bluuebunny/crossref_metadata_2025 huggingface dataset:
Determination of ddATP Levels in Human Immunodeficiency Virus-Infected Patients Treated with Dideoxyinosine
Authors: Le Saint Cecile , Terreux Raphael , Duval Daniele , Durant Jacques , Ettesse Helene , Dellamonica Pierre , Guedj Roger , Vincent Jean Pierre , Cupo Anny
Published: January 2004

Clinical failures of the highly active antiretroviral therapy could result from inefficient intracellular concentrations of antiviral drugs. The determination of drug contents in target cells of each patient would be useful in clinical investigations and trials. The purpose of this work was to quantify the intracellular concentration of ddATP, the active metabolite of dideoxyinosine (ddI), in peripheral blood mononuclear cells (PBMCs) of human immunodeficiency virus (HIV)-infected patients treated with ddI. We have raised antibodies against ddA-citrate, a stable isostere of ddATP selected on the basis of its structural and electronic analogies with ddATP. The anti-ddA-citrate antibodies recognized ddATP and ddA with nanomolar affinities and cross-reacted neither with any of the nucleotide reverse transcriptase inhibitors used in HIV therapy nor with their phosphorylated metabolites. The three phosphorylated metabolites of ddI (ddAMP, ddADP, and ddATP) were purified by anion exchange chromatography and the amount of each metabolite was determined by radioimmunoassay with or without prior phosphatase treatment. The intracellular levels of the three ddI metabolites were measured both in an in vitro model and in PBMCs of HIV-infected patients under ddI treatment. The possibility to measure intracellular levels of ddATP from small blood samples of HIV-infected patients treated with ddI could be exploited to develop individual therapeutic monitoring.
Multiplex PCR Amplimer Conformation Analysis for Rapid Detection of
gyrA
Mutations in Fluoroquinolone-Resistant
Mycobacterium tuberculosis
Clinical Isolates
Authors: Cheng Augustine F. B. , Yew Wing W. , Chan Edward W. C. , Chin Miu L. , Hui Mamie M. M. , Chan Raphael C. Y.
Published: January 2004

A new strategy known as multiplex PCR amplimer conformation was developed for detection of mutation in the
gyrA
gene of 138 clinical isolates of
Mycobacterium tuberculosis
. The method generated a single-stranded and heteroduplex DNA banding pattern of multiplex PCR amplimers of the region of interest that was extremely sensitive to specific mutations, thus enabling much more sensitive and reliable mutation analysis compared to the standard single-stranded conformation polymorphism technique. The genetic profiles of the
gyrA
gene of the 138 isolates as detected by MPAC were confirmed by nucleotide sequencing and were found to correlate strongly with the in vitro susceptibilities of the mutant strains to six fluoroquinolones (ofloxacin, levofloxacin, sparfloxacin, moxifloxacin, gatifloxacin, and sitafloxacin). All 32 isolates that contained
gyrA
mutations exhibited cross-resistance to the six fluoroquinolones (ofloxacin MIC for 90% of strains > 16 mg/liter), although moxifloxacin, gatifloxacin, and sitafloxacin (MIC for 90% of strains ≤ 4 mg/liter) were apparently more active than ofloxacin, levofloxacin, and sparfloxacin (MIC for 90% of strains ≥ 16 mg/liter). All
gyrA
mutations were clustered in codons 90, 91, and 94, and aspartic acid 94 was most frequently mutated. Twenty-three isolates without
gyrA
mutations were also found to exhibit reduced susceptibility to ofloxacin (MIC for 90% of strains = 4 mg/liter), but largely remained susceptible to other drugs (MIC for 90% of strains ≤ 1 mg/liter). Another 83 isolates without mutations were fully susceptible to all six fluoroquinolones (ofloxacin MIC for 90% of strains = 1 mg/liter). In conclusion, high-level phenotypic resistance to fluoroquinolones among
M. tuberculosis
clinical isolates, which appears to be predominantly due to
gyrA
mutations, may be readily detected by genotyping techniques such as multiplex PCR amplimer conformation.
In Vitro Activities of New Antimicrobials against
Nocardia brasiliensis
Authors: Vera-Cabrera Lucio , Gonzalez Eva , Choi Sung H. , Welsh Oliverio
Published: January 2004

The in vitro sensitivities of 30 strains of
Nocardia brasiliensis
to DA-7867, gatifloxacin, moxifloxacin, and BMS-284756 (garenoxacin) were determined using the broth microdilution method. All
N. brasiliensis
strains were sensitive to these antimicrobials. The most active drug in vitro was DA-7867, with a MIC at which 90% of the isolates tested were inhibited of 0.03 μg/ml and a MIC at which 50% of the isolates tested were inhibited of 0.06 μg/ml.
Activities of Cethromycin and Telithromycin against Recent North American Isolates of
Streptococcus pneumoniae
Authors: Jorgensen James H. , Crawford Sharon A. , McElmeel M. Leticia , Whitney Cynthia G.
Published: January 2004

The in vitro activities of two investigational ketolides, cethromycin (formerly ABT-773) and telithromycin, were determined for a selected group of 312
Streptococcus pneumoniae
isolates from a national surveillance program. The MIC of cethromycin at which 50% of the isolates were inhibited was 0.008 μg/ml, and the MIC at which 90% of the isolates were inhibited was 0.06 μg/ml; the corresponding values for telithromycin were ≤0.015 and 0.25 μg/ml, respectively. For six quinupristin-dalfopristin-resistant strains, the cethromycin MICs were 0.25 to 16 μg/ml and the telithromycin MICs were 1 to 4 μg/ml. However, there was only 0.3% resistance to telithromycin.
Replacement of ParC α4 Helix with That of GyrA Increases the Stability and Cytotoxicity of Topoisomerase IV-Quinolone-DNA Ternary Complexes
Authors: Pfeiffer Emily S. , Hiasa Hiroshi
Published: January 2004

