Lenvatinib plus Pembrolizumab for Advanced Endometrial Cancer.

BACKGROUND: Standard therapy for advanced endometrial cancer after failure of platinum-based chemotherapy remains unclear. METHODS: In this phase 3 trial, we randomly assigned, in a 1:1 ratio, patients with advanced endometrial cancer who had previously received at least one platinum-based chemotherapy regimen to receive either lenvatinib (20 mg, administered orally once daily) plus pembrolizumab (200 mg, administered intravenously every 3 weeks) or chemotherapy of the treating physician's choice (doxorubicin at 60 mg per square meter of body-surface area, administered intravenously every 3 weeks, or paclitaxel at 80 mg per square meter, administered intravenously weekly [with a cycle of 3 weeks on and 1 week off]). The two primary end points were progression-free survival as assessed on blinded independent central review according to the Response Evaluation Criteria in Solid Tumors, version 1.1, and overall survival. The end points were evaluated in patients with mismatch repair-proficient (pMMR) disease and in all patients. Safety was also assessed. RESULTS: A total of 827 patients (697 with pMMR disease and 130 with mismatch repair-deficient disease) were randomly assigned to receive lenvatinib plus pembrolizumab (411 patients) or chemotherapy (416 patients). The median progression-free survival was longer with lenvatinib plus pembrolizumab than with chemotherapy (pMMR population: 6.6 vs. 3.8 months; hazard ratio for progression or death, 0.60; 95% confidence interval [CI], 0.50 to 0.72; P<0.001; overall: 7.2 vs. 3.8 months; hazard ratio, 0.56; 95% CI, 0.47 to 0.66; P<0.001). The median overall survival was longer with lenvatinib plus pembrolizumab than with chemotherapy (pMMR population: 17.4 vs. 12.0 months; hazard ratio for death, 0.68; 95% CI, 0.56 to 0.84; P<0.001; overall: 18.3 vs. 11.4 months; hazard ratio, 0.62; 95% CI, 0.51 to 0.75; P<0.001). Adverse events of grade 3 or higher occurred in 88.9% of the patients who received lenvatinib plus pembrolizumab and in 72.7% of those who received chemotherapy. CONCLUSIONS: Lenvatinib plus pembrolizumab led to significantly longer progression-free survival and overall survival than chemotherapy among patients with advanced endometrial cancer. (Funded by Eisai and Merck Sharp and Dohme [a subsidiary of Merck]; Study 309-KEYNOTE-775 ClinicalTrials.gov number, NCT03517449.).

Optimizing the transport and storage conditions of current Good Manufacturing Practice -grade human umbilical cord mesenchymal stromal cells for transplantation (HUC-HEART Trial).

BACKGROUND: The HUC-HEART Trial is a clinical study of intramyocardial delivery of current Good Manufacturing Practice (cGMP)-grade human umbilical cord multipotent stromal cells (HUC-MSCs) in ischemic cardiomyopathy where 2 × 107 cells are administered to peri-infarcted myocardium. Prior to the onset of the trial, we aimed to optimize the transport/storage conditions for obtaining the highest cell viability and proliferation rate of cells to be transplanted. METHODS: Cells were tested after being transported in phosphate-buffered saline (PBS) or Ringer's lactate-based (RL) transport media supplemented with human serum albumin (HSA) and/or hydroxyethyl starch (HES) at two temperatures (2-10°C or 22-24°C). RESULTS: The effects of transport conditions on cell viability following 6 h were found highest (93.4 ± 1.5) in RL-based media at 2-10°C. Karyotypes were found normal upon transportation in any of the formulations and temperatures. However, the highest proliferation rate was noted (3.1-fold increase) in RL (1% HSA) media at 2-10°C over 6 days in culture. From that point, RL (1% HSA) media at 2-10°C was used for further experiments. The maximum cell storage time was detected around 24 h at 2-10°C. Extended storage periods resulted in a decrease in cell viability but not in MSC marker expression. An increase in actin quantity was detected in hypoxia (5% O2) groups in early culture days; no difference was noted between hypoxic versus normoxic (21% O2) conditions in later days. DISCUSSION: The overall results suggest that non-commercial, simple media formulations with extended storage intervals at 2-10°C temperatures are capable of retaining the characteristics of clinical-grade HUC-MSCs. The above findings led us to use RL (1% HSA) media at 2-10°C for transport and storage in the HUC-HEART Trial; 23 patients received HUC-MSCs by August 2018; no adverse effects were noted related to cell processing and transplantation.

