Ollivier Hyrien

Plasma tissue factor may be predictive of venous thromboembolism in pancreatic cancer

Pancreatic cancer is the fourth leading cause of cancer death in the US [1]. It is frequently complicated by venous thromboembolism (VTE), with published incidence rates varying from 17% to 57% [2]. VTE can directly contribute to mortality, and has been associated with poor outcomes in pancreatic cancer [3]. Tissue factor (TF) is the principal physiologic initiator of coagulation and is commonly expressed in a variety of cancers. In pancreatic cancer, high grade TF expression is an adverse prognostic factor [4]. We recently published findings showing that TF expression occurs early in malignant transformation of the pancreas, is associated with angiogenesis and may be predictive of subsequent VTE [5]. Patients with high-grade TF expression had a symptomatic VTE rate of 26.3% compared with 4.5% in patients with low expression (P = 0.04). Similar findings have been reported in ovarian cancer [6]. Patients with metastatic pancreatic cancer have been shown to have increased levels of microparticle (MP)-associated TF activity compared with healthy controls [7]. We hypothesized that elevated circulating levels of TF would be associated with development of VTE in pancreatic cancer. We, therefore, measured TF in samples obtained from patients with locally advanced or metastatic pancreatic cancer enrolled in a prospective trial conducted by the University of Rochester Community Clinical Oncology Program. Selected sites participating in the trial also offered participation in a laboratory companion study from 2 October 2002 to 29 December 2006. The study was approved by an Institutional Review Board. All patients provided written informed consent. All patients were randomized to single-agent gemcitabine alone or with dalteparin 5000 anti-Xa units until progression. Blood collection was planned at baseline (prior to initiating chemotherapy) and every 4 weeks while on the study. Citrated plasma was processed within 1 h of collection by centrifuging at 2000 × g for 15 min at 8 °C to produce platelet-poor plasma (PPP; <10 000 platelets μL−1) and then stored at −80 °C. TF antigen (Ag) levels were determined by an in-house ELISA from citrated plasma stored at −80 °C. Briefly, 100 μL of the mouse anti-human TF antibody, hTF1 (5 μg mL−1 in PBS), was coated onto 96-well ELISA plates (Nunc No. 439454) by overnight incubation at 4 °C. All subsequent washes and incubations were done at 25 °C. Plates were washed with PBS-Tween 20 and blocked with casein (Vector Labs No. SP-5020) for 15 min. Stored plasma samples were thawed, mixed, centrifuged at 350 × g for 1 min, and diluted 1:1 in casein. Serial dilutions of a recombinant soluble TF1–219 (sTF) standard were added to the plates in duplicate. One hundred microliters of the diluted plasma samples were added to the plates in triplicate, incubated at room temperature for 2 h and then washed. One hundred microliters of an sTF immuno-purified, biotinylated, F(ab′)2-fragmented rabbit anti-sTF anti-body (0.12 μg mL−1 in casein) was added to each well. The plates were incubated for 2 h and then washed. Streptavidin (Pierce No. 21126) (Pierce, Rockford, IL, USA) was diluted 1:10 000 and 100 μL was added to each well. The plates were incubated at room temperature for 30 min and then washed. One hundred microliters of the 1-Step Ultra TMB-ELISA reagent (Pierce No. 34028) was added to each well. The plates were incubated for 8 min and the reaction was stopped with 100 μL of 2 M sulfuric acid before reading the optical density (OD) at 450 nm in a 96-well reader (Spectramax190, Molecular Devices, Sunnyvale, CA, USA) from Molecular Devices using SoftmaxPro software (Molecular Devices, Sunnyvale, CA). TF concentrations were determined from a standard plot of the sTF protein concentrations and reported as pg mL−1 of sTF. To determine a normal level for TF Ag, we measured TF in plasma from 15 healthy volunteers and from a commercially available pooled plasma control (Innovative Research, Inc., Southfield, MI, USA) repeated six times in separate assays. Mean plasma TF was 27.0 pg mL−1 (SD 12.9) in healthy volunteers, and 26.9 pg mL−1 (SD 3.94) in pooled plasma control, with an interassay coefficient of variability (CV) of 14.7%. TF was also measured using a procoagulant activity (PCA) assay in a similar manner to a recent study [7]. MPs were pelleted from 200 μL of PPP by centrifugation at 20 000 × g for 15 min at 4 °C, washed twice with HBSA (137 mM NaCl, 5.38 mM KCl, 5.55 mM glucose, 10 mM HEPES, 0.1% bovine serum albumin, pH 7.5), and re-suspended in 100 μL of HBSA. Samples were incubated with either hTF1 (4 μg mL−1; 1 μL) or a control antibody (mouse IgG: 4 μg mL−1; 1 μL) for 15 min at 25 °C and then 50 μL aliquots were added to duplicate wells of a 96-well plate. Next, 50 μL of HBSA containing 10 nM FVIIa, 300 