Replacement of the α4 helix of ParC with that of GyrA increased the stability of topoisomerase IV-quinolone-DNA ternary complexes. This mutant topoisomerase IV-mediated cell killing was more efficient than topoisomerase IV-mediated cell killing in
Escherichia coli
. Thus, the α4 helix plays critical roles in determining the stability and the cytotoxicity of ternary complexes.
fexA
, a Novel
Staphylococcus lentus
Gene Encoding Resistance to Florfenicol and Chloramphenicol
Authors: Kehrenberg Corinna , Schwarz Stefan
Published: January 2004

The
Staphylococcus lentus
plasmid pSCFS2 carries a novel florfenicol-chloramphenicol resistance gene, designated
fexA
, encoding a protein of 475 amino acids with 14 transmembrane domains. The FexA protein differs from all previously known proteins involved in the efflux of chloramphenicol and florfenicol. Induction of
fexA
expression by chloramphenicol and florfenicol occurs via translational attenuation.
Ribosomal Alterations Contribute to Bacterial Resistance against the Dipeptide Antibiotic TAN 1057
Authors: Limburg E. , Gahlmann R. , Kroll H.-P. , Beyer D.
Published: January 2004

TAN 1057-resistant
Staphylococcus aureus
and
Escherichia coli
strains were selected to elucidate the mechanism of resistance and the mode of action of this dipeptide antibiotic. Cell-free translation with isolated ribosomes and S150 fractions from sensitive and resistant
S. aureus
strains demonstrated that alterations in the ribosomes contribute to the resistance of the bacteria.
Intact
mutS
in Laboratory-Derived and Clinical Glycopeptide-Intermediate
Staphylococcus aureus
Strains
Authors: Muthaiyan Arunachalam , Jayaswal Radheshyam K. , Wilkinson Brian J.
Published: January 2004

The
mutS
gene of the methyl-directed mismatch repair system was sequenced in 10 parent and glycopeptide-intermediate
Staphylococcus aureus
strains. The
mutS
gene was intact in all strains studied. Hence, mutations in this gene had played no role in the development of vancomycin resistance in these strains.
Class 1 Integron Containing Metallo-β-Lactamase Gene
bla
VIM-2
in
Pseudomonas aeruginosa
Clinical Strains Isolated in Japan
Authors: Yatsuyanagi Jun , Saito Shioko , Harata Seizaburo , Suzuki Noriyuki , Ito Yuko , Amano Ken-ichi , Enomoto Katsuhiko
Published: January 2004

Four
bla
VIM-2
gene-harboring
Pseudomonas aeruginosa
strains were identified. These strains possessed a class 1 integron harboring
ORF1
,
bla
VIM-2
, and
aacA4
gene cassettes. The transposon-mediated horizontal spread of the
bla
VIM-2
gene among these strains was suggested, which increases the threat that the
bla
VIM-2
gene will disseminate among diverse genera of bacteria.
CTX-M-12 β-Lactamase in a
Klebsiella pneumoniae
Clinical Isolate in Colombia
Authors: Villegas Maria Virginia , Correa Adriana , Perez Federico , Zuluaga Tania , Radice Marcela , Gutkind Gabriel , Casellas José María , Ayala Juan , Lolans Karen , Quinn John P. ,   the Colombian Nosocomial Resistance Study Group
Published: January 2004

We describe the detection of the CTX-M-12 β-lactamase from a clinical isolate of
Klebsiella pneumoniae
in Colombia. Screening of nosocomial
Klebsiella
spp. and
Escherichia coli
isolates from a network of teaching hospitals revealed the presence of CTX-M enzymes in multiple cities. This is the first description of CTX-M in Colombia.
Differential Diffusions of Indinavir and Lopinavir in Genital Secretions of Human Immunodeficiency Virus-Infected Women
Authors: Launay Odile , Tod Michel , Louchahi Kamel , Belarbi Linda , Bouchaud Olivier , Mémain Nathalie , Petitjean Olivier , Robineau Michel , Guillevin Loïc , Lortholary Olivier
Published: January 2004

Plasma and cervicovaginal secretion (CVS) samples were collected from 19 human immunodeficiency virus type 1-infected women on lopinavir- or indinavir-containing regimens. Lopinavir and indinavir were detectable in 29 and 93% of CVS samples, respectively, a finding that may be ascribed to these drugs' differences in protein binding and pK
a
. The relationship between lopinavir and indinavir pharmacodynamics and viral evolution in the female genital tract should be assessed over time.
Plasma Pharmacokinetics of Sulfadiazine Administered Twice Daily versus Four Times Daily Are Similar in Human Immunodeficiency Virus-Infected Patients
Authors: Jordan M. Kelli , Burstein Aaron H. , Rock-Kress Diane , Alfaro Raul M. , Pau Alice K. , Kovacs Joseph A. , Piscitelli Stephen C.
Published: January 2004

The pharmacokinetics of 2,000 mg of sulfadiazine administered twice daily (BID) versus those of 1,000 mg administered four times a day were compared in eight human immunodeficiency virus-infected patients. No differences in pharmacokinetic parameters were detected between the regimens. These data provide a pharmacokinetic rationale for BID dosing of sulfadiazine for the treatment and suppression of toxoplasmosis.
Moxifloxacin Penetration in Bronchial Secretions of Mechanically Ventilated Patients with Pneumonia
Authors: Leone Marc , Albanèse Jacques , Sampol-Manos Emmanuelle , Simon Nicolas , Lacarelle Bruno , Bruguerolle Bernard , Martin Claude
Published: January 2004

The pharmacokinetics of moxifloxacin was studied in 17 mechanically ventilated patients with pneumonia. Patients were given 400 mg of moxifloxacin intravenously. Blood samples and bronchial secretions were taken on days 1 and 4. A dose of 400 mg of moxifloxacin allows one to achieve efficient concentrations in bronchial secretions and plasma.
Safety and Efficacy of Antimicrobial Peptides against Naturally Acquired Leishmaniasis
Authors: Alberola J. , Rodríguez A. , Francino O. , Roura X. , Rivas L. , Andreu D.
Published: January 2004

