Gelatinase activity imaged by activatable cell-penetrating peptides in cell-based and in vivo models of stroke.

Matrix metalloproteinases (MMPs), particularly gelatinases (MMP-2/-9), are involved in neurovascular impairment after stroke. Detection of gelatinase activity in vivo can provide insight into blood-brain barrier disruption, hemorrhage, and nerve cell injury or death. We applied gelatinase-activatable cell-penetrating peptides (ACPP) with a cleavable l-amino acid linker to examine gelatinase activity in primary neurons in culture and ischemic mouse brain in vivo We found uptake of Cy5-conjugated ACPP (ACPP-Cy5) due to gelatinase activation both in cultured neurons exposed to n-methyl-d-aspartate and in mice after cerebral ischemia. Fluorescence intensity was significantly reduced when cells or mice were treated with MMP inhibitors or when a cleavage-resistant ACPP-Cy5 was substituted. We also applied an ACPP dendrimer (ACPPD) conjugated with multiple Cy5 and/or gadolinium moieties for fluorescence and magnetic resonance imaging (MRI) in intact animals. Fluorescence analysis showed that ACPPD was detected in sub-femtomole range in ischemic tissues. Moreover, MRI and inductively coupled plasma mass spectrometry revealed that ACPPD produced quantitative measures of gelatinase activity in the ischemic region. The resulting spatial pattern of gelatinase activity and neurodegeneration were very similar. We conclude that ACPPs are capable of tracing spatiotemporal gelatinase activity in vivo, and will therefore be useful in elucidating mechanisms of gelatinase-mediated neurodegeneration after stroke.

Assessment and validation of a defined fluid restriction protocol in the use of subcutaneous desmopressin for children with inherited bleeding disorders.

INTRODUCTION: Despite the availability of subcutaneous desmopressin (1-deamino-8-d-arginine vasopressin, SC-DDAVP) as a haemostatic agent for children with mild bleeding disorders, few publications specifically address the safety or efficacy of this mode of administration. AIM: Our aim was to assess whether a defined fluid restriction protocol was effective in preventing hyponatremia in children receiving perioperative SC-DDAVP, and to document adequate biological and clinical response in this setting. METHODS: We retrospectively analysed a cohort of children with mild bleeding disorders prescribed SC-DDAVP over a 5-year period following institution of a 'two-thirds maintenance' fluid restriction protocol. RESULTS: Sixty-nine patients received SC-DDAVP following this protocol, including 15 with mild haemophilia A, 49 with von Willebrand disease (VWD) and five with platelet storage pool disorder. In patients who underwent formal preoperative assessment a complete or partial response was observed in 28/29 with type 1 VWD and 14/15 with mild haemophilia A. Perioperative SC-DDAVP provided excellent haemostasis in all patients, with no requirement for factor concentrate or blood products. Mild asymptomatic hyponatremia was detected in seven children who received multiple doses of DDAVP (lowest sodium 129 mmol L(-1) ); however, adherence to the prescribed fluid restriction protocol was questionable in six of these cases. Symptomatic hyponatremia was not observed. CONCLUSION: Subcutaneous desmopressin was well-tolerated, with no serious side-effects observed, and good biological responses in preoperative trials. A two-thirds maintenance fluid regimen was effective at preventing symptomatic hyponatremia in our cohort, and is now the standard protocol for fluid restriction post-DDAVP administration in our centre.

Olaparib monotherapy in patients with advanced relapsed ovarian cancer and a germline BRCA1/2 mutation: a multistudy analysis of response rates and safety.

