bluuebunny/crossref_metadata_2025 huggingface dataset:
Phase II Study of First-Line Sequential Chemotherapy with Gemcitabine-Carboplatin Followed by Docetaxel in Patients with Advanced Non-Small Cell Lung Cancer
Authors: Chiappori Alberto , Simon George , Williams Charles , Haura Eric , Rocha-Lima Caio , Wagner Henry , Bepler Gerold , Antonia Scott
Published: July 2005

Rationale: Despite the use of novel chemotherapeutic agents, patients with advanced non-small cell lung cancer (NSCLC) continue to show a poor survival. Objectives: To assess the safety and efficacy of a novel sequential and putatively non-cross-resistant chemotherapy regimen. Methods: Eligibility included: stages IV and IIIB (malignant pleural effusion), performance status 0–1, and adequate renal, hepatic and bone marrow function. Patients with previously treated and controlled brain metastases were not excluded. Responses were determined according to the Response Evaluation Criteria in Solid Tumors. Treatment consisted of gemcitabine, 1,000 mg/m2, on days 1 and 8, and carboplatin, AUC = 5, on day 1 every 4 weeks (2–4 cycles) followed by docetaxel, 75 mg/m2, on day 1 every 3 weeks (4 cycles). Docetaxel was given after four cycles of gemcitabine-carboplatin or if progression of disease occurred, after the first two cycles. Results: Forty patients were enrolled. All patients received at least one cycle of gemcitabine-carboplatin. Due to PD, 15 patients received fewer than four cycles and only 1 received docetaxel subsequently. Of the 25 patients who completed four cycles of gemcitabine-carboplatin, 23 received docetaxel.In total, 24 patients received at least one cycle of docetaxel, and 12 patients completed four cycles of both regimens. The overall response rate was 23.6% (9/38 patients, 95% confidence interval, CI, 11–40%), with 15.8% (6/38 patients, 95% CI, 6–31%) and 12.5% (3/24 patients, 95% CI, 3–32%) response rates to gemcitabine-carboplatin and docetaxel, respectively. No patient with PD on gemcitabine-carboplatin responded to docetaxel. Toxicities were tolerable and mostly hematologic. Median survival time and progression-free survival were 6.7 and 4.9 months, respectively, with a 1-year survival of 37.5%. Conclusion: Sequential gemcitabine-carboplatin and docetaxel can be safely administered in advanced NSCLC. Our results are comparable to those achieved with other similar regimens and do not represent a significant improvement in the treatment of advanced NSCLC.
Biweekly Docetaxel-Irinotecan Treatment with Filgrastim Support Is Highly Active in Antracycline-Paclitaxel-Refractory Breast Cancer Patients
Authors: Frasci Giuseppe , D’Aiuto Giuseppe , Thomas Renato , Comella Pasquale , Di Bonito Maurizio , Lapenta Liliana , D’Aiuto Massimiliano , Botti Gerardo , Vallone Paolo , De Rosa Vincenzo , D’Aniello Roberta , Giordano Renato , Comella Giuseppe
Published: July 2005

Purpose: To evaluate the feasibility and activity of combination treatment with docetaxel (DTX) and irinotecan (CPT-11), given together every other week, combined with filgrastim support, in anthracycline- and paclitaxel-pretreated breast cancer (BC) patients. Patients and Methods: Advanced BC patients pretreated with anthracycline- and paclitaxel-based chemotherapy were eligible. DTX (80 mg/m2) and CPT-11 (100 mg/m2) were given biweekly with filgrastim support (300 µg/day on days 4–7). Results: Fifty patients (48 with metastatic and 2 with locally advanced cancer) were enrolled, with a total of 318 cycles being delivered. Thirty-one patients had visceral localizations. All patients had received epirubicin plus paclitaxel, with or without cisplatin, as front-line treatment for advanced disease. Overall, fatigue and diarrhea were the main chemotherapy-related toxicities in this study, being severe in 10 (20%) and 4 (8%) patients. Grade 3 or 4 neutropenia and thrombocytopenia occurred in 18 (36%) and 6 (12%) patients, respectively. Red blood cell transfusions were required in 4 patients. A total of 32 objective responses were registered (overall response rate, ORR = 64%, 95% confidence interval = 49–77%), including 8 complete responses (16%). An additional 8 patients showed stable disease. After a median follow-up of 18 (range 4–29) months, 30 patients were still alive, and 19 were progression free; median progression-free and overall survivals were 10 and 23 months, respectively. Conclusions: Biweekly DTX/CPT-11 with G-CSF support is a well-tolerated and highly effective approach in anthracycline-/paclitaxel-pretreated patients. The very promising ORR and survival outcome observed in this subset of patients with a poor prognosis suggest that this regimen might play a major role in the management of this disease.
Reduced Expression of PTEN Protein and Its Prognostic Implications in Invasive Ductal Carcinoma of the Breast
Authors: Tsutsui Shinichi , Inoue Hiroshi , Yasuda Kazuhiro , Suzuki Kosuke , Higashi Hidefumi , Era Shoichi , Mori Masaki
Published: July 2005

