bluuebunny/crossref_metadata_2025 huggingface dataset:
Pharmacodynamic Properties of the Low Molecular Weight Heparin, Tinzaparin: Effect of Molecular Weight Distribution on Plasma Tissue Factor Pathway Inhibitor in Healthy Human Subjects
Authors: Mousa Shaker A. , Bozarth Jeffrey , Barrett Jeffrey S.
Published: September 2003

Low molecular weight heparins (LMWHs) stimulate the release of endothelial tissue factor pathway inhibitor (TFPI). An inhibitor of tissue factor—induced coagulation, TFPI may contribute to the efficacy of LMWHs. The release of TFPI may be affected by characteristics of LMWHs, such as molecular weight (MW) distribution. The authors measured plasma levels of TFPI after single, subcutaneous injections of tinzaparin (mean MW = 6500 Daltons [Da]; 3.5% of fractions < 2000 Da) or a tinzaparin‐like LMWH (mean MW = 5650 Da; 18.3% of fractions < 2000 Da) in 30 healthy subjects. Maximum mean plasma levels of TFPI approached 200 to 230 ng/mL 0.8 hours after administration of either drug. Plasma levels of TFPI remained elevated compared with baseline values for 12 to 16 hours after drug administration. These results suggest that LMWH fractions < 2000 Da do not affect the release of TFPI.
Safety, Pharmacokinetics, and Pharmacodynamics of E5564, a Lipid A Antagonist, during an Ascending Single‐Dose Clinical Study
Authors: Wong Y. Nancy , Rossignol Daniel , Rose Jeffrey R. , Kao Richard , Carter Alison , Lynn Melvyn
Published: September 2003

E5564, a structural analog of the lipid A portion of lipopolysaccharide (LPS), is a potent antagonist of the biochemical and physiologic effects of LPS in several in vitro and in vivo models and is currently under clinical development as a possible therapeutic for the treatment of sepsis and septic shock. The objectives of this study were to (1) assess the safety and tolerability of E5564 following a 30‐minute intravenous (IV) infusion, (2) evaluate the pharmacokinetic profile of E5564, and (3) measure the ability of E5564 to block LPS stimulation ex vivo in blood taken from subjects up to 8 hours after ending the infusion. Healthy male volunteers (n= 7/dose group) were randomly assigned to each of four dose levels (350, 1000, 2000, or 3500 μg). Within each dose group, 5 subjects received drug and 2 received placebo. E5564 or matching placebo was administered by a 30‐minute infusion, and blood samples were collected at predetermined time points. All doses of E5564 were demonstrated to be safe and well tolerated. E5564 plasma concentrations were determined using a validated LC/MS/MS method. The Cmax and AUC of E5564 increased in a dose‐proportional manner. E5564 pharma‐ cokinetics were characterized by a slow clearance (0.67–0.95 mL/h/kg), a small volume of distribution (41–54 mL/kg), and a relatively long elimination half‐life (42–51 h). As measured in the ex vivo assay, E5564 inhibited LPS‐induced tumor necrosis factor–α (TNF‐α) in a dose‐dependent manner, and at the higher doses (2 and 3.5 mg), antagonistic activity was measurable up to 8 hours postinfusion. E5564 lacked LPS‐like agonist activity at doses up to 3.5 mg. Taken together, we believe that E5564 is a safe, potent antagonist of LPS in blood and will likely benefit patients in the treatment of LPS‐related diseases.
Medication Errors: Experience of the United States Pharmacopeia (USP) MEDMARX Reporting System
Authors: Santell John P. , Hicks Rodney W. , McMeekin Judy , Cousins Diane D.
Published: September 2003

Medication errors are pervasive in America's health care system. MEDMARX is an Internet‐accessible, anonymous medication error reporting program designed for hospitals and health systems to systematically collect, analyze, and report medication errors. This study examined 154,816 medication error reports that were submitted to MEDMARX between January 1, 1999, and December 31, 2001. Data fields analyzed were error category (based on the National Coordinating Council for Medication Error Reporting and Prevention's Error Category Index), type(s) of error, cause(s) of error, contributing factor(s), and product(s) involved. Approximately two‐thirds of the errors reported reached the patient, with relatively few causing harm. Death was reported in 19 occurrences. Errors of omission and improper dose/quantity were the most commonly reported. Performance deficit and procedure/protocol not followed were consistently identified as causes of error. Distractions and workload increase were often cited as contributing factors. There was a similar pattern of products reported in each of the years. Implications for quality assurance, clinical practice, and health policy are presented.
Gastric Function in the Elderly: Effects on Absorption of Ketoconazole
Authors: Hurwitz Aryeh , Ruhl Constance E. , Kimler Bruce F. , Topp Elizabeth M. , Mayo Matthew S.
Published: September 2003

The authors studied effects of age‐related changes in gastric function on absorption of ketoconazole. Eighteen men and women age 65 years or older swallowed 200 mg ketoconazole on two occasions, once as tablets with water and once as tablets crushed in acidic juice. The sequence was randomly determined. Gastric pH was measured by radiotelemetry and gastric emptying rate by radiolabeled technetium with a gamma camera. Plasma ketoconazole was assayed by high‐performance liquid chromatography (HPLC). Subjects with gastric pH less than or equal to 4.5 absorbed ketoconazole equally well from intact tablets and tablets crushed in acid. When pH was 5.0 or higher, ketoconazole was absorbed well from acid‐crushed tablets but not from intact tablets. Gastric emptying was shown to be rapid in all subjects. Since the prevalence of such hypoacidity is approximately 5% in the elderly, and other parameters of gastric function are usually normal, impaired absorption of drugs such as ketoconazole should be uncommon with normal aging.
Evaluation of In Vitro Drug Interactions with Karenitecin, a Novel, Highly Lipophilic Camptothecin Derivative in Phase II Clinical Development
Authors: Smith Judith A. , Newman Robert A. , Hausheer Frederick H. , Madden Timothy
Published: September 2003