BACKGROUND: The PARP inhibitor olaparib (Lynparza™) demonstrates antitumor activity in women with relapsed ovarian cancer and a germline BRCA1/2 mutation (gBRCAm). Data from olaparib monotherapy trials were used to explore the treatment effect of olaparib in patients with gBRCAm ovarian cancer who had received multiple lines of prior chemotherapy. PATIENTS AND METHODS: This analysis evaluated pooled data from two phase I trials [NCT00516373 (study 2); NCT00777582 (study 24)] and four phase II trials [NCT00494442 (study 9); NCT00628251 (study 12); NCT00679783 (study 20); NCT01078662 (study 42)] that recruited women with relapsed ovarian, fallopian tube or peritoneal cancer. All patients had a documented gBRCAm and were receiving olaparib 400 mg monotherapy twice daily (capsule formulation) at the time of relapse. Objective response rate (ORR) and duration of response (DoR) were evaluated using original patient outcomes data for patients with measurable disease at baseline. RESULTS: Of the 300 patients in the pooled population, 273 had measurable disease at baseline, of whom 205 (75%) had received ≥3 lines of prior chemotherapy. In the pooled population, the ORR was 36% [95% confidence interval (CI) 30-42] and the median DoR was 7.4 months (95% CI 5.7-9.1). The ORR among patients who had received ≥3 lines of prior chemotherapy was 31% (95% CI 25-38), with a DoR of 7.8 months (95% CI 5.6-9.5). The safety profile of olaparib was similar in patients who had received ≥3 lines of prior chemotherapy compared with the pooled population; grade ≥3 adverse events were reported in 54% and 50% of patients, respectively. CONCLUSION: Durable responses to olaparib were observed in patients with relapsed gBRCAm ovarian cancer who had received ≥3 lines of prior chemotherapy. CLINICALTRIALSGOV: NCT00516373; NCT00494442; NCT00628251; NCT00679783; NCT00777582; NCT01078662.

Pediatric transplantation: preventing thrombosis.

Due to progressive advances in surgical techniques, immunosuppressive therapies, and supportive care, outcomes from both solid organ transplantation and hematopoietic stem cell transplantation continue to improve. Thrombosis remains a challenging management issue in this context, with implications for both graft survival and long-term quality of life. Unfortunately, there remains a general paucity of pediatric-specific data regarding thrombosis incidence, risk stratification, and the safety or efficacy of preventative strategies with which to guide treatment algorithms. This review summarizes the available evidence and rationale underlying the spectrum of current practices aimed at preventing thrombosis in the transplant recipient, with a particular focus on risk factors, pathophysiology, and described antithrombotic regimens.

Serum protein profiling reveals baseline and pharmacodynamic biomarker signatures associated with clinical outcome in mCRC patients treated with chemotherapy ± cediranib.

BACKGROUND: This study evaluated soluble serum proteins as biomarkers to subset patients with metastatic colorectal cancer (mCRC) treated with chemotherapy±cediranib, a vascular endothelial growth factor (VEGF) signalling inhibitor (VEGFi). Exploring biomarkers at pre- and on-treatment may identify patient subgroups showing clinical benefit on cediranib combination. METHODS: Two hundred and seven serum proteins were analysed in 588 mCRC patients at pre- and on-treatment with chemotherapy (FOLFOX/CAPOX)±cediranib 20 mg. Patients were enrolled in the phase III trial HORIZON II. We correlated baseline biomarker signatures and pharmacodynamic (PD) biomarkers with PFS and OS. RESULTS: We identified a baseline signature (BS) of 47 biomarkers that included VEGFA, VEGFD, VEGFR2, VEGFR3 and TIE-2, which defined two distinct subgroups of patients. Patients treated with chemotherapy plus cediranib who had 'high' BS had shorter PFS (HR=1.82, P=0.003) than patients with 'low' BS. This BS did not correlate with PFS of the patients treated with chemotherapy plus placebo. In addition, we identified a profile of 16 PD proteins on treatment associated with PFS (HR=0.58, P<0.001) and OS (HR=0.52, P<0.001) in patients treated with chemotherapy plus cediranib. This PD profile did not correlate with PFS and OS in patients treated with chemotherapy plus placebo. CONCLUSIONS: Serum proteins may represent relevant biomarkers to predict the outcome of patients treated with VEGFi-based therapies. We report a BS and PD biomarkers that may identify mCRC patients showing increased benefit of combining cediranib with chemotherapy. These exploratory findings need to be validated in future prospective studies.