The effect of CYP1A1 and GSTP1 isozymes on the occurrence of aortic aneurysms.

BACKGROUND: Aortic aneurysms are vascular diseases that are associated with high mortality and morbidity. Cytochrome P450 CYP1A1 and glutathione S-transferase (GSTP1) isozymes were searched and compared with the patients who had experienced aortic surgery due to aortic aneurysm and atherosclerotic patients without aneurysm to find the relation of the oxidative stress with the aneurysms. MATERIALS AND METHODS: Study group consisted of the patients with the diagnosis of aortic aneurysm (group I, n: 12) and control group who were operated for coronary bypass surgery: preoperatively drug users (group II, n: 21) and nonusers (group III, n: 15). Paraffin sections (4 µm thick) of aortic biopsy materials were stained with hematoxylin and eosine, CYP1A1 and GSTP1 immunohistochemical markers. The specimens were evaluated using light microscopy at 40- to 400-fold magnification. RESULTS: The expressions of CYP1A1 and GSTP1 isozymes were found statistically significantly higher in the patients who have an aortic aneurysm than both the control groups (p < 0.05). There was no significant association between protein expressions, drugs and duration of usage, patient's demographic variables, and smoking (p > 0.05). CONCLUSIONS: In this pioneering study, CYP1A1 and GSTP1 isozymes are related with the aneurysms. The strategy that prevents the oxidative stress for the patients who had aortic aneurysms could be a valuable choice of searching to effect the aneurysmal progression.

Which type of conditioning method protects the spinal cord from the ischemia-reperfusion injury in 24 hours?

OBJECTIVE: This study was designed to test the effects of different types of preconditioning and postconditioning methods on spinal cord protection following aortic clamping. METHODS: The animals (rabbits) were divided into sham-operated, ischemic preconditioning, remote ischemic preconditioning, simultaneous aortic and ischemic remote preconditioning, and ischemic postconditioning groups. After neurological evaluations, ultrastructural analysis and immunohistochemical staining for caspase-3 were evaluated after 24 h following ischemia. RESULTS: The neurological outcomes of the remote ischemic preconditioning (4.2 ± 0.4) and ischemic postconditioning (4.6 ± 0.8) groups were significantly improved when compared with the ischemia group (2.2 ± 04). The immunohistochemical analysis revealed that the lowest percentage of apoptosis was in-group ischemic preconditioning at 12.5 ± 30.6%. In the comparison of intracellular edema in an ultrastructural analysis, the ischemic preconditioning and ischemic postconditioning groups had significantly lower values than the ischemia group. CONCLUSION: The conditioning methods attenuate ischemia-reperfusion injury for spinal cord injury. Ischemic and remote preconditioning and also postconditioning methods are simple to perform and inexpensive.

Effect of conditioning on visceral organs during indirect ischemia/reperfusion injury.

BACKGROUND: The mortality and morbidity rates of even extensive thoracoabdominal replacement have improved markedly in recent years. We investigated the effects of a temporary occlusion of the aorta as a direct precondition and temporary occlusion of the axillary artery for remote preconditioning to determine any effects that preconditioning may have on indirect (nonischemic) injuries to visceral organs (indirect effects of remote ischemia/reperfusion injury). METHODS: Thirty-seven New Zealand white rabbits were divided into five groups: controls (sham-operated; group 1); direct ischemia to the infrarenal aorta without preconditioning (group 2); direct ischemic preconditioning to the infrarenal aorta (group 3); remote ischemic preconditioning before clamping the infrarenal aorta (group 4); and simultaneous direct aortic and remote ischemic preconditioning before the clamping and during clamping of the infrarenal aorta (group 5). We used a 30-minute ischemia period for aortic occlusion for spinal cord ischemia/reperfusion. The axillary artery was used for remote preconditioning. After 24 hours, tissue specimens of the internal organs were obtained. RESULTS: Myocardial congestion was the main pathology detected in all groups. Histopathologic evaluation of tissue samples taken from the hearts showed no significant differences in terms of the degree of polymorphonuclear leukocyte (PMNL) infiltration and edema between the groups. Lung congestion and pneumonic cell infiltration were detected in all the groups. Pneumonic cell infiltration was significantly high in groups 2 and 3. Cell infiltration was lowest in group 4 at 71.4% of normal values, which differed from the normal values of 25-33.3% in the other groups (P < 0.05). Although there is a difference between the groups in case of renal congestion, there is not any difference as tubular damage and PMN. There was a significant difference with regard to renal congestion between groups 2 and 3. Renal congestion was normal in 80% of the kidneys in group 3. This differed from the normal values observed in the other groups (14.3-57.1%, P < 0.05). Liver congestion was detected in all groups. CONCLUSIONS: Different preconditioning methods may play an important role in distinct organ injuries during aortic cross-clamping. The visceral organs that exhibited positive and constructive results with direct and remote preconditioning included the lungs and kidneys during indirect ischemia/reperfusion injury. Remote ischemic conditioning was determined to be especially advantageous as a protection method, due to the fact that it is easy to use and effective for indirect ischemia/reperfusion injury.