Objective: The PTEN tumor suppressor gene has been demonstrated to be inactivated in a variety of human tumors. In breast cancer, the PTEN gene mutation is not commonly found whereas loss of heterozygosity affecting the PTEN locus is frequently found. The aim of this study was to analyze PTEN protein expression in breast cancer and to evaluate the prognostic significance of PTEN protein expression. Methods: Paraffin-embedded sections ofinvasive ductal carcinoma of the breast were immunohistochemically stained for PTEN protein expression in 236 breast cancers. The immunohistochemical expression of breast cancer cells was judged to be either normal or reduced compared with the PTEN protein expression of the normal mammary gland. Results: The expression of PTEN protein was found to have decreased in 67 (28%) of 236 breast cancers. The reduced expression correlated with lymph node metastasis (p = 0.0371), but not with tumor size, nuclear grade, MIB-1 counts or p53 protein expression. Univariate analysis indicated that patients with a reduced PTEN expression had a shorter disease-free survival (DFS) than those with a normal PTEN expression (p = 0.0174). Univariate analyses also determined tumor size, lymph node metastases, nuclear grade, MIB-1 counts, p53 protein as well as PTEN protein expression to be significant factors for DFS, while multivariate analysis determined lymph node metastases and the MIB-1 counts to be independent significant factors for DFS. Conclusions: The inactivation of PTEN, demonstrated by a reduced expression of PTEN protein by immunohistochemistry, was found in about one third of all breast cancers. The reduced expression of PTEN protein correlated with lymph node metastases and a worse prognosis in the patients with breast cancer.
Loss of Growth Hormone Secretagogue Receptor 1a and Overexpression of Type 1b Receptor Transcripts in Human Adrenocortical Tumors
Authors: Barzon Luisa , Pacenti Monia , Masi Giulia , Stefani Anna-Lisa , Fincati Karina , Palù Giorgio
Published: July 2005

Objective and Methods: Quantitative analysis of mRNA expression of ghrelin and its receptors GHS-R1a and -R1b in a large series of normal and neoplastic human adrenocortical tissues. Evaluation of the effects of ghrelin on GHS-R expression and proliferation of human adrenocortical carcinoma (ACC) cell lines. Results: Ghrelin and GHS-R transcripts are expressed in normal adrenal cortex, with GHS-R1b mRNA levels being 5- to 10-fold higher than GHS-R1amRNA. A significant increase in ghrelin expression was observed in adrenocortical adenomas, but not in carcinomas. GHS-R1a was undetectable in about 60% of both benign and malignant tumor samples, except for cortisol-producing adenomas, which showed increased receptor expression. At variance, GHS-R1b was overexpressed in both benign and malignant adrenocortical tumors. In vitro studies in human ACC cell lines demonstrated that GHS-R1a is downregulated and GHS-R1bmRNA expression is upregulated by ghrelin, while inhibiting cell proliferation. Conclusion: Downregulation ofGHS-R1a in adrenal tumors and the presence of high levels of GHS-R1b transcripts in adrenocortical tissue suggest a role for these receptors in adrenal function and growth. In this regard, ghrelin inhibits cell proliferation and modulates GHS-R expression in ACC cells in vitro.
Immunohistochemical Expression of Multidrug Resistance Proteins as a Predictor of Poor Response to Chemotherapy and Prognosis in Patients with Nodal Diffuse Large B-Cell Lymphoma
Authors: Ohsawa Masahiko , Ikura Yoshihiro , Fukushima Hiroko , Shirai Nobuyuki , Sugama Yoshimi , Suekane Takehisa , Hirayama Michihiko , Hino Masayuki , Ueda Makiko
Published: July 2005

Background/Aims: The aim of this study was to determine whether expression of P-glycoprotein (P-gp), multidrug-resistance-related protein 1 (MRP1), and lung resistance protein (LRP) was related to the response to induction chemotherapy and prognosis in untreated diffuse large B-cell lymphoma (DLBCL). Methods: We assessed immunohistochemical expression of P-gp, MRP1 and LRP, using formalin-fixed and paraffin-embedded specimens of lymph node in 41 patients with DLBCL. Association between expression of these three proteins and their impact on clinical outcome and prognosis was statistically evaluated. Results: P-gp was positive in 37% of subjects, MRP1 in 63%, and LRP in 68%. The complete remission rates achieved in the group expressing these multidrug resistance (MDR) proteins was significantly lower than in the group not expressing them (20 versus 58%; p = 0.025 in P-gp, 23 versus 80%; p < 0.001 in MRP1 and 32 versus 69%, p = 0.043 in LRP, respectively). Furthermore, the patients expressing LRP had a shorter overall survival rate than those that did not (median of 26 months versus median not reached; p = 0.013). Conclusions: These findings suggest that the three MDR proteins are important predictive factors for the clinical outcome and prognosis in patients with DLBCL.
Calcium Flux in Neuroblastoma Cells Is a Coupling Mechanism between Non-Genomic and Genomic Modes of Estrogens
Authors: Zhao Xing , MacBride Megan M. , Peterson Blake R. , Pfaff Donald W. , Vasudevan Nandini
Published: July 2005

Estrogens have been demonstrated to rapidly modulate calcium levels in a variety of cell types. However, the significance of estrogen-mediated calcium flux in neuronal cells is largely unknown. The relative importance of intra- and extracellular sources of calcium in estrogenic effects on neurons is also not well understood. Previously, we have demonstrated that membrane-limited estrogens, such as E-BSA given before an administration of a 2-hour pulse of 17β-estradiol (E2), can potentiate the transcription mediated by E2 from a consensus estrogen response element (ERE)-driven reporter gene. Inhibitors to signal transduction cascades given along with E-BSA or E2 demonstrated that calcium flux is important for E-BSA-mediated potentiation of transcription in a transiently transfected neuroblastoma cell line. In this report, we have used inhibitors to different voltage-gated calcium channels (VGCCs) and to intracellular store receptors along with E-BSA in the first pulse or with E2 in the second pulse to investigate the relative importance of these channels to estrogen-mediated transcription. Neither L- nor P-type VGCCs seem to play a role in estrogen action in these cells; while N-type VGCCs are important in both the non-genomic and genomic modes of estrogen action. Specific inhibitors also showed that the ryanodine receptor and the inositol trisphosphate receptor are important to E-BSA-mediated transcriptional potentiation. This report provides evidence that while intracellular stores of calcium are required to couple non-genomic actions of estrogen initiated at the membrane to transcription in the nucleus, extracellular sources of calcium are also important in both non-genomic and genomic actions of estrogens.
Relation between the Hypothalamic-Pituitary-Thyroid (HPT) Axis and the Hypothalamic-Pituitary-Adrenal (HPA) Axis during Repeated Stress
Authors: Helmreich Dana L. , Parfitt D.B. , Lu X.-Y. , Akil H. , Watson S.J.
Published: July 2005