Measurement of U and Pu isotope ratios in hair and nail samples using extraction chromatography and multi-collector inductively coupled plasma mass spectrometry.

A bioassay capable of monitoring occupational or environmental exposure to special nuclear materials would be a useful tool for nuclear nonproliferation programs. Hair and nail are potential biomonitors of exposure to U and Pu. A method is described to measure isotope ratios of ultra-trace concentrations of U and Pu in hair and nail samples. The method uses multiple extraction chromatography resins to separate U and Pu fractions from the sample matrix. The U recovery was quantitative while the Pu recovery ranged from 81% to 109%, with a U decontamination factor of 5×10(4). Following the separation (234)U/(238)U, (235)U/(238)U and (240)Pu/(239)Pu were measured in human hair and hair and nail samples using multi-collector inductively coupled plasma mass spectrometry (MC-ICPMS). The human hair and nail samples had elevated ratios of (234)U/(238)U which could reflect exposure to naturally fractionated U.

Screen of whole blood responses to flagellin identifies TLR5 variation associated with outcome in melioidosis.

Melioidosis is a severe infection caused by the flagellated bacterium Burkholderia pseudomallei. The nonsense polymorphism TLR51174C>T is associated with improved outcome in Thais with melioidosis. We hypothesized that other TLR5 variants may modulate the host response and determine outcome in melioidosis. We genotyped 12 TLR5 variants selected de novo from the HapMap database and examined the association of each with cytokines induced by flagellin stimulation of whole blood from healthy Thai subjects. We found a blunted cytokine response for three related markers that were in linkage disequilibrium (LD) with a non-synonymous variant, TLR51846T>C. Carriers of TLR51846T>C had broadly impaired cytokine responses induced by flagellin. TLR51846T>C was associated with protection against death in melioidosis patients (odds ratio: 0.62, 95% confidence interval: 0.42-0.93, P=0.021). We observed no impairment in TLR51846C-dependent nuclear factor κB activation, however, suggesting an alternative mechanism for the effect. We found that TLR51846T>C was in strong LD with TLR51174C>T. Many of the blunted cytokine responses observed and the association of TLR51846T>C with survival in melioidosis patients may be attributable to TLR51174C>T, but we could not exclude an independent effect of TLR51846T>C. These data identify novel associations for TLR51846T>C, enhance our understanding of TLR5 genetic architecture in Thais and highlight the role of TLR5 in melioidosis.

Prognostic/predictive value of 207 serum factors in colorectal cancer treated with cediranib and/or chemotherapy.

BACKGROUND: The prognostic and predictive value of multiple serum biomarkers was evaluated using samples from a randomised phase III study (HORIZON II) investigating chemotherapy with or without cediranib in metastatic colorectal cancer (mCRC). METHODS: Baseline levels of 207 protein markers were measured in serum samples from 582 HORIZON II (FOLFOX/XELOX plus cediranib 20 mg (n=330) or placebo (n=252)) patients. Median baseline values of each biomarker were used to categorise patients as high or low. Markers were then assessed for their association with efficacy, defined by progression-free survival (PFS) and overall survival (OS). A generalised boosted regression model identified markers of particular interest. RESULTS: Correlation of protein levels with PFS and OS suggested that multiple factors had a prognostic value, independent of treatment arm, including IL-6, IL-8, C-reactive protein (CRP), ICAM-1 and carcinoembryonic antigen (CEA). Among the angiogenesis regulators, low levels of vascular endothelial growth factor (VEGF), VEGF-D, VEGFR-1, VEGFR-3, NRP1 and Tie-2 correlated with better outcome. CONCLUSION: This large data set generated using serum samples from mCRC patients treated with chemotherapy and VEGF inhibitors, defines baseline characteristics for 207 serum proteins. Multiple prognostic factors were identified that could be disease related or predict which patients derive most benefit from 5-fluorouracil (5-FU)-based chemotherapy, meriting further exploration in prospective studies.

Prognostic and predictive value of VEGF, sVEGFR-2 and CEA in mCRC studies comparing cediranib, bevacizumab and chemotherapy.

