bluuebunny/crossref_metadata_2025 huggingface dataset:
Robust Control Design for Retarded Discrete-Time Systems
Authors: Tissir El Houssaine
Published: December 2006

This paper focuses on the analysis and synthesis of a robust stabilizing controller for linear discrete time systems with norm-bounded time varying uncertainties. Delay independent robust stability conditions are derived and two synthesis methods are presented. One method is to construct a robust memoryless state feedback control law from the solutions of linear matrix inequalities. The other method consists of designing robust observer-based output feedback controller. The results are expressed in termes of linear matrix inequalities. A comparison with μ∕LDI tests is presented. Furthermore, numerical examples are given for illustration.
Adaptive Servoregulation of a Projectile Fin Using Piezoelectric Actuator
Authors: Mani Smitha , Singh Sahjendra N. , Parimi Surya Kiran , Yim Woosoon
Published: December 2006

This brief paper treats the question of adaptive control of a projectile fin using a piezoelectric actuator. The hollow projectile fin is rigid, within which a flexible cantilever beam with a piezoelectric active layer is mounted. The model of the fin-beam system includes the aerodynamic moment, which is a function of angle of attack of the projectile. The rotation angle of the fin is controlled by deforming the flexible beam, which is hinged at the tip of the rigid fin. An adaptive servoregulator is designed for the control of the fin angle using the fin angle and its derivative for feedback. Interestingly, the knowledge of the dimension and parameters of the system is not essenstial for the synthesis of the control law. In the closed-loop system, the fin angle asymptotically converges to the desired set point and the elastic modes converge to their equilibrium values. Computer simulation and laboratory test results are presented to show the performance of the controller.
Input Selection for Modeling and Diagnostics With Application to Diesel Engines
Authors: Martin Brett , Meckl Peter
Published: December 2006

A theoretical and experimental approach to the use of information theory in input space selection for modeling and diagnostic applications is examined. The assumptions and test cases used throughout the paper are specifically tailored to diesel engine diagnostic and modeling applications. This work seeks to quantify the amount of information about an output contained within an input space. The information theoretic quantity, conditional entropy, is shown to be an accurate predictor of model and diagnostic algorithm performance and therefore is a good choice for an input vector selection metric. Methods of estimating conditional entropy from collected data, including the amount of needed data, are also discussed.
Evaluating the impact of x‐ray spectral shape on image quality in flat‐panel CT breast imaging
Authors: Glick Stephen J. , Thacker Samta , Gong Xing , Liu Bob
Published: January 2007

In recent years, there has been an increasing interest in exploring the feasibility of dedicated computed tomography (CT) breast imaging using a flat‐panel digital detector in a truncated cone‐beam imaging geometry. Preliminary results are promising and it appears as if three‐dimensional tomographic imaging of the breast has great potential for reducing the masking effect of superimposed parenchymal structure typically observed with conventional mammography. In this study, a mathematical framework used for determining optimal design and acquisition parameters for such a CT breast imaging system is described. The ideal observer signal‐to‐noise ratio (SNR) is used as a figure of merit, under the assumptions that the imaging system is linear and shift invariant. Computation of the ideal observer SNR used a parallel‐cascade model to predict signal and noise propagation through the detector, as well as a realistic model of the lesion detection task in breast imaging. For all evaluations, the total mean glandular dose for a CT breast imaging study was constrained to be approximately equivalent to that of a two‐view conventional mammography study. The framework presented was used to explore the effect of x‐ray spectral shape across an extensive range of kVp settings, filter material types, and filter thicknesses. The results give an indication of how spectral shape can affect image quality in flat‐panel CT breast imaging.
Combination of Multiplex PCRs for Staphylococcal Cassette Chromosome
mec
Type Assignment: Rapid Identification System for
mec
,
ccr
, and MajorDifferences in Junkyard Regions
Authors: Kondo Yoko , Ito Teruyo , Ma Xiao Xue , Watanabe Shinya , Kreiswirth Barry N. , Etienne Jerome , Hiramatsu Keiichi
Published: December 2006

Staphylococcal
cassette chromosome
mec
(SCC
mec
) typing, in
combination with genotyping of the
Staphylococcus
aureus
chromosome, has become essential for defining
methicillin-resistant
S. aureus
(MRSA) clones in
epidemiological studies. We have developed a convenient system for
SCC
mec
type assignment. The system consists of six multiplex
PCRs (M-PCRs) for identifying the
ccr
gene complex
(
ccr
), the
mec
gene complex (
mec
), and
specific structures in the junkyard (J) regions: M-PCR with primer set
1 (M-PCR 1) identified five types of
ccr
genes; M-PCR 2
identified class A to class C
mec
; M-PCRs 3 and 4 identified
specific open reading frames in the J1 regions of type I and IV and of
type II, III, and V SCC
mec
elements, respectively; M-PCR 5
identified the transposons Tn
554
and ΨTn
554
integrated into the J2 regions of type II and III SCC
mec
elements; and M-PCR 6 identified plasmids pT181 and pUB110 integrated
into J3 regions. The system was validated with 99 MRSA strains carrying
SCC
mec
elements of different types. The SCC
mec
types
of 93 out of the 99 MRSA strains could be assigned. The SCC
mec
type assignments were identical to those made with a PCR system that
uses numerous primer pairs to identify genes or gene alleles. Our
system of six M-PCRs is thus a convenient and reliable method for
typing SCC
mec
elements.
In Vitro Activities of the Lipopeptides Palmitoyl (Pal)-Lys-Lys-NH
2
and Pal-Lys-Lys Alone and in Combination with Antimicrobial Agents against Multiresistant Gram-Positive Cocci
Authors: Kamysz Wojciech , Silvestri Carmela , Cirioni Oscar , Giacometti Andrea , Licci Alberto , Della Vittoria Agnese , Okroj Marcin , Scalise Giorgio
Published: December 2006