nM FX and 10 mM CaCl2 was added to each sample and the mixture incubated for 2 h at 37 °C. FXa generation was stopped by the addition of 25 μL of 25 mM EDTA buffer and 25 μL of the chromogenic substrate S2765 (4 mM) was added and incubated at 37 °C for 15 min. Finally, absorbance at 405 nm was measured using a VERSAmax microplate reader (Molecular Devices). TF activity was calculated by reference to a standard curve generated using relipidated recombinant human TF (0–55 pg mL−1). The TF-dependent FXa generation (pg mL−1) was determined by subtracting the amount of FXa generated in the presence of hTF1 from the amount of FXa generated in the presence of the control antibody. Mean TF PCA in healthy controls was 0.21 pg mL−1 (SD 0.11) with an interassay CV of 21%. Eleven patients provided at least one blood specimen (range, 1–8; median five samples per patient). TF Ag was measured in all 11 patients and TF MP PCA in 10 patients with available specimens. The mean TF Ag level (± SD) for all timepoints was 30.9 pg mL−1 (±15.4) and mean TF PCA was 0.85 pg mL−1 (±1.26). Mean TF Ag levels and MP-PCA at baseline were 56.6 and 0.95 pg mL−1, respectively. However, there was a wide variation, with baseline TF Ag levels ranging from 0 to 269 pg mL−1 (Fig. 1A) and TF MP PCA from 0 to 3.1 pg mL−1. Nine patients had levels of plasma TF Ag (32 ± 19 pg mL−1 SD) that were similar to those seen in healthy volunteers although TF MP PCA was elevated in these patients compared with controls (0.46 ± 1.26 pg mL−1, P = 0.01). In these patients, both TF levels and PCA remained stable throughout treatment. None of these patients developed VTE. Four of these nine patients had been randomized to dalteparin. In contrast, patient 5, randomized to dalteparin, had a rapid increase in TF Ag and PCA after visit 2, and patient 10 had TF Ag and MP PCA that were high at baseline and increased progressively through 3 months of chemotherapy (Fig. 1A). Both of these patients developed VTE. Patient 5 developed an occlusive popliteal deep venous thrombosis (DVT), confirmed by ultrasound, 15 days after discontinuing both gemcitabine and dalteparin due to progression of cancer. The patient subsequently presented 12 days later with sudden-onset shortness of breath while on anticoagulation with warfarin, was admitted and died during the hospital admission. An autopsy confirmed the presence of a massive pulmonary embolism (PE). The last TF Ag and MP PCA for patient 5, taken 15 days prior to the initial episode of DVT, were 377 and 4.4 pg mL−1, respectively. Patient 10, who was randomized to no prophylaxis, developed an ultrasound-confirmed peroneal vein DVT 56 days after starting chemotherapy. The patient then had extension into the femoral vein, confirmed by ultrasound, 26 days later while on therapeutic anticoagulation with warfarin. The last TF Ag and MP PCA for patient 10 taken on the day of diagnosis of DVT were 504 and 5.5 pg mL−1, respectively. When evaluating the entire study population, there was a significant association between TF Ag levels (P = 0.036) as well as TF MP PCA levels and development of VTE (P = 0.044). There was also a significant correlation between TF Ag and MP PCA assays (linear correlation coefficient 0.89, P < 0.0001; Fig. 1B). Fig. 1 (A) Serial plasma TF Ag levels in 11 patients with locally advanced or metastatic pancreatic cancer receiving systemic chemotherapy. Patients 5 and 10, marked with an asterisk, developed VTE. TF MP PCA levels for these two patients are displayed as dashed ... This analysis suggests a role for TF in the pathogenesis of VTE in pancreatic cancer. Furthermore, these data suggest that a rise in plasma TF measured either by TF Ag or MP PCA during the course of chemotherapy may be predictive of subsequent symptomatic VTE events in patients with pancreatic cancer. The source of circulating TF is an area of controversy. Studies have identified TF-containing microparticles that also possess endothelial cell-, platelet- and macrophage-derived surface antigens [8–10]. In patients with cancer, tumor cells may be a potential source of TF as well. The findings reported here are in agreement with previous reports, including ours, suggesting an association between TF expression by tumor cells and subsequent VTE in pancreatic and ovarian cancer [5,6]. Although our findings are of statistical significance, given the small sample size and small number of events, we consider this to be a preliminary report requiring confirmation. This study was also inadequately powered to evaluate the impact of thromboprophylaxis and response to chemotherapy. Our findings suggest, however, that future prospective studies should investigate the potential role of TF as a biomarker for VTE in pancreatic and other cancers known to have high levels of TF expression by tumor cells. Both TF Ag and MP PCA assays deserve further investigation in this regard.

