bluuebunny/crossref_metadata_2025 huggingface dataset:
Single-Dose Pharmacokinetics of Intravenous Itraconazole and Hydroxypropyl-β-Cyclodextrin in Infants, Children, and Adolescents
Authors: Abdel-Rahman Susan M. , Jacobs Richard F. , Massarella Joseph , Kauffman Ralph E. , Bradley John S. , Kimko Hui C. , Kearns Gregory L. , Shalayda Kevin , Curtin Christopher , Maldonado Samuel D. , Blumer Jeffrey L.
Published: May 2007

This investigation was designed to evaluate the single-dose pharmacokinetics of itraconazole, hydroxyitraconazole, and hydroxypropyl-β-cyclodextrin (HP-β-CD) after intravenous administration to children at risk for fungal infection. Thirty-three children aged 7 months to 17 years received a single dose of itraconazole (2.5 mg/kg in 0.1-g/kg HP-β-CD) administered over 1 h by intravenous infusion. Plasma samples for the determination of the analytes of interest were drawn over 120 h and analyzed by high-pressure liquid chromatography, and the pharmacokinetics were determined by traditional noncompartmental analysis. Consistent with the role of CYP3A4 in the biotransformation of itraconazole, a substantial degree of variability was observed in the pharmacokinetics of this drug after IV administration. The maximum plasma concentrations (
C
max
) for itraconazole, hydroxyitraconazole, and HP-β-CD averaged 1,015 ± 692 ng/ml, 293 ± 133 ng/ml, and 329 ± 200 μg/ml, respectively. The total body exposures (area under the concentration-time curve from 0 to 24 h) for itraconazole, hydroxyitraconazole, and HP-β-CD averaged 4,922 ± 6,784 ng·h/ml, 3,811 ± 2,794 ng·h/ml, and 641.5 ± 265.0 μg·h/ml, respectively, with no significant age dependence observed among the children evaluated. Similarly, there was no relationship between age and total body clearance (702.8 ± 499.4 ml/h/kg); however, weak associations between age and the itraconazole distribution volume (
r
2
= 0.18,
P
= 0.02),
C
max
(r
2
= 0.14,
P
= 0.045), and terminal elimination rate (
r
2
= 0.26,
P
< 0.01) were noted. Itraconazole infusion appeared to be well tolerated in this population with a single adverse event (stinging at the site of infusion) deemed to be related to study drug administration. Based on the findings of this investigation, it appears that intravenous itraconazole can be administered to infants beyond 6 months, children, and adolescents using a weight-normalized approach to dosing.
Measurement of Voriconazole Activity against
Candida albicans
,
C. glabrata
, and
C. parapsilosis
Isolates Using Time-Kill Methods Validated by High-Performance Liquid Chromatography
Authors: Li Yanjun , Nguyen M. Hong , Derendorf Hartmut , Cheng Shaoji , Clancy Cornelius J.
Published: May 2007

We developed a high-performance liquid chromatography (HPLC) assay to validate time-kill and postantifungal-effect (PAFE) experiments for voriconazole against
Candida albicans
,
Candida glabrata
, and
Candida parapsilosis
isolates. Voriconazole exerted prolonged fungistatic activity but no PAFE at concentrations achievable in human sera. HPLC confirmed that experiments were conducted at the desired steady-state voriconazole concentrations.
Effects of the Translocation Status of Human Immunodeficiency Virus Type 1 Reverse Transcriptase on the Efficiency of Excision of Tenofovir
Authors: Marchand Bruno , White Kirsten L. , Ly John K. , Margot Nicolas A. , Wang Ruth , McDermott Martin , Miller Michael D. , Götte Matthias
Published: May 2007