Right ventricular and septal function in patients with pulmonary hypertension.

OBJECTIVE: Pulmonary hypertension (PHT) exacerbates the functions of both ventricles. This prospective, randomised study was planned to investigate the effects of PHT on kinetics of both ventricles and the septum. METHODS: Twenty-five patients were randomly selected among the patients who had been planned to undergo mitral valve replacement (MVR) because of isolated mitral stenosis and divided into two groups according to their preoperative pulmonary artery pressure (PAP) values. Blood pool gated single photon emission tomography (BPGS) and transthoracic echocardiography were performed. Ventricles' regional, global and functional parameters were also assessed by using pulsed wave Doppler tissue imaging (DTI). RESULTS: Preoperative and postoperative PAP of the group 1 (PAP < 50 mmHg) were 40.0 ± 2.8 and 30.0 ± 2.6 mmHg (p = 0.03), group 2 (PAP ≥ 50 mmHg) were 71.9 ± 4.7 and 50.6 ± 3.5 mmHg (p < 0.05). The global right and left ventricle scores were decreased after the operation. The decrement was only significant in group 2. Considering the septal kinetics, right ventricle septal score was decreased from 7.6 to 3.3 (p < 0.05) in group 1, from 3.8 to 1.6 (p < 0.05) in group 2 postoperatively. CONCLUSION: Following MVR, a decrement in PAP values, and an improvement in ventricular function, especially in the right ventricular and septal kinetics were achieved. Furthermore, it was found that both DTI and BPGS techniques are beneficial to investigate the functional changes postoperatively and in the follow-up period of the patients who undergo mitral valve surgery.

Lenvatinib Plus Pembrolizumab in Previously Treated Advanced Endometrial Cancer: Updated Efficacy and Safety From the Randomized Phase III Study 309/KEYNOTE-775.

Clinical trials frequently include multiple end points that mature at different times. The initial report, typically based on the primary end point, may be published when key planned co-primary or secondary analyses are not yet available. Clinical Trial Updates provide an opportunity to disseminate additional results from studies, published in JCO or elsewhere, for which the primary end point has already been reported.We report the final prespecified analysis for overall survival (OS), along with updated progression-free survival (PFS) and objective response rate (ORR), and safety from the open-label, randomized, phase III Study 309/KEYNOTE-775. In total, 827 patients with advanced, recurrent, or metastatic endometrial cancer (EC) were randomly assigned to receive lenvatinib 20 mg orally once daily plus pembrolizumab 200 mg intravenously once every 3 weeks (n = 411) or chemotherapy of the treating physician's choice (doxorubicin 60 mg/m2 intravenously once every 3 weeks or paclitaxel 80 mg/m2 intravenously once weekly [3 weeks on; 1 week off] [n = 416]). Efficacy was reported for patients with mismatch repair proficient (pMMR) tumors and all-comers, and by subgroups (histology, prior therapy, MMR status). Updated safety was also reported.Lenvatinib plus pembrolizumab showed benefits in OS (pMMR HR, 0.70; 95% CI, 0.58 to 0.83; all-comer HR, 0.65; 95% CI, 0.55 to 0.77), PFS (pMMR HR, 0.60; 95% CI, 0.50 to 0.72; all-comer HR, 0.56; 95% CI, 0.48 to 0.66), and ORR (pMMR patients, 32.4% v 15.1%; all-comers, 33.8% v 14.7%) versus chemotherapy. OS, PFS, and ORR favored lenvatinib plus pembrolizumab in all subgroups of interest. No new safety signals were observed. Lenvatinib plus pembrolizumab continued to show improved efficacy versus chemotherapy and manageable safety in patients with previously treated advanced EC.

