bluuebunny/crossref_metadata_2025 huggingface dataset:
Hyperhomocysteinemia in l‐dopa treated Parkinson's disease patients: effect of cobalamin and folate administration
Authors: Lamberti P. , Zoccolella S. , Armenise E. , Lamberti S. V. , Fraddosio A. , de Mari M. , Iliceto G. , Livrea P.
Published: March 2005

Homocysteine (Hcy) is a risk factor for vascular diseases, cognitive impairment and dementia. l‐dopa treatment may represent an acquired cause of hyperhomocysteinemia (HHcy), as evidenced by studies in rats as well as in Parkinson's disease (PD) patients. Folate and cobalamin status also seems to influence the effects of l‐dopa on plasma Hcy levels; therefore B‐vitamins supplementation has been proposed to reduce the HHcy in l‐dopa treated PD patients. Plasma Hcy, folate, and cobalamin levels were evaluated in 20 PD patients treated with l‐dopa in the baseline condition and following a 5‐week period of treatment with cobalamin and folate; results were compared with 35 controls. Analysis of data revealed that Hcy levels were higher in l‐dopa treated PD patients when compared with age‐ and sex‐matched controls and that supplementation of the diet with cobalamin and folate is effective in reducing Hcy concentrations; these findings may have important implications in the treatment of PD patients who are potentially at risk for vascular diseases and cognitive impairment or dementia.
Autonomic nervous system laboratories: a European survey
Authors: Lahrmann H. , Magnifico F. , Haensch C. A. , Cortelli P.
Published: March 2005

We present a questionnaire‐based survey on methods adopted in European autonomic nervous system (ANS) laboratories in the year 2002. Seventy‐nine laboratories from 16 countries in Western and Eastern Europe provided information on test methods, equipment, staff, educational programme and research activities. The results confirmed the anticipated heterogeneity of techniques used to investigate ANS disorders. However, cardiovascular (84.8%) and sudomotor (43.0%) tests were the most common. There were no standards regarding equipment, but devices used varied widely, in part commercially available, in part self‐developed. Some ANS laboratories performed cardiovascular tests using standard EMG devices with implemented routines to measure heart rate variability. Many investigators used published normative data (75.4%). Most of the responders were involved in scientific work, half were active in education of trainees. A wide variation was found in number and profession of staff members. As more than 60% of questionnaires were returned by neurologists our results may be limited to neurological ANS laboratories. Given the wide variation of methods and equipment used in European ANS laboratories, minimal standards for routine ANS testing are desirable. Standards may help to compare results of diagnostic tests, evaluate equipment and test laboratory methods and commercial devices and set‐up educational programmes.
Protein L‐agarose for Adsorption of Autoantibodies: A Potential Tool for Extracorporeal Treatment
Authors: Duarte Isa Santos , Zollner Ricardo de Lima , Bueno Sonia Maria Alves
Published: March 2005

Abstract:  This work investigated the adsorption of autoantibodies such as anti‐SS‐A/Ro, anti‐SS‐B/La, anti‐Sm, and anti‐dsDNA on protein L‐agarose gel. In order to determine better conditions for IgG adsorption on this matrix, some buffer systems were tested. Adsorption data were analyzed using the Langmuir and Langmuir–Freundlich isotherm models. The experimental isotherms were best described by the Langmuir–Freundlich model, which indicated negative and positive cooperativities for binding in the presence of PBS and HEPES buffers, respectively. The Kd values for phosphate buffered saline solution (PBS) and hydroxyethylpiperazine ethanesulfonic acid (HEPES) were 2.8 × 10−7 M and 3.2 × 10−7 M, respectively, which indicate a high affinity between IgG and the immobilized protein L. The amount of protein adsorbed per amount of protein loaded was high for anti‐Sm (44%) and anti‐dsDNA (46%), but low for anti‐SS‐B/La (9%). The amount of albumin adsorbed was lower than 0.06 mg/mL, which may  remove  the  need  for  a  plasma  replacement  solution  in   clinical   apheresis.
Influence of Growth Hormone on Maintenance of Capillary‐like Structures in an In Vitro Model of Stromal Vascular Tissue—Results From Morphometric Analysis
Authors: Frerich Bernhard , Kurtz‐Hoffmann Jan , Lindemann Nico
Published: March 2005

Abstract:  The in vitro development of a vascular stroma might be a solution for the engineering of vascularized tissues, however, in vitro stability of capillary‐like structures is limited. In order to test the influence on maintenance of capillary‐like structures, human growth hormone (hGH) was added in concentrations of 0.5, 5, 50, and 500 ng/mL in an in vitro model of stromal vascular tissue. The angiogenic response and maintenance of capillary‐like structures were analyzed by means of confocal laser scanning microscopy (CLSM) and image analysis after 8, 16, and 32 days of culture. The highest angiogenic response was observed with a concentration of 50 ng/mL hGH. With the addition of 50 and 500 ng/mL, the length of capillary‐like structures could be maintained on high levels up to the 32nd day of culture, whereas with 5 ng/mL values dropped to the level of the control group. The proposed technique of analysis allows quantification of capillary‐like network formation and might be useful for tissue engineering applications.
A Study of the Emergence of Cooperation in Mediation
Authors: Poitras Jean
Published: March 2005

All mediators confront the challenge of how to encourage cooperation among parties in a mediation. Based on a phenomenological study of workplace mediation, this article explores the variables that are linked to the emergence of cooperation between parties. In the first part of the study, factors influencing the desire to cooperate are identified and categorized, based upon whether they help or hinder cooperation. The second part of the study compares the characteristics of cooperative and antagonistic mediation climates in order to better understand how cooperation is established during the mediation process. Based on the findings, strategies are proposed to help mediators facilitate the transition of an antagonistic climate into a cooperative one and thereby encourage the emergence of cooperation.
Occludin as direct target for glucocorticoid‐induced improvement of blood–brain barrier properties in a murinein vitrosystem
Authors: Förster Carola , Silwedel Christine , Golenhofen Nikola , Burek Malgorzata , Kietz Silke , Mankertz Joachim , Drenckhahn Detlev
Published: March 2005

Homeostasis of the central nervous system (CNS) microenvironment is essential for its normal function. It is maintained by the blood–brain barrier (BBB) which regulates the transport of molecules from blood into brain and backwards. The integrity of the BBB is compromised in many disorders of the human CNS; therapeutical strategies for several of these diseases include treatment with glucocorticoids, but the molecular basis of how glucocorticoids regulate BBB permeability is not understood. Here, we report the generation and characterization of a murine immortalized brain (cerebral) capillary endothelial (cEND) cell line which expresses the BBB marker occludin at intercellular tight junctions (TJ). Hydrocortisone at physiological concentrations induced upregulation of occludin, accompanied by a threefold enhancement of transendothelial electrical resistance to values up to 1000 Ωcm2. Insulin enhanced the glucocorticoid response. At the molecular level, hydrocortisone induces increase of occludin at protein and mRNA levels by activation of the glucocorticoid receptor (GR) and its binding to putative glucocorticoid responsive elements in the occludin promoter. At the same time, insulin potentiated the ligand‐dependent GR transactivation via induction of the GR in thisin vitrosystem. This study thus provides insights into the molecular processes of barrier genesis, and may help to elucidate mechanisms of brain pathology at the microvascular level.
Null mutation of myeloperoxidase in mice prevents mechanical activation of neutrophil lysis of muscle cell membranes in vitro and in vivo
Authors: Nguyen Hal X. , Lusis Aldons J. , Tidball James G.
Published: March 2005

Membrane lysis is a common and early defect in muscles experiencing acute injuries or inflammation. Although increased mechanical loading of muscles can induce inflammation and membrane lysis, whether mechanical loads applied to muscle can promote the activation and cytolytic capacity of inflammatory cells and thereby increase muscle damage is unknown. We tested whether mechanical loads applied to mouse muscle cells in vitro can increase membrane lysis, and whether neutrophil‐mediated lysis of muscle cells is promoted by mechanical loads applied in vitro and in vivo. Cyclic loads applied to muscle cells for 24 h in vitro produced little muscle cell lysis. Similarly, the addition of neutrophils to muscle cell cultures in the presence of superoxide dismutase (SOD) produced little muscle cell lysis. However, when cyclic mechanical loads were applied to neutrophil–muscle co‐cultures in the presence of SOD, there was a synergistic effect on muscle cell lysis, suggesting that mechanical loading activates neutrophil cytotoxicity. However, application of mechanical loads to co‐cultures of muscle cells and neutrophils that are null mutants for myeloperoxidase (MPO) showed no mechanical activation of neutrophil cytotoxicity. This indicates that loading promotes neutrophil cytotoxicity via MPO. Activity assays confirmed that mechanical loading of neutrophil–muscle co‐cultures significantly increased MPO activity. We further tested whether muscle membrane lysis in vivo was mediated by neutrophils when muscle was subjected to modified loading by using a mouse model of muscle reloading following a period of unloading. We observed that MPO −/− soleus muscles showed a significant 52% reduction in membrane lysis compared to wild‐type mice, although the mutation did not decrease inflammatory cell extravasation. Together, these in vitro and in vivo findings show that mechanical loading activates neutrophil‐mediated lysis of muscle cells through an MPO‐dependent pathway.
Simplified Kinematic Analysis of Bevel Epicyclic Gear Trains With Application to Power-Flow and Efficiency Analyses
Authors: Nelson Carl A. , Cipra Raymond J.
Published: March 2005

A kinematic analysis technique is introduced to find the angular velocities of all links in bevel epicyclic gear trains. The method relies on previous work in graph theory. It improves on existing techniques used for analysis of planar geared mechanisms in its ability to accurately solve the kinematics of spatial geared mechanisms, particularly bevel gear trains, in a simpler manner. Usefulness of the method is demonstrated through its application to power-flow and efficiency analyses as well as its implementation in computer software. This discussion is limited to gear trains whose input and output axes are collinear, such as automotive automatic transmissions.
Meshing Analysis of the Planetary Indexing Cam Mechanisms
Authors: Wang Hao , Zhang Ce , Lin and Zhongqin , Chen Guanlong
Published: March 2005

The planetary indexing cam mechanism is a type of indexing cam mechanism that employs planetary transmission structure. The mechanism is unusual compared to other planar cam mechanisms for the self-intersection occurs inevitably on the pitch curve of the cam. This results in the loss of contacts between a few rollers and cam profile. This paper investigates the effects of such loss of contacts on the transmission by analyzing the meshing procedure of the mechanism. The number of the self-intersection points on the pitch curve is determined in terms of the rotary index of the continuous curve. The meshing procedure is analyzed in detail and the pressure angles are calculated. The effects of the design parameters on the meshing status are studied and illustrated. At the end, it is pointed out that loss of contacts will not hamper the rightness of motion transfer in the mechanism if the design parameters are well selected.
Dynamic Modeling and Simulation of a Dual-Clutch Automated Lay-Shaft Transmission
Authors: Zhang Y. , Chen X. , Zhang X. , Jiang H. , Tobler W.
Published: March 2005

This paper presents a systematic model for the simulation and analysis of a power-shift transmission that features a dual-clutch design. The paper models the kinematics, dynamics, and control of the transmission for the analysis of powertrain overall performance and shift transient characteristics. The model is implemented in an object-oriented software tool. Analytical formulations and look-up tables are both used for modeling of powertrain components. The vehicle system model is established by integrating the various components and subsystem models according to the transmission power flow and control logic. The input to the simulation model is the vehicle speed-time profile and the output provides the speed follow-up, engine, and clutch operation status for the drive range and shift processes involved. As a numerical example, the model is used for a vehicle equipped with the power-shift transmission to simulate the speed follow-up over a specified drive range and the dynamic transients during shifts.© 2004 American Institute of Physics.
Automated Hierarchical Assembly System Construction in Automobile Body Assembly Planning
Authors: Wang Wurong , Chen Guanlong , Lin Zhonqin , Lai Xinmin
Published: March 2005

DFA (design for assembly) is an approach to designing products with ease of assembly in mind. Multilayer assembly system construction and possible assembly sequencing are important information for DFA, especially at the prototype design stage of an automobile. This paper aims to help automobile designers construct a hierarchical assembly system, develop a systematic approach to automated subassembly detection, and assembly sequencing in automobile body assembly planning. Modeling of an automobile assembly is given, based on precedence knowledge among automobile parts. Algorithms to detect possible subassemblies with two-step verification and to generate all available sequences are also developed in this study.
Simulation of Nanoscale Multidimensional Transient Heat Conduction Problems Using Ballistic-Diffusive Equations and Phonon Boltzmann Equation
Authors: Yang Ronggui , Chen Gang , Laroche Marine , Taur Yuan
Published: March 2005

Heat conduction in micro- and nanoscale and in ultrafast processes may deviate from the predictions of the Fourier law, due to boundary and interface scattering, the ballistic nature of the transport, and the finite relaxation time of heat carriers. The transient ballistic-diffusive heat conduction equations (BDE) were developed as an approximation to the phonon Boltzmann equation (BTE) for nanoscale heat conduction problems. In this paper, we further develop BDE for multidimensional heat conduction, including nanoscale heat source term and different boundary conditions, and compare the simulation results with those obtained from the phonon BTE and the Fourier law. The numerical solution strategies for multidimensional nanoscale heat conduction using BDE are presented. Several two-dimensional cases are simulated and compared to the results of the transient phonon BTE and the Fourier heat conduction theory. The transient BTE is solved using the discrete ordinates method with a two Gauss-Legendre quadratures. Special attention has been paid to the boundary conditions. Compared to the cases without internal heat generation, the difference between the BTE and BDE is larger for the case studied with internal heat generation due to the nature of the ballistic-diffusive approximation, but the results from BDE are still significantly better than those from the Fourier law. Thus we conclude that BDE captures the characteristics of the phonon BTE with much shorter computational time.
Heat Transfer in Two-Pass Rotating Rectangular Channels (AR=1:2 and AR=1:4) With Smooth Walls
Authors: Fu Wen-Lung , Wright Lesley M. , Han Je-Chin
Published: March 2005

This paper reports the heat transfer coefficients in two-pass rotating rectangular channels [aspect ratio (AR=1:2 and AR=1:4)] with smooth walls. The experiments are conducted at four Reynolds numbers: 5000, 10,000, 25,000, and 40,000. The rotation numbers vary from 0.0 to 0.21 and 0.0 to 0.3 for AR=1:2 and AR=1:4, respectively. For each channel, two channel orientations are studied, 90° and 45° with respect to the plane of rotation. The results showed that the 180° sharp turn significantly enhanced heat transfer on both the leading and trailing surfaces in the turn region for both nonrotating and rotating channels. The results also showed that the rotation effect increased the heat transfer on the trailing surface in the first pass, but reduced the heat transfer on the leading surface. However, the heat transfer difference between the leading and trailing walls in the second pass is relatively small compared to the first pass due to strong turn effect.
Absence of Oscillations and Resonance in Porous Media Dual-Phase-Lagging Fourier Heat Conduction
Authors: Vadasz Peter
Published: March 2005

The approximate equivalence between the dual-phase-lagging heat conduction model and the Fourier heat conduction in porous media subject to lack of local thermal equilibrium suggested the possibility of thermal oscillations and resonance. The present investigation demonstrates that the physical conditions necessary for such thermal waves and, possibly resonance, to materialize are not attainable in a porous slab subject to constant temperature conditions applied on the boundaries.
In Vitro Susceptibility Testing ofBorrelia burgdorferiSensu Lato Isolates Cultured from Patients with Erythema Migrans before and after Antimicrobial Chemotherapy
Authors: Hunfeld Klaus-Peter , Ruzic-Sabljic Eva , Norris Douglas E. , Kraiczy Peter , Strle Franc
Published: March 2005

Clinical treatment failures have been reported to occur in early Lyme borreliosis (LB) for many suitable antimicrobial agents. Investigations of possible resistance mechanisms of theBorrelia burgdorfericomplex must analyze clinical isolates obtained from LB patients, despite their receiving antibiotic treatment. Here, borrelial isolates obtained from five patients with erythema migrans (EM) before the start of antibiotic therapy and again after the conclusion of treatment were investigated. The 10 isolates were characterized by restriction fragment length polymorphism analysis and plasmid profile analysis and subjected to susceptibility testing against a variety of antimicrobial agents including those used for initial chemotherapy. Four out of five patients were infected by the same genospecies (Borrelia afzelii,n= 3;Borrelia garinii,n= 1) at the site of the EM lesion before and after antimicrobial therapy. In one patient the genospecies of the initial isolate (B.afzelii) differed from that of the follow-up isolate (B.garinii). No significant changes in the in vitro susceptibilities became obvious for corresponding clinical isolates before the start and after the conclusion of antimicrobial therapy. This holds true for the antimicrobial agents used for specific chemotherapy of the patients, as well as for any of the additional agents tested in vitro. Our study substantiates borrelial persistence in some EM patients at the site of the infectious lesion despite antibiotic treatment over a reasonable time period. Borrelial persistence, however, was not caused by increasing MICs or minimal borreliacidal concentrations in these isolates. Therefore, resistance mechanisms other than acquired resistance to antimicrobial agents should be considered in patients with LB resistant to treatment.
Is In Vitro Antibiotic Combination More Effective than Single-Drug Therapy against Anthrax?
Authors: Athamna Abed , Athamna Muhammad , Nura Aburashed , Shlyakov Eli , Bast Darrin J. , Farrell David , Rubinstein Ethan
Published: March 2005

Antibiotic combinations are used to enhance antibacterial efficacy and to prevent the development of resistance. We have tested a possible synergistic effect of several antibacterial combinations on
Bacillus anthracis
. The in vitro activities of antibiotic combinations against two strains of
B. anthracis
, strain Sterne and the Russian anthrax vaccine strain STi, were tested by the fractional inhibitory concentration (FIC) method, derived from the MICs of the agents in combination, and by measuring the rate of bacterial killing over time by several antibiotic combinations. The FIC results showed that synergism against both
B. anthracis
strains was observed only with the combination of rifampin and clindamycin. The telithromycin-amoxicillin combination showed synergism against strain Sterne only. All other combinations were either indifferent or antagonistic. The results of the bacterial time-kill study demonstrated indifferent effects for all combinations. These in vitro results demonstrate the difficulties in obtaining synergistic combinations of antibiotics against
B. anthracis
.
Testing of the In Vitro Susceptibilities ofMadurella mycetomatisto Six Antifungal Agents by Using the Sensititre System in Comparison with a Viability-Based 2,3-Bis(2-Methoxy-4-Nitro-5-Sulfophenyl)-5- [(Phenylamino)Carbonyl]-2H-Tetrazolium Hydroxide (XTT) Assay and a Modified NCCLS Method
Authors: van de Sande Wendy W. J. , Luijendijk Ad , Ahmed Abdalla O. A. , Bakker-Woudenberg Irma A. J. M. , van Belkum Alex
Published: March 2005

The in vitro susceptibilities of 36 clinical isolates ofMadurella mycetomatis, the prime agent of eumycetoma in Africa, to ketoconazole, itraconazole, fluconazole, voriconazole, amphotericin B, and flucytosine were determined by the Sensititre YeastOne system. This system appeared to be a rapid and easy test, and by use of hyphal suspensions it generated results comparable to those of a modified NCCLS method. After 10 days of incubation, the antifungal activities of ketoconazole (MIC at which 90% of isolates were inhibited [MIC90], 0.125 μg/ml), itraconazole (MIC90, 0.064 μg/ml), and voriconazole (MIC90, 0.125 μg/ml) appeared superior to those of fluconazole (MIC90, 128 μg/ml) and amphotericin B (MIC90, 1 μg/ml), with MICs in the clinically relevant range. All isolates were resistant to flucytosine (all MICs above 64 μg/ml). Based on the relatively broad range of MICs obtained for the antifungal agents, routine testing ofM. mycetomatisisolates for susceptibility to antifungal agents seems to be relevant to adequate therapeutic management.
Development of Cell-Based Assays for In Vitro Characterization of Hepatitis C Virus NS3/4A Protease Inhibitors
Authors: Chung Victoria , Carroll Anthony R. , Gray Norman M. , Parry Nigel R. , Thommes Pia A. , Viner K. Claire , D'Souza Eric A.
Published: March 2005

A recombinant vaccinia virus, expressing the NS3-to-NS5 region of the N clone of hepatitis C virus (HCV), was generated and utilized both in a gel-based assay and in an enzyme-linked immunosorbent assay (ELISA) to evaluate the pyrrolidine-5,5-
trans
-lactams, a series of inhibitors of the HCV NS3/4A protease. The absolute levels of processed, mature HCV nonstructural proteins in this system were found to decrease in the presence of the
trans-
lactams. Monitoring of this reduction enabled end points and 50% inhibitory concentrations to be calculated in order to rank the active compounds according to potency. These compounds had no effect on the transcription or translation of the NS3-5 polyprotein at concentrations shown to inhibit NS3/4A protease, and they were shown to be specific inhibitors of this protease. The ELISA, originally developed using the vaccinia virus expression system, was modified to utilize Huh-7 cells containing an HCV replicon. Results with this assay correlated well with those obtained with the recombinant vaccinia virus assays. These results demonstrate the utility of these assays for the characterization of NS3/4A protease inhibitors. In addition, inhibitors of other viral targets, such as polymerase and helicase, can be evaluated in the context of the replicon ELISA.
Silencing of Glycopeptide Resistance in
Enterococcus faecalis
BM4405 by Novobiocin
Authors: Patiño Lorena Abadía , Chippaux Marc , Courvalin Patrice , Périchon Bruno
Published: March 2005

Enterococcus faecalis
BM4405-1, a susceptible derivative of the VanE-type vancomycin-resistant
E. faecalis
strain BM4405, was obtained after growth in the presence of novobiocin, an inhibitor of the GyrB subunit of DNA gyrase. In contrast to findings for BM4405, UDP-MurNAc-
l
-Ala-γ-
d
-Glu-
l
-Lys-
d
-Ala-
d
-Ala (pentapeptide[
d
-Ala]) was the only peptidoglycan precursor found in BM4405-1, and no VanXY
E
d
,
d
-peptidase or VanT serine racemase activities were detected in that strain, even after induction by subinhibitory concentrations of vancomycin. Sequencing of the
vanE
operon of BM4405-1 revealed two mutations leading to substitutions in VanE (D200N) and in the C-terminal amino acid of VanR
E
(Y225F). Cloning of the
vanE
,

vanXY
E

, and

vanT
E

genes of BM4405-1 into the susceptible
E. faecalis
strain JH2-2 conferred resistance to vancomycin, indicating that the mutation in
vanE
was not responsible for susceptibility. Transcriptional analysis of the
vanE
operon in BM4405 by quantitative reverse transcription-PCR indicated that novobiocin did not affect the expression level of the
vanE
operon. Sequencing of the
gyrB
gene of BM4405-1 revealed a mutation responsible for substitution of a residue (K337Y) required for ATPase activity and thus implicated in DNA supercoiling. Cloning of the
gyrB
gene of BM4405 restored vancomycin resistance to BM4405-1. Taken together, these data suggest that alteration of DNA supercoiling following a mutation in GyrB was responsible for lack of expression of the
vanE
operon and thus for vancomycin susceptibility in BM4405-1.
Acquisition of Resistance to Carbapenems in Multidrug-Resistant Clinical Strains of
Acinetobacter baumannii
: Natural Insertional Inactivation of a Gene Encoding a Member of a Novel Family of β-Barrel Outer Membrane Proteins
Authors: Mussi María A. , Limansky Adriana S. , Viale Alejandro M.
Published: March 2005

The outer membrane proteins responsible for the influx of carbapenem β-lactam antibiotics in the nonfermentative gram-negative pathogen
Acinetobacter baumannii
are still poorly characterized. Resistance to both imipenem and meropenem in multidrug-resistant clinical strains of
A. baumannii
is associated with the loss of a heat-modifiable 29-kDa outer membrane protein, designated CarO. The chromosomal locus containing the
carO
gene was cloned and characterized from different clinical isolates. Only one
carO
copy, present in a single transcriptional unit, was found in the
A. baumannii
genome. The
carO
gene encodes a polypeptide of 247 amino acid residues with a typical N-terminal signal sequence and a predicted transmembrane β-barrel topology. Its absence from different carbapenem-resistant clinical isolates of
A. baumannii
resulted from the disruption of
carO
by distinct insertion elements. The overall data thus support the notion that CarO participates in the influx of carbapenem antibiotics in
A. baumannii
. Moreover, database searches identified the presence of
carO
homologs only in species of the genera
Acinetobacter
,
Moraxella
, and
Psychrobacter
, disclosing the existence of a novel family of outer membrane proteins restricted to the family
Moraxellaceae
of the class γ
-Proteobacteria
.
CTX-M-10 Linked to a Phage-Related Element Is Widely Disseminated among
Enterobacteriaceae
in a Spanish Hospital
Authors: Oliver Antonio , Coque Teresa M. , Alonso Diana , Valverde Aránzazu , Baquero Fernando , Cantón Rafael
Published: March 2005

CTX-M-10 has been widely disseminated among multiple clones of several species of
Enterobacteriaceae
, harboring seemingly different plasmids, for over a decade in Ramón y Cajal University Hospital, Madrid, Spain. Cloning and sequencing of a 12.2-kb DNA fragment from plasmid pRYCE21 from
Klebsiella pneumoniae
strain KP4aC revealed a novel phage-related element immediately upstream of
bla
CTX-M-10
conserved among different CTX-M-10-producing strains. This is the first report showing an extended-spectrum-β-lactamase gene linked to a phage-related element.
Occurrence of CTX-M-3, CTX-M-15, CTX-M-14, and CTX-M-9 Extended-Spectrum β-Lactamases in
Enterobacteriaceae
Clinical Isolates in Korea
Authors: Kim Jungmin , Lim Yu-Mi , Jeong Young-Sook , Seol Sung-Yong
Published: March 2005

Among 603 isolates of
Enterobacteriaceae
collected between June and November 2003 from three university hospitals within Korea,
bla
CTX-M-3
,
bla
CTX-M-15
,
bla
CTX-M-14
, and
bla
CTX-M-9
were detected in 41 isolates of species from five different genera of
Enterobacteriaceae
,
Escherichia coli
,
Klebsiella pneumoniae
,
Citrobacter freundii
,
Enterobacter
spp., and
Serratia marcescens
.
Capsule Homology Does Not Increase the Frequency of Transformation of Linked Penicillin Binding Proteins PBP 1a and PBP 2x in
Streptococcus pneumoniae
Authors: Trzciński Krzysztof , MacNeil Adam , Klugman Keith P. , Lipsitch Marc
Published: March 2005

Penicillin resistance is mainly confined to a limited number of
Streptococcus pneumoniae
serotypes. Given linkage between the capsular biosynthesis locus and two penicillin binding proteins, we tested whether capsule homology increases transformation rates of penicillin resistance. Transformation rates in homologous donor-recipient pairs were no higher than expected, falsifying this hypothesis.
Sensititre YeastOne Caspofungin Susceptibility Testing of
Candida
Clinical Isolates: Correlation with Results of NCCLS M27-A2 Multicenter Study
Authors: Cantón Emilia , Pemán Javier , Gobernado Miguel , Álvarez Elena , Baquero Fernando , Cisterna Ramón , Gil Joaquina , Martín-Mazuelos Estrella , Rubio Carmen , Sánchez-Sousa Aurora , Serrano Carmen
Published: March 2005

The ability of Sensititre YeastOne to discriminate isolates with reduced caspofungin susceptibility was determined against 36
Candida
spp. (6 with a known
FKS1
mutation). Results were compared with those of M27-A2. The MIC endpoint was 100% growth inhibition. Overall agreement (±2 log
2
) was 87.16%. Sensititre YeastOne detected strains with reduced caspofungin susceptibility.
Genetic Basis of Antibiotic Resistance in Clinical Isolates ofStreptococcus gallolyticus(Streptococcus bovis)
Authors: Leclercq Roland , Huet Corinne , Picherot Mélanie , Trieu-Cuot Patrick , Poyart Claire
Published: March 2005

Among 128Streptococcus gallolyticus(Streptococcus bovis) isolates, 77.7% were resistant to tetracyclines and containedtet(M) and/ortet(L) and/ortet(O). A total of 59.4% had macrolide resistance and containederm(B) and, rarely,mef(A). Among the one-third of isolates highly resistant to kanamycin and streptomycin, most harboredaphA3andaad-6genes.
Effects of Age and Sex on Single-Dose Pharmacokinetics of Tigecycline in Healthy Subjects
Authors: Muralidharan Gopal , Fruncillo Richard J. , Micalizzi Marlynne , Raible Donald G. , Troy Steven M.
Published: March 2005

The pharmacokinetics of tigecycline was evaluated in 46 healthy young and elderly men and women. Except for the volumes of distribution at steady state (approximately 350 liters in women versus 500 liters in men), there were no significant differences in tigecycline pharmacokinetic parameters. Based on pharmacokinetics, no dosage adjustment is warranted based on age or sex.
Chlamydial Infection Induces Pathobiotype-Specific Protein Tyrosine Phosphorylation in Epithelial Cells
Authors: Virok Dezso P. , Nelson David E. , Whitmire William M. , Crane Deborah D. , Goheen Morgan M. , Caldwell Harlan D.
Published: March 2005

Members of the genus
Chlamydia
are strict obligate intracellular pathogens that exhibit marked differences in host range and tissue tropism despite sharing a remarkable level of genomic synteny. These pathobiotype differences among chlamydiae are also mirrored in their early interactions with cultured mammalian host cells. Chlamydial attachment and entry is known to trigger protein tyrosine phosphorylation. In this study, we examined the kinetics and pattern of protein tyrosine phosphorylation induced by infection with a comprehensive collection of chlamydial strains exhibiting diversity in host, tissue, and disease tropisms. We report new findings showing that protein tyrosine phosphorylation patterns induced by infection directly correlate with the pathobiotype of the infecting organism. Patterns of protein tyrosine phosphorylation were induced following early infection that unambiguously categorized chlamydial pathobiotypes into four distinct groups: (i)
Chlamydia trachomatis
trachoma biovars (serovars A to H), (ii)
C. trachomatis
lymphogranuloma venereum biovars (serovars L1 to L3), (iii)
C. muridarum
, and (iv)
C. pneumoniae
and
C. caviae
. Notably, chlamydia-infected murine and human epithelial cells exhibited the same protein tyrosine phosphorylation patterns; this is indirect evidence suggesting that the phosphorylated protein(s) is of chlamydial origin. If our hypothesis is correct, these heretofore-uncharacterized proteins may represent a novel class of bacterial molecules that influence pathogen-host range or tissue tropism.
Antibiotic-Free Plasmid Stabilization by Operator-Repressor Titration for Vaccine Delivery by Using Live
Salmonella enterica
Serovar Typhimurium
Authors: Garmory Helen S. , Leckenby Matthew W. , Griffin Kate F. , Elvin Stephen J. , Taylor Rosa R. , Hartley M. Gill , Hanak Julian A. J. , Williamson E. Diane , Cranenburgh Rocky M.
Published: March 2005

Live, attenuated bacteria are effective vectors for heterologous antigen delivery. However, loss of heterologous gene-bearing plasmids is problematic, and antibiotics and their resistance genes are not desirable for in vivo DNA vaccine delivery due to biosafety and regulatory concerns. To solve this problem, we engineered the first vaccine delivery strain that has no requirement for antibiotics or other selectable marker genes to maintain the recombinant plasmid. This model strain of
Salmonella enterica
serovar Typhimurium, SLDAPD, uses operator-repressor titration (ORT) technology, which requires only the short, nonexpressed
lacO
sequence for selection and maintenance. SLDAPD, recovered from the spleens and Peyer's patches of mice following oral inoculation, was shown to maintain a plasmid that, in contrast, was lost from parental strain SL3261. We also demonstrated successful application of this technology to vaccine development, since SLDAPD carrying a plasmid without an antibiotic resistance gene that expressed the
Yersinia pestis
F1 antigen was as efficacious in protecting vaccinated mice against plague as the parental SL3261 strain carrying an antibiotic-selected version of this plasmid. Protection of mice against plague by immunization with
Salmonella
expressing F1 has previously required two or more doses; here we demonstrated for the first time protective immunity after a single oral immunization. This technology can easily be used to convert any suitable attenuated strain to an antibiotic-free ORT strain for recombinant protein vaccine delivery in humans.
Process Development and Analysis of Liver-Stage Antigen 1, a Preerythrocyte-Stage Protein-Based Vaccine forPlasmodium falciparum
Authors: Hillier Collette J. , Ware Lisa A. , Barbosa Arnoldo , Angov Evelina , Lyon Jeffrey A. , Heppner D. Gray , Lanar David E.
Published: March 2005

Plasmodium falciparumliver-stage antigen 1 (LSA-1) is expressed solely in infected hepatocytes and is thought to have a role in liver schizogony and merozoite release. Specific humoral, cellular, and cytokine immune responses to LSA-1 are well documented, with epitopes identified that correlate with antibody production, proliferative T-cell responses, or cytokine induction. With the goal of developing a vaccine against this preerythrocyte-stage protein, we undertook the good manufacturing practices (GMP) manufacture of a recombinant LSA-1 construct, LSA-NRC, incorporating the N- and C-terminal regions of the protein and two of the centrally placed 17-amino-acid repeats. To improve the protein yield, a method of codon harmonization was employed to reengineer the gene sequence for expression inEscherichia coli. A 300-liter GMP fermentation produced 8 kg of bacterial cell paste, and a three-step column chromatographic method yielded 8 mg of purified antigen per g of paste. The final bulk protein was >98% pure, demonstrated long-term stability, and contained <0.005 endotoxin units per 50 μg of protein. To accomplish the initial stages of evaluation of this protein as a human-use vaccine against malaria, we immunized rabbits and mice with LSA-NRC in Montanide ISA 720. New Zealand White rabbits and A/J (H-2K) mice produced high-titer antibodies that recognized liver-stage parasites in infected cultured human hepatocytes. Gamma interferon-producing cells, which have been associated with LSA-1-mediated protection, were detected in splenocytes harvested from immunized mice. Finally, sera taken from people living in a region where malaria is holoendemic recognized LSA-NRC by Western blotting.
Effect of B7-2 and CD40 Signals from Activated Antigen-Presenting Cells on the Ability of Zwitterionic Polysaccharides To Induce T-Cell Stimulation
Authors: Stephen Tom Li , Niemeyer Marcus , Tzianabos Arthur O. , Kroenke Martin , Kasper Dennis L. , Kalka-Moll Wiltrud M.
Published: March 2005

Carbohydrates have been thought to stimulate immune responses independently of T cells; however, zwitterionic polysaccharides (ZPSs) from the capsules of some bacteria elicit potent CD4+-T-cell responses in vivo and in vitro. We demonstrated that HLA-DR on professional antigen-presenting cells (APCs) is required for ZPS-induced T-cell proliferation in vitro (15). Recently, it was shown that ZPSs are processed to low-molecular-weight carbohydrates by a nitric oxide-mediated mechanism in endosomes and locate in the major histocompatibility complex class II pathway (5, 15). The effect of the ZPS-mediated expression of HLA-DR and costimulatory molecules on the APC and T-cell engagement and subsequent T-cell activation has not been elucidated. Herein, we report that ZPS-mediated induction of HLA-DR-surface expression and T-cell proliferation are maximally enhanced after incubation of APCs for 8 h with ZPS. Treatment of APCs with bafilomycin A inhibits the up-regulation of ZPS-mediated HLA-DR surface expression and leads to inhibition of T-cell proliferation. Monoclonal antibodies (MAbs) to the costimulatory molecules B7-2 and CD40L specifically block ZPS-mediated T-cell activation, while a MAb to B7-1 does not. Surface expression of B7-2 and B7-1 but not of CD40 is maximally enhanced at 8 to 16 h of treatment of APCs with ZPS. The results demonstrate that the cellular immune response to ZPS depends on the translocation of HLA-DR to the cell surface and requires costimulation via B7-2 and CD40 on activated APCs. The implication is that activation of ZPS-specific T cells requires an orchestrated arrangement of both presenting and costimulatory molecules to form an immunological synapse.
Role of SraP, a Serine-Rich Surface Protein of
Staphylococcus aureus
, in Binding to Human Platelets
Authors: Siboo Ian R. , Chambers Henry F. , Sullam Paul M.
Published: March 2005

The binding of bacteria to platelets is a postulated central event in the pathogenesis of infective endocarditis. Platelet binding by
Streptococcus gordonii
is mediated in large part by GspB, a high-molecular-mass cell wall glycoprotein. Although
Staphylococcus aureus
has a GspB homolog (SraP), little is known about its function. SraP has a calculated molecular mass of 227 kDa and, like GspB, is predicted to contain an atypical N-terminal signal sequence, two serine-rich repeat regions (srr1 and srr2) separated by a nonrepeat region, and a C-terminal cell wall anchoring motif (LPDTG). To assess whether SraP contributes to platelet binding, we compared the binding to human platelets of
S. aureus
strain ISP479C and of an isogenic variant (strain PS767) in which
sraP
had been disrupted by allelic replacement. Platelet binding in vitro by PS767 was 47% ± 17% (mean ± standard deviation) lower than that of ISP479C (
P
< 0.001). In addition, a recombinant fragment of SraP containing srr1 and the nonrepeat region was found to bind platelets directly. Binding was saturable, suggesting a receptor-ligand interaction. When tested in a rabbit model of endocarditis, in which each animal was simultaneously infected with ISP479C and PS767 at a ratio of approximately 1:1, the titers of the mutant strain within vegetations were significantly lower than those of the parent strain at 1 and 24 h postinfection. These results indicate that SraP can mediate the direct binding of
S. aureus
to platelets and that the platelet-binding domain of this glycoprotein is located within its N-terminal region. Moreover, the expression of SraP appears to be a virulence determinant in endovascular infection.
Intranasal Interleukin-12 Treatment for Protection against Respiratory Infection with theFrancisella tularensisLive Vaccine Strain
Authors: Duckett Nathalie S. , Olmos Sofia , Durrant Douglas M. , Metzger Dennis W.
Published: March 2005

Francisella tularensisis a gram-negative intracellular bacterium that can induce lethal respiratory infection in humans and rodents. However, little is known about the role of innate or adaptive immunity in protection from respiratory tularemia. In the present study, the role of interleukin-12 (IL-12) in inducing protective immunity in the lungs against intranasal infection of mice with the live vaccine strain (LVS) ofF. tularensiswas investigated. It was found that gamma interferon (IFN-γ) and IL-12 were strictly required for protection, since mice deficient in IFN-γ, IL-12 p35, or IL-12 p40 all succumbed to LVS doses that were sublethal for wild-type mice. Furthermore, exogenous IL-12 treatment 24 h before intranasal infection with a lethal dose of LVS (10,000 CFU) significantly decreased bacterial loads in the lungs, livers, and spleens of wild-type BALB/c and C57BL/6 mice and allowed the animals to survive infection; such protection was not observed in IFN-γ-deficient mice. The resistance induced by IL-12 to LVS infection was still observed in NK cell-deficient beige mice but not in CD8−/−mice. These results demonstrate that exogenous IL-12 delivered intranasally can prevent respiratory tularemia through a mechanism that is at least partially dependent upon the expression of IFN-γ and CD8 T cells.
Lipopolysaccharide Is Transferred from High-Density to Low-Density Lipoproteins by Lipopolysaccharide-Binding Protein and Phospholipid Transfer Protein
Authors: Levels J. H. M. , Marquart J. A. , Abraham P. R. , van den Ende A. E. , Molhuizen H. O. F. , van Deventer S. J. H. , Meijers J. C. M.
Published: March 2005

Lipopolysaccharide (LPS), the major outer membrane component of gram-negative bacteria, is a potent endotoxin that triggers cytokine-mediated systemic inflammatory responses in the host. Plasma lipoproteins are capable of LPS sequestration, thereby attenuating the host response to infection, but ensuing dyslipidemia severely compromises this host defense mechanism. We have recently reported thatEscherichia coliJ5 and Re595 LPS chemotypes that contain relatively short O-antigen polysaccharide side chains are efficiently redistributed from high-density lipoproteins (HDL) to other lipoprotein subclasses in normal human whole blood (ex vivo). In this study, we examined the role of the acute-phase proteins LPS-binding protein (LBP) and phospholipid transfer protein (PLTP) in this process. By the use of isolated HDL containing fluorescent J5 LPS, the redistribution of endotoxin among the major lipoprotein subclasses in a model system was determined by gel permeation chromatography. The kinetics of LPS and lipid particle interactions were determined by using Biacore analysis. LBP and PLTP were found to transfer LPS from HDL predominantly to low-density lipoproteins (LDL), in a time- and dose-dependent manner, to induce remodeling of HDL into two subpopulations as a consequence of the LPS transfer and to enhance the steady-state association of LDL with HDL in a dose-dependent fashion. The presence of LPS on HDL further enhanced LBP-dependent interactions of LDL with HDL and increased the stability of the HDL-LDL complexes. We postulate that HDL remodeling induced by LBP- and PLTP-mediated LPS transfer may contribute to the plasma lipoprotein dyslipidemia characteristic of the acute-phase response to infection.
Generation of Attenuated
Mycobacterium bovis
Strains by Signature-Tagged Mutagenesis for Discovery of Novel Vaccine Candidates
Authors: Collins Desmond M. , Skou Bronwyn , White Stefan , Bassett Shalome , Collins Lauren , For Raewyn , Hurr Kathryn , Hotter Grant , de Lisle Geoffrey W.
Published: March 2005

Mycobacterium bovis
, a member of the
Mycobacterium tuberculosis
complex, has a particularly wide host range and causes tuberculosis in most mammals, including humans. A signature tag mutagenesis approach, which employed illegitimate recombination and infection of guinea pigs, was applied to
M. bovis
to discover genes important for virulence and to find potential vaccine candidates. Fifteen attenuated mutants were identified, four of which produced no lesions when inoculated separately into guinea pigs. One of these four mutants had nine deleted genes including
mmpL4
and
sigK
and, in guinea pigs with aerosol challenge, provided protection against tuberculosis at least equal to that of
M. bovis
BCG. Seven mutants had mutations near the
esxA
(
esat-6
) locus, and immunoblot analysis of these confirmed the essential role of other genes at this locus in the secretion of EsxA (ESAT-6) and EsxB (CFP10). Mutations in the eight other attenuated mutants were widely spread through the chromosome and included
pks1
, which is naturally inactivated in clinical strains of
M. tuberculosis
. Many genes identified were different from those found by signature tag mutagenesis of
M. tuberculosis
by use of a mouse infection model and illustrate how the use of different approaches enables identification of a wider range of attenuating mutants.
Sweat rate and fluid turnover in American football players compared with runners in a hot and humid environment
Authors: Godek S Fowkes , Bartolozzi A R , Godek J J
Published: March 2005

Objectives:To determine sweat rate (SwR) and fluid requirements for American footballers practicing in a hot, humid environment compared with cross country runners in the same conditions.

Methods:Fifteen subjects, 10 footballers and five runners, participated. On the 4th and 8th day of preseason two a day practices, SwR during exercise was determined in both morning and afternoon practices/runs from the change in body mass adjusted for fluids consumed and urine produced. Unpairedttests were used to determine differences between groups.

Results:Overall SwR measured in litres/h was higher in the footballers than the cross country runners (2.14 (0.53)v1.77 (0.4); p<0.01). Total sweat loss in both morning (4.83 (1.2)v1.56 (0.39) litres) and afternoon (4.8 (1.2)v1.97 (0.28) litres) practices/runs, and daily sweat losses (9.4 (2.2)v3.53 (0.54) litres) were higher in the footballers (p<0.0001). The footballers consumed larger volumes of fluid during both morning and afternoon practices/runs (23.9 (8.9)v5.5 (3.1) ml/min and 23.5 (7.3)v13.6 (5.6) ml/min; p<0.01). For complete hydration, the necessary daily fluid consumption calculated as 130% of daily sweat loss in the footballers was 12.2 (2.9) litres compared with 4.6 (0.7) litres in the runners (p<0.0001). Calculated 24 hour fluid requirements in the footballers ranged from 8.8 to 19 litres.

Conclusions:The American footballers had a high SwR with large total daily sweat losses. Consuming large volumes of hypotonic fluid may promote sodium dilution. Recommendations for fluid and electrolyte replacement must be carefully considered and monitored in footballers to promote safe hydration and avoid hyponatraemia.