Leishmaniases, which are important causes of morbidity and mortality in humans and dogs, are extremely difficult to treat. Antimicrobial peptides are rarely used as alternative treatments for naturally acquired parasitic diseases. Here we report that the acylated synthetic antimicrobial peptide Oct-CA(1-7)M(2-9) is safe and effective for treating naturally acquired canine leishmaniasis.
Comparison of the Dynamics of Resistance-Associated Mutations to Nucleoside Reverse Transcriptase Inhibitors, Nonnucleoside Reverse Transcriptase Inhibitors, and Protease Inhibitors after Cessation of Antiretroviral Combination Therapy
Authors: Wirden Marc , Delaugerre Constance , Marcelin Anne Genevieve , Ktorza Nadine , Ait Mohand Hocine , Dominguez Stephanie , Schneider Luminita , Ghosn Jade , Pauchard Michele , Costagliola Dominique , Katlama Christine , Calvez Vincent
Published: January 2004

The dynamics of mutations associated with resistance to antiretroviral drugs were analyzed after cessation of therapy. The results showed that the kinetics of the shift to wild-type amino acid residues were significantly faster for protease inhibitors, intermediate for nonnucleoside reverse transcriptase inhibitors, and slower for nucleoside reverse transcriptase inhibitors.
Emergence in
Klebsiella pneumoniae
and
Enterobacter cloacae
Clinical Isolates of the VIM-4 Metallo-β-Lactamase Encoded by a Conjugative Plasmid
Authors: Luzzaro Francesco , Docquier Jean-Denis , Colinon Céline , Endimiani Andrea , Lombardi Gianluigi , Amicosante Gianfranco , Rossolini Gian Maria , Toniolo Antonio
Published: January 2004

Resistance to carbapenems is an emerging problem among gram-negative hospital pathogens. A transferable plasmid encoding the VIM-4 metallo-β-lactamase was detected in isolates of
Klebsiella pneumoniae
and
Enterobacter cloacae
obtained from a single patient under carbapenem therapy. Thus, enterobacteria appear to increasingly contribute to the spread of VIM-type enzymes.
Inhibition of the Subgenomic Hepatitis C Virus Replicon in Huh-7 Cells by 2′-Deoxy-2′-Fluorocytidine
Authors: Stuyver Lieven J. , McBrayer Tamara R. , Whitaker Tony , Tharnish Phillip M. , Ramesh Mangala , Lostia Stefania , Cartee Leanne , Shi Junxing , Hobbs Ann , Schinazi Raymond F. , Watanabe Kyoichi A. , Otto Michael J.
Published: January 2004

2′-Deoxy-2′-fluorocytidine (FdC) is a potent inhibitor of the hepatitis C virus RNA replicon in culture, and FdC-5′-triphosphate is an effective inhibitor of the NS5B polymerase. Dynamic profiling of cell growth in an antiviral assay showed that FdC caused cytostasis due to an S-phase arrest. These observations demonstrate that FdC treatment is affecting both a viral target and a cellular target.
Effects of Ascorbic Acid, Glutathione, Thiocyanate, and Iodide on Antimicrobial Activity of Acidified Nitrite
Authors: Fite Alemu , Dykhuizen Rolf , Litterick Audrey , Golden Michael , Leifert Carlo
Published: January 2004

It has been hypothesized that dietary nitrite augments the antimicrobial activity of gastric acid after conversion to nitric oxide and other reactive nitrogen intermediates, thus resulting in increased resistance against gastrointestinal infection. In this study, we showed that the reducing agents ascorbic acid and glutathione reduced the activity of acidified nitrite against
Yersinia enterocolitica
(
P
< 0.001). In contrast, iodide and thiocyanate increased the antimicrobial activity (
P
< 0.001), whereas hydroxyacids (citrate, lactate, and tartarate) had no measurable effects.
Pharmacokinetics of DA-7867, a New Oxazolidinone, after Intravenous or Oral Administration to Rats: Intestinal First-Pass Effect
Authors: Bae Soo K. , Chung Won-Suk , Kim Eun J. , Rhee Jae K. , Kwon Jong W. , Kim Won B. , Lee Myung G.
Published: January 2004

Pharmacokinetic parameters of DA-7867 were dose independent after both intravenous administration and oral administration (at doses of 1 to 20 mg/kg of body weight) to rats. After oral administration of DA-7867 to rats at a dose of 10 mg/kg, approximately 8.27% of oral dose was not absorbed from the gastrointestinal tract,Fwas 70.8%, and approximately 21.8% of the oral dose was eliminated by the intestine (intestinal first-pass effect).
Bifunctional Anti-Human Immunodeficiency Virus Type 1 Small Molecules with Two Novel Mechanisms of Action
Authors: Huang Li , Yuan Xiong , Aiken Christopher , Chen Chin Ho
Published: January 2004

A class of betulinic acid derivatives was synthesized to target two critical steps in the human immunodeficiency virus type 1 (HIV-1) replication cycle, entry and maturation. Each mechanism of HIV-1 inhibition is distinct from clinically available anti-HIV therapeutics. The viral determinants of the antientry and antimaturation activities are the bridging sheet of HIV-1 gp120 and the P24/p2 cleavage site, respectively.
Class 1 Integrons Increase Trimethoprim-Sulfamethoxazole MICs against Epidemiologically Unrelated
Stenotrophomonas maltophilia
Isolates
Authors: Barbolla Raquel , Catalano Mariana , Orman Betina E. , Famiglietti Angela , Vay Carlos , Smayevsky Jorgelina , Centrón Daniela , Piñeiro Silvia A.
Published: January 2004

Twenty-five plasmid-specified antimicrobial resistance determinants common to gram-negative bacilli from nosocomial infection were investigated from 31
Stenotrophomonas maltophilia
isolates. Twenty-four clones were identified by pulsed-field gel electrophoresis, and in three clones that exhibited an increased trimethoprim-sulfamethoxazole MIC, the
sul1
determinant was found. These results support not only the higher spread of class 1 integrons compared to other mechanisms but also the potential limitation of using trimethoprim-sulfamethoxazole for therapy of severe
S. maltophilia
infections.
Clonal Multidrug-Resistant
Shigella dysenteriae
Type 1 Strains Associated with Epidemic and Sporadic Dysenteries in Eastern India
Authors: Pazhani Gururaja Perumal , Sarkar Bhaswati , Ramamurthy Thandavarayan , Bhattacharya S. K. , Takeda Yoshifumi , Niyogi S. K.
Published: January 2004