Influence of blood collection systems on coagulation tests.

OBJECTIVE: Coagulation tests are influenced by pre-analytic conditions such as blood collection systems. Change of glass collection tubes with plastic ones will cause alteration of the test results. The aim of this study was to compare three plastic blood collection tubes with a standard glass blood collection tube and each plastic collection tube with the other two for possible additional tube-to- tube differences. MATERIAL AND METHODS: A total of 284 blood samples were obtained from 42 patients receiving warfarin during their routine controls, besides 29 healthy volunteers. Subgroup analyses were done according to health status. RESULTS: Our study demonstrated that different blood collection tubes have a statistically significant influence on coagulation tests. The magnitude of the effect depends on the tube used. However most of the tests performed on samples obtained from any tube correlated significantly with results obtained from other tube samples. CONCLUSION: Although blood collection tubes with different brands or properties will have distinct effects on coagulation tests, the influence of these blood collection tubes may be relatively small to interfere with decision-making on dose prescription, therefore lack clinical importance. Correlations between the results showed that, one of these plastic blood collection tubes tested in our study, can be used interchangably for a wide variety of coagulation assays. CONFLICT OF INTEREST: None declared.

Development and inter-laboratory validation of the VISAGE enhanced tool for age estimation from semen using quantitative DNA methylation analysis.

The analysis of DNA methylation has become an established method for chronological age estimation. This has triggered interest in the forensic community to develop new methods for age estimation from biological crime scene material. Various assays are available for age estimation from somatic tissues, the majority from blood. Age prediction from semen requires different DNA methylation markers and the only assays currently developed for forensic analysis are based on SNaPshot or pyrosequencing. Here, we describe a new assay using massively parallel sequencing to analyse 13 candidate CpG sites targeted in two multiplex PCRs. The assay has been validated by five consortium laboratories of the VISible Attributes through GEnomics (VISAGE) project within a collaborative exercise and was tested for reproducible quantification of DNA methylation levels and sensitivity with DNA methylation controls. Furthermore, DNA extracts and stains on Whatman FTA cards from two semen samples were used to evaluate concordance and mimic casework samples. Overall, the assay yielded high read depths (> 1000 reads) at all 13 marker positions. The methylation values obtained indicated robust quantification with an average standard deviation of 2.8% at the expected methylation level of 50% across the 13 markers and a good performance with 50 ng DNA input into bisulfite conversion. The absolute difference of quantifications from one participating laboratory to the mean quantifications of concordance and semen stains of remaining laboratories was approximately 1%. These results demonstrated the assay to be robust and suitable for age estimation from semen in forensic investigations. In addition to the 13-marker assay, a more streamlined protocol combining only five age markers in one multiplex PCR was developed. Preliminary results showed no substantial differences in DNA methylation quantification between the two assays, indicating its applicability with the VISAGE age model for semen developed with data from the complete 13-marker tool.

Relation between the frequency of CD34⁺ bone marrow derived circulating progenitor cells and the number of diseased coronary arteries in patients with myocardial ischemia and diabetes.

BACKGROUND: Bone marrow-derived circulating progenitor cells (BM-CPCs) in patients with coronary heart disease are impaired with respect to number and mobilization. However, it is unknown whether the mobilization of BM-CPCs depends on the number of diseased coronary arteries. Therefore, in our study, we analysed the correlation between the diseased coronary arteries and the frequency of CD34/45+ BM-CPCs in peripheral blood (PB) in patients with ischemic heart disease (IHD). METHODS: The frequency of CD34/45+ BM-CPCs was measured by flow cytometry in 120 patients with coronary 1 vessel (IHD1, n = 40), coronary 2 vessel (IHD2, n = 40), coronary 3 vessel disease (IHD3, n = 40) and in a control group of healthy subjects (n = 40). There was no significant difference of the total number of cardiovascular risk factors between IHD groups, beside diabetes mellitus (DM), which was significantly higher in IHD3 group compared to IHD2 and IHD1 groups. RESULTS: The frequency of CD34/45+ BM-CPCs was significantly reduced in patients with IHD compared to the control group (CD34/45+; p < 0.001). The frequency of BM-CPCs was impaired in patients with IHD3 compared to IHD1 (CD34/45+; p < 0.001) and to IHD2 (CD34/45+; p = 0.001). But there was no significant difference in frequency of BM-CPCs between the patients with IHD2 and IHD1 (CD34/45+; p = 0.28). In a subgroup we observed a significant negative correlation between levels of hemoglobin AIc (HbAIc) and the frequency of BM-CPCs (CD34/45+; p < 0.001, r = -0.8). CONCLUSIONS: The frequency of CD34/45+ BM-CPCs in PB is impaired in patients with IHD. This impairment may augment with an increased number of diseased coronary arteries. Moreover, the frequency of CD34/45+ BM-CPCs in ischemic tissue is further impaired by diabetes in patients with IHD.