Previous work has indicated that acute and repeated stress can alter thyroid hormone secretion. Corticosterone, the end product of hypothalamic-pituitary-adrenal (HPA) axis activation and strongly regulated by stress, has been suggested to play a role in hypothalamic-pituitary-thyroid (HPT) axis regulation. In the current study, we sought to further characterize HPT axis activity after repeated exposure to inescapable foot-shock stress (FS), and to examine changes in proposed regulators of the HPT axis, including plasma corticosterone and hypothalamic arcuate nucleus agouti-related protein (AGRP) mRNA levels. Adult male Sprague-Dawley rats were subjected to one daily session of inescapable FS for 14 days. Plasma corticosterone levels were determined during and after the stress on days 1 and 14. Animals were killed on day 15, and trunk blood and brains were collected for measurement of hormone and mRNA levels. Repeated exposure to FS led to a significant decrease in serum levels of 3,5,3′-triiodothyronine (T3) and 3,5,3′,5′-tetraiodothyronine (T4). Stress-induced plasma corticosterone levels were not altered by repeated exposure to the stress. Despite the decrease in peripheral hormone levels, thyrotropin-releasing hormone (TRH) mRNA levels within the paraventricular nucleus of the hypothalamus were not altered by the stress paradigm. Arcuate nucleus AGRP mRNA levels were significantly increased in the animals exposed to repeated FS. Additionally, we noted significant correlations between stress-induced plasma corticosterone levels and components of the HPT axis, including TRH mRNA levels and free T4 levels. Additionally, there was a significant correlation between AGRP mRNA levels and total T3 levels. Changes in body weight were also correlated with peripheral corticosterone and TRH mRNA levels. These results suggest that repeated exposure to mild-electric foot-shock causes a decrease in peripheral thyroid hormone levels, and that components of the HPA axis and hypothalamic AGRP may be involved in stress regulation of the HPT.
Estrogen Increases Nociception-Evoked Brain-Derived Neurotrophic Factor Gene Expression in the Female Rat
Authors: Allen Amy L. , McCarson Kenneth E.
Published: July 2005

Chronic pain induces plastic changes in nociceptive sensory pathways, and is often accompanied and exacerbated by depression. Estrogen can influence nociceptive sensory processing, but the molecular mechanisms underlying sex differences in pain remain unclear. Brain-derived neurotrophic factor (BDNF) may orchestrate changes occurring during persistent pain or depression by increasing spinal nociceptive signaling and altering neuronal growth in higher brain structures. This study addressed whether estrogen regulates BDNF gene expression in central systems associated with nociceptive processing and/or affective state, which may in turn influence sex differences in pain sensitivity. Thus, BDNF gene expression was quantified in intact female rats in proestrus and diestrus, and in ovariectomized (OVX) rats with or without 17β-estradiol (E2) replacement following intraplantar injection of dilute formalin as an inflammatory nociceptive stimulus. Twenty-four hours after formalin injection, central nervous system (CNS) tissues were removed and solution hybridization-nuclease protection assays used to quantify BDNF mRNA levels. Results demonstrated that estrogen replacement increased BDNF mRNA levels in the hippocampus, cortex and spinal cord. Cortical BDNF mRNA levels were significantly decreased by nociception, in the hippocampus this decrease was only evident in estrogen-treated rats. Spinal BDNF expression was robustly increased by nociception. The pain-evoked up-regulation of spinal BDNF gene expression was significantly potentiated by concomitant estrogen treatment. Results demonstrate that BDNF gene expression in certain brain structures is inhibited by inflammatory pain, yet estrogen may enhance central nervous system sensitization associated with sensory processing. Since alterations in BDNF gene expression in higher brain centers may be relevant to cognitive changes that occur in recurrent depression, these results may provide insights into the coincidence of chronic pain and depression.
Eradication of Helicobacter pylori Can Be Accurately Confirmed 14 Days after Termination of Triple Therapy Using a High-Dose Citric Acid-Based 13C Urea Breath Test
Authors: Shirin Haim , Levine Arie , Shevah Orit , Shabat-Sehayek Vered , Aeed Hussein , Wardi Joram , Birkenfeld Shlomo , Eliakim Rami , Avni Yona
Published: July 2005

