroquine on Ca2+ oscillations in human induced pluripotent stem cell-derived cardiomyocytes.

All currently employed pharmaceutical formulations of hydroxychloroquine (HCQ) sulfate are a racemate, consisting of equal parts mixture of two stereoisomers: R(-)HCQ and S(+)HCQ sulfates. The aims of the current study were first, to obtain and characterize pure HCQ enantiomers. The separation and purification of free base HCQ enantiomers from the racemate form were performed using semi-preparative chiral high-performance liquid chromatography. Second, we compared the pharmacological properties of both optical isomers and racemic mixture on the intracellular Ca2+ oscillations employing an in vitro model of human cardiomyocytes derived from induced pluripotent stem cells (iPSCs). The results of the pharmacological investigations indicate that the racemic and pure stereoisomer forms of HCQ sulfate exhibited a dose-dependent inhibition of spontaneous Ca2+ oscillations (as measured from their frequency and Ca2+ peak widths) in cardiomyocytes 5-45 min following exposure. In addition, the concentration-response relationships for all three compounds indicated that the rank order of potency (IC50 ) was R(-)HCQ >racemic HCQ >S(+)HCQ for the frequency of the Ca2+ oscillations and width of Ca2+ peaks for all time points examined. These studies indicate that both R(-) and S(+) stereoisomers exhibit differing pharmacological actions on hiPSC cardiomyocytes, with the former effecting a greater potency on cell Ca2+ handling.

Use of a third-generation perfluorocarbon for preservation of rat DCD liver grafts.

BACKGROUND: Cold storage in any of the commonly used preservation solutions is not always adequate for donation after cardiac death (DCD) liver grafts due to prolonged warm ischemic time. In this study, we used a third-generation perfluorocarbon (PFC), Oxycyte, for DCD liver graft preservation in a rat model. MATERIALS AND METHODS: Twenty-eight rats (14 in each group) were used. Thirty minutes after cardiopulmonary arrest, livers were harvested and flushed with a cold and pre-oxygenated solution of either University of Wisconsin (UW) or UW + 20% PFC. After 8 h of cold preservation in either of the investigated solutions, liver graft specimens were analyzed for evidence of ischemic injury. Hemotoxylin and eosin staining (H and E), as well as immunohistochemical analysis with anti-cleaved caspase 3 antibody, was performed. Levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in the preservation solution were analyzed at 1 and 8 h during preservation. RESULTS: In the PFC group, the degree of cell congestion, vacuolization and necrosis were all significantly less than in the UW group (P = 0.002-0.004). The number of cells with a positive cleaved caspase 3 antibody reaction was reduced by about 50% in comparison with the UW group (P < 0.006). The AST level in the PFC group was significantly less than in the UW group after 8 h of preservation (P < 0.048). CONCLUSION: The addition of PFC to UW solution significantly decreases the degree of histologic damage in rat DCD liver grafts. This preservation strategy can be potentially helpful for organ preservation after prolonged warm ischemia.

Release of cytokines and hemodynamic instability during the reperfusion of a liver graft.

The objectives of this prospective, observational study were (1) to determine whether a transplanted liver graft releases proinflammatory cytokines into the systemic circulation upon reperfusion and (2) to determine whether they contribute to any subsequent hemodynamic instability observed after graft reperfusion (if this release occurs). Blood samples from 17 consecutive patients undergoing liver transplantation were analyzed for cytokines, including tumor necrosis factor α (TNF-α), interleukin-1ß (IL-1ß), IL-2, IL-6, and IL-8. Blood samples were obtained from the radial artery, portal vein, and flush blood (a sample taken from a catheter placed above the infrahepatic inferior vena cava clamp). The amount of catecholamines necessary to maintain a mean arterial pressure between 65 and 75 mm Hg during graft reperfusion was compared with the level of cytokines. A statistical analysis was performed with the least squares method, Kendall's tau-b test, and regression analysis. We demonstrated that flush blood from the liver grafts contained a significant amount and variety of cytokines. Most of these were removed by graft irrigation. The concentration of TNF-α in samples obtained from flush blood at the end of liver irrigation was significantly higher than the concentration in samples obtained from the radial artery (P = 0.0067) or portal vein (P = 0.0003) before reperfusion. This correlated directly with the amount of catecholamines used to treat hemodynamic instability. Although there were increased levels of IL-1ß, IL-2, and IL-8 in the flush blood, there was no statistically significant correlation between the levels of these cytokines and the amount of catecholamines used.