Resveratrol shows vasoprotective effect reducing oxidative stress without affecting metabolic disturbances in insulin-dependent diabetes of rabbits.

PURPOSE: Resveratrol has been shown to have vasoprotective effects by upregulating oxidative defense mechanisms in a variety of pathophysiological conditions. However, the effect of resveratrol on diabetic oxidative stress and vascular and metabolic abnormalities is not completely understood. Therefore, this study was designed to evaluate whether long-term resveratrol supplementation has a protective effect on vascular function and integrity in association with metabolic parameters and oxidative stress in insulin-dependent diabetes. METHODS: Diabetes was induced in rabbits with alloxan and maintained for 8 weeks. We used a resveratrol dose of 5 mg/L (10 weeks, starting 14 days before alloxan injection) and 50 mg/L (8 or 10 weeks, starting concomitantly or 14 days before alloxan injection) in the drinking water of rabbits. RESULTS: Relaxation to acetylcholine was impaired (control 75.6 ± 3.59%, versus diabetic 42.23 ± 2.53%) and contractions to phenylephrine increased (control 136.89 ± 2.27%, versus diabetic 159.37 ± 6.27%) in aortas from diabetic animals. These changes were associated with increased basal or NAD(P)H-induced superoxide production, as well as lipid peroxide and superoxide dismutase (SOD) levels in the aortic samples. The maximal relaxation to acetylcholine improved by 75.74 ± 9.04% in diabetic rabbits treated with resveratrol. The increased contractions to phenylephrine were not restored to control values after resveratrol treatments, but sensitivity to the contractions tended to decrease. Resveratrol increased nitrite/nitrate levels and suppressed basal or NAD(P)H-induced superoxide production and lipid peroxide levels in the aortas. Importantly, resveratrol increased serum insulin levels without affecting blood glucose and the lipid profile in diabetic rabbits. Using electron microscopic examinations, resveratrol was found to markedly protect the endothelial integrity from diabetes. CONCLUSION: Overall, there was no noticeable difference between resveratrol treatment groups on the recovery from diabetes. Our results indicate that resveratrol alleviates type 1 diabetes-induced vasculopathy by decreasing vascular oxidative stress and thereby increasing the bioavailability of nitric oxide without changing metabolic abnormalities.

Overcoming drug resistance in hormone- and drug-refractory prostate cancer cell line, PC-3 by docetaxel and gossypol combination.

Drug resistance is a significant challenge of daily oncology practice. Docetaxel and gossypol both have antitumoral activity in hormone-refractory prostate cancer (HRPC). Our results revealed that docetaxel and gossypol were synergistically cytotoxic and apoptotic in PC-3 cells in a dose- and time-dependent manner. We further investigated the expression profiles of genes involved in drug resistance and metabolism with a Human Cancer Drug Resistance and Metabolism PCR Array (SuperArray). Six of the 84 genes that are known to regulate drug resistance, metabolism, cell cycle, DNA repair and oncogenesis were downregulated >or=3-fold change by the combination treatment. These results may be important in devising mechanism-based and targeted therapeutic strategies for prostate cancer, especially in devising combination therapy for drug resistant prostate cancers.

Umbilical cord mesenchymal stromal cells engraft and transdifferentiate into cardiomyocyte-like cells following acute myocardial ischemia⋆.