Background: Confirmation of Helicobacter pylori eradication by urea breath test (UBT) is currently performed 4–6 weeks after completion of therapy because of unacceptable false-negative results in UBTs performed earlier. Use of a high-dose citric acid test meal appears to enable accurate detection of H. pylori even during short term therapy with proton pump inhibitors. Aim: To evaluate if use of a high dose citric acid (4.0 g) test meal can decrease the interval required for confirmation of eradication after triple therapy. Methods: 233 patients positive for H. pylori were randomized to undergo UBT at 7 days or 14 days after triple therapy, and again at 6 weeks. The latter test was considered the gold standard test. Results: The UBT performed 6 weeks after the end of treatment found that 79.9% were cured. The same test 7 days after therapy found false-negative detection of H. pylori in 7.3% patients compared to 3.2% patients examined after 14 days. The sensitivity, specificity, positive and negative predictive values and accuracy for evaluation on day 14 were 80, 100, 100, 96.3 and 96.7%, respectively. Conclusions: High-dose citric acid-based UBT is a valid test for the assessment of H. pylori status 14 days after triple therapy. This may obviate the delay in instituting second-line eradication therapy, or further evaluation of the symptomatic patient unresponsive to therapy despite eradication.
Focus on the Conditions of Resection and Reconstruction in Gastric Cancer
Authors: Ogoshi Kyoji , Okamoto Yuichi , Nabeshima Kazuhito , Morita Mari , Nakamura Kenji , Iwata Kunihiro , Soeda Jinichi , Kondoh Yasumasa , Makuuchi Hiroyasu
Published: July 2005

To assess the roles of the extent of gastric resection and duodenal food passage reconstruction in gastric cancer, we examined a consecutive series of 1,061 patients who underwent total or partial (proximal and distal) gastrectomies with or without duodenal food passage reconstruction between August of 1974 and January of 2002, and received gastrectomies with D2–3 lymph node dissection. Patients who underwent distal or proximal gastrectomy were found to have significantly better survival rates than those who underwent total gastrectomy in stages 1A (10-year survival: 86.6 and 78.9 vs. 61.6%), 2 (56.5 and 65.6 vs. 34.4%), 3A (45.9 and 33.3 vs. 15.2%), and 4 (5-year survival rates: 23.7 and 50.0 vs. 7.1%). Additionally, patients with duodenal food passage reconstruction or double tract reconstruction also showed significantly better survival rates than those without duodenal food reconstruction in stages 1A (10-year survival: 86.4 and 82.5 vs. 61.7%), 1B (69.9 and 90.6 vs. 54.1%), 2 (60.5 and 63.3 vs. 16.5%), and 3A (39.9 and 47.4 vs. 23.1%). In multivariate analysis, the independent prognostic factors were age at operation, depth of tumor, duodenal food passage reconstruction, and lymph node metastasis. Our results indicate that both the extent of gastric resection and duodenal food passage reconstruction were important factors in the outcome of gastric cancer patients, and that surgeons should perform minimal gastric resection with preservation of the duodenal food passage when the gastric stump is tumor-free.
Symptoms and Pathophysiological Correlations in Patients with Constipation and Functional Dyspepsia
Authors: Sarnelli Giovanni , Grasso Raffaella , Ierardi Enzo , De Giorgi Francesco , Savarese Maria Flavia , Russo Luigi , Budillon Gabriele , Cuomo Rosario
Published: July 2005

Introduction: Patients with constipation often report dyspeptic symptoms, but whether constipation is associated with specific dyspeptic symptoms and altered gastrointestinal (GI) motility, remains to be established. Our aim was to study symptoms association and GI motility parameters in patients with constipation and functional dyspepsia. Patients and Method: 42 patients with different symptoms and severity of constipation and dyspepsia were enrolled. Scintigraphic gastric emptying, colonic transit time and gallbladder contraction were studied in all subjects. Results: No significant association was observed between individual symptoms of constipation and dyspepsia. Patients with more severe constipation did not have higher dyspepsia severity scores. Colonic transit time, gastric half emptying and gallbladder contraction were not significantly correlated. Although patients with severe nausea had faster colonic transit than those with absent/mild symptom (19 ± 2 vs. 48 ± 7 h; p < 0.05), the multivariate analysis only revealed a significant association between severe postprandial fullness, delayed t1/2 (OR 1.05, CI 1–1.1) and impaired gallbladder contraction (OR 0.94, CI 0.89–0.99). Conclusions: Constipation was not associated with severity, or any particular dyspeptic symptom. Although motor abnormalities of both colon and proximal GI tract regions existed in the subset of constipated dyspeptic patients, they did not seem associated with the genesis of different dyspeptic symptoms.
Smoking and Alcohol Habits as Risk Factors for Benign Digestive Diseases in a Japanese Population: The Radiation Effects Research Foundation Adult Health Study
Authors: Yamada Michiko , Wong F.Lennie , Fujiwara Saeko , Tatsukawa Yoshimi , Suzuki Gen
Published: July 2005

Background: Although an association between benign digestive diseases and smoking or drinking habits was reported, consistent results have not been obtained either in European, American or Japanese populations. Methods: Smoking and alcohol habits as risk factors for the incidence of gastric ulcer, duodenal ulcer, chronic liver disease and cirrhosis as well as cholelithiasis were examined using the longitudinal data of the Adult Health Study collected biennially between 1958 and 1998. During 1958–1998, 1,093 gastric ulcers, 437 duodenal ulcers, 2,054 chronic liver diseases and cirrhoses, and 1,136 cholelithiasis cases were newly detected based on medical history, fluoroscopy or endoscopy and ultrasonography. Smoking and drinking histories were obtained from five and three questionnaires, respectively, administered during different periods. The relative risks (RRs) for ever smoked to never smoked and that for ever drank to never drank were estimated after adjustment for city, sex, age, birth cohort, calendar time and radiation dose. Results: The analysis showed a positive association of smoking with gastric ulcer (RR: 2.03, 95% CI: 1.71–2.41), duodenal ulcer (RR: 1.31, 95% CI: 0.99–1.72), chronic liver disease and cirrhosis (RR: 1.23, 95% CI: 1.08–1.39) and cholelithiasis (RR: 1.19, 95% CI: 1.02–1.40), and a positive association of drinking with chronic liver disease and cirrhosis (RR: 1.10, 95% CI: 0.99–1.23). Conclusions: Thepeptic ulcer, chronic liver disease and cholelithiasis incidence increased significantly with smoking, and the chronic liver disease incidence increased significantly with drinking simultaneously in a prospective study of a Japanese population.
In vivo rabbit acute model tests of polyurethane catheters coated with a novel antithrombin-heparin covalent complex
Authors: Du Ying Jun , Klement Petr , Berry Leslie R. , Tressel Paul , Chan Anthony K. C.
Published: July 2005