The Spectrum of Neurobehavioral Sequelae after Repetitive Mild Traumatic Brain Injury: A Novel Mouse Model of Chronic Traumatic Encephalopathy

There has been an increased focus on the neurological sequelae of repetitive mild traumatic brain injury (TBI), particularly neurodegenerative syndromes, such as chronic traumatic encephalopathy (CTE); however, no animal model exists that captures the behavioral spectrum of this phenomenon. We sought to develop an animal model of CTE. Our novel model is a modification and fusion of two of the most popular models of TBI and allows for controlled closed-head impacts to unanesthetized mice. Two-hundred and eighty 12-week-old mice were divided into control, single mild TBI (mTBI), and repetitive mTBI groups. Repetitive mTBI mice received six concussive impacts daily for 7 days. Behavior was assessed at various time points. Neurological Severity Score (NSS) was computed and vestibulomotor function tested with the wire grip test (WGT). Cognitive function was assessed with the Morris water maze (MWM), anxiety/risk-taking behavior with the elevated plus maze, and depression-like behavior with the forced swim/tail suspension tests. Sleep electroencephalogram/electromyography studies were performed at 1 month. NSS was elevated, compared to controls, in both TBI groups and improved over time. Repetitive mTBI mice demonstrated transient vestibulomotor deficits on WGT. Repetitive mTBI mice also demonstrated deficits in MWM testing. Both mTBI groups demonstrated increased anxiety at 2 weeks, but repetitive mTBI mice developed increased risk-taking behaviors at 1 month that persist at 6 months. Repetitive mTBI mice exhibit depression-like behavior at 1 month. Both groups demonstrate sleep disturbances. We describe the neurological sequelae of repetitive mTBI in a novel mouse model, which resemble several of the neuropsychiatric behaviors observed clinically in patients sustaining repetitive mild head injury.

Integration of mutation and chromosomal damage endpoints into 28-day repeat dose toxicology studies.