OBJECTIVE: Human umbilical cord-derived mesenchymal stromal cells (hUC-MSCs) gained importance in acute/chronic ischemic cardiomyopathy because of their outstanding regenerative potential in various pathologic conditions. The present study was designed to determine to what extent hUC-MSCs contribute to myocardial regeneration in acute experimental myocardial infarction (MI) in rats. METHODS: Animals were assigned into two groups; the control group received intramyocardial PBS injections, while the hUC-MSC group received calcein-AM-labeled 8.8 × 106/kg hUC-MSCs. Three weeks following the acute MI induction, rats were sacrificed after assessing the left ventricular (LV) function using echocardiography. For the assessment of infarct size, the triphenyl tetrazolium chloride (TTC) test was used in isolated hearts. Collagen-rich scar tissue was demonstrated using Masson's trichrome staining, followed by the detection of cardiac troponin I (cTnI), α-sarcomeric actin (α-SA), von Willebrand factor (vWF), CD68 and CD206 expressions in control and cell-injected sections. RESULTS: Echocardiography revealed a significant difference (P = 0.037) in the LV ejection fraction between groups. TTC assays demonstrated a significant difference (P = 0.006) between the groups regarding the ratio of the infarcted LV area. Calcein-AM-loaded cells were identified mostly in ischemic myocardium. Transplanted cells also expressed human-specific cTnI, providing concrete proof of transdifferentiation into cardiomyocytes, and α-SA. vWF+ cells verified the neovascularization in the ischemic myocardium. Finally, a slight shift from pro-inflammatory to anti-inflammatory macrophages (CD68+/CD206+) was noted in both groups. CONCLUSIONS: We found that the intramyocardial transplanted hUC-MSCs engrafted and partially transdifferentiated into cardiomyocytes, reduced scar formation, and induced angiogenesis through the association of pro/anti-inflammatory macrophages.

Intramyocardial Transplantation of Umbilical Cord Mesenchymal Stromal Cells in Chronic Ischemic Cardiomyopathy: A Controlled, Randomized Clinical Trial (HUC-HEART Trial).

BACKGROUND AND OBJECTIVES: The HUC-HEART Trial (ClinicalTrials.gov Identifier: NCT02323477) was a controlled, prospective, phase I/II, multicenter, single-blind, three-arm randomized study of intramyocardial delivery of human umbilical cord-derived mesenchymal stromal cells (HUC-MSCs) combined with coronary artery bypass-grafting (CABG) in patients with chronic ischemic cardiomyopathy (CIC). The trial aimed to assess (i) the safety and the efficacy of cell transplantation during one-year follow-up, (ii) to compare the efficacy of HUC-MSCs with autologous bone-marrow- derived mononuclear cells (BM-MNCs) in the same clinical settings. METHODS AND RESULTS: Fifty-four patients who were randomized to receive HUC-MSCs (23×106) (n=26) or BM-MNCs (70×107) (n=12) in combination with CABG surgery. The control patients (n=16) received no cells/vehicles but CABG intervention. All patients were screened at baseline and 1, 3, 6, 12 months after transplantation. Forty-six (85%) patients completed 12 months follow-up. No short/mid-term adverse events were encountered. Decline in NT-proBNP (baseline∼ 6 months) in both cell-treated groups; an increase in left ventricular ejection fraction (LVEF) (5.4%) and stroke volume (19.7%) were noted (baseline∼6 or 12 months) only in the HUC-MSC group. Decreases were also detected in necrotic myocardium as 2.3% in the control, 4.5% in BM-MNC, and 7.7% in the HUC-MSC groups. The 6-min walking test revealed an increase in the control (14.4%) and HUC-MSC (23.1%) groups. CONCLUSIONS: Significant findings directly related to the intramyocardial delivery of HUC-MSCs justified their efficacy in CIC. Stricter patient selection criteria with precisely aligned cell dose and delivery intervals, rigorous follow-up by detailed diagnostic approaches would further help to clarify the responsiveness to the therapy.

Body fluid identification and assignment to donors using a targeted mRNA massively parallel sequencing approach - results of a second EUROFORGEN / EDNAP collaborative exercise.

In a previous EUROFORGEN/EDNAP collaborative exercise, we tested two assays for targeted mRNA massively parallel sequencing for the identification of body fluids/tissues, optimized for the Illumina MiSeq/FGx and the Ion Torrent PGM/S5 platforms, respectively. The task of the second EUROFORGEN/EDNAP collaborative exercise was to analyze dried body fluid stains with two different multiplexes, the former Illumina 33plex mRNA panel for body fluid/tissue identification and a 35plex cSNP panel for assignment of body fluids/tissues to donors that was introduced in a proof-of-concept study recently. The coding region SNPs (cSNPs) are located within the body fluid specific mRNA transcripts and represent a direct link between the body fluid and the donor. We predicted the origin of the stains using a partial least squares discriminant analysis (PLS-DA) model, where most of the single source samples were correctly predicted. The mixed body fluid stains showed poorer results, however, at least one component was predicted correctly in most stains. The cSNP data demonstrated that coding region SNPs can give valuable information on linking body fluids/tissues with donors in mixed body fluid stains. However, due to the unfavorable performance of some cSNPs, the interpretation remains challenging. As a consequence, additional markers are needed to increase the discrimination power in each body fluid/tissue category.