BACKGROUND: The prognostic/predictive value of potential vascular endothelial growth factor (VEGF) signalling biomarkers was evaluated retrospectively using samples from two randomized Phase III studies (HORIZON II and III) investigating cediranib in metastatic colorectal cancer (mCRC). METHODS: Baseline levels of VEGF, soluble VEGF receptor-2 (sVEGFR-2) and carcinoembryonic antigen (CEA) were measured in plasma/serum samples collected from patients participating in HORIZON II (n=860; FOLFOX/XELOX plus cediranib 20 mg (n=502) or placebo (n=358)) and HORIZON III (n=1422; mFOLFOX6 plus cediranib 20 mg (n=709) or bevacizumab (n=713)). Median biomarker baseline levels determined cutoff values for the patient subgroups. RESULTS: Baseline data were available for 88-97% of patients/study (>2000 patients). In both the studies, high baseline VEGF and CEA were associated with worse outcomes for progression-free survival (PFS) and overall survival (OS) independent of treatment (HORIZON II OS: VEGF, hazard ratio (HR)=1.35 (95% confidence interval (CI): 1.12-1.63); CEA, HR=1.63 (1.36-1.96); HORIZON III OS: VEGF, HR=1.32 (1.12-1.54); CEA, HR=1.50 (1.29-1.76)). sVEGFR-2 was not prognostic for PFS/OS. Baseline VEGF and CEA were not predictive for PFS/OS outcome to cediranib treatment; low sVEGFR-2 was associated with a trend towards improved cediranib effect in HORIZON II. CONCLUSION: Baseline VEGF and CEA levels were treatment-independent prognostic biomarkers for PFS and OS in both the studies.

Expanded phenotype-genotype correlations in a pediatric population with type 1 von Willebrand disease.

BACKGROUND: Recent phenotype-genotype studies have provided valuable insights into the pathophysiology of type 1 von Willebrand disease (VWD); however, no study has examined an exclusively pediatric cohort. OBJECTIVES: To describe phenotype-genotype correlations in a selected pediatric cohort with a historical diagnosis of type 1 VWD, using first-degree family members as controls. METHODS: Comprehensive phenotypic assessment included standard assays of von Willebrand factor (VWF) level and function, bleeding score, desmopressin response, VWF propeptide (VWFpp) level, and platelet-derived VWF mRNA level. RESULTS: Fourteen VWF mutations were identified in 17 of 23 index cases (ICs) (aged 5-17 years), including four that were previously unreported (L60P, nt1658 insT, Q1388X, and C2237F). VWFpp levels were lower in ICs than in unaffected controls (median 49 vs. 86 U dL(-1) , P < 0.0001). A VWFpp/VWF antigen ratio of > 1.6 was observed in eight of nine ICs with a suboptimal response to desmopressin, including four of four with the R1205H (Vicenza) mutation (median 7.9), and three of four IC with the R1315C mutation (median 1.9). The R1315C mutation was also associated with a reduced absolute VWFpp level (median 32 U dL(-1) ), a previously unreported finding. The amount of platelet-derived VWF mRNA was significantly reduced in individuals with nonsense mutations. CONCLUSIONS: Increased VWF clearance and intracellular retention are important mechanisms underlying type 1 VWD in pediatric patients, concordant with the observations of larger, predominantly adult, cohort studies. Additionally, in some patients, nonsense-mediated decay of mutant mRNA transcripts may be contributory. Several mechanisms underlie the variable phenotype associated with the R1315C mutation. The potential utility of VWFpp as an independent marker of VWF biosynthesis and release warrants further research.

Evaluation of satraplatin in dogs with spontaneously occurring malignant tumors.