The objective of this study was to describe the potential metabolism and protein‐binding interactions with karenitecin, a novel computer‐engineered, highly lipophilic camptothecin. Individual cloned cytochrome P450 (CYP450) isoenzymes were used to determine, in vitro, the metabolism of karenitecin. Known substrates and inhibitors of each isoenzyme were employed to evaluate CYP450 drug interactions with karenitecin. To assess the extent, variability, and role of various drug‐binding proteins, the authors examined, in vitro, the effects of both albumin (Alb) and α‐acidic glycoprotein (AAG) on karenitecin plasma protein binding (PPB). Equilibrium dialysis techniques were used to measure the free fraction of karenitecin in the presence of varying ratios of Alb and AAG. Artificial plasma, spiked with karenitecin, was dialyzed for 72 hours at 37°C against a Sorensen's buffer solution using regenerated cellulose membranes having a molecular weight cutoff of 12 to 14 kDa. Additional protein‐binding experiments were conducted to assess the potential PPB drug interactions between karenitecin and other highly protein‐bound drugs commonly used in the treatment of cancer patients. In vitro experiments suggested that karenitecin is metabolized by CYP450 3A4, 2C8, and 2D6 isoenzymes and is an inhibitor of the CYP450 3A4 and 2C8 isoenzymes. The mean (± SD) percentage of karenitecin bound to plasma proteins was 99.1% ± 0.27%. The extent of karenitecin protein binding was directly proportional to the plasma concentration of AAG. Protein‐binding displacement interactions were observed in the in vitro experiments with phenobarbital, phenytoin, mitoxantrone, and salicylic acid. It was concluded that karenitecin has the potential to alter CYP450 3A4 and 2C8 drug‐metabolizing activity. In addition, in vitro PPB evaluations have demonstrated that karenitecin may displace other highly PPB drugs and that slight variations in plasma AAG concentration may result in large variations in free drug exposure. Each of these interactions could potentially result in increasing the toxicity or alter the efficacy of combination anticancer drug therapy if they are significant in patients. Future karenitecin clinical trials should include studies to monitor or evaluate the effects of these potential drug interactions on the overall toxicity of karenitecin when used in combination with other drugs.
A Review and Assessment of Potential Sources of Ethnic Differences in Drug Responsiveness
Authors: Bjornsson Thorir D. , Wagner John A. , Donahue Stephen R. , Harper Dawn , Karim Aziz , Khouri Marlene S. , Murphy William R. , Roman Kristin , Schneck Dennis , Sonnichsen Daryl S. , Stalker Dennis J. , Wise Stephen D. , Dombey Stuart , Loew Caroline
Published: September 2003

The International Conference on Harmonization (ICH) E5 guidelines were developed to provide a general framework for evaluating the potential impact of ethnic factors on the acceptability of foreign clinical data, with the underlying objective to facilitate global drug development and registration. It is well recognized that all drugs exhibit significant intersubject variability in pharmacokinetics and pharmacologic response and that such differences vary considerably among individual drugs and depend on a variety of factors. One such potential factor involves ethnicity. The objective of the present work was to perform an extensive review of the world literature on ethnic differences in drug disposition and responsiveness to determine their general significance in relation to drug development and registration. A few examples of suspected ethnic differences in pharmacokinetics or pharmacodynamics were identified. The available literature, however, was found to be heterologous, including a variety of study designs and research methodologies, and most of the publications were on drugs that were approved a long time ago.
Pharmacokinetics of Hydroxyurea in Plasma and Cerebrospinal Fluid of HIV‐1‐Infected Patients
Authors: Gwilt Peter R. , Manouilov Konstantine K. , McNabb JoCarol , Swindells Susan S.
Published: September 2003

Hydroxyurea has been shown to potentiate the activity of the antiretroviral nucleoside analogs. A significant complication of AIDS is invasion of the virus into the CNS, resulting in HIV‐associated dementia (HAD). Because of the polar nature of these nucleosides and the presence of efflux pumps in the blood‐brain barrier, only low CNS drug concentrations are achieved. Introduction of hydroxyurea into the CNS may therefore increase the antiviral activity of these drugs. This study evaluates the accessibility of hydroxyurea to the CNS following oral drug administration. Twelve HIV patients received 800 mg, 1000 mg, or 1200 mg oral hydroxyurea. Cerebrospinal fluid (CSF) and plasma drug concentrations were measured over 8 hours and simultaneously fitted to a pharmacokinetic model. It was determined that CSF hydroxyurea concentrations, corresponding to those found to increase antiretroviral nucleoside activity in vitro, were achieved.
Relationship of Arachidonic Acid Concentration to Cyclooxygenase‐Dependent Human Platelet Aggregation
Authors: Burke Joanne , Kraft Walter K. , Greenberg Howard E. , Gleave Melanie , Pitari Giovanni M. , VanBuren Sandi , Wagner John A. , Waldman Scott A.
Published: September 2003