Catheter use has been associated with an incresed risk of thrombotic complications. The objective was to make catheters less thrombogenic with the use of antithrombin-heparin covalent complex (ATH). The antithrombotic activity of ATH-coated catheters was compared to uncoated (control) and heparincoated catheters in an acute rabbit model of accelerated occluding clot formation. Anaesthetized rabbits were pre-injected with rabbit 125I-fibrinogen,followed by insertion of test catheters into the jugular vein. Blood was drawn and held in a syringe, reinjected, then flushed with saline. The experiment was terminated when blood could no longer be withdrawn (occluding clot). Efficacy was defined as the ability of catheters to remain unoccluded. Clot formation, determined by measuring deposition of radiolabeled fibrin, was a secondary endpoint. ATH-coated catheters were resistant to clotting for the full 240-minute duration, while uncoated and heparin-coated catheters had an average clotting time of 78 and 56 minutes, respectively. The patency ofATH coating was dependant on intact heparin pentasaccharide sequences, rather than the chemistries of the basecoat, the PEO spacer arm, or the antithrombin (AT) protein. The ATH coating was stable to ethylene oxide sterilization, modest abrasion, protease attack, and the coating did not appear to leach off the catheter. Surface tension measurements showed that the ATH modified surface was more hydrophilic than uncoated control catheters or heparin-coated catheters. Thus, ATH-coated catheters are better at preventing clots than uncoated or heparin-coated catheters and show promise as an alternative to the currently available catheters in reducing thrombotic complications associated with its use.
Effect of surface modified liposomes on the aggregation of platelets and tumor cells
Authors: Keil Claudia , Fichtner Iduna , Zeisig Reiner
Published: July 2005

Metastasis is still the most serious reason for the high mortality of cancer patients. It is a complex process in which platelets play a crucial role. Several attempts have been performed to inhibit the metastatic process, some of these using modified liposomes. The aggregation behaviour of human platelets and HT29 colon carcinoma cells in the presence of liposomes with a modified surface has been investigated in the present study. Liposomes (PC/CH/DMPE) were unmodified, sterically stabilized by polyethylene glycol (PEG-DSPE), or equipped with the carbohydrate ligand sialyl LewisX (conjugated to PEG-DMPE or DMPE as anchor) intended to specifically compete with ligands expressed by HT29 cells. We found in vitro that an addition of surface modified liposomes to human platelets in plasma caused an up to 2.9-fold increase in platelet aggregation. In addition, when HT29 tumor cells were mixed with platelets and surface modified lipo somes, the number of tumor cells found in aggregates increased significantly from 8.3 % (only tumor cells) to 30.2 %. This result was supported by fluorescence micrographs demonstrating a strong association of platelets and liposomes around the tumor cells. In addition, a clear decrease in number and a change in the distribution of metastases after intravenous injection of HT29 cells in combination with liposomes was observed in vivo. While in control mice metastases in lung, liver and in intestine were prevailing, liposomal treatment resulted in a new localization of metastases in muscles. Taking together, the ability of surface modified liposomes to enhance aggregate formation of platelets and tumor cells has been demonstrated for the first time. The capability of these vesicles to interfere with the metastatic process might have implications for the use of such liposomes for therapeutic applications.
Streptococcus pneumoniae R6x induced p38 MAPK JNK-mediated Caspase-dependent apoptosis in human endothelial cells
Authors: N’Guessan Philippe Dje , Schmeck Bernd , Ayim Abena , Hocke Andreas C. , Brell Bernhard , Hammerschmidt Sven , Rosseau Simone , Suttorp Norbert , Hippenstiel Stefan
Published: July 2005

Streptococcus pneumoniae is the major pathogen of communipnetyacquired umonia and a common cause of otitis, meningitis and sepsis. During pneumococci infection accompanied with bacterial invasion and hematogenous spreading, the endothelium is directly targeted by pneumococci and their virulence factors. Therefore, we tested the hypothesis that pneumococci induced endothelial apoptosis. Unencapsulated R6x pneumococci strongly induced apoptosis of human endothelial cells both from lung microvasculature and umbilical vein, whereas an encapsulated strain D39 mainly led to necrotic cell death. Deletion of the gene coding for pneumolysin reduced pneumococci-induced apoptosis in HUVEC. Furthermore, N-acetyl-L-cysteine, an antioxidant thiol, significantly reduced apoptosis caused by R6x, and LDH release induced by D39, pointing to a role for reactive oxygen species in the pathogenesis. Apoptotic cells showed increased cleavage and activity of caspases 6 and 9 but only late activation of caspase 3. Programmed cell death could be strongly reduced by pan-caspase inhibitor zVAD. Reduced levels of Bcl2 and cytosolic increase of apoptosis-inducing factor in pneumococci-infected cells implicated involvement of mitochondrial death pathways. Caspase activation and apoptosis were abolished by cAMP elevation. Moreover, p38 mitogen-activated protein kinase and c-Jun NH2-terminal kinase were activated in pneumococci-infected cells and inhibitors of both kinases strongly reduced pneumococci-induced caspase activation and apoptosis. Hence,kinase- and caspase-dependence of pneumococci-induced endothelial apoptosis may bear relevance to novel therapeutic approaches to pneumococci-related disease.
Abciximab therapy is associated with increased platelet activation and decreased heparin dosage in patients with acute myocardial infarction
Authors: Piorkowski Michael , Priess Jana , Weikert Ulf , Jaster Markus , Schwimmbeck Peter-Lothar , Schultheiss Heinz-Peter , Rauch Ursula
Published: July 2005