The in vitro activities of the lipopeptides palmitoyl (Pal)-Lys-Lys-NH
2
and Pal-Lys-Lys against gram-positive cocci were investigated. Enterococci and streptococci demonstrated higher susceptibilities than staphylococci and
Rhodococcus equi
. A positive interaction was shown when the lipopeptides were combined with beta-lactams and vancomycin. These results suggest that lipopeptides are promising candidates for antimicrobial therapy for infections caused by gram-positive organisms.
Action of Disinfectant Quaternary Ammonium Compounds againstStaphylococcus aureus
Authors: Ioannou Christopher J. , Hanlon Geoff W. , Denyer Stephen P.
Published: December 2006

Mode-of-action studies concluded that alkyldimethylbenzylammonium chloride (ADBAC) (a blend of C12, C14and C16alkyl homologues) and didecyldimethylammonium chloride (DDAC) are both membrane-active agents, possessing subtly different modes of action reflecting early cell interactions againstStaphylococcus aureus. ADBAC and DDAC exhibited similar MIC behaviors from 0.4 ppm to 1.8 ppm over an inoculum range of 1 × 105to 1 × 109CFU/ml at 35°C. For ADBAC and DDAC, an increased rapidity of killing againstS. aureus(final concentration, 2 × 109CFU/ml) was observed at 35°C compared to 25°C. Concentration exponents (η) for killing were <2.5 for both agents, and temperature influenced the η value. Examination of leakage and kill data suggested that a single leakage marker was not indicative of cell death. ADBAC and DDAC possessed Langmuir (L4) and high-affinity (H3/4) uptake isotherms, respectively. ADBAC molecules formed a single monolayer of coverage of cells at the end of primary uptake, and DDAC formed a double monolayer. Rapid cell leakage occurred at bactericidal concentrations, with total depletion of the intracellular potassium and 260-nm-absorbing pools released in this strict order. Autolysis was observed for ADBAC and DDAC at concentrations of 9 μg/ml (0.0278 mM and 0.0276 mM, respectively) and above, together with the depletion of approximately 30% of the internal potassium pool. Autolysis contributed to ADBAC and DDAC lethality, although high biocide concentrations may have inhibited autolytic enzyme activity.
Pharmacokinetic Interaction between Voriconazole and Methadone at Steady State in Patients on Methadone Therapy
Authors: Liu Ping , Foster Grover , LaBadie Robert , Somoza Eugene , Sharma Amarnath
Published: December 2006

This trial was aimed to estimate the pharmacokinetic interaction between voriconazole and methadone at steady state in male patients on methadone therapy and to characterize the safety and tolerability profile during the coadministration. Twenty-three patients on individualized methadone therapy (30 to 100 mg once daily) were enrolled into this randomized, patient- and investigator-blind, placebo-controlled, parallel-group study. Methadone pharmacokinetic samples were collected from patients receiving methadone alone as the baseline before they were randomized to coadminister either 200 mg voriconazole twice daily (BID) (400-mg BID loading doses on the first day) (n= 16) or matching placebo (n= 7) for the next 5 days. Pharmacokinetic samples for methadone and voriconazole were collected on the last day of voriconazole dosing. The safety data were collected throughout the study. Voriconazole increased the steady-state exposure of pharmacologically active enantiomer (R)-methadone: the mean area under the concentration-time curve from 0 to 24 h (AUC0-24) was increased by 47.2% (90% confidence intervals [CI]: 37.7%, 57.4%), and the mean peak concentration (Cmax) was increased by 30.7% (90% CI: 22.2%, 39.8%). The magnitude of increase in (S)-methadone exposure was greater than that of (R)-methadone: the AUC0-24was increased by 103.4% (90% CI: 85.0%, 123.6%), and theCmaxwas increased by 65.4% (90% CI: 52.6%, 79.2%). Methadone appeared to have no effect on the steady-state voriconazole pharmacokinetics compared to the historical data for voriconazole alone. Methadone patients receiving voriconazole showed no signs or symptoms of significant opioid withdrawal or overdose. Coadministration of 200 mg voriconazole BID with methadone was generally safe and well tolerated. Nevertheless, caution should be exercised when voriconazole is coadministered with methadone due to the increase in (R)-methadone exposure, which in turn may require a dose reduction of methadone.
Effect of Neutropenia and Treatment Delay on the Response to Antifungal Agents in Experimental Disseminated Candidiasis
Authors: Hope William W. , Drusano George L. , Moore Caroline B. , Sharp Andrew , Louie Arnold , Walsh Thomas J. , Denning David W. , Warn Peter A.
Published: December 2006

Disseminated candidiasis is associated with a high rate of morbidity and mortality. The presence of neutrophils and the timely administration of antifungal agents are likely to be critical factors for a favorable therapeutic outcome of this syndrome. The effect of neutropenia on the temporal profile of the burden ofCandida albicansin untreated mice and those treated with amphotericin B was determined using a pharmacodynamic model of disseminated candidiasis. A mathematical model was developed to describe the rate and extent of theC. albicanskilling attributable to neutrophils and to amphotericin B. The consequences of a delay in the administration of amphotericin B, flucytosine, or micafungin were studied by defining dose-response relationships. Neutrophils caused a logarithmic decline in fungal burden in treated and untreated mice. The combination of amphotericin B and neutrophils resulted in a high rate ofCandidakilling and a sustained anti-C. albicanseffect. In neutropenic mice, 5 mg/kg of body weight of amphotericin B was required to prevent progressive logarithmic growth. An increased delay in drug administration resulted in a reduction in the maximum effect to a point at which no drug effect could be observed. Neutrophils and the timely initiation of antifungal agents are critical determinants in the treatment of experimental disseminated candidiasis.
Semimechanistic Pharmacokinetic/Pharmacodynamic Model for Assessment of Activity of Antibacterial Agents from Time-Kill Curve Experiments
Authors: Nielsen Elisabet I. , Viberg Anders , Löwdin Elisabeth , Cars Otto , Karlsson Mats O. , Sandström Marie
Published: December 2006