Multidrug-resistant strains of
Shigella dysenteriae
type 1 were implicated in three outbreaks and sporadic cases of dysentery in eastern India in 2002 and 2003. After a hiatus of 14 years, this pathogen reemerged with an altered antibiotic resistance pattern. In addition to ampicillin, co-trimoxazole, tetracycline, chloramphenicol, and nalidixic acid, all the recent strains were resistant to norfloxacin, lomefloxacin, pefloxacin, and ofloxacin and showed reduced susceptibility to ciprofloxacin. Pulsed-field gel electrophoresis identified a new clone of
S. dysenteriae
type 1 that was associated with the recent outbreaks and sporadic cases. Based on the spatial and temporal spread of multidrug-resistant
S. dysenteriae
type 1, we predict that this clonal type may spread further in this region.
Effects of Positively Selected Sequence Variations in Human and
Macaca fascicularis
β-Defensins 2 on Antimicrobial Activity
Authors: Antcheva Nikolinka , Boniotto Michele , Zelezetsky Igor , Pacor Sabrina , Falzacappa Maria Vittoria Verga , Crovella Sergio , Tossi Alessandro
Published: January 2004

The evolution of orthologous genes coding for β-defensin 2 (BD2) in primates has been subject to positive selection during the divergence of the platyrrhines from the catarrhines and of the
Cercopithecidae
from the
Hylobatidae
, great apes, and humans. Three peptides have been selected for a functional analysis of the effects of sequence variations on the direct antimicrobial activity: human BD2 (hBD2),
Macaca fascicularis
BD2 (mfaBD2), and a variant of the human peptide lacking Asp
4
, (−D)hBD2, which is characteristic only of the human/great ape peptides. hBD2 and mfaBD2 showed a significant difference in specificity, the former being more active towards
Escherichia coli
and the later towards
Staphylococcus aureus
and
Candida albicans.
Asp
4
in the human peptide appears to be important, as (−D)hBD2 was less structured and had a markedly lower antimicrobial activity. The evolution of β-defensin 2 in primates may thus have been driven, at least in part, by different environmental pressures so as to modulate antimicrobial activity.
Mucosal Vaccination Increases Endothelial Expression of Mucosal Addressin Cell Adhesion Molecule 1 in the Human Gastrointestinal Tract
Authors: Lindholm Catharina , Naylor Andrew , Johansson Eva-Liz , Quiding-Järbrink Marianne
Published: January 2004

Homing of leukocytes to various tissues is dependent on the interaction between homing receptors on leukocytes and their ligands, addressins, on endothelial cells. Mucosal immunization results in homing of antigen-specific lymphocytes back to the mucosa where they first encountered the antigen. However, it is unknown whether this homing of antigen-specific cells is mediated by an altered endothelial addressin expression after vaccination. Using different immunization routes with an oral cholera vaccine, we show that the endothelial expression of mucosal addressin cell adhesion molecule 1 (MAdCAM-1) is increased in the gastric and upper small intestinal mucosae after immunization through various local routes in the upper gastrointestinal tract. In contrast, rectal immunization did not influence the levels of MAdCAM-1 in the gastric or duodenal mucosa. Furthermore, we show that MAdCAM-1 can be induced on human endothelial cells by tumor necrosis factor alpha (TNF-α) and gamma interferon. The vaccine component cholera toxin B subunit (CTB) increased MAdCAM-1 expression on endothelial cells in cultured human gastric explants, an effect that seemed to be mediated by TNF-α. In conclusion, MAdCAM-1 expression is increased in the upper gastrointestinal tract after local immunizations with a vaccine containing CTB. This strongly suggests the involvement of MAdCAM-1 in the preferential homing of mucosal lymphocytes to their original site of activation.
Members of the Merozoite Surface Protein 7 Family with Similar Expression Patterns Differ in Ability To Protect againstPlasmodium yoeliiMalaria
Authors: Mello Kerrianne , Daly Thomas M. , Long Carole A. , Burns James M. , Bergman Lawrence W.
Published: January 2004

Previously, we described the isolation of thePlasmodium yoeliisequence-related moleculesP. yoeliiMSP-7 (merozoite surface protein 7) andP. yoeliiMSRP-2 (MSP-7-related protein 2) by their ability to interact with the amino-terminal end ofP. yoeliiMSP-1 in a yeast two-hybrid system. One of these molecules was the homologue ofPlasmodium falciparumMSP-7, which was biochemically isolated as part of the shed MSP-1 complex. In the present study, with antibodies directed against recombinant proteins, immunoprecipitation analyses of the rodent system demonstrated that bothP. yoeliiMSP-7 andP. yoeliiMSRP-2 could be isolated from parasite lysates and from parasite culture supernatants. Immunofluorescence studies colocalizedP. yoeliiMSP-7 andP. yoeliiMSRP-2 with the amino-terminal portion of MSP-1 and with each other on the surface of schizonts. Immunization withP. yoeliiMSRP-2 but notP. yoeliiMSP-7 protected mice against a lethal infection withP. yoeliistrain 17XL. These results establish that bothP. yoeliiMSP-7 andP. yoeliiMSRP-2 are expressed on the surface of merozoites and released from the parasite and thatP. yoeliiMSRP-2 may be the target of a protective immune response.
Transcutaneous Immunization with Combined Cholera Toxin and CpG Adjuvant Protects againstChlamydia muridarumGenital Tract Infection
Authors: Berry Linda J. , Hickey Danica K. , Skelding Kathryn A. , Bao Shisan , Rendina Amanda M. , Hansbro Philip M. , Gockel Christine M. , Beagley Kenneth W.
Published: January 2004