The inhibition of the glycoprotein (GP) IIb/IIIa receptor for reducing periprocedural ischemic events in patients undergoing coronary intervention is known to influence platelet reactivity. Suboptimal doses of GP IIb/IIIa antagonists have been suggested to be prothrombotic and proinflammatory. This study was performed to observe platelet activation markers, whole blood aggregation and the dosage of unfractionated heparin (UFH) in the presence or absence of the GP IIb/IIIa inhibitor abciximab. Patients with acute myocardial infarction undergoing percutaneous coronary intervention were treated with (n=15) or without (n=15) abciximab. Platelet activation markers were flow cytometrically measured before and after PCI. Whole blood platelet aggregation was tested by a platelet function assay. The patients with abciximab showed a significant increase in platelet activation markers (P-selectin: 7.12 ± 0.36 AU vs 11.05 ± 0.79 AU) and a lower requirement of UFH to prolong aPTT > 60 sec during the infusion. 12 hours after infusion P-selectin level decreased (7.20 ± 0.58 AU), whereas whole blood aggregation was increasing again. After stopping abciximab, requirement of UFH to prolong aPTT increased in the treated group to a greater extent to a level similar to the untreated group even when most of the platelets were still inhibited. The increased platelet activation found at the end of abciximab treatment points to a procoaguable condition that should be carefully monitored and treated by adapting anticoagulation and antiplatelet drugs.
Regulation of platelet guanylyl cyclase by collagen: evidence that Glycoprotein VI mediates platelet nitric oxide synthesis in response to collagen
Authors: Riba Rocio , Sharifi Marzieh , Farndale Richard W. , Khalid M. Naseem
Published: July 2005

The molecular regulation of nitric oxide synthase (NOS) in blood platelets is an uncharacterised area of platelet biology. We investigated the mechanism of collagen-stimulated NO synthesis in platelets. Our aim was to identify the key collagen receptor and downstream signalling mechanisms linking collagen to NOS activation. Collagen and the GpVI-specific platelet activator collagen-related peptide (CRP-XL) stimulated NO synthesis, as evidenced by increased [3H]L-citrulline production, and cyclic GMP (cGMP) formation. After platelet activation by collagen and CRP-XL was normalised, we found no differences in NOS activation or cGMP formation in response to these agonists. Blocking the interaction of collagen with integrin α2β1, a second collagen receptor, failed to affect NOS activation by collagen. These data indicate that collagen-induced NO synthesis is linked to GpVI activation. cGMP formation in response to collagen and CRP-XL required increased intracellular Ca2+, Src family kinases,phosphatidylinositol 3-kinase (PI3-K) and protein kinase C. By comparison, Gp VI-independent cGMP formation induced by thrombin was Src kinase-dependent, but was independent of PI3-K and PKC. Thus the mechanisms of collagen- and CRP-XL-induced NOS activation were identical, but distinct from that of thrombin. Platelet activation in response to collagen leads to secretion of adenosine diphosphate (ADP) and thromboxaneA2 (TxA2). Our results demonstrate that collagenstimulated cGMP synthesis was enhanced significantly by platelet- derived ADP and TxA2. These results reveal that collagen stimulates platelet NOS activation through a specific Ca2+-dependent GpVI receptor signalling cascade,and demonstrate that collagen-induced cGMP accrual requires the release of secondary platelet agonists.
Platelets express steroidogenic 17β–hydroxysteroid dehydrogenases
Authors: Gnatenko Dmitri V. , Cupit Lisa D. , Huang Emily C. , Dhundale Anilkumar , Peter L. Perrotta , Bahou Wadie F.
Published: July 2005

Human blood platelets have important, regulatory functions in diverse hemostatic and pathological disorders, including vascular remodeling, inflammation, and wound repair. Microarray analysis was used to study the molecular basis of essential thrombocythemia, a myeloproliferative disorder with quantitative and qualitative platelet defects associated with cardiovascular and thrombohemorrhagic symptoms, not infrequently neurological. A platelet-expressed gene (HSD17B3) encoding type 3 17β-hydroxysteroid dehydrogenase (previously characterized as a testis-specific enzyme catalyzing the final step in gonadal synthesis of testosterone) was selectively down-regulated in ET platelets, with reciprocal induction of the type 12 enzyme (HSD17B12). Functional 17β-HSD3 activity corresponding to ∼10% of that found in murine testis was demonstrated in normal platelets. The induction of HSD17B12 in ET platelets was unassociated with a concomitant increase in androgen biosynthesis, suggesting distinct functions and/or substrate specificities of the types 3 and 12 enzymes. Application of a molecular assay distinguished ET from normal platelets in 20 consecutive patients (p < 0.0001). These data provide the first evidence that distinct subtypes of steroidogenic 17β−HSDs are functionally present in human blood platelets, and that the expression patterns of HSD17B3 and HSD17B12 are associated with an uncommon platelet disorder manifest by quantitative and qualitative platelet defects.
Role of human brain microvascular endothelial cells during central nervous system infection
Authors: Adam Rüdiger , Rüssing Daniel , Adams Ortwin , Ailyati Aziz , Kim Kwang Sik , Schroten Horst , Däubener Walter
Published: July 2005