Regulation of growth factors in hormone- and drug-resistant prostate cancer cells by synergistic combination of docetaxel and octreotide.

OBJECTIVE To evaluate the effects of combined treatment with docetaxel and octreotide, a somatostatin analogue, on human hormone- and drug-refractory prostate cancer cell lines, PC-3 and DU-145, and on some growth factors related to tumour growth and angiogenesis in prostate cancer. MATERIALS AND METHODS A cell proliferation assay was used to assess the cytotoxicity of the drugs. To verify apoptosis, both DNA fragmentation (by enzyme-linked immunosorbent assay) and caspase 3/7 activity were measured. We also investigated the effect of combined docetaxel and octreotide on growth factors secreted from prostate cancer cells using a human growth factor antibody array. RESULTS The combination of docetaxel and octreotide resulted in significant synergistic cytotoxic activity and apoptosis, which was dose- and time-dependent. The combined treatment also resulted in significantly less secretion of stem cell factor and platelet-derived growth factor-AB in PC-3 cells, and transforming growth factor-beta and basic fibroblast growth factor in DU-145 cells, than in untreated controls. CONCLUSION Octreotide, a somatostatin analogue, combined with docetaxel might provide a rationale treatment option for hormone-refractory prostate cancer cells, not only by direct inhibition of cell proliferation but also by inhibiting the secretion of growth factors.

Alterations in cerebrospinal fluid PO(2), PCO(2), and pH measurements during and after experimental thoracic aortic cross-clamping.

In a model of aortic cross-clamping, we studied the use of a multiparameter sensor for measurement of cerebrospinal fluid (CSF) PO(2), PCO(2), and pH during and after aortic cross-clamping. The present study addressed the above-mentioned alterations and their relation according to time intervals. In 31 pigs, a sensor was introduced into the intrathecal space and epidural laser Doppler was used to measure spinal cord blood flow (SCF). By placing the aortic clamp at different levels, three different spinal cord ischemia groups were obtained (mild, moderate, and severe). CSF variables with SCF were studied for 25%, 50%, and 100% changes according to baseline level. In the clamping period, SCF decreased 71.5%, 40.0%, and 33.3% in groups 1, 2, and 3, respectively. CSF O(2) tension reached 0 in group 1, decreased 74.8% in group 2, and was 12.7% in group 3. CSF CO(2) tension increased 247.2% and 202.0% in groups 1 and 2, respectively, but slightly increased in group 3. The maximum reaction time of CSF O(2) tension was about 16.7-26.9min, although this range was 34.5-49.8min in CSF CO(2) tension. We recognized that O(2) tension reacts faster than PCO(2) and pH. It is possible for O(2) tension to be used faster than produced CO(2) in the ischemic medium, although it is known that the diffusion rate of CO(2) is much higher. Spinal cord O(2) tension monitoring is an important method to detect ischemic changes.

The protective effects of resveratrol and L-NAME on visceral organs following aortic clamping.

BACKGROUND: This study investigated the effect of temporary occlusion of the aorta on the development of ischemia-reperfusion (I/R) injury of the visceral organs, the optimal timing of administration of resveratrol, and its mechanism of protection via inhibiting nitric oxide (NO) release with an NO synthase inhibitor. METHODS: Rabbits were divided into seven groups according to the administration period of resveratrol and/or N(G)-nitro-L-arginine methyl ester (L-NAME): control group; group 1, resveratrol during ischemic period; group 2, resveratrol during reperfusion period; group 3, L-NAME during ischemic period; group 4, L-NAME during reperfusion period; group 5, resveratrol during ischemic period and L-NAME during reperfusion period; group 6, L-NAME during ischemic period and resveratrol during reperfusion period. The infrarenal aorta was clamped for 30 min. Blood samples were taken for the biochemical assessment, and organ specimens were taken for pathological assessment at 24hr of reperfusion. RESULTS: In groups 5 and 6, the renal I/R injury was comparatively milder (I/R injury score 1.04+/-0.29 in control group, 0.25+/-0.17 in group 5, and 0.33+/-0.13 in group 6 [p<0.05]). The I/R injury of bowel was milder in group 5 (I/R injury score 1.8+/-0.80 in control group vs. 0.0+/-0.0 in group 5 [p<0.05]). CONCLUSION: The protective effects of resveratrol on organs that have high metabolic rate like kidney and bowel was proven histopathologically. It may be beneficial to use different pharmacological medications in different periods of the I/R damage as they represent different characteristics with and without oxygen. The combination of resveratrol and L-NAME against I/R injury appears to be an effective option in the near future.