Chlamydia trachomatisis a pathogen of the genital tract and ocular epithelium. Infection is established by the binding of the metabolically inert elementary body (EB) to epithelial cells. These are taken up by endocytosis into a membrane-bound vesicle termed an inclusion. The inclusion avoids fusion with host lysosomes, and the EBs differentiate into the metabolically active reticulate body (RB), which replicates by binary fission within the protected environment of the inclusion. During the extracellular EB stage of theC. trachomatislife cycle, antibody present in genital tract or ocular secretions can inhibit infection both in vivo and in tissue culture. The RB, residing within the intracellular inclusion, is not accessible to antibody, and resolution of infection at this stage requires a cell-mediated immune response mediated by gamma interferon-secreting Th1 cells. Thus, an ideal vaccine to protect againstC. trachomatisgenital tract infection should induce both antibody (immunoglobulin A [IgA] and IgG) responses in mucosal secretions to prevent infection by chlamydial EB and a strong Th1 response to limit ascending infection to the uterus and fallopian tubes. In the present study we show that transcutaneous immunization with major outer membrane protein (MOMP) in combination with both cholera toxin and CpG oligodeoxynucleotides elicits MOMP-specific IgG and IgA in vaginal and uterine lavage fluid, MOMP-specific IgG in serum, and gamma interferon-secreting T cells in reproductive tract-draining caudal and lumbar lymph nodes. This immunization protocol resulted in enhanced clearance ofC. muridarum(C. trachomatis, mouse pneumonitis strain) following intravaginal challenge of BALB/c mice.
Systemic Th1 Immunization of Mice againstHelicobacter pyloriInfection with CpG Oligodeoxynucleotides as Adjuvants Does Not Protect from Infection but Enhances Gastritis
Authors: Sommer Frank , Wilken Henning , Faller Gerhard , Lohoff Michael
Published: January 2004

Recent reports have suggested that oral vaccination of mice againstHelicobacter pyloriis dependent on a Th1-mediated immune response. However, oral vaccination in mice neither induces sterilizing immunity nor leads to complete protection from disease. Therefore, in this study we investigated whether a systemic subcutaneous immunization againstH. pyloriby using CpG oligodeoxynucleotides as a Th1 adjuvant could achieve protection in a mouse model ofH. pyloriinfection. CpG oligodeoxynucleotides are known for their ability to induce nearly entirely Th1-biased immune responses and may be approved for human use in future. Immunization of mice withH. pylorilysate and CpG induced a strong local and systemic Th1 immune response. Despite this strong Th1 response, mice were not protected from infection withH. pyloriyet had a 10-fold reduction in the number ofH. pyloriin the gastric mucosa compared to nonimmunized mice. Of note, reduction of the bacterial density in immunized mice was accompanied by a significantly enhanced gastritis. Hence, systemic Th1 immunization of mice, even though being able to reduce the bacterial load in the stomach, is associated with aggravated pathology.
Salmonella enterica
Serovar Typhimurium Requires Nonsterol Precursors of the Cholesterol Biosynthetic Pathway for Intracellular Proliferation
Authors: Catron Drew M. , Lange Yvonne , Borensztajn Jayme , Sylvester Matthew D. , Jones Bradley D. , Haldar Kasturi
Published: January 2004

We have previously shown that
Salmonella enterica
serovar Typhimurium infection perturbs the host cholesterol biosynthetic pathway. Here we show that inhibiting the first step of this pathway (3-hydroxy-3-methylglutaryl coenzyme A reductase) reduces the growth of intracellular
S. enterica
serovar Typhimurium and has no effect on extracellular bacterial growth. Selectively inhibiting synthesis of downstream sterol components has no effect on infection, suggesting that the effect of statins on host nonsterol intermediates is detrimental to bacterial growth. Furthermore, statins also reduce bacterial proliferation in the
S. enterica
serovar Typhimurium mouse model. This suggests that blocking the production of nonsterol precursors in the host cell can be used to reduce infection.
Functional Analysis of thecagPathogenicity Island inHelicobacter pyloriIsolates from Patients with Gastritis, Peptic Ulcer, and Gastric Cancer
Authors: Backert Steffen , Schwarz Tobias , Miehlke Stephan , Kirsch Christian , Sommer Christian , Kwok Terry , Gerhard Markus , Goebel Ulf B. , Lehn Norbert , Koenig Wolfgang , Meyer Thomas F.
Published: January 2004

Helicobacter pyloriis the causative agent of a variety of gastric diseases, but the clinical relevance of bacterial virulence factors is still controversial. Virulent strains carrying thecagpathogenicity island (cagPAI) are thought to be key players in disease development. Here, we have comparedcagPAI-dependent in vitro responses inH. pyloriisolates obtained from 75 patients with gastritis, peptic ulcer, and gastric cancer (n= 25 in each group). AGS gastric epithelial cells were infected with each strain and assayed for (i) CagA expression, (ii) translocation and tyrosine phosphorylation of CagA, (iii) c-Src inactivation, (iv) cortactin dephosphorylation, (v) induction of actin cytoskeletal rearrangements associated with cell elongation, (vi) induction of cellular motility, and (vii) secretion of interleukin-8. Interestingly, we found high but similar prevalences of all of thesecagPAI-dependent host cell responses (ranging from 56 to 80%) among the various groups of patients. This study revealed CagA proteins with unique features, CagA subspecies of various sizes, and new functional properties for the phenotypic outcomes. We further showed that induction of AGS cell motility and elongation are two independent processes. Our data corroborate epidemiological studies, which indicate a significant association ofcagPAI presence and functionality with histopathological findings in gastritis, peptic ulcer, and gastric cancer patients, thus emphasizing the importance of thecagPAI for the pathogenicity ofH. pylori. Nevertheless, we found no significant association of the specificH. pylori-induced responses with any particular patient group. This may indicate that the determination of disease development is highly complex and involves multiple bacterial and/or host factors.
Chemokine Receptor 5 Is Dispensable for Innate and Adaptive Immune Responses toListeria monocytogenesInfection
Authors: Zhong Maggie X. , Kuziel William A. , Pamer Eric G. , Serbina Natalya V.
Published: January 2004