The cerebral endothelium is involved both in regulatinthinflux g e of immune cells into the brain and in modifying immunological reactions within the CNS. A number of human pathogens may cause encephalitis or meningitis when this important protective barrier is impaired. We have previously shown that interferon- γ activated human brain microvascular endothelial cells (HBMEC) restrict the growth of bacteria and parasites. We now provide evidence that HBMEC are also capable of inhibiting viral replication after stimulation with IFN-γ, an effect further augmented by costimulation with IL-1. This antiviral effect was completely blocked in the presence of L-tryptophan, indicating the induction of the tryptophan degrading enzyme indoleamine 2,3-dioxygenase (IDO) to be responsible for the observed antiviral effect. Apart from exerting antimicrobial effects tryptophan depletetion has also been described as a regulatory mechanism in T cell responses to both allo- and autoantigens. We were able to demonstrate that IDO mediated degradation of L-tryptohan in HBMEC is responsible for a significant reduction inT lymphocyte proliferation. Resupplementation of L-tryptophan and restoration of initial T cell responses demonstrated the central role of this essential amino acid in the reduction of T-cell proliferation. Brain endothelial cells appear to limit microbial expansion in the CNS by local degradation of tryptophan, thus acting in concert with other IDO-positive cell populations on the parenchymal side of the blood-brain barrier such as astrocytes, microglia and neurons. Since all dietary tryptophan must cross the blood-brain barrier, the microvascular endothelial cells may play a key role in restricting tryptophan influx from the bloodstream into the brain. As deleterious effects of brain infections can often be attributed to subsequently invading immune cells, an IDO-mediated reduction of lymphocyte proliferation may be beneficial for preventing collateral brain damage.
Influence of the 4600A/G and 4678G/C polymorphisms in the endothelial protein C receptor (EPCR) gene on the risk of venous thromboembolism in carriers of factor V Leiden
Authors: Medina Pilar , Navarro Silvia , Estellés Amparo , Vayá Amparo , Bertina Rogier M. , España Francisco
Published: July 2005

Two polymorphisms in the endothelial protein C receptor (EPCR) gene, 4600A/G and 4678G/C, have been reported to influence the risk of venous thromboembolism (VTE). The objective of this study was to assess whether these polymorphisms modify the risk of VTE in carriers of factor V (FV) Leiden. We genotyped 295 carriers of FV Leiden for these polymorphisms: 100 unrelated patients with a history ofVTE (propositi) and 195 relatives (14 of them symptomatic) of 81 of the propositi. Spontaneous VTE events occurred in 71% of propositi carrying the 4678GG genotype, 65% carrying the GC, and 43% with the CC genotype. The mean age at the first onset was significantly higher in propositi carrying the 4678CC than in those with the GC or GG genotype (p=0.046). Among the 276 carriers of FV Leiden from the 81 families studied, the 95 symptomatic members had similar 4600G allele and 4600AG genotype frequencies but significantly lower 4678C allele (p=0.002) and 4678CC genotype (p=0.004) frequencies than the 181 asymptomatic members. The probability of being free of thrombosis at age 40 was significantly higher in the 66 carriers of the 4678CC genotype (94%) than in the 138 carrying the GC (72%) or in the 72 with the 4678GG genotype (60%) (p<0.001). Multivariate analysis showed that the 4678CC genotype reduced the risk of thrombosis in carriers of FV Leiden (OR=0.31;95% CI=0.16–0.83). The incidence ofVTE was higher in the 195 relatives with FV Leiden than in the 133 without FV Leiden (OR=4.7; CI=1.3–7.2). These results show that carriers of FV Leiden with the 4678CC genotype have a significantly reduced risk of VTE compared with those carrying the 4678GG or GC genotype, probably due to the higherAPC levels previously observed in individuals carrying the 4678CC genotype.
The transcriptional response of human endothelial cells to infection with Bartonella henselae is dominated by genes controlling innate immune responses, cell cycle, and vascular remodelling
Authors: Québatte Maxime , Foser Stefan , Certa Ulrich , Dehio Michaela
Published: July 2005

The bacterial pathogen Bartonella henselae (Bh) is for respona sible broad range of clinical manifestations, including the formation of vascular tumours as the result of pathogen-triggered vasoproliferation. In vitro, the interaction of Bh with human umbilical vein endothelial cells (Huvec) involves (i) cytoskeletal rearrangements in conjunction with bacterial internalization, (ii) nuclear factor κB (NFκB)-dependent proinflammatory activation, (iii) the inhibition of apoptosis, and (iv) the modulation of angiogenic properties such as proliferation, migration, and tubular differentiation. To study the transcriptional signature of these pathogen- triggered changes of Huvec, we performed transcriptional profiling with Affymetrix U133 GeneChips. At 6 h or 30 h of infection, a total of 706 genes displayed a clear and statistically significant change of expression (>2.5-fold, t-test p-value<0.05). These included 314 up-regulated genes dominated by the innate immune response. The gene list comprises subsets of tumour necrosis factor α (TNFα, 99 genes) and interferon α (IFNα, 30 genes) inducible genes, which encode components of the NF-κB-dependent proinflammatory response and the type I IFN-dependent anti-infective response, respectively. The remaining set of 197 up-regulated genes mirrors other cellular changes induced by Bh, in particular proliferation and proangiogenic activation. The set of 362 down-regulated genes includes 41TNFα- or IFNα-suppressible genes, and 52 genes involved in cell cycle control or progression. This comprehensive analysis of Bh-triggered changes of the Huvec transcriptome identified candidate genes putatively involved in controlling innate immune responses, cell cycle, and vascular remodelling, and may thus provide the basis for functional studies of the molecular mechanisms underlying these pathogen-induced cellular processes.
Rapid identification of female haemophilia A carriers with deletions in the factor VIII gene by quantitative Real-Time PCR analysis
Authors: Barceló María J. , Baena Manel , Cornet Mónica , Venceslá Adoración , Mateo José , Fontcuberta Jordi , Baiget Montserrat , Tizzano Eduardo F.
Published: July 2005