Two endpoints of genetic toxicity, mutation at the X-linked Pig-a gene and chromosomal damage in the form of micronucleated reticulocytes (MN-RETs), were evaluated in blood samples obtained from 28-day repeat-dosing studies typical of those employed in toxicity evaluations. Male Wistar Han rats were treated at 24-h intervals on days 1 through 28 with one of five prototypical genotoxicants: N-ethyl-N-nitrosourea, 7,12-dimethyl-12-benz[a]anthracene, 4-nitroquinoline-1-oxide (4NQO), benzo(a)pyrene, and N-methyl-N-nitrosourea. Flow cytometric scoring of CD59-negative erythrocytes (indicative of glycosylphosphatidylinositol anchor deficiency and hence Pig-a mutation) was performed using blood specimens obtained on days -1, 15, 29, and 56. Blood specimens collected on days 4 and 29 were evaluated for MN-RET frequency using flow cytometry-based MicroFlow Kits. With the exception of 4NQO, each chemical induced significant increases in the frequency of MN-RETs on days 4 and 29. All five agents increased the frequency of mutant phenotype (CD59 negative) reticulocytes (RETs) and erythrocytes. Mutation responses in RETs occurred earlier than in erythrocytes and tended to peak, or nearly peak, at day 29. In contrast, the mutant phenotype erythrocyte responses were modest on day 29 and required additional time to reach their maximal value. The observed kinetics were expected based on the known turnover of RETs and erythrocytes. The data show that RETs can serve as an appropriate indicator cell population for 28-day studies. Collectively, these data suggest that blood-based genotoxicity endpoints can be effectively incorporated into routine toxicology studies, a strategy that would reduce animal usage while providing valuable genetic toxicity information within the context of other toxicological endpoints.

The pathophysiology underlying repetitive mild traumatic brain injury in a novel mouse model of chronic traumatic encephalopathy.

Background: An animal model of chronic traumatic encephalopathy (CTE) is essential for further understanding the pathophysiological link between repetitive head injury and the development of chronic neurodegenerative disease. We previously described a model of repetitive mild traumatic brain injury (mTBI) in mice that encapsulates the neurobehavioral spectrum characteristic of patients with CTE. We aimed to study the pathophysiological mechanisms underlying this animal model. Methods: Our previously described model allows for controlled, closed head impacts to unanesthetized mice. Briefly, 12-week-old mice were divided into three groups: Control, single, and repetitive mTBI. Repetitive mTBI mice received six concussive impacts daily, for 7 days. Mice were then subsequently sacrificed for macro- and micro-histopathologic analysis at 7 days, 1 month, and 6 months after the last TBI received. Brain sections were immunostained for glial fibrillary acidic protein (GFAP) for astrocytes, CD68 for activated microglia, and AT8 for phosphorylated tau protein. Results: Brains from single and repetitive mTBI mice lacked macroscopic tissue damage at all time-points. Single mTBI resulted in an acute rea ctive astrocytosis at 7 days and increased phospho-tau immunoreactivity that was present acutely and at 1 month, but was not persistent at 6 months. Repetitive mTBI resulted in a more marked neuroinflammatory response, with persistent and widespread astrogliosis and microglial activation, as well as significantly elevated phospho-tau immunoreactivity to 6-months. Conclusions: The neuropathological findings in this new model of repetitive mTBI resemble some of the histopathological hallmarks of CTE, including increased astrogliosis, microglial activation, and hyperphosphorylated tau protein accumulation.

Late Residual γ-H2AX Foci In Murine Skin are Dose Responsive and Predict Radiosensitivity In Vivo

Abstract Accurate biodosimetry is needed to estimate radiation doses received in vivo from accidental or unwarranted radiation exposures. We investigated the use of DNA repair foci (e.g. γ-H2AX) at late times after irradiation in vivo as a biodosimeter of initial ionizing radiation dose. Two radiosensitive strains (SCID and BALB/c) and two radioresistant strains (C57BL/6 and C3H/HeJ) were used to quantify γ-H2AX foci in a skin tissue microarray after doses of 1 to 10 Gy at early and late times after irradiation (1 and 7 days). Using a 3D quantitative immunofluorescence microscopy analysis, we observed a dose response for γ-H2AX foci for all strains at 30 min, 24 h and 7 days after irradiation. The numbers of residual foci were significantly different between each of the four strains and reflected the relative radiosensitivity in vivo. In comparing γ-H2AX focus and micronucleus formation after irradiation, we also observed association between the number of micronuclei and number of foci after 1 and 7 days between radiosensitive and radioresistant strains. We conclude that 3D image analysis of γ-H2AX in skin can be used to detect relative radiosensitivity based on late residual γ-H2AX foci. This technique may be a useful biodosimeter to determine dose at times up to 1 week after accidental or catastrophic radiation exposure in vivo.