Chemokine receptor 5 (CCR5) binds macrophage inflammatory protein 1α (MIP-1α), MIP-1β, RANTES, and members of the monocyte chemotactic protein family and is also a receptor for human immunodeficiency virus (HIV). CCR5 ligands can suppress HIV-1 entry into cells. In humans, homozygous mutations of theccr5gene confer resistance to HIV-1 infection. The role of CCR5 in defense against microbial infection is unclear. In this study we examined the innate and adaptive immune responses of CCR5-deficient mice to the intracellular bacterial pathogenListeria monocytogenes.We found that migration of monocytic cells, formation ofL. monocytogenes-containing lesions, and bacterial clearance occurred normally in the spleens and livers of CCR5-deficient animals. Activation of macrophages and dendritic cells during the first 3 days postinfection was normal in the absence of CCR5, as demonstrated by intact expression of inducible nitric oxide synthase (iNOS) and production of the cytokines tumor necrosis factor alpha, gamma interferon, and interleukin-12. Priming ofL. monocytogenes-specific CD8 T cells also occured independently of CCR5 expression. Previously immunized, CCR5-deficient animals mounted normal secondary CD8 T-cell responses and cleared bacteria from infected organs similarly to wild-type controls, suggesting that CCR5 is dispensable for migration and activation of memory CD8 T cells. Our data indicate that CCR5-mediated chemotaxis is not required for defense against infection withL. monocytogenes.
Paucibacillary Tuberculosis in Mice after Prior Aerosol Immunization with
Mycobacterium bovis
BCG
Authors: Nuermberger E. L. , Yoshimatsu T. , Tyagi S. , Bishai W. R. , Grosset J. H.
Published: January 2004

To develop a murine model of paucibacillary tuberculosis for experimental chemotherapy of latent tuberculosis infection, mice were immunized with viable
Mycobacterium bovis
BCG by the aerosol or intravenous route and then challenged six weeks later with virulent
Mycobacterium tuberculosis
. The day after immunization, the counts were 3.71 ± 0.10 log
10
CFU in the lungs of aerosol-immunized mice and 3.65 ± 0.11 and 4.93 ± 0.07 log
10
CFU in the lungs and spleens of intravenously immunized mice, respectively. Six weeks later, the lungs of all BCG-immunized mice had many gross lung lesions and splenomegaly; the counts were 5.97 ± 0.14 and 3.54 ± 0.07 log
10
CFU in the lungs and spleens of aerosol-immunized mice, respectively, and 4.36 ± 0.28 and 5.12 ± 0.23 log
10
CFU in the lungs and spleens of intravenously immunized mice, respectively. Mice were then aerosol challenged with
M. tuberculosis
by implanting 2.37 ± 0.13 log
10
CFU in the lungs. Six weeks after challenge,
M. tuberculosis
had multiplied so that the counts were 6.41 ± 0.27 and 4.44 ± 0.14 log
10
CFU in the lungs and spleens of control mice, respectively. Multiplication of
M. tuberculosis
was greatly limited in BCG-immunized mice. Six weeks after challenge, the counts were 4.76 ± 0.24 and 3.73 ± 0.34 log
10
CFU in the lungs of intravenously immunized and aerosol-immunized mice, respectively. In contrast to intravenously immunized mice, there was no detectable dissemination to the spleen in aerosol-immunized mice. Therefore, immunization of mice with BCG by the aerosol route prior to challenge with a low dose of
M. tuberculosis
resulted in improved containment of infection and a stable paucibacillary infection. This model may prove to be useful for evaluation of new treatments for latent tuberculosis infection in humans.
New Aspects Regarding Evolution and Virulence of
Listeria monocytogenes
Revealed by Comparative Genomics and DNA Arrays
Authors: Doumith Michel , Cazalet Christel , Simoes Natalie , Frangeul Lionel , Jacquet Christine , Kunst Frank , Martin Paul , Cossart Pascale , Glaser Philippe , Buchrieser Carmen
Published: January 2004

Listeria monocytogenes
is a food-borne bacterial pathogen that causes a wide spectrum of diseases, such as meningitis, septicemia, abortion, and gastroenteritis, in humans and animals. Among the 13
L. monocytogenes
serovars described, invasive disease is mostly associated with serovar 4b strains. To investigate the genetic diversity of
L. monocytogenes
strains with different virulence potentials, we partially sequenced an epidemic serovar 4b strain and compared it with the complete sequence of the nonepidemic
L. monocytogenes
EGDe serovar 1/2a strain. We identified an unexpected genetic divergence between the two strains, as about 8% of the sequences were serovar 4b specific. These sequences included seven genes coding for surface proteins, two of which belong to the internalin family, and three genes coding for transcriptional regulators, all of which might be important in different steps of the infectious process. Based on the sequence information, we then characterized the gene content of 113
Listeria
strains by using a newly designed
Listeria
array containing the “flexible” part of the sequenced
Listeria
genomes. Hybridization results showed that all of the previously identified virulence factors of
L. monocytogenes
were present in the 93
L. monocytogenes
strains tested. However, distinct patterns of the presence or absence of other genes were identified among the different
L. monocytogenes
serovars and
Listeria
species. These results allow new insights into the evolution of
L. monocytogenes
, suggesting that early divergence of the ancestral
L. monocytogenes
serovar 1/2c strains from the serovar 1/2b strains led to two major phylogenetic lineages, one of them including the serogroup 4 strains, which branched off the serovar 1/2b ancestral lineage, leading (mostly by gene loss) to the species
Listeria innocua
. The identification of 30
L. monocytogenes
-specific and several serovar-specific marker genes, such as three
L. monocytogenes
serovar 4b-specific surface protein-coding genes, should prove powerful for the rapid tracing of listeriosis outbreaks, but it also represents a fundamental basis for the functional study of virulence differences between
L. monocytogenes
strains.
Molecular Characterization of a Glucose-Inhibited Division Gene,
gidA
, That Regulates Cytotoxic Enterotoxin of
Aeromonas hydrophila
Authors: Sha Jian , Kozlova E. V. , Fadl A. A. , Olano J. P. , Houston C. W. , Peterson J. W. , Chopra A. K.
Published: January 2004