Inhibition of ex vivo arachidonic acid (AA)–induced aggregation is a biomarker for the isotype selectivity of cyclooxygenase (COX) inhibitors since platelets express COX‐1 but not COX‐2. At low concentrations, there is broad inter‐ and intrasubject variability in AA‐induced aggregation of platelets ex vivo. This study defined a concentration that reliably induces aggregation without overcoming inhibition by therapeutic aspirin therapy (ASA, 81‐mg) treatment. Logistic regression analysis of ex vivo aggregation, induced with increasing concentrations of AA in platelet‐rich plasma (PRP), estimated that platelets from ≥ 90% of subjects would aggregate at ≥ 1.5 mM AA (95% confidence interval [CI], 1.1, 2.1). A concentration of 1.6 mM AA failed to aggregate platelets from 26 healthy volunteers, who had previously aggregated at this concentration, following six daily oral doses of 81 mg of ASA. These data demonstrate that 1.6 mM AA reproducibly induces platelet aggregation in PRP from healthy volunteers without overcoming the antiplatelet effect of daily low‐dose aspirin therapy.
Valdecoxib, a COX‐2‐Specific Inhibitor, Does Not Affect Cardiac Repolarization
Authors: Sarapa Nenad , Britto Margaret R. , Cotton Barbara , Cox Steven R. , Francom Steven F. , Jin Na , Polasek Emery D. , Sainati Stephen M. , Crosby‐Sessoms Sharon L. , Fleishaker Joseph C.
Published: September 2003

This double‐blind, four‐way crossover study assessed the effect of valdecoxib on the QTc interval duration in 25 male and 9 female healthy adults. Subjects received placebo or 40 mg, 80 mg, or 120 mg valdecoxib once daily for 5 days. Serial ECGs were obtained for 24 hours before the first treatment (baseline) and on the 5th day of each treatment. The study was statistically powered to detect a difference of ≥ 5.6 ms in the average daily QTc change from baseline and a ≥ 7.8‐ms difference in the average maximal daily change from baseline. No QTc prolongation versus placebo (Fridericia's or Bazett's correction) was observed for any valdecoxib dose. A 22% greater than proportional increase in valdecoxib AUC0–24 was observed over the 40‐ to 120‐mg dose range, supporting the conclusiveness of the negative QTc risk assessment even at supratherapeutic doses (up to three times the maximum recommended dose of 40 mg per day) and concentrations. In conclusion, repeated administration of doses up to 120 mg valdecoxib had no effect on cardiac repolarization in healthy volunteers, suggesting that chronic administration of valdecoxib to patients would not increase the risk from cardiac arrhythmia associated with QT prolongation.
Lack of Bioequivalence of Levofloxacin When Coadministered with a Mineral‐Fortified Breakfast of Juice and Cereal
Authors: Amsden Guy W. , Whitaker Anne‐Marie , Johnson Patrick W.
Published: September 2003

Previous studies have demonstrated that the chelation interactions demonstrated between fluoroquinolones and antacids also occur when they are coadministered with mineral‐fortified foods. This study was conducted to evaluate the bioequivalence of levofloxacin when administered in a fasting state as compared to when it was administered with a common breakfast of calcium‐fortified orange juice and ready‐to‐eat cereal. Fourteen of 16 healthy volunteers completed this study and received 500 mg of levofloxacin with each of the following: (1) 12 ounces of water, (2) subject‐measured portions of juice and cereal, and (3) subject‐measured portions of juice and cereal with milk. Plasma samples were collected prior to dosing and for up to 48 hours after. The results demonstrated that neither fed phase was bioequivalent to the fasting arm in terms of Cmax (with milk, 79.2% [72.6%, 85.7%]; without milk, 79.1% [73.3%, 84.9%]). In addition, a weak correlation was identified between the amount of change in 24‐hour exposure and mineral fortification. The results of this study further demonstrate a need to require additional fed‐fasted bioequivalence studies for drugs that demonstrate no interaction with the FDA meal but have significant interactions with drugs or supplements that contain large amounts of multivalent ions.
Universal prestorage leukoreduction in Canada decreases platelet alloimmunization and refractoriness
Authors: Seftel Matthew D. , Growe Gershon H. , Petraszko Tanya , Benny W. Barrett , Le Alan , Lee Chao-Yong , Spinelli John J. , Sutherland Heather J. , Tsang Peter , Hogge Donna E.
Published: September 2003

Randomized controlled trials have shown a reduction in platelet alloimmunization and refractoriness in patients with acute leukemia (AL) with the use of poststorage leukoreduction of blood products. Universal prestorage leukoreduction (ULR) of red cell and platelet products has been performed in Canada since August 1999. We conducted a retrospective analysis of 13 902 platelet transfusions in 617 patients undergoing chemotherapy (CT) for AL or stem cell transplantation (SCT) before (n = 315) and after (n = 302) the introduction of ULR. Alloimmunization was significantly reduced (19% to 7%, P < .001) in the post-ULR group. Alloimmune platelet refractoriness was similarly reduced (14% to 4%, P < .001). Fewer patients in the post-ULR group received HLA-matched platelets (14% vs 5%, P < .001). Alloimmunization and alloimmune refractoriness in the 318 patients who were previously pregnant and/or transfused were also reduced after ULR (P = .023 and P = .005, respectively). In a Cox regression model, the 3 independent factors that predicted for alloimmune refractoriness were nonleukoreduced blood products (relative risk [RR], 2.2 [95% CI, 1.2-4.3]), a history of pregnancy and/or transfusion (RR, 2.3 [95% CI, 1.3-4.2]), and receipt of 13 or more platelet transfusions (RR, 6.0 [95% CI, 2.4-15.3]). In conclusion, ULR reduces alloimmunization, refractoriness, and requirements for HLA-matched platelets when applied as routine transfusion practice to patients receiving CT or SCT.
Ephrin receptor, EphB4, regulates ES cell differentiation of primitive mammalian hemangioblasts, blood, cardiomyocytes, and blood vessels
Authors: Wang Zhengyu , Cohen Kenneth , Shao Ying , Mole Pamela , Dombkowski David , Scadden David T.
Published: September 2003

Differentiation of pluripotent embryonic stem (ES) cells is associated with expression of fate-specifying gene products. Coordinated development, however, must involve modifying factors that enable differentiation and growth to adjust in response to local microenvironmental determinants. We report here that the ephrin receptor, EphB4, known to be spatially restricted in expression and critical for organized vessel formation, modifies the rate and magnitude of ES cells acquiring genotypic and phenotypic characteristics of mesodermal tissues. Hemangioblast, blood cell, cardiomyocyte, and vascular differentiation was impaired in EphB4–/– ES cells in conjunction with decreased expression of mesoderm-associated, but not neuroectoderm-associated, genes. Therefore, EphB4 modulates the response to mesoderm induction signals. These data add differentiation kinetics to the known effects of ephrin receptors on mammalian cell migration and adhesion. We propose that modifying sensitivity to differentiation cues is a further means for ephrin receptors to contribute to tissue patterning and organization.
Microarray analysis reveals that TP53- and ATM-mutant B-CLLs share a defect in activating proapoptotic responses after DNA damage but are distinguished by major differences in activating prosurvival responses
Authors: Stankovic Tatjana , Hubank Mike , Cronin Debbie , Stewart Grant S. , Fletcher Danielle , Bignell Colin R. , Alvi Azra J. , Austen Belinda , Weston Victoria J. , Fegan Christopher , Byrd Philip J. , Moss Paul A. H. , Taylor A. Malcolm R.
Published: September 2003

The ATM/p53-dependent DNA damage response pathway plays an important role in the progression of lymphoid tumors. Inactivation of the ATM or TP53 gene is frequent in B-cell lymphocytic leukemia (B-CLL) and leads to aggressive disease. Although the ATM and p53 pathways overlap, they are not congruent, and it is unclear how the mechanism of tumor progression differs between ATM- and p53-deficient tumors. Using microarray analysis of ATM-mutant, TP53-mutant, and ATM/TP53 wild-type B-CLLs, we show that after exposure to DNA damage transcriptional responses are entirely dependent on ATM function. The p53 proapoptotic responses comprise only a part of ATM-regulated transcription; additionally, ATM regulates prosurvival responses independently of p53. Consequently, the greater severity of the TP53-mutant B-CLLs compared with ATM-mutant B-CLLs is consistent with the additive effect of defective apoptotic and elevated survival responses after DNA damage in these tumors. We also show that transcription expression profiles of ATM-deficient, TP53-deficient, and wild-type B-CLLs are indistinguishable before irradiation. Therefore, damage-induced transcriptional fingerprinting can be used to stratify tumors according to their biologic differences and simultaneously identify potential targets for treating refractory tumors.
Skewed representation of functionally distinct populations of virus-specific CD4 T cells in HIV-1–infected subjects with progressive disease: changes after antiretroviral therapy
Authors: Harari Alexandre , Petitpierre Stéphanie , Vallelian Florence , Pantaleo Giuseppe
Published: September 2003

HIV-1- and cytomegalovirus (CMV)-specific CD4 T-cell-mediated antiviral immunity was evaluated by assessing the frequency of interleukin 2 (IL-2)- and interferon γ (IFN-γ)-secreting cells following antigen-specific stimulation in blood and lymph node. HIV-1-infected subjects with progressive disease at early stage of infection with no previous history of antiretroviral therapy (ART), subjects with nonprogressive disease, and HIV-negative subjects were studied. On the basis of the ability to secrete IL-2 and IFN-γ, 3 functionally distinct populations of CD4 T cells were identified: (1) IL-2-secreting cells; (2) IL-2/IFN-γ-secreting cells; and (3) IFN-γ-secreting cells. CMV-specific CD4 T cells were almost equally distributed within the 3 functionally distinct cell populations in the 3 study groups as well as HIV-1-specific CD4 T cells in subjects with nonprogressive disease. However, a skewing toward IFN-γ-secreting cells (70% of HIV-1-specific CD4 T cells) was observed in subjects with progressive disease, and IL-2- and IL-2/IFN-γ-secreting cells were almost absent. The frequencies of IL-2- and of IL-2/IFN-γ-secreting HIV-1-specific CD4 T cells were negatively correlated with the levels of viremia. Interestingly, prolonged ART was able to correct the skewed representation of different populations of HIV-1-specific CD4 T cells but was associated with only a partial recovery of IL-2-secreting cells. These results indicate that the composition of the pool of functionally distinct virus-specific CD4 T cells is important for virus control. (Blood. 2004;103:966-972)
BEAM-alemtuzumab reduced-intensity allogeneic stem cell transplantation for lymphoproliferative diseases: GVHD, toxicity, and survival in 65 patients
Authors: Faulkner Rowena D. , Craddock Charles , Byrne Jennifer L. , Mahendra Prem , Haynes Andrew P. , Prentice Hugh G. , Potter Michael , Pagliuca Antonio , Ho Aloysius , Devereux Stephen , McQuaker Grant , Mufti Ghulam , Yin John Liu , Russell Nigel H.
Published: September 2003

We report the outcomes of reduced-intensity allogeneic stem cell transplantation using BEAM-alemtuzumab conditioning (carmustine, etoposide, cytosine arabinoside, melphalan, and alemtuzumab 10 mg/d on days –5 to –1) in 6 United Kingdom transplant centers. Sixty-five patients with lymphoproliferative diseases underwent sibling (n = 57) or matched unrelated donor (n = 8) transplantation. Sustained donor engraftment occurred in 60 (97%) of 62 patients. Of the 56 patients undergoing chimerism studies, 35 (63%) had full donor chimerism. Overall, 73% were in complete remission (CR) after transplantation. At a median follow-up of 1.4 years (range, 0.1-5.6 years), 37 remain alive and in CR. Acute graft-versus-host disease (GVHD) occurred in 11 (17%) of 64, grades I-II only. Estimated 1-year transplantation-related mortality (TRM) was 8% for patients undergoing first transplantation but was significantly worse for those who had previously undergone autologous transplantation. Six patients relapsed (estimated 2-year relapse risk, 20%). Histologic diagnosis (mantle cell lymphoma and high-grade non-Hodgkin lymphoma) and age at transplantation (> 46 years) were significantly associated with higher relapse risk and worse event-free survival. Relapse did not occur in any patient who developed acute or chronic GVHD. This study demonstrates that reduced-intensity allogeneic stem cell transplantation for lymphoproliferative diseases using a BEAM-alemtuzumab preparative regimen is associated with sustained donor engraftment, a high response rate, minimal toxicity, and a low incidence of GVHD.
ST1926, a novel and orally active retinoid-related molecule inducing apoptosis in myeloid leukemia cells: modulation of intracellular calcium homeostasis
Authors: Garattini Enrico , Parrella Edoardo , Diomede Luisa , Gianni' Maurizio , Kalac Yesim , Merlini Lucio , Simoni Daniele , Zanier Romina , Ferrara Fabiana Fosca , Chiarucci Ilaria , Carminati Paolo , Terao Mineko , Pisano Claudio
Published: September 2003

Retinoid-related molecules (RRMs) are derivatives of retinoic acid and promising antileukemic agents with a mechanism of action different from that of other common chemotherapeutics. Here, we describe a novel chemical series designed against the RRM prototype, CD437. This includes molecules with apoptotic effects in acute promyelocytic leukemia and other myelogenous leukemia cell lines, as well as ST2065, an RRM with antagonistic properties. The most interesting apoptotic agent is ST1926, a compound more powerful than CD437 in vitro and orally active in vivo on severe combined immunodeficiency (SCID) mice that received transplants of NB4 cells. ST1926 has the same mechanism of action of CD437, as indicated by the ability to trans-activate retinoic acid receptor γ, to induce the phosphorylation of p38 and JNK, and to down-regulate the expression of many genes negatively modulated by CD437. ST1926 causes an immediate increase in the cytosolic levels of calcium that are directly related to the apoptotic potential of the RRMs considered. The intracellular calcium elevation is predominantly the result of an inhibition of the mitochondrial calcium uptake. The phenomenon is blocked by the ST2065 antagonist, the intracellular calcium chelator BAPTA (1,2 bis (2-aminophenoxy) ethane-N, N, N′,N′-tetraacetic acid tetrakis (acetoxymethyl ester), and by high concentrations of calcium blockers of the dihydropyridine type, compounds that suppress ST1926-induced apoptosis.
Transplanted adult hematopoietic stems cells differentiate into functional endothelial cells
Authors: Bailey Alexis S. , Jiang Shuguang , Afentoulis Michael , Baumann Christina I. , Schroeder David A. , Olson Susan B. , Wong Melissa H. , Fleming William H.
Published: September 2003

During early embryogenesis, blood vessels and hematopoietic cells arise from a common precursor cell, the hemangioblast. Recent studies have identified endothelial progenitor cells in the peripheral blood, and there is accumulating evidence that a subset of these cells is derived from precursors in the bone marrow. Here we show that adult bone marrow–derived, phenotypically defined hematopoietic stem cells (c-kit+, Sca-1+, lineage–) give rise to functional endothelial cells. With the exception of the brain, donor-derived cells are rapidly integrated into blood vessels. Durably engrafted endothelial cells express CD31, produce von Willebrand factor, and take up low-density lipoprotein. Analysis of DNA content indicates that donor-derived endothelial cells are not the products of cell fusion. Self-renewal of stem cells with hematopoietic and endothelial cell potential was revealed by serial transplantation studies. The clonal origin of both hematopoietic and endothelial cell outcomes was established by the transfer of a single cell. These results suggest that adult bone marrow–derived hematopoietic stem cells may serve as a reservoir for endothelial cell progenitors.
FOP-FGFR1 tyrosine kinase, the product of a t(6;8) translocation, induces a fatal myeloproliferative disease in mice
Authors: Guasch Géraldine , Delaval Bénédicte , Arnoulet Christine , Xie Min-Jue , Xerri Luc , Sainty Danielle , Birnbaum Daniel , Pébusque Marie-Josèphe
Published: September 2003

Constitutive activation of aberrant fibroblast growth factor receptor 1 (FGFR1) kinase as a consequence of gene fusion such as FOP-FGFR1 associated with t(6; 8)(q27;p11-12) translocation, is the hallmark of an atypical aggressive stem cell myeloproliferative disorder (MPD) in humans. In this study, we show that expression of FOP-FGFR1 in primary bone marrow cells induced by retroviral transduction generates a MPD in mice. Constitutive FOP-FGFR1 kinase activity was both essential and sufficient to cause a chronic myeloproliferative syndrome in the murine bone marrow transplantation model. In contrast to the human disorder, lymphoproliferation and progression to acute phase were not observed. Lymphoid symptoms, however, appeared when onset of the disease was delayed as the result of mutation of FOP-FGFR1 at tyrosine 511, the phospholipase C γ (PLCγ) binding site.
Role of MTHFR genetic polymorphisms in the susceptibility to childhood acute lymphoblastic leukemia
Authors: Krajinovic Maja , Lamothe Stéphanie , Labuda Damian , Lemieux-Blanchard Émilie , Théorêt Yves , Moghrabi Albert , Sinnett Daniel
Published: September 2003

The central role of methylenetetrahydrofolate reductase (MTHFR) in the folate metabolism renders MTHFR gene polymorphisms (C677T and A1298C) potential modulators of a variety of disorders whose development depends on folate/homocysteine imbalance. Here, we provide additional evidence on the protective role of these polymorphisms in acute lymphoblastic leukemia (ALL), the most common pediatric cancer. A case-control study was conducted in 270 ALL patients and 300 healthy controls of French-Canadian origin. The TT677/AA1298 and CC677/CC1298 individuals were associated with reduced risk of ALL (crude odds ratio [OR] = 0.4; 95% confidence interval [CI], 0.2-0.9; and OR = 0.3; 95% CI, 0.1-0.6; respectively). Further stratification in patients born before and after January 1996 (approximate time of Health Canada recommendation for folic acid supplement in pregnancy) revealed that the protective effect of MTHFR variants is accentuated and present only in children born before 1996. Similar results were obtained when a transmission disequilibrium test was performed on a subset of children (n = 95) in a family-based study. This finding suggests gene-environment interaction and its role in the susceptibility to childhood ALL, which is consistent with previous findings associating either folate deficiency or MTHFR polymorphisms with risk of leukemia.
Jun N-terminal kinase activity and early growth-response factor-1 gene expression are down-regulated in Fanconi anemia group A lymphoblasts
Authors: Pipaon Carlos , Casado Jose Antonio , Bueren Juan Antonio , Fernandez-Luna Jose Luis
Published: September 2003

Fanconi anemia (FA) is an autosomal recessive cancer susceptibility syndrome characterized by cellular sensitivity to genotoxic agents. In recent years, FA proteins have been associated with different molecules involved in signal transduction, which has raised the interest in FA-dependent signaling pathways. Here, we report that the c-Jun N-terminal kinase (JNK) fails to phosphorylate in response to UV radiation and treatment with mitomycin C in FA lymphoblast cells derived from type A patients (FA-A). Furthermore, defective kinase activity seems to be specific for JNK, because extracellular signal-regulated kinase (ERK) responded to the proper stimuli in FA-A cells. We also demonstrate that the early growth-response factor-1 (Egr-1), a JNK downstream target gene that is normally induced by genotoxic stress, is not upregulated in UV-treated FA-A cells. Moreover, FA-A cells are more sensitive to apoptosis than control lymphoblasts. Both JNK and Egr-1 may be part of a pathway triggered by FA proteins, because functional correction of FA-A cells by gene transfer restores, at least in part, JNK activation and Egr-1 expression after UV exposure. Together, our data suggest that activation of JNK and expression of Egr-1 gene in B lymphoblasts mediate a cellular response to genotoxic agents that may be induced by FA proteins.
Discrepancies between clinical and postmortem diagnoses in critically ill patients: an observational study
Authors: Perkins Gavin D , McAuley Danny F , Davies Sarah , Gao Fang
Published: September 2003

Introduction

The autopsy has long been regarded as an important tool for confirming the clinical cause of death, education and quality assurance. Concerns surrounding informed consent and the retention of organs have heightened clinicians' anxieties in requesting permission to perform an autopsy. The present study was conducted to determine whether the autopsy still has a role to play in extending knowledge about the cause of death in a group of patients who died while receiving intensive care.

Method

We retrospectively investigated trends in postmortem examination rates and discrepancies between premortem clinical and postmortem diagnoses in a population of critically ill patients admitted to a 13 bed, general medical/surgical intensive care unit between January 1998 and June 2001. Agreement between diagnoses before death and postmortem findings were compared using the Goldman system.

Results

Out of total 636 deaths, 49 (7.7%) underwent postmortem examinations. Of these, 38 (78%) cases were available for review. We found that postmortem findings were in complete agreement with predeath diagnoses in fewer than half of the cases (n = 17, 45%). Major missed diagnoses were present in 15 cases (39%). Myocardial infarction, carcinoma and pulmonary embolism represented the most frequently missed diagnoses.

Conclusion

Postmortem examinations remain a useful tool in confirming diagnostic accuracy and should be considered in all patients who die in the intensive care unit. Recognition of the diagnoses missed before death may improve outcome or avoid unnecessary prolongation of life where terminal disease is present.
Smooth and rough lipopolysaccharide phenotypes ofBrucellainduce different intracellular trafficking and cytokine/chemokine release in human monocytes
Authors: Rittig Michael G , Kaufmann Andreas , Robins Adrian , Shaw Barry , Sprenger Hans , Gemsa Diethard , Foulongne Vincent , Rouot Bruno , Dornand Jacques
Published: September 2003

Virulence of the intracellular pathogen Brucella for humans is mainly associated with its lipopolysaccharide (LPS) phenotype, with smooth LPS phenotypes generally being virulent and rough ones not. The reason for this association is not quite understood. We now demonstrate by flow cytometry, electron microscopy, and ELISA that human peripheral blood monocytes interact both quantitatively and qualitatively different with smooth and rough Brucella organisms in vitro. We confirm that considerably higher numbers of rough than smooth brucellae attach to and enter the monocytes in nonopsonic conditions; but only smooth brucellae replicate in the host cells. We show for the first time that rough brucellae induce higher amounts than smooth brucellae of several CXC (GRO-α, IL-8) and CC (MIP-1α, MIP-1β, MCP-1, RANTES) chemokines, as well as pro- (IL-6, TNF-α) and anti-inflammatory (IL-10) cytokines released by challenged monocytes. Upon uptake, phagosomes containing rough brucellae develop selective fusion competence to form spacious communal compartments, whereas phagosomes containing smooth brucellae are nonfusiogenic. Collectively, our data suggest that rough brucellae attract and infect monocytes more effectively than smooth brucellae, but only smooth LPS phenotypes establish a specific host cell compartment permitting successful parasitism. These novel findings link the LPS phenotype of Brucella and its virulence for humans at the level of the infected host cells. Whether this is due to a direct effect of the LPS molecules or to upstream bacterial mechanisms remains to be established.
Neutrophil activation by fMLP regulates FOXO (forkhead) transcription factors by multiple pathways, one of which includes the binding of FOXO to the survival factor Mcl-1
Authors: Crossley Lisa J
Published: September 2003

Activation signals from bacterial stimuli set into motion a series of events that alter the abbreviated lifespan of neutrophils. These studies show that the bacterial chemoattractant, formyl-Met-Leu-Phe (fMLP), promotes the phosphorylation/inactivation of the FOXO subfamily of forkhead transcription factors (FKHR, FKHR-L1, and AFX) through the phosphatidylinositol-3-kinase/Akt (protein kinase B) and the RAS mitogen-activated protein kinase pathways. Furthermore, fMLP stimulation causes the inducible expression of the prosurvival Bcl-2 family member Mcl-1, which then binds to a complex containing FKHR. These studies show that fMLP-stimulated neutrophils coordinate the regulation of FOXO transcription factors and the survival factor Mcl-1, a mechanism that may allow neutrophils to alter their survival.
Surfactant protein A enhances Mycobacterium avium ingestion but not killing by rat macrophages
Authors: Lopez Joseph P , Clark Emily , Shepherd Virginia L
Published: September 2003

Mycobacterium avium complex (MAC) is a significant cause of opportunistic infection in patients with acquired immunodeficiency syndrome. Although the major route of entry of MAC is via the gastrointestinal tract, MAC can infect humans through the respiratory tract and eventually encounter alveolar macrophages within the lung. Once in the lung, MAC can potentially interact with surfactant protein A (SP-A), an important component of the pulmonary innate-immune response. Previous work on other pulmonary pathogens including Mycobacterium bovis Bacillus Calmette-Guerin (BCG) suggests that SP-A participates in promoting efficient clearance of these organisms by alveolar macrophages. In the present study, we investigated the role of SP-A in clearance of MAC by cultured rat macrophages. SP-A bound to MAC organisms and enhanced the ingestion of the mycobacteria by macrophages. Infection of macrophages with SP-A-MAC complexes induced the production of nitric oxide (NO) and tumor necrosis factor-α. However, intracellular survival of MAC was not altered by preopsonization with SP-A. In addition, inhibitors of inducible NO synthase did not alter MAC clearance. These results suggest that SP-A can bind to and enhance the uptake of MAC by alveolar macrophages, similar to previous findings with BCG and Mycobacterium tuberculosis.However, unlike BCG and other pulmonary pathogens that are cleared effectively in the presence of SP-A via a NO-dependent pathway, macrophage-mediated clearance of MAC is not enhanced by SP-A.
Eosinophil peroxidase catalyzes JNK-mediated membrane blebbing in a Rho kinase-dependent manner
Authors: McElhinney Brian , Poynter Matthew E , Shrivastava Punya , Hazen Stanley L , Janssen-Heininger Yvonne M W
Published: September 2003

Eosinophilic influx is characteristic of numerous inflammatory conditions. Eosinophil peroxidase (EPO) is a major enzyme present in eosinophils and upon degranulation, becomes released into the airways of asthmatics. As a result of its cationic nature and its ability to catalyze the formation of highly toxic oxidants, EPO has significant potential to induce cellular injury. The focus of the present study was to determine the cell-signaling events important in EPO-induced death of lung epithelial cells. In the presence of hydrogen peroxide and nitrite (NO2−; hereafter called EPO with substrates), EPO catalyzes the formation of nitrogen dioxide. EPO with substrates induced rapid and sustained activation of c-Jun-NH2-terminal kinase (JNK) and led to cell death, as was evidenced by enhanced mitochondrial depolarization, cytochrome c release, cleavage of caspases 9 and 3, poly-adenosine 5′-diphosphate ribosylation of proteins, the formation of single-stranded DNA, and membrane permeability. Moreover, EPO with substrates caused Rho-associated coiled coil-containing kinase-1-dependent dynamic membrane blebbing. Inhibition of JNK activity in cells expressing a dominant-negative JNK-1 construct (JNK-APF) prevented mitochondrial membrane depolarization and substantially decreased the number of cells blebbing compared with vector controls. The cellular responses to EPO with substrates were independent of whether NO2−, bromide, or thiocyanide was used as substrates. Our findings demonstrate that catalytically active EPO is capable of causing significant damage to lung epithelial cells in vitro and that this involves the activation of JNK.
CCR4 blockade does not inhibit allergic airways inflammation
Authors: Conroy Dolores M , Jopling Louise A , Lloyd Clare M , Hodge Martin R , Andrew David P , Williams Timothy J , Pease James E , Sabroe Ian
Published: September 2003

The CC chemokine receptor 4 (CCR4) shows selectivity for the recruitment of memory T cell subsets, including those of the T helper cell type 2 (Th2) phenotype. In humans, CCR4+ T cells are recruited to the asthmatic lung in response to allergen challenge; however, the contribution of this pathway to allergic disease remains uncertain. We therefore investigated the role of CCR4 in allergic airways inflammation in the guinea pig. Blockade of CCR4 with a specific antibody resulted in only minor changes in numbers of CCR4+ Th cells in the bronchoalveolar lavage fluid of allergen-challenged guinea pigs and failed to inhibit the generation of eotaxin/CC chemokine ligand (CCL)11 or macrophage-derived chemokine/CCL22 or the recruitment of inflammatory leukocytes to the lung. These data suggest that although CCR4 was originally proposed as a marker of Th2 status, antigen-specific Th2 cells are recruited to the lung predominantly by other pathways. This study casts doubts on the validity of CCR4 as a therapeutic target in the treatment of asthma.
The role of TNF–TNFR2 interactions in generation of CTL responses and clearance of hepatic adenovirus infection
Authors: Kafrouni Michel I , Brown Geri R , Thiele Dwain L
Published: September 2003

Deficiency or inhibition of tumor necrosis factor (TNF) significantly prolongs hepatic expression of recombinant adenoviral vectors. To explore mechanisms responsible for this observation, the present studies examined the effects of TNF versus TNF receptor 1 (TNFR1) or TNFR2 deficiency on the course of antiviral-immune responses to a replication-deficient, β-galactosidase-encoding recombinant adenovirus (AdCMV-lacZ). Clearance of AdCMV-lacZ was significantly delayed in TNF-deficient mice. Less pronounced but significant delays in AdCMV-lacZ clearance were observed in TNFR2-deficient but not TNFR1-deficient mice. Numbers of interferon-γ expressing intrahepatic lymphocytes (IHL) were similar in AdCMV-lacZ-infected, TNF-deficient, TNFR1-deficient, TNFR2-deficient, and control mice. However, IHL isolated from AdCMV-lacZ-infected, TNF-deficient or AdCMV-lacZ-infected, TNFR2-deficient mice exhibited decreased levels of FasL expression and adenovirus-specific cytolytic T lymphocyte (CTL) activity. Similar defects in allo-specific killing of Fas-sensitive hepatocyte targets by TNF-deficient or TNFR2-deficient but not TNFR1-deficient CTL were also noted. No defects in generation of allo-specific cytotoxicity directed against perforin-sensitive target cells were noted in TNF-, TNFR1-, or TNFR2-deficient lymphocytes. These findings indicate that TNF/TNFR2 interactions facilitate generation of FasL-dependent CTL effector pathways that play an important role in in vivo antiviral-immune responses in the liver.
Convergent alteration of granulopoiesis, chemotactic activity, and neutrophil apoptosis during mouse selection for high acute inflammatory response
Authors: Ribeiro Orlando G , Maria Durvanei A , Adriouch Sahil , Pechberty Séverine , Cabrera Wafa H K , Morisset Jean , Ibañez Olga M , Seman Michel
Published: September 2003

Neutrophil homeostasis was investigated in two mouse lines, AIRmax and AIRmin, genetically selected for high or low acute inflammatory response (AIR) and compared with unselected BALB/c mice. Mature neutrophil phenotype and functions appeared similar in the three mouse lines. However, an unprecedented phenotype was revealed in AIRmax animals characterized by a high neutrophil production in bone marrow (BM), a high number of neutrophils in blood, a high concentration of chemotactic agents in acrylamide-induced inflammatory exudates, and an increased resistance of locally infiltrated neutrophils to spontaneous apoptosis. In vitro, BM production of neutrophils and eosinophils was accompanied by an unusual high up-regulation of cytokine receptors as assessed by antibodies to CD131, which bind the common β chain of receptors to interleukin (IL)-3, IL-5, and granulocyte macrophage-colony stimulating factor. An accelerated neutrophil maturation was also observed in response to all-trans retinoic acid. Several candidate genes can be proposed to explain this phenotype. Yet, more importantly, the results underline that genetic selection, based on the degree of AIR and starting from a founding population resulting from the intercross of eight inbred mouse lines, which display a continuous range of inflammatory responses, can lead to the convergent selection of alleles affecting neutrophil homeostasis. Similar gene combinations may occur in the human with important consequences in the susceptibility to inflammatory or infectious diseases and cancer.
TNF revisited: TNF-independent antitumor activity in sera of mice sensitized withPropionibacterium acnesand challenged with lipopolysaccharide
Authors: Schwamberger Günter , Hammerl Peter , Ferber Ernst , Freudenberg Marina , Galanos Chris
Published: September 2003

Sera of mice sensitized with bacteria and subsequently challenged with lipopolysaccharide promote hemorrhagic necrosis of tumors in vivo and display cytotoxic activity against tumor cells in vitro, which has been attributed to the induction of tumor necrosis factor (TNF). Here, we describe the induction of a previously unrecognized antitumor activity in such sera, which is distinct from TNF but displays tumor-specific cytocidal activity in vitro as well as potent tumor-regressing activity in vivo. Biochemical analysis of this activity yielded a molecular mass of ∼150 kDa, closely resembling a novel tumoricidal factor of murine macrophages (Mφ) termed MTC 170 (Mφ tumor cytotoxin, approximate molecular mass 170 kDa), which we have previously proposed to constitute a major effector pathway for the destruction of tumor cells by activated Mφ.
CD40-mediated up-regulation of Toll-like receptor 4-MD2 complex on the surface of murine dendritic cells
Authors: Frleta Davor , Noelle Randolph J , Wade William F
Published: September 2003

Toll-like receptors (TLRs) recognize pathogen-associated molecular patterns, which are non-self macromolecular components of pathogens that allow the innate-immune system to recognize infection. TLRs are expressed on macrophages and dendritic cells (DC). TLR stimulation or CD40 agonists can induce inflammatory cytokine secretion from macrophages and DC, and promote DC maturation. The regulation of TLR expression by inflammation has begun to be explored. Our studies have focused on the regulation of TLR4 surface expression on DC. TLR4, along with the adaptor molecule MD2, is involved in the recognition of lipopolysaccharide (LPS). CD40 stimulation via cross-linked anti-CD40 monoclonal antibody (mAb) up-regulates TLR4-MD2 surface expression on a DC cell line (DC2.4) and on ex vivo-cultured splenic DC. LPS treatment down-regulated surface TLR4-MD2 on DC2.4 cells, but if combined with anti-CD40 mAb, increased TLR4-MD2 expression was observed. The increased TLR4-MD2 surface expression by any treatment did not correlate with TLR4 mRNA levels. The functional consequence of increased TLR4-MD2 expression following LPS and anti-CD40 treatment was examined. Although CD40 prestimulation did slightly enhance interleukin-12p70 secretion after LPS restimulation, simultaneous anti-CD40 mAb and LPS treatment, which up-regulates TLR4-MD2 complex, does not restore DC responsiveness to subsequent LPS.