Large deletions of the factorVIII gene account for approximately 5% of severe haemophilia A patients. Although deletions are readily detectable in males, the identification of heterozygosity in possible carriers of these families still constitutes a challenge. In order to identify a deleted allele over the background of the normal allele in these carriers, we developed a rapid real-time quantitative PCR approach by means of LightCycler technology and SYBR green I for monitoring product formation. The method was applied to families with independent deletions (one in exon 14 and the other in exons 23-24) of the Factor VIII gene, thereby allowing a reliable determination of carrier or non-carrier status. The method is extremely versatile and can be adapted to other deletions within the factor VIII gene as well as to other diseases whose molecular pathology consists of deletions or duplications.
A murine model of deep vein thrombosis Characterization and validation in transgenic mice
Authors: Szema Linda , Chen Chao-Ying , Schwab Jeffrey P. , Schmeling Gregory , Cooley Brian C.
Published: July 2005

Deep vein thrombosis (DVT) occurs with high prevalence in association with a number of risk factors, including major surgery, trauma, obesity, bed rest (>5 days), cancer, a previous history of DVT, and several predisposing prothrombotic mutations. A novel murine model of DVT was developed for applications to preclinical studies of transgenically constructed prothrombotic lines and evaluation of new antithrombotic therapies. A transient direct-current electrical injury was induced in the common femoral vein of adult C57Bl/6 mice. A non-occlusive thrombus grew, peaking in size at 30 min, and regressing by 60 min, as revealed by histomorphometric volume reconstruction of the clot. Pre-heparinization greatly reduced clot formation at 10, 30, and 60 min (p<0.01 versus non-heparinized). Homozygous FactorV Leiden mice (analogous to the clinical FactorV Leiden prothrombotic mutation) on a C57Bl/6 background had clot volumes more than twice those of wild-types at 30 min (0.121±0.018 mm3 vs. 0.052±0.008 mm3, respectively; p<0.01). Scanning electron microscopy revealed a clot surface dominated by fibrin strands, in contrast to arterial thrombi which showed a platelet-dominated structure. This new model of DVT presents a quantifiable approach for evaluating thrombosis-related murine transgenic lines and for comparatively evaluating new pharmacologic approaches for prevention of DVT.
ClfA221–550, a fibrinogen-binding segment of Staphylococcus aureus clumping factor A, disrupts fibrinogen function
Authors: Shih Mei-Hsu , Tsai Pei-Jane , Liu Chao-Zong
Published: July 2005

Clumping factor A (ClfA) is a surface of protein Staphylococcusaureus bacteria known for its ability to bind the C-terminus of plasma fibrinogen γ chain, which participates in mediating fibrinogen- platelet interaction and fibrin cross-linking, resulting in thrombus formation. With an aim to develop agents that block fibrinogen γ chain C-terminus,the fibrinogen-binding segment of ClfA locating at residues 221–550 was produced by recombinant technology and tested for its ability to inhibit platelet functions and fibrin clot formation. Recombinant ClfA221–550 bound fibrinogen and blocked fibrinogen-platelet interaction, resulting in the inhibition of both ADP- and collagen-induced platelet aggregations. ClfA221–550 also affected fibrin clot formation, in which factor XIIIa-mediated cross-linking of fibrinogen γ chains was abrogated by ClfA221–550 leaving the release of fibrinopeptides A and B from fibrinogen by thrombin unaltered, indicating that ClfA221–550 interfered with fibrin clot formation without affecting thrombin’s catalytic activity. Plateletmediated clot retraction depends on both platelet-fibrinogen interaction and fibrin clot formation, which makes platelet thrombus less susceptible to fibrinolysis. At the concentration that reduced platelet aggregation by 40%, ClfA221–550 prevented platelet- mediated clot retraction, whereas the glycoprotein IIb/IIIa antagonist tirofiban needed a higher concentration in inhibiting clot retraction than inhibiting platelet aggregation. By virtue of the multiple effects of ClfA221–550 on platelet aggregation, fibrin clot formation and platelet-mediated clot retraction,the binding of ClfA221–550 to fibrinogen merits further investigation for its potential as a new antithrombotic agent.
Chlamydophila pneumoniae
Authors: Maass Matthias , Suttorp Norbert , Rupp Jan , Krüll Matthias
Published: July 2005

Chlamydophila (Chlamydia) pneumoniae, a gram-negative obligate intracellular bacterium, is a widespread respiratory pathogen. Chronic C. pneumoniae infection has been suggested as a trigger/ promoter of inflammation that may result in vascular lesions. Although the genome of C. pneumoniae has been sequenced completely this information has not yet led to an understanding of the mechanisms of acute infection and target cell activation nor to the identification of potential chlamydial virulence factors. Intriguingly, current antibiotic treatment options for acute chlamy- dial infection were proven to be ineffective with respect to clinical outcome in different groups of atherosclerotic patients. The reason might be that primary infection of vascular smooth muscle cells and blood monocytes with C. pneumoniae resembles rather a persistent, antibiotic-resistant, than an active infection. In this review we will focus on the importance of putative host cell receptors for C.pneumoniae and subsequently activated signal transduction pathways.