Human Follicular Lymphoma CD39+-Infiltrating T Cells Contribute to Adenosine-Mediated T Cell Hyporesponsiveness

Our previous work has demonstrated that human follicular lymphoma (FL) infiltrating T cells are anergic, in part due to suppression by regulatory T cells. In this study, we identify pericellular adenosine, interacting with T cell-associated G protein-coupled A2A/B adenosine receptors (AR), as contributing to FL T cell hyporesponsiveness. In a subset of FL patient samples, treatment of lymph node mononuclear cells (LNMC) with specific A2A/B AR antagonists results in an increase in IFN-γ or IL-2 secretion upon anti-CD3/CD28 Ab stimulation, as compared with that seen without inhibitors. In contrast, treatment with an A1 AR antagonist had no effect on cytokine secretion. As the rate limiting step for adenosine generation from pericellular ATP is the ecto-ATPase CD39, we next show that inhibition of CD39 activity using the inhibitor ARL 67156 partially overcomes T cell hyporesponsiveness in a subset of patient samples. Phenotypic characterization of LNMC demonstrates populations of CD39-expressing CD4+ and CD8+ T cells, which are overrepresented in FL as compared with that seen in normal or reactive nodes, or normal peripheral blood. Thirty percent of the FL CD4+CD39+ T cells coexpress CD25high and FOXP3 (consistent with regulatory T cells). Finally, FL or normal LNMC hydrolyze ATP in vitro, in a dose- and time-dependent fashion, with the rate of ATP consumption being associated with the degree of CD39+ T cell infiltration. Together, these results support the finding that the ATP-ectonucleotidase-adenosine system mediates T cell anergy in a human tumor. In addition, these studies suggest that the A2A/B AR as well as CD39 are novel pharmacological targets for augmenting cancer immunotherapy.

Comparative in vitro and in vivo assessment of genotoxic effects of etoposide and chlorothalonil by the comet assay

The alkaline single cell gel electrophoresis (comet) assay was used to assess in vitro and in vivo genotoxicity of etoposide, a topoisomerase II inhibitor known to induce DNA strand breaks, and chlorothalonil, a fungicide widely used in agriculture. For in vivo studies, rats were sacrificed at various times after treatment and the induction of DNA strand breaks was assessed in whole blood, bone marrow, thymus, liver, kidney cortex and in the distal part of the intestine. One hour after injection, etoposide induced DNA damage in all organs studied except kidney, especially in bone marrow, thymus (presence of HDC) and whole blood. As observed during in vitro comet assay on Chinese hamster ovary (CHO) cells, dose- and time-dependent DNA effects occurred in vivo with a complete disappearance of damage 24 h after administration. Even though apoptotic cells were detected in vitro 48 h after cell exposure to etoposide, such a result was not found in vivo. After chlorothalonil treatment, no DNA strand breaks were observed in rat organs whereas a clear dose-related DNA damage was observed in vitro. The discrepancy between in vivo and in vitro models could be explained by metabolic and mechanistic reasons. Our results show that the in vivo comet assay is able to detect the target organs of etoposide and suggest that chlorothalonil is devoid of appreciable in vivo genotoxic activity under the protocol used.

Phase II study of gemcitabine, oxaliplatin in combination with panitumumab in KRAS wild-type unresectable or metastatic biliary tract and gallbladder cancer

Background:Current data suggest that platinum-based combination therapy is the standard first-line treatment for biliary tract cancer. EGFR inhibition has proven beneficial across a number of gastrointestinal malignancies; and has shown specific advantages among KRAS wild-type genetic subtypes of colon cancer. We report the combination of panitumumab with gemcitabine (GEM) and oxaliplatin (OX) as first-line therapy for KRAS wild-type biliary tract cancer.Methods:Patients with histologically confirmed, previously untreated, unresectable or metastatic KRAS wild-type biliary tract or gallbladder adenocarcinoma with ECOG performance status 0–2 were treated with panitumumab 6 mg kg−1, GEM 1000 mg m−2 (10 mg m−2 min−1) and OX 85 mg m−2 on days 1 and 15 of each 28-day cycle. The primary objective was to determine the objective response rate by RECIST criteria v.1.1. Secondary objectives were to evaluate toxicity, progression-free survival (PFS), and overall survival.Results:Thirty-one patients received at least one cycle of treatment across three institutions, 28 had measurable disease. Response rate was 45% and disease control rate was 90%. Median PFS was 10.6 months (95% CI 5–24 months) and median overall survival 20.3 months (95% CI 9–25 months). The most common grade 3/4 adverse events were anaemia 26%, leukopenia 23%, fatigue 23%, neuropathy 16% and rash 10%.Conclusions:The combination of gemcitabine, oxaliplatin and panitumumab in KRAS wild type metastatic biliary tract cancer showed encouraging efficacy, additional efforts of genetic stratification and targeted therapy is warranted in biliary tract cancer.