By using a mini-transposon, we obtained two mutated strains of a diarrheal isolate, SSU, of
Aeromonas hydrophila
that exhibited a 50 to 53% reduction in the hemolytic activity and 83 to 87% less cytotoxic activity associated with the cytotoxic enterotoxin (Act). Act is a potent virulence factor of
A. hydrophila
and has been shown to contribute significantly to the development of both diarrhea and septicemia in animal models. Subsequent cloning and DNA sequence analysis revealed that transposon insertion occurred at different locations in these two mutants within the same 1,890-bp open reading frame for the glucose-inhibited division gene (
gidA
). A similar reduction in hemolytic (46%) and cytotoxic (81%) activity of Act was noted in the
gidA
isogenic mutant of
A. hydrophila
that was generated by marker exchange mutagenesis. Northern blot analysis revealed that the transcription of the cytotoxic enterotoxin gene (
act
) was not altered in the
gidA
transposon and isogenic mutants. However, by generating a chromosomal
act
::alkaline phosphatase gene (
phoA
) reporter construct, we demonstrated significantly reduced phosphatase activity in these mutants, indicating the effect of glucose-inhibited division (GidA) protein in modulating
act
gene expression at the translational level. The biological effects of Act in the
gidA
mutants were restored by complementation. The virulence of the
gidA
mutants in mice was dramatically reduced compared to the those of the wild-type (WT) and complemented strains of
A. hydrophila
. The histopathological examination of lungs, in particular, indicated severe congestion, alveolar hemorrhage, and acute inflammatory infiltrate in the interstitial compartment and the alveolar spaces when mice were infected with the WT and complemented strains. Minimal-to-mild changes were noted in the lungs with the
gidA
mutants. Taken together, our data indicate for the first time that GidA regulates the most-potent virulence factor of
A. hydrophila
, Act.
Conservation ofBabesia bovisSmall Heat Shock Protein (Hsp20) among Strains and Definition of T Helper Cell Epitopes Recognized by Cattle with Diverse Major Histocompatibility Complex Class II Haplotypes
Authors: Norimine Junzo , Mosqueda Juan , Palmer Guy H. , Lewin Harris A. , Brown Wendy C.
Published: January 2004

Babesia bovissmall heat shock protein (Hsp20) is recognized by CD4+T lymphocytes from cattle that have recovered from infection and are immune to challenge. This candidate vaccine antigen is related to a protective antigen ofToxoplasma gondii, Hsp30/bag1, and both are members of the α-crystallin family of proteins that can serve as molecular chaperones. In the present study, immunofluorescence microscopy determined that Hsp20 is expressed intracellularly in all merozoites. Importantly, Hsp20 is also expressed by tick larval stages, including sporozoites, so that natural tick-transmitted infection could boost a vaccine-induced response. The predicted amino acid sequence of Hsp20 from merozoites is completely conserved among differentB. bovisstrains. To define the location of CD4+T-cell epitopes for inclusion in a multiepitope peptide or minigene vaccine construct, truncated recombinant Hsp20 proteins and overlapping peptides were tested for their ability to stimulate T cells from immune cattle. Both amino-terminal (amino acids [aa] 1 to 105) and carboxy-terminal (aa 48 to 177) regions were immunogenic for the majority of cattle in the study, stimulating strong proliferation and IFN-γ production. T-cell lines from all individuals with distinctDRB3haplotypes responded to aa 11 to 62 of Hsp20, which contained one or more immunodominant epitopes for each animal. One epitope, DEQTGLPIKS (aa 17 to 26), was identified by T-cell clones. The presence of strain-conserved T helper cell epitopes in aa 11 to 62 of the ubiquitously expressed Hsp20 that are presented by major histocompatibility complex class II molecules represented broadly in the Holstein breed supports the inclusion of this region in vaccine constructs to be tested in cattle.
Protection against CD95-Induced Apoptosis by Chlamydial Infection at a Mitochondrial Step
Authors: Fischer Silke F. , Harlander Thomas , Vier Juliane , Häcker Georg
Published: January 2004

Chlamydiae are obligate intracellular bacteria that infect human epithelial and myeloid cells. Previous work has established that chlamydiae are able to protect a cell against apoptosis induced by certain experimentally applied stimuli. Here we provide an analysis of this protective activity against the signal transduction during CD95-induced apoptosis. In HeLa cells overexpressing CD95, infection withChlamydia trachomatisinhibited the appearance of apoptotic morphology, effector caspase activity, the activation of caspase-9 and -3, and the release of cytochromecfrom mitochondria. However, caspase-8-processing and activity (measured as cleavage of Bid) were unaffected by the chlamydial infection. Similarly, infection with the speciesC. pneumoniaedid not prevent the activation of caspase-8 but inhibited the appearance of effector caspase activity upon signaling through CD95. Furthermore, infection withC. trachomatiswas able to inhibit CD95-induced apoptosis in Jurkat lymphoid cells, where a mitochondrial contribution is required, but not in SKW6.4 lymphoid cells, where caspase-8 directly activates caspase-3. Taken together, these data show that chlamydial infection can protect cells against CD95-induced apoptosis but only where a mitochondrial signaling step is necessary for apoptotic signal transduction.
Gastroenteritis in NF-κB-Deficient Mice Is Produced with Wild-TypeCamplyobacter jejunibut Not withC. jejuniLacking Cytolethal Distending Toxin despite Persistent Colonization with Both Strains
Authors: Fox James G. , Rogers Arlin B. , Whary Mark T. , Ge Zhongming , Taylor Nancy S. , Xu Sandy , Horwitz Bruce H. , Erdman Susan E.
Published: January 2004