Collaborative therapy with nebivalol and L-NAME for spinal cord ischemia/reperfusion injury.

Postoperative neurologic deficit is the most devastating complication after thoracoabdominal aortic aneurysm repair. Our aim was to investigate whether nebivolol has protective effects during ischemia or reperfusion and the most effective mechanism of protection via inhibiting nitric oxide (NO) release with an NO synthase inhibitor in an experimental model of spinal cord ischemia/reperfusion injury. Spinal cord ischemia was induced by occlusion of the infrarenal aorta for 30 min. Thirty-one rabbits were divided into five groups according to the administration period of nebivolol and/or N(G)-nitro-L-arginine methyl ester (L-NAME): control group; group NI, nebivolol during ischemic period; group NR, nebivolol during reperfusion period; group NILR, nebivolol during ischemic period and L-NAME during reperfusion period; and group LINR, L-NAME during ischemic period and nebivolol during reperfusion period. Blood samples were taken at both ischemia and reperfusion periods to obtain nitrite/nitrate levels. After neurologic evaluation at 24 hr of reperfusion, malondialdehyde (MDA) levels were measured. Neurologic impairment was significantly lower in group LINR (Tarlov score 3.4 +/- 0.6, p < 0.05). MDA levels were lower in nebivolol-treated animals, but the lowest value was achieved in the NR group, 35.6 +/- 2.7 nmol/g (p < 0.001). Nitrite levels were decreased significantly in all nebivolol-treated animals in the reperfusion period, but the lowest value was measured in the LINR group (455 +/- 137 vs. 1,760 +/- 522 nmol/mL, p < 0.001). Prophylactic use of nebivolol reduced neurologic injury, and combining with L-NAME provided the best clinical improvement by attenuating the inflammatory mileu in this experimental model. Combination of nebivolol and L-NAME appears to be an effective option for spinal cord protection against ischemia/reperfusion injury.

Dobutamine stress echocardiography and left ventricular mass changes of mechanical aortic valve replacement in four years follow-up.

AIM: The aim of this study was to investigate the differences in cardiac response to stress according to the size of the prosthetic valve in patients who underwent aortic valve replacement (AVR) and to evaluate the relationship between the size of the prosthetic valve and cardiac recovery-remodeling after the operation. METHODS: Thirty patients who had undergone AVR (12 patients) or double valve replacement (18 patients) underwent dobutamine-stress echocardiography 4.2 years after the operation to evaluate response to stress . They were divided into 2 groups according to valve prosthesis size. The small-size AVR group (group 1, n=17) had prosthetic aortic valves 21 pounds mm; the large-size AVR group (group 2, n=13) had valves >21 mm. Response to stress and preoperative and postoperative echocardiographic findings were compared. Pulsed and continuous-wave Doppler studies were performed at rest and at the end of each stage. Peak and mean aortic gradients, left ventricular diastolic and systolic functions were measured for each group. RESULTS: Dobutamine stress increased heart rate and blood pressure in both groups. Peak pressure gradient across the aortic valve prostheses was 42.1 mm Hg in group 1 and 20.9 mm Hg in group 2 (P<0.05) at rest. After dobutamine infusion, the peak pressure gradient across the aortic valve prostheses increased to 85.1 mm Hg in group 1 and 54 mm Hg in group 2 (P<0.05). Isovolumetric relaxation time returned to normal in both groups following dobutamine infusion; this decrease was significant only in group 1. Patients achieved a decrease in left atrium and left ventricular diameters and volumes, as evidence of remodeling following AVR. Left ventricular mass index (LVMI) decreased from 127.6+/-47.6 to 98.1+/-36.9 and from 159.9+/-16.1 to 125.3+/-10.1 in groups 1 and 2, respectively, but this decline was not statistically significant. CONCLUSIONS: Smaller valves have higher gradients and this significant difference increases under stress. Significant improvement in echocardiographic diameters, cardiac filling volumes and LVMI reflects the benefit of the operation. Cardiac remodeling is independent of valve size, although high transprosthetic gradients occur during stress conditions.