Modulation of Single-Cell IgG Secretion Frequency and Rates in Human Memory B Cells by CpG DNA, CD40L, IL-21 and Cell Division

During the recall response by CD27+ IgG class-switched human memory B cells, total IgG secreted is a function of the following: 1) the number of IgG-secreting cells (IgG-SC), and 2) the secretion rate of each cell. In this study, we report the quantitative ELISPOT method for simultaneous estimation of single-cell IgG secretion rates and secreting cell frequencies in human B cell populations. We found that CD27+ IgM− memory B cells activated with CpG and cytokines had considerable heterogeneity in the IgG secretion rates, with two major secretion rate subpopulations. BCR cross-linking reduced the frequency of cells with high per-cell IgG secretion rates, with a parallel decrease in CD27high B cell blasts. Increased cell death may account for the BCR-stimulated reduction in high-rate IgG-SC CD27high B cell blasts. In contrast, the addition of IL-21 to CD40L plus IL-4-activated human memory B cells induced a high-rate IgG-SC population in B cells with otherwise low per-cell IgG secretion rates. The profiles of human B cell IgG secretion rates followed the same biphasic distribution and range irrespective of division class. This, along with the presence of non-IgG-producing, dividing B cells in CpG plus cytokine-activated B memory B cell populations, is suggestive of an on/off switch regulating IgG secretion. Finally, these data support a mixture model of IgG secretion in which IgG secreted over time is modulated by the frequency of IgG-SC and the distribution of their IgG secretion rates.

Improving the Detection of Subtle IKr-Inhibition : Assessing Electrocardiographic Abnormalities of Repolarization Induced by Moxifloxacin

AbstractBackground: QT prolongation is an incomplete measure of drug-induced changes in repolarization. In this study, we investigated a novel, automatic ECG technique for describing ventricular repolarization morphology and we compared these results to corrected QT (QTc) prolongation for identifying ECGs of healthy individuals on moxifloxacin. Methods: We analysed data from the US FDA ECG Warehouse involving 160 standard ECGs from 40 healthy subjects enrolled in a randomized, parallel, placebo-controlled, ‘thorough QT’ study. Computerized ECG analysis included a series of scalar and vectorial parameters describing duration of repolarization segments and T-wave/loop morphology including its symmetry, amplitude and shape. Binary logistic models for the identification of moxifloxacin-induced abnormalities of the repolarization were developed. Results: Moxifloxacin induced significant changes in several ECG parameters including QT and QT apex and early repolarization duration (ERD)30% T-wave amplitude and slopes of the ascending and descending arm of the T-wave. The logistic model based only on T-wave morphology parameters outperformed the model based on QTc interval for identifying the presence of moxifloxacin. Combining information about repolarization interval duration with T-wave morphology significantly improved the detection of presence of moxifloxacin (p < 0.01). The increased sensitivity of our novel ECG method contributes to a >40% reduction in the sample size required to detect significant QTc prolongation induced by moxifloxacin. Conclusions: Repolarization morphology is significantly altered by moxifloxacin. The computerized ECG technique provides a novel method for quantifying morphological changes of repolarization segment. Our new parameters reflecting the morphology of the T-wave outperformed QTc measurements when identifying moxifloxacin-induced blockade of the outward rapid components of the delayed rectifier repolarizing potassium current (Ikr). These data indicate that the analysis of T-wave morphology could play a role in the assessment of drug toxicity.