Campylobacter jejunicontinues to be a leading cause of bacterial enteritis in humans. However, because there are no readily available animal models to study the pathogenesis ofC. jejuni-related diseases, the significance of potential virulence factors, such as cytolethal distending toxin (CDT), in vivo are poorly understood. Mice deficient in NF-κB subunits (p50−/−p65+/−) in a C57BL/129 background are particularly susceptible to colitis induced by another enterohepatic microaerobe,Helicobacter hepaticus, which, likeC. jejuni, produces CDT. Wild-typeC. jejuni81-176 and an isogenic mutant lacking CDT activity (cdtBmutant) were inoculated into NF-κB-deficient (3X) and C57BL/129 mice. Wild-typeC. jejunicolonized 29 and 50% of the C57BL/129 mice at 2 and 4 months postinfection (p.i.), respectively, whereas theC. jejuni cdtBmutant colonized 50% of the C57BL/129 mice at 2 p.i. but none of the mice at 4 months p.i. Although the C57BL/129 mice developed mild gastritis and typhlocolitis, they had robust immunoglobulin G (IgG) and Th1-promoted IgG2a humoral responses to both the wild-type strain and theC. jejuni cdtBmutant. In contrast, 75 to 100% of the 3X mice were colonized with both the wild type and theC. jejuni cdtBmutant at similar levels at all times examined. Wild-typeC. jejunicaused moderately severe gastritis and proximal duodenitis in 3X mice that were more severe than the gastrointestinal lesions caused by theC. jejuni cdtBmutant. Persistent colonization of NF-κB-deficient mice with the wild type and theC. jejuni cdtBmutant was associated with significantly impaired IgG and IgG2a humoral responses (P< 0.001), which is consistent with an innate or adaptive immune system defect(s). These results suggest that the mechanism of clearance ofC. jejuniis NF-κB dependent and that CDT may have proinflammatory activity in vivo, as well as a potential role in the ability ofC. jejunito escape immune surveillance. NF-κB-deficient mice should be a useful model to further study the role of CDT and other aspects ofC. jejunipathogenesis.
Antibodies to Malaria Peptide Mimics Inhibit
Plasmodium falciparum
Invasion of Erythrocytes
Authors: Casey Joanne L. , Coley Andrew M. , Anders Robin F. , Murphy Vince J. , Humberstone Karen S. , Thomas Alan W. , Foley Michael
Published: January 2004

Apical membrane antigen 1 (AMA1) is expressed on the surfaces of
Plasmodium falciparum
merozoites and is thought to play an important role in the invasion of erythrocytes by malaria parasites. To select for peptides that mimic conformational B-cell epitopes on AMA1, we screened a phage display library of >10
8
individual peptides for peptides bound by a monoclonal anti-AMA1 antibody, 4G2dc1, known to inhibit
P. falciparum
invasion of erythrocytes. The most reactive peptides, J1, J3, and J7, elicited antibody responses in rabbits that recognized the peptide immunogen and both recombinant and parasite AMA1. Human antibodies in plasma samples from individuals exposed to chronic malaria reacted with J1 and J7 peptides and were isolated using immobilized peptide immunoadsorbents. Both rabbit and human antibodies specific for J1 and J7 peptides were able to inhibit the invasion of erythrocytes by
P. falciparum
merozoites. This is the first example of phage-derived peptides that mimic an important epitope of a blood-stage malaria vaccine candidate, inducing and isolating functional protective antibodies. Our data support the use of J1 and J7 peptide mimics as in vitro correlates of protective immunity in future AMA1 vaccine trials.
Platelet-Activating Factor Receptor Deficiency Delays Elimination of Adult Worms but Reduces Fecundity inStrongyloides venezuelensis-Infected Mice
Authors: Negrão-Corrêa Deborah , Souza Danielle G. , Pinho Vanessa , Barsante Michele M. , Souza Adriano L. S. , Teixeira Mauro M.
Published: January 2004

We describe the parasitological kinetics and histopathological and immunological alterations in platelet-activating factor receptor-deficient (PAFR−/−) and wild-type mice after a singleStrongyloides venezuelensisinfection (subcutaneous inoculation of 500 L3 larvae). There was no difference in the numbers of worms that reached and became established in the small intestines of PAFR−/−and wild-type mice. However, at 12 days after infection, significantly more worms were recovered from PAFR−/−mice. Although PAFR−/−infected mice showed a delay in elimination of adult worms, worms established in the small intestine of these mice produced a significantly lower number of eggs due to a reduction in worm fecundity. There were also significant reductions in the number of circulating and tissue eosinophils and tumor necrosis factor levels in the small intestines of PAFR−/−mice infected for 7 days compared to the number and level in wild-type mice. Histological analysis confirmed the reduced inflammatory process and revealed that the PAFR−/−mice had a smaller number of goblet cells. The concentrations of the type 2 cytokines interleukin-4 (IL-4), IL-5, and IL-10 were lower in small intestine homogenates and in supernatants of antigen-stimulated lymphocytes from spleens or mesenteric lymph nodes of PAFR−/−mice than in the corresponding preparations from wild-type mice. Thus, inS. venezuelensis-infected PAFR−/−mice, decreased intestinal inflammation is associated with enhanced worm survival but decreased fecundity. We suggest that although a Th2-predominant inflammatory response decreases worm survival, the worm may use factors produced during this response to facilitate egg output and reproduction. PAFR-mediated responses appear to modulate these host-derived signals that are important for worm fecundity.
Haemophilus ducreyi
Strain ATCC 27722 Contains a Genetic Element with Homology to the
Vibrio
RS1 Element That Can Replicate as a Plasmid and Confer NAD Independence on
Haemophilus influenzae
Authors: Munson Robert S. , Zhong Huachun , Mungur Rachna , Ray William C. , Shea Robin J. , Mahairas Gregory G. , Mulks Martha H.
Published: January 2004

The nucleotide sequence of pNAD1, a plasmid from
Haemophilus ducreyi
identified on the basis of its ability to confer NAD independence on
Actinobacillus pleuropneumoniae
and
H. influenzae
, has been determined. In addition to containing the
nadV
gene, the plasmid contains homologues of the
rstR
and
rstA
genes, genes encoding repressor and replication proteins, respectively, in the
Vibrio
CTXφ and the
Vibrio
RS1 element, suggesting a single-stranded bacteriophage origin for pNAD1. Tandem copies of the plasmid are integrated into the
H. ducreyi
35000HP genome.