The ATP-dependent phosphorolytic excision of nucleoside analogue reverse transcriptase inhibitors can diminish their inhibitory effects on human immunodeficiency virus replication. Previous studies have shown that excision can occur only when the reverse transcriptase complex exists in its pretranslocational state. Binding of the next complementary nucleotide causes the formation of a stable dead-end complex in the posttranslocational state, which blocks the excision reaction. To provide mechanistic insight into the excision of the acyclic phosphonate nucleotide analog tenofovir, we compared the efficiencies of the reaction in response to changes in the translocation status of the enzyme. We found that rates of excision of tenofovir with wild-type reverse transcriptase can be as high as those seen with 3′-azido-3′-deoxythymidine monophosphate (AZT-MP). Thymidine-associated mutations, which confer >100-fold and 3-fold decreased susceptibility to AZT and tenofovir, respectively, caused substantial increases in the efficiency of excision of both inhibitors. However, in contrast to the case for AZT-MP, the removal of tenofovir was highly sensitive to dead-end complex formation. Site-specific footprinting experiments revealed that complexes with AZT-terminated primers exist predominantly pretranslocation. In contrast, complexes with tenofovir-terminated primers are seen in both configurations. Low concentrations of the next nucleotide are sufficient to trap the complex posttranslocation despite the flexible, acyclic character of the compound. Thus, the relatively high rate of excision of tenofovir is partially neutralized by the facile switch to the posttranslocational state and by dead-end complex formation, which provides a degree of protection from excision in the cellular environment.
Target Gene Sequencing To Characterize the Penicillin G Susceptibility of
Neisseria meningitidis
Authors: Taha Muhamed-Kheir , Vázquez Julio A. , Hong Eva , Bennett Desiree E. , Bertrand Sophie , Bukovski Suzana , Cafferkey Mary T. , Carion Françoise , Christensen Jens Jørgen , Diggle Mathew , Edwards Giles , Enríquez Rocío , Fazio Cecilia , Frosch Matthias , Heuberger Sigrid , Hoffmann Steen , Jolley Keith A. , Kadlubowski Marcin , Kechrid Amel , Kesanopoulos Konstantinos , Kriz Paula , Lambertsen Lotte , Levenet Ileanna , Musilek Martin , Paragi Metka , Saguer Aouatef , Skoczynska Anna , Stefanelli Paola , Thulin Sara , Tzanakaki Georgina , Unemo Magnus , Vogel Ulrich , Zarantonelli Maria Leticia
Published: May 2007

Clinical isolates of
Neisseria meningitidis
with reduced susceptibility to penicillin G (intermediate isolates, Pen
I
) harbor alterations in the
penA
gene encoding the penicillin binding protein 2 (PBP2). A 402-bp DNA fragment in the 3′ half of
penA
was sequenced from a collection of 1,670 meningococcal clinical isolates from 22 countries that spanned 60 years. Phenotyping, genotyping, and the determination of MICs of penicillin G were also performed. A total of 139 different
penA
alleles were detected with 38 alleles that were highly related, clustered together in maximum-likelihood analysis and corresponded to the penicillin G-susceptible isolates. The remaining 101
penA
alleles were highly diverse, corresponded to different genotypes or phenotypes, and accounted for 38% of isolates, but no clonal expansion was detected. Analysis of the altered alleles that were represented by at least five isolates showed high correlation with the Pen
I
phenotype. The deduced amino acid sequence of the corresponding PBP2 comprised five amino acid residues that were always altered. This correlation was not complete for rare alleles, suggesting that other mechanisms may also be involved in conferring reduced susceptibility to penicillin. Evidence of mosaic structures through events of interspecies recombination was also detected in altered alleles. A new website was created based on the data from this work (
http://neisseria.org/nm/typing/penA
). These data argue for the use of
penA
sequencing to identify isolates with reduced susceptibility to penicillin G and as a tool to improve typing of meningococcal isolates, as well as to analyze DNA exchange among
Neisseria
species.
Inhaled Large Porous Particles of Capreomycin for Treatment of Tuberculosis in a Guinea Pig Model
Authors: Garcia-Contreras L. , Fiegel J. , Telko M. J. , Elbert K. , Hawi A. , Thomas M. , VerBerkmoes J. , Germishuizen W. A. , Fourie P. B. , Hickey A. J. , Edwards D.
Published: May 2007

Capreomycin is used for the treatment of multidrug-resistant tuberculosis (MDR-TB), but it is limited therapeutically by its severe side effects. The objectives of the present studies were (i) to design low-density porous capreomycin sulfate particles for efficient pulmonary delivery to improve local and systemic drug bioavailability and capacity to reduce the bacillary load in the lungs in a manner similar to that achieved with intramuscular injections; (ii) to determine pharmacokinetic parameters after pulmonary administration of these capreomycin particles; and (iii) to evaluate the efficacy of these particles in treating animals in a small-aerosol-inoculum guinea pig model of TB. Capreomycin particles were manufactured by spray drying and characterized in terms of size and drug content. Pharmacokinetic parameters were determined by noncompartmental methods with healthy guinea pigs after administration of capreomycin particles by insufflation. The efficacy of the particles was evaluated by histopathological analysis and in terms of wet organ weight and bacterial burden in TB-infected animals. Lungs of animals receiving a 14.5-mg/kg dose of capreomycin particles showed significantly lower wet weights and smaller bacterial burdens than those of animals receiving any other treatment. These results were supported by histopathological analysis. The feasibility of inhaling capreomycin in a novel powder form, with the ultimate objective of the treatment of MDR-TB, is demonstrated by pharmacokinetic and pharmacodynamic studies with guinea pigs. If applied to humans with