BACKGROUND: Satraplatin is the 1st orally bioavailable platinum anticancer drug. OBJECTIVE: Our objectives were to evaluate efficacy in vitro against a canine cancer cell line, to determine the maximally tolerated dose (MTD) of satraplatin in tumor-bearing dogs, to identify the dose-limiting and other toxicities in dogs, and to record pharmacokinetics (PK). ANIMALS: Dogs with macro- or microscopic malignant neoplasia. METHODS: D17 canine osteosarcoma cells first were evaluated in a clonogenic survival assay. Then, dogs with a diagnosis of malignant neoplasia were prospectively entered in standard 3 + 3 cohorts. Additional patients were entered at the MTD to assess efficacy. Total and free platinum (by ultrafiltrate) concentrations were determined with inductively coupled plasma mass spectroscopy. RESULTS: Satraplatin inhibited clonogenic survival in vitro at clinically relevant and achievable concentrations. Twenty-three dogs were treated, 14 with PK evaluation. The MTD was 35 mg/m(2)/d for 5 days, repeated every 3-4 weeks. Bioavailability was 41%. PK variables (mean ± SD) at the MTD included T(max) 1.8 (± 0.7) hours, C(max) 72 (± 26) ng/mL, area under concentration (AUC)(0-24 h) 316 (± 63) h × ng/mL, and MRT 7 (± 1.3) hours. Higher AUC after the 5th versus the 1st dose suggested drug accumulation. Interestingly, platelets consistently reached nadir sooner than did neutrophils (day 14 versus 19). Myelosuppression was dose-limiting and gastrointestinal toxicity was mild. CONCLUSIONS AND CLINICAL IMPORTANCE: Satraplatin was well tolerated in tumor-bearing dogs, thus warranting further investigation in a phase II trial.

Efficiency of Pm-147 direct charge radioisotope battery.

A theoretical analysis is presented here of the efficiency of direct charge radioisotope batteries based on the efficiency of the radioactive source, the system geometry, electrostatic repulsion of beta particles from the collector, the secondary electron emission, and backscattered beta particles from the collector. Efficiency of various design batteries using Pm-147 sources was experimentally measured and found to be in good agreement with calculations. The present approach can be used for predicting the efficiency for different designs of direct charge radioisotope batteries.

Review of chromium (VI) apoptosis, cell-cycle-arrest, and carcinogenesis.

Hexavalent chromium combines with glutathione in chloride intracellular channel carrier to form tetravalent and pentavalent chromium in plasma and organelle membranes. It also combines with NADH/NADPH to form pentavalent chromium in mitochondria. Tetravalent- and pentavalent- chromium (directly and indirectly) mediated DNA double strand breaks activate DNA damage signaling sensors: DNA-dependent-protein-kinase signals p53-dependent intrinsic mitochondrial apoptosis, and ataxia-telangiectasia-mutated and ataxia-telangiectasia-Rad3-related signal cell-arrest for DNA repair. Tetravalent chromium may be the most potent species since it causes DNA breaks and somatic recombination, but not apoptosis. Upon further failure of apoptosis and senescence/DNA-repair, damaged cells may become immortal with loss-of-heterozygosity and genetic plasticity.

Use of species other than oak to flavor wine: an exploratory survey.

The concept of geographical exclusivity prompted an exploratory survey with toasted woods other than oak to flavor wine. A total of 11 woods and oak (Quercus alba) were cut into chips 20 × 10 × 2.5 mm and toasted at 200 °C for 2 h (light toast) or 210 °C for 3 h (dark). Weight losses and changes in CIE color space were monitored. The toasted chips were infused in unoaked chardonnay (5 g L(-1)) for 2 wk. Parallel infusions were done with model wine (water, ethanol, tartaric acid) adjusted to pH 3.5. Ultraviolet absorbances due to infusion were recorded on the basis that that lignin compounds and their potential pyrolysates are based on phenolic structures that absorb in the ultraviolet range. Weight losses on light and dark toasting were highly variable between species as were color changes, suggesting potential for different flavor outcomes from chemical changes. Ultraviolet absorbance curves were also highly variable showing that different species yielded different quantities of potentially flavor-active phenolic compounds in real and model wine. More absorbing matter was extracted from the light toast treatments, and light toast oak, which demonstrated the greatest weight loss on light toasting, yielded the widest range of ultraviolet-absorbing matter. In an informal sensory trial with the 24 species/toast combinations infused in chardonnay all but one wood, Cupressus macrocarpa, resulted in flavors reminiscent of oaked wines. A hedonic consumer trial with 4 species and oak compared with uninfused chardonnay showed that each of the 4 had potential as a flavorant. Thus, woods unsuited to barrel construction could provide unrealized flavor opportunities in the wine industry, and could extend to flavoring spirits.

Promethium-147 capacitor.

Beta particle surface fluxes for tritium, Ni-63, Pm-147, and Sr-90 sources were calculated in this work. High current density was experimentally achieved from Pm-147 oxide in silica-titana glass. A 96 GBq (2.6 Ci) Pm-147 4pi-source with flux efficiency greater than 50% was used for constructing a direct charge capacitor with a polyimide coated collector and vacuum as electrical insulation. The capacitor connected to high resistance (TOmega) loads produced up to 35 kV. Overall conversion efficiency was over 10% (on optimal load).

Analysis of k0 neutron activation analysis at the University of Missouri Research Reactor.

k(0) Instrumental Neutron Activation Analysis (k(0) INAA) was evaluated at the University of Missouri Research Reactor (MURR). The MURR, unlike many other research reactors that employ k(0) INAA, is refueled on a weekly basis. To determine if the neutron spectrum is stable enough for routine k(0) INAA analysis, the neutron spectrum parameters alpha and f were measured over a 9 month period. The average values of f and alpha measured using the Cd ratio multi monitor technique were 57.4+/-4.5 and 0.039+/-0.012, respectively. It was determined that k(0) INAA could be used at the MURR by employing the average value of alpha and f. However, significant measurement bias could occur for elements with high Q(0) values if the true spectral parameters deviate by more than 10% from the average value.

Autologous peripheral blood stem cell transplantation in first remission adult acute myeloid leukaemia--an intention to treat analysis and comparison of outcome using a predictive model based on the MRC AML10 cohort.

The role of autologous peripheral blood stem cell transplantation (APBSCT) in acute myeloid leukaemia (AML) remains controversial. The current study evaluated the application of APBSCT in a large consecutive series of patients with untreated AML, and compared outcome with a predictive model based on MRC AML10 data. Of 148 evaluable patients, 118 patients entered complete remission (CR) after induction therapy comprising three cycles of daunorubicin, cytosine arabinoside and oral 6-thioguanine. Of these patients, 68 (57%) proceeded to consolidation therapy with two courses of intermediate dose cytosine arabinoside, and stem cell mobilisation, and 40 of these patients (34%) underwent the APBSCT procedure after high dose busulphan conditioning. Harvest quality was the main factor precluding APBSCT. Five-year event-free survival (EFS) in patients who achieved CR was 38% and in APBSCT patients was 57%. There were no transplant-related deaths. No significant differences were demonstrated between observed and expected outcomes at 1 and 2 years, based on the predictive model derived from the MRC AML10 study. These data therefore indicate that only a third of eligible adult patients will undergo APBSCT. However, the results demonstrate favourable survival in such patients, with no transplant-related mortality.

Long-term stability of a coronary stent coating post-implantation.

A coronary stent possessing a phosphorylcholine-based polymer coating was removed from a human patient 6 months after implantation and analyzed for the presence of the coating. An atomic force microscopy (AFM) technique has been employed to scrape away several 10- micro m(2) areas on the struts of the explanted stent. Scanning-electron microscopy (SEM) and tapping-mode AFM confirmed a surface coating had been removed in each case. Cross-sectional analysis and force-of-removal measurements showed that both coating depth and hardness were characteristic of that for the phosphorylcholine- (PC-) based coating prior to implantation. AFM amplitude-phase and distance curves from the explanted stent were comparable to those obtained when an unused stent was analyzed. Furthermore, laser ablation high-resolution inductively coupled-plasma mass spectometery (LA-HR-ICP-MS) was used to detect the low level of silicon present in the PC coating after explantation. The results from these techniques confirm that the stent coating is the original PC polymer and is not of biological origin, and support the long-term stability of the coating in vivo.