Dosing of antibacterial agents is generally based on point estimates of the effect, even though bacteria exposed to antibiotics show complex kinetic behaviors. The use of the whole time course of the observed effects would be more advantageous. The aim of the present study was to develop a semimechanistic pharmacokinetic (PK)/pharmacodynamic (PD) model characterizing the events seen in a bacterial system when it is exposed to antibacterial agents with different mechanisms of action. Time-kill curve experiments were performed with a strain of
Streptococcus pyogenes
exposed to a wide range of concentrations of the following antibiotics: benzylpenicillin, cefuroxime, erythromycin, moxifloxacin, and vancomycin. Bacterial counts were monitored with frequent sampling during the experiment. A simultaneous fit of all data was accomplished. The degradation of the drugs was monitored and corrected for in the model, and a link model was used to account for an effect delay. In the final PK/PD model, the total bacterial population was divided into two subpopulations: one growing drug-susceptible population and one resting insusceptible population. The drug effect was included as an increase of the killing rate of bacteria in the susceptible state, according to a maximum-effect (
E
max
) model. An internal model validation showed that the model was robust and had good predictability. In conclusion, for all drugs, the final PK/PD model successfully described bacterial growth and killing kinetics when the bacteria were exposed to different antibiotic concentrations. The semimechanistic model that was developed might, after further refinement, serve as a tool for the development of optimal dosing strategies for antibacterial agents.
Quinolone Resistance in
Helicobacter pylori
Isolates in Germany
Authors: Glocker Erik , Stueger Hans-Peter , Kist Manfred
Published: December 2006

We show that quinolone resistance in
Helicobacter pylori
has reached an alarming level in Germany. Our data suggest that the use of quinolones requires prior antimicrobial susceptibility testing, especially for isolates from patients who have already undergone previous unsuccessful eradication treatments, and also underline the further need for surveillance studies to monitor antibiotic resistance in
H. pylori
.
Poly-γ-Glutamate Capsule-Degrading Enzyme Treatment Enhances Phagocytosis and Killing of EncapsulatedBacillus anthracis
Authors: Scorpio Angelo , Chabot Donald J. , Day William A. , O'Brien David K. , Vietri Nicholas J. , Itoh Yoshifumi , Mohamadzadeh Mansour , Friedlander Arthur M.
Published: December 2006

The poly-γ-d-glutamic acid capsule confers antiphagocytic properties onBacillus anthracisand is essential for virulence. In this study, we showed that CapD, a γ-polyglutamic acid depolymerase encoded on theB. anthraciscapsule plasmid, degraded purified capsule and removed the capsule from the surface of anthrax bacilli. Treatment with CapD induced macrophage phagocytosis of encapsulatedB. anthracisand enabled human neutrophils to kill encapsulated organisms. A second glutamylase, PghP, a γ-polyglutamic acid hydrolase encoded byBacillus subtilisbacteriophage ΦNIT1, had minimal activity in degradingB. anthraciscapsule, no effect on macrophage phagocytosis, and only minimal enhancement of neutrophil killing. Thus, the levels of both phagocytosis and killing corresponded to the degree of enzyme-mediated capsule degradation. The use of enzymes to degrade the capsule and enable phagocytic killing ofB. anthracisoffers a new approach to the therapy of anthrax.
Influence on Mitochondria and Cytotoxicity of Different Antibiotics Administered in High Concentrations on Primary Human Osteoblasts and Cell Lines
Authors: Duewelhenke N. , Krut O. , Eysel P.
Published: December 2006

Osteomyelitis, osteitis, spondylodiscitis, septic arthritis, and prosthetic joint infections still represent the worst complications of orthopedic surgery and traumatology. Successful treatment requires, besides surgical débridement, long-term systemic and high-concentration local antibiotic therapy, with possible local antibiotic concentrations of 100 μg/ml and more. In this study, we investigated the effect of 20 different antibiotics on primary human osteoblasts (PHO), the osteosarcoma cell line MG63, and the epithelial cell line HeLa. High concentrations of fluoroquinolones, macrolides, clindamycin, chloramphenicol, rifampin, tetracycline, and linezolid during 48 h of incubation inhibited proliferation and metabolic activity, whereas aminoglycosides and inhibitors of bacterial cell wall synthesis did not. Twenty percent inhibitory concentrations for proliferation of PHO were determined as 20 to 40 μg/ml for macrolides, clindamycin, and rifampin, 60 to 80 μg/ml for chloramphenicol, tetracylin, and fluoroquinolones, and 240 μg/ml for linezolid. The proliferation of the cell lines was always less inhibited. We established the measurement of extracellular lactate concentration as an indicator of glycolysis using inhibitors of the respiratory chain (antimycin A, rotenone, and sodium azide) and glycolysis (iodoacetic acid) as reference compounds, whereas inhibition of the respiratory chain increased and inhibition of glycolysis decreased lactate production. The measurement of extracellular lactate concentration revealed that fluoroquinolones, macrolides, clindamycin, rifampin, tetracycline, and especially chloramphenicol and linezolid impaired mitochondrial energetics in high concentrations. This explains partly the observed inhibition of metabolic activity and proliferation in our experiments. Because of differences in the energy metabolism, PHO provided a more sensitive model for orthopedic antibiotic usage than stable cell lines.
Synergistic Activities of Azithromycin and Amphotericin B against
Naegleria fowleri
In Vitro and in a Mouse Model of Primary Amebic Meningoencephalitis
Authors: Soltow Shannon M. , Brenner George M.
Published: December 2006

Naegleria fowleri
is responsible for producing a rapidly fatal central nervous system infection known as primary amebic meningoencephalitis (PAM). To date, amphotericin B, an antifungal agent, is the only agent with established clinical efficacy in the treatment of PAM. However, amphotericin B is not always successful in treating PAM and is associated with severe adverse effects. We previously found azithromycin to be more effective than amphotericin B in a mouse model of PAM. We therefore investigated the combination of amphotericin B and azithromycin in vitro and in a mouse model of PAM. For the in vitro studies, 50% inhibitory concentrations were calculated for each drug alone and for the drugs in fixed combination ratios of 1:1, 3:1, and 1:3. We found amphotericin B and azithromycin to be synergistic at all three of the fixed combination ratios. In our mouse model of PAM, a combination of amphotericin B (2.5 mg/kg of body weight) and azithromycin (25 mg/kg) protected 100% of the mice, whereas amphotericin B alone (2.5 mg/kg) protected only 27% of mice and azithromycin alone (25 mg/kg) protected 40% of mice. This study indicates that amphotericin B and azithromycin are synergistic against the Lee strain of
N. fowleri
, suggesting that the combined use of these agents may be beneficial in treating PAM.
Aph(3′)-IIc, an Aminoglycoside Resistance Determinant from
Stenotrophomonas maltophilia
Authors: Okazaki Aki , Avison Matthew B.
Published: December 2006

We report the characterization of an intrinsic, chromosomally carried
aph(3′)-IIc
gene from
Stenotrophomonas maltophilia
clinical isolate K279a, encoding an aminoglycoside phosphotransferase enzyme that significantly increases MICs of kanamycin, neomycin, butirosin, and paromomycin when expressed in
Escherichia coli
. Disruption of
aph(3′)-IIc
in K279a results in decreased MICs of these drugs.
Association of QnrB Determinants and Production of Extended-Spectrum β-Lactamases or Plasmid-Mediated AmpC β-Lactamases in Clinical Isolates of
Klebsiella pneumoniae
Authors: Pai Hyunjoo , Seo Mi-Ran , Choi Tae Yeal
Published: December 2006

Clinical isolates of
Escherichia coli
and
Klebsiella pneumoniae
producing extended-spectrum β-lactamases or plasmid-mediated AmpC β-lactamases were screened for
qnrA
and
qnrB
genes.
QnrB
was present in 54 of 54 DHA-1-producing
K. pneumoniae
isolates and 10 of 45 SHV-12-producing ones, suggesting that the distribution of plasmids conferring resistance to extended-spectrum cephalosporins and quinolones in clinical isolates of
K. pneumoniae
is widespread.
Comparative Pharmacokinetics of Azithromycin in Serum and White Blood Cells of Healthy Subjects Receiving a Single-Dose Extended-Release Regimen versus a 3-Day Immediate-Release Regimen
Authors: Liu Ping , Allaudeen Hameed , Chandra Richa , Phillips Kem , Jungnik Arvid , Breen Jeanne D. , Sharma Amarnath
Published: December 2006

The pharmacokinetic profiles of azithromycin given as a single-dose regimen (2.0-g extended-release microspheres) were characterized in serum and white blood cells (WBC) and compared with those of a 3-day regimen (a 500-mg immediate-release tablet once daily; total dose, 1.5 g) in an open-label, randomized, parallel-group study of 24 healthy adult subjects. Serial blood samples were collected up to 5 days after the start of dosing for both regimens. Safety assessments were conducted throughout the study. A single 2.0-g dose of azithromycin microspheres achieved significantly higher exposures in serum and WBC during the first 24 h after the start of dosing than a 3-day regimen: an approximately threefold higher area under the curve from time zero to 24 h postdose (AUC0-24) and an approximately twofold higher mean peak concentration on day 1. The single-dose regimen provided total azithromycin exposures in serum and WBC similar to those of the 3-day regimen, as evidenced by the similar AUC0-120and trough azithromycin concentrations in serum and WBC (mononuclear leukocytes [MNL] and polymorphonuclear leukocytes [PMNL]). For both regimens, the average total azithromycin exposures in MNL and PMNL were approximately 300- and 600-fold higher than those in serum. Azithromycin concentrations in MNL and PMNL remained above 10 μg/ml for at least 5 days after the start of dosing for both regimens. This “front-loading” of the dose on day 1 is safely achieved by the extended-release microsphere formulation, which maximizes the drug exposure at the time when the bacterial burden is likely to be highest.
The Phytotoxin Albicidin is a Novel Inhibitor of DNA Gyrase
Authors: Hashimi Saeed M. , Wall Melisa K. , Smith Andrew B. , Maxwell Anthony , Birch Robert G.
Published: December 2006

Xanthomonas albilineans
produces a family of polyketide-peptide compounds called albicidins which are highly potent antibiotics and phytotoxins as a result of their inhibition of prokaryotic DNA replication. Here we show that albicidin is a potent inhibitor of the supercoiling activity of bacterial and plant DNA gyrases, with 50% inhibitory concentrations (40 to 50 nM) less than those of most coumarins and quinolones. Albicidin blocks the religation of the cleaved DNA intermediate during the gyrase catalytic sequence and also inhibits the relaxation of supercoiled DNA by gyrase and topoisomerase IV. Unlike the coumarins, albicidin does not inhibit the ATPase activity of gyrase. In contrast to the quinolones, the albicidin concentration required to stabilize the gyrase cleavage complex increases 100-fold in the absence of ATP. The slow peptide poisons microcin B17 and CcdB also access ATP-dependent conformations of gyrase to block religation, but in contrast to albicidin, they do not inhibit supercoiling under routine assay conditions. Some mutations in
gyrA
, known to confer high-level resistance to quinolones or CcdB, confer low-level resistance or hypersensitivity to albicidin in
Escherichia coli
. Within the albicidin biosynthesis region in
X. albilineans
is a gene encoding a pentapeptide repeat protein designated AlbG that binds to
E. coli
DNA gyrase and that confers a sixfold increase in the level of resistance to albicidin in vitro and in vivo. These results demonstrate that DNA gyrase is the molecular target of albicidin and that
X. albilineans
encodes a gyrase-interacting protein for self-protection. The novel features of the gyrase-albicidin interaction indicate the potential for the development of new antibacterial drugs.
Mosaic Staphylococcal Cassette Chromosome
mec
Containing Two Recombinase Loci and a New
mec
Complex, B2
Authors: Heusser R. , Ender M. , Berger-Bächi B. , McCallum N.
Published: December 2006

A novel staphylococcal cassette chromosome (SCC)
mec
from a clinical methicillin-resistant
Staphylococcus aureus
isolate (ST100/CC5) had a mosaic structure, composed of SCC DNA from several different backgrounds. It harbored two complete
ccr
loci and a new variant of
mec
complex B, with Δ
mecR1
interrupted by the aminoglycoside resistance transposon Tn
4001
.
Inhibition of Antiassociation Activity of Translation Initiation Factor 3 by Paromomycin
Authors: Hirokawa Go , Kaji Hideko , Kaji Akira
Published: December 2006

The effect of paromomycin on the interaction of ribosomal subunits was studied. Paromomycin inhibited the antiassociation activity of initiation factor 3 (IF3). Furthermore, ribosomal subunits were associated to form 70S ribosomes by paromomycin even in the presence of 1 mM Mg
2+
. Paromomycin did not inhibit the binding of IF3 to the 30S ribosomal subunits. On the other hand, IF3 bound to the 30S subunits was expelled by paromomycin-induced subunit association (70S formation). These results indicate that the stabilization of 70S ribosomes by paromomycin may in part be responsible for its inhibitory effects on translocation and ribosome recycling.
Antibacterial Activity and Mechanism of Action of a Novel Anilinouracil-Fluoroquinolone Hybrid Compound
Authors: Butler Michelle M. , LaMarr William A. , Foster Kimberly A. , Barnes Marjorie H. , Skow Donna J. , Lyden Patrick T. , Kustigian Lauren M. , Zhi Chengxin , Brown Neal C. , Wright George E. , Bowlin Terry L.
Published: December 2006

The anilinouracils (AUs) such as 6-(3-ethyl-4-methylanilino)uracil (EMAU) are a novel class of gram-positive, selective, bactericidal antibacterials which inhibit pol IIIC, the gram-positive-specific replicative DNA polymerase. We have linked various fluoroquinolones (FQs) to the N-3 position of EMAU to generate a variety of AU-FQ “hybrids” offering the potential for targeting two distinct steps in DNA replication. In this study, the properties of a hybrid, “251D,” were compared with those of representative AUs and FQs in a variety of in vitro assays, including pol IIIC and topoisomerase/gyrase enzyme assays, antibacterial, bactericidal, and mammalian cytotoxicity assays. Compound 251D potently inhibited pol IIIC and topoisomerase/gyrase, displayed gram-positive antibacterial potency at least 15 times that of the corresponding AU compound, and as expected, acted selectively on bacterial DNA synthesis. Compound 251D was active against a broad panel of antibiotic-resistant gram-positive pathogens as well as several gram-negative organisms and was also active against both AU- and FQ-resistant gram-positive organisms, demonstrating its capacity for attacking both of its potential targets in the bacterium. 251D also was bactericidal for gram-positive organisms and lacked toxicity in vitro. Although we obtained strains of
Staphylococcus aureus
resistant to the individual parent compounds, spontaneous resistance to 251D was not observed. We obtained 251D resistance in multiple-passage experiments, but resistance developed at a pace comparable to those for the parent compounds. This class of AU-FQ hybrids provides a promising new pharmacophore with an unusual dual mechanism of action and potent activity against antibiotic-sensitive and -resistant gram-positive pathogens.
Inhibition of Autoimmune Type 1 Diabetes by Gastrointestinal Helminth Infection
Authors: Saunders Karin A. , Raine Tim , Cooke Anne , Lawrence Catherine E.
Published: December 2006

Gastrointestinal nematode infections are prevalent worldwide and are potent inducers of T helper 2 responses with the capacity to modulate the immune response to heterologous antigens. Parasitic helminth infection has even been shown to modulate the immune response associated with autoimmune diseases. Nonobese diabetic (NOD) mice provide a model for studying human autoimmune diabetes; as in humans, the development of diabetes in NOD mice has been linked to the loss of self-tolerance to beta cell autoantigens. Previous studies with the NOD mouse have shown that helminth and bacterial infection appears to inhibit type 1 diabetes by disrupting the pathways leading to the Th1-mediated destruction of insulin-producing beta cells. The aim of our study was to examine whether infection with the gastrointestinal helminthsTrichinella spiralisorHeligmosomoides polygyruscould inhibit the development of autoimmune diabetes in NOD mice and to analyze the mechanisms involved in protection and the role of Th2 responses. Protection from diabetes was afforded by helminth infection, appeared to inhibit autoimmune diabetes by disrupting pathways leading to the destruction of beta cells, and was mediated by seemingly independent mechanisms depending on the parasite but which may be to be related to the capacity of the host to mount a Th2 response.
Biological Trojan Horse: Antigen 43 Provides Specific Bacterial Uptake and Survival in Human Neutrophils
Authors: Fexby Sara , Bjarnsholt Thomas , Jensen Peter Østrup , Roos Viktoria , Høiby Niels , Givskov Michael , Klemm Per
Published: December 2006

Escherichia coliis a versatile pathogen causing millions of infections in humans every year. This bacterium can form multicellular aggregates when it expresses a self-associating protein, antigen 43 (Ag43), on its surface. We have discovered that Ag43-expressingE. colicells are efficiently taken up by human defense cells, polymorphonuclear neutrophils (PMNs), in an opsonin-independent manner. Surprisingly, the phagocytosed bacteria were not immediately killed but resided as tight aggregates within the PMNs. Our observations indicate that Ag43-mediated uptake and survival in PMNs constitute a mechanism to subvert one of the primary defense mechanisms of the human body.
Escherichia coli
from Urine of Female Patients with Urinary Tract Infections Is Competent for Intracellular Bacterial Community Formation
Authors: Garofalo Corinne K. , Hooton Thomas M. , Martin Steven M. , Stamm Walter E. , Palermo Joseph J. , Gordon Jeffrey I. , Hultgren Scott J.
Published: December 2006

Nearly 50% of women experience at least one urinary tract infection (UTI) in their lifetime. Studies with mice have revealed that uropathogenic
Escherichia coli
(UPEC) isolates invade superficial umbrella cells that line the bladder, allowing them to find a safe haven and subvert clearance by innate host responses. Rapid intracellular replication results in the formation of distinctive intracellular bacterial communities (IBCs). In this study, we evaluated whether UPEC strains cultured from the urine of women and classified as causing acute cystitis, recurrent cystitis, asymptomatic bacteriuria, or pyelonephritis could progress through the IBC cascade in a well-characterized mouse model of cystitis. Of 18 UPEC isolates collected from women, 15 formed IBCs. Variations in the size, number, and kinetics of IBC formation were observed with strains isolated from women with different clinical syndromes. Two of the three isolates that did not form IBCs when inoculated alone were able to do so when coinoculated with an isolate that was capable of generating IBCs. The mixed infections dramatically altered the behavior of the coinfecting bacteria relative to their behavior in a single infection. The study also showed that mice with five different genetic backgrounds can support IBC formation. Although UPEC isolates differ genetically in their virulence factors, the majority of UPEC isolates from different types of UTI proceed through the IBC pathway, confirming the generality of IBCs in UTI pathogenesis in mice.
Differential Clearance and Immune Responses to Tick Cell-Derived versus Macrophage Culture-DerivedEhrlichia chaffeensisin Mice
Authors: Ganta Roman R. , Cheng Chuanmin , Miller Elizabeth C. , McGuire Bridget L. , Peddireddi Lalitha , Sirigireddy Kamesh R. , Chapes Stephen K.
Published: December 2006

Human monocytic ehrlichiosis is caused by a tick-transmitted rickettsia,Ehrlichia chaffeensis. We recently reported thatE. chaffeensisgrown in tick cells expresses different proteins than bacteria grown in macrophages. Therefore, we tested the hypothesis that immune responses againstE. chaffeensiswould be different if the mice are challenged with bacteria grown in macrophages or tick cells. We assessed theE. chaffeensisclearance from the peritoneum, spleen, and liver by C57BL/6J mice using a TaqMan-based real-time reverse transcription-PCR assay. Macrophage-grownE. chaffeensiswas cleared in 2 weeks from the peritoneum, whereas the pathogen from tick cells persisted for nine additional days and included three relapses of increasing bacterial load separated by three-day intervals. Tick cell-grown bacteria also persisted in the livers and spleens with higher bacterial loads compared to macrophage-grown bacteria and fluctuated over a period of 35 days. Three-day periodic cycles were detected in T-cell CD62L/CD44 ratios in the spleen and bone marrow in response to infections with both tick cell- and macrophage-grown bacteria and were accompanied by similar periodic cycles of spleen cell cytokine secretions and nitric oxide and interleukin-6 by peritoneal macrophages. TheE. chaffeensis-specific immunoglobulin G response was considerably higher and steadily increased in mice infected with the tick cell-derivedE. chaffeensiscompared to DH82-grown bacteria. In addition, antigens detected by the immunoglobulins were significantly different between mice infected with theE. chaffeensisoriginating from tick cells or macrophages. The differences in the immune response to tick cell-grown bacteria compared to macrophage-grown bacteria reflected a delay in the shift of gene expression from the tick cell-specific Omp 14 gene to the macrophage-specific Omp 19 gene. These data suggest that the host response toE. chaffeensisdepends on the source of the bacteria and that this experimental model requires the most natural inoculum possible to allow for a realistic understanding of host resistance.
Staphylococcal Enterotoxin B In Vivo Modulates both Gamma Interferon Receptor Expression and Ligand-Induced Activation of Signal Transducer and Activator of Transcription 1 in T Cells
Authors: Plaza R. , Rodriguez-Sanchez J. L. , Juarez C.
Published: December 2006

Superantigens (SAg) are bacterial exotoxins that provoke extreme responses in the immune system; for example, the acute hyperactivation of SAg-reactive T cells that leads to toxic shock syndrome is followed within days by strong immunosuppression. The gamma interferon (IFN-γ) response is deeply affected in both extremes. The implication of IFN-γ in the pathophysiology of lethal shock induced in mice after a secondary challenge with the SAg staphylococcal enterotoxin B (SEB) prompted us to study the regulation of IFN-γ secretion and the intracellular response. We demonstrate in this study that a rechallenge with SEB becomes lethal only when given inside a critical time window after SEB priming and is associated with an increase of IFN-γ serum release 72 h after priming. However, at this time, a selective blockade of IFN-γ/STAT1 signaling develops in spleen cells, correlating with a lack of expression of the IFN-γ receptor beta subunit and STAT1 in the T-cell population. Selective blockade of the STAT1 signaling pathway—while simultaneously maintaining STAT3 signaling and expression—may be a protective mechanism that shortens IFN-γ production during the Th1 effector response. This blockade may also have consequences on switching towards a suppressor phenotype with chronic exposure to the superantigen.
High Levels of CXCL10 Are Produced by Intestinal Epithelial Cells in AIDS Patients with Active Cryptosporidiosis but Not after Reconstitution of Immunity
Authors: Wang Heuy-Ching , Dann Sara M. , Okhuysen Pablo C. , Lewis Dorothy E. , Chappell Cynthia L. , Adler Douglas G. , White A. Clinton
Published: December 2006

Chemokines play key roles in attracting immune cells to sites of infections. However, few data on chemokine expression in the gut during human infections are available. We examined expression of chemokines in intestinal tissues of AIDS patients during active
Cryptosporidium
infection and during resolution of such an infection. The chemokines and cytokines in cell lysates from jejunal biopsy tissues were assayed by a 22-multiplex bead immunoassay. CXCL10 (IP-10) and its receptor, CXCR3, in sections were studied by immunohistochemistry. In biopsies from AIDS patients with active cryptosporidiosis, four chemokines (CXCL10, CCL11 [eotaxin], CCL5 [RANTES], and CCL2 [monocyte chemoattractant protein 1]) and three cytokines (interleukin-1α [IL-1α], IL-10, and granulocyte colony-stimulating factor) were detected. The level of CXCL10 was significantly increased in AIDS patients with cryptosporidiosis compared to the level in AIDS patients without cryptosporidiosis or in normal volunteers (median in AIDS patients with cryptosporidiosis, 508 pg/mg protein, compared to 111 pg/mg and 72 pg/mg protein in AIDS patients without cryptosporidiosis and in normal volunteers, respectively [
P
< 0.05 and
P
< 0.005, respectively, as determined by a Mann-Whitney test]). The level of CXCL10 correlated with the parasite burden (as measured by the number of
Cryptosporidium
oocysts in the stools) and also with the IL-1α concentration (Pearson correlation values, 0.961 [
P
< 0.01] and 0.737 [
P
< 0.05]). As determined by immunohistochemistry, CXCL10 localized to epithelial cells at the site of infection. Following effective antiparasite and antiretroviral therapy,
Cryptosporidium
infections resolved, and the levels of CXCL10 decreased to normal levels. We hypothesized that CXCL10 plays an important role in the resolution of cryptosporidiosis by attracting immune effector cells to the site of infection. By contrast, in AIDS patients lacking effector cells, CXCL10 may contribute to the immunopathogenesis by recruiting inflammatory cells.
Serratia marcescensSerralysin Induces Inflammatory Responses through Protease-Activated Receptor 2
Authors: Kida Yutaka , Inoue Hiroyoshi , Shimizu Takashi , Kuwano Koichi
Published: December 2006

TheSerratia marcescens-derived protease serralysin is considered to play an important role in the pathogenesis of infection. Protease-activated receptor 2 (PAR-2) is activated by trypsin and also several other trypsin-like serine proteases, leading to the modulation of inflammatory and immune responses. However, little is known about the activation of PAR-2 by bacterial proteases and its roles in bacterial infection. In this study, we investigated whetherS. marcescensserralysin activates host inflammatory responses through PAR-2. Our results demonstrated that serralysin induces interleukin-6 (IL-6) and IL-8 mRNA expression in a human lung squamous cell carcinoma, EBC-l cells. In addition, serralysin activated activator protein 1 (AP-1)-, CCAAT/enhancer-binding protein (C/EBP)-, and nuclear factor-κB (NF-κB)-driven promoters in EBC-1 cells. An electrophoretic mobility shift assay showed that serralysin activates the binding of AP-1, C/EBPβ, and NF-κB in the cells. Inactivation of serralysin resulted in the failure of transactivation of AP-1-, C/EBP-, and NF-κB-driven promoters in the cells. Furthermore, serralysin activated AP-1-, C/EBP-, and NF-κB-driven promoters via PAR-2 in HeLa cells. PAR-2 antagonist peptides decreased serralysin-induced transactivation of AP-1-, C/EBP-, and NF-κB-driven promoters in EBC-1 cells. Considered together, these results suggest that serralysin requires PAR-2 to activate the critical transcription factors AP-1, C/EBPβ, and NF-κB for host inflammatory responses.
Respiratory Tract Infection withMycoplasma pneumoniaein Interleukin-12 Knockout Mice Results in Improved Bacterial Clearance and Reduced Pulmonary Inflammation
Authors: Salvatore C. M. , Fonseca-Aten M. , Katz-Gaynor K. , Gomez A. M. , Mejias A. , Somers C. , Chavez-Bueno S. , McCracken G. H. , Hardy R. D.
Published: December 2006

Mycoplasma pneumoniaeis a leading cause of pneumonia and is associated with asthma. Evidence linksM. pneumoniaerespiratory disease severity with interleukin-12 (IL-12) concentration in respiratory secretions. We evaluated the microbiologic, inflammatory, and pulmonary function indices ofM. pneumoniaepneumonia in IL-12 (p35) knockout (KO) mice and wild-type (WT) mice to determine the role of IL-12 inM. pneumoniaerespiratory disease. Eight-week-old wild-type BALB/c mice and 8-week-old IL-12 (p35) KO BALB/c mice were inoculated once intranasally with 107CFU ofM. pneumoniae. Mice were evaluated at days 2, 4, and 7 after inoculation. Outcome variables included quantitative bronchoalveolar lavage (BAL)M. pneumoniaeculture, lung histopathologic scores (HPS), BAL cytokine concentrations determined by enzyme-linked immunosorbent assay (tumor necrosis factor alpha [TNF-α], gamma interferon [IFN-γ], IL-1β, IL-2, IL-4, IL-5, IL-6, IL-10, and granulocyte-macrophage colony-stimulating factor) and plethysmography, before and after methacholine, to assess airway obstruction (AO) and airway hyperreactivity (AHR). IL-12 (p35) KO mice infected withM. pneumoniaewere found to have significantly lower BALM. pneumoniaeconcentrations compared withM. pneumoniae-infected WT mice. Lung HPS and the parenchymal pneumonia subscores (neutrophilic alveolar infiltrate), as well as AO, were significantly lower in infected KO mice. No difference was found for AHR. Infected KO mice had significantly lower BAL concentrations of IFN-γ than WT mice; a trend toward lower BAL concentrations was observed for IL-10 (P= 0.065) and TNF-α (P= 0.078). No differences were found for IL-1β, IL-2, IL-4, IL-5, or IL-6. The lack of IL-12 in experimentalM. pneumoniaepneumonia was associated with less severe pulmonary disease and more rapid microbiologic and histologic resolution.
In Vivo Gene Expression Analysis Identifies Genes Required for Enhanced Colonization of the Mouse Urinary Tract by Uropathogenic
Escherichia coli
Strain CFT073
dsdA
Authors: Haugen Brian J. , Pellett Shahaireen , Redford Peter , Hamilton Holly L. , Roesch Paula L. , Welch Rodney A.
Published: December 2006

Deletional inactivation of the gene encoding
d
-serine deaminase,
dsdA
, in uropathogenic
Escherichia coli
strain CFT073 results in a hypermotile strain with a hypercolonization phenotype in the bladder and kidneys of mice in a model of urinary tract infection (UTI). The in vivo gene expression profiles of CFT073 and CFT073
dsdA
were compared by isolating RNA directly from the urine of mice challenged with each strain individually. Hybridization of cDNAs derived from these samples to CFT073-specific microarrays allowed identification of genes that were up- or down-regulated in the
dsdA
deletion strain during UTI. Up-regulated genes included the known
d
-serine-responsive gene
dsdX
, suggesting in vivo intracellular accumulation of
d
-serine by CFT073
dsdA.
Genes encoding F1C fimbriae, both copies of P fimbriae, hemolysin, OmpF, a dipeptide transporter DppA, a heat shock chaperone IbpB, and clusters of open reading frames with unknown functions were also up-regulated. To determine the role of these genes as well as motility in the hypercolonization phenotype, mutants were constructed in the CFT073
dsdA
background and tested in competition against the wild type in the murine model of UTI. Strains with deletions of one or both of the two P fimbrial operons,
hlyA
,
fliC
,
ibpB
, c0468, locus c3566 to c3568, or c2485 to c2490 colonized mouse bladders and kidneys at levels indistinguishable from wild type. CFT073
dsdA
c2398 and CFT073
dsdA focA
maintained a hypercolonization phenotype. A CFT073
dsdA dppA
mutant was attenuated 10- to 50-fold in its colonization ability compared to CFT073. Our results support a role for
d
-serine catabolism and signaling in global virulence gene regulation of uropathogenic
E. coli
.
Microbial Metalloproteinases Mediate Sensing of Invading Pathogens and Activate Innate Immune Responses in the Lepidopteran Model Host
Galleria mellonella
Authors: Altincicek Boran , Linder Monica , Linder Dietmar , Preissner Klaus T. , Vilcinskas Andreas
Published: December 2006

Thermolysin-like metalloproteinases such as aureolysin, pseudolysin, and bacillolysin represent virulence factors of diverse bacterial pathogens. Recently, we discovered that injection of thermolysin into larvae of the greater wax moth,
Galleria mellonella
, mediated strong immune responses. Thermolysin-mediated proteolysis of hemolymph proteins yielded a variety of small-sized (<3 kDa) protein fragments (protfrags) that are potent elicitors of innate immune responses. In this study, we report the activation of a serine proteinase cascade by thermolysin, as described for bacterial lipopolysaccharides (LPS), that results in subsequent prophenoloxidase activation leading to melanization, an elementary immune defense reaction of insects. Quantitative real-time reverse transcription-PCR analyses of the expression of immune-related genes encoding the inducible metalloproteinase inhibitor, gallerimycin, and lysozyme demonstrated increased transcriptional rates after challenge with purified protfrags similar to rates after challenge with LPS. Additionally, we determined the induction of a similar spectrum of immune-responsive proteins that were secreted into the hemolymph by using comparative proteomic analyses of hemolymph proteins from untreated larvae and from larvae that were challenged with either protfrags or LPS. Since
G. mellonella
was recently established as a valuable pathogenicity model for
Cryptococcus neoformans
infection, the present results add to our understanding of the mechanisms of immune responses in
G. mellonella
. The obtained results support the proposed danger model, which suggests that the immune system senses endogenous alarm signals during infection besides recognition of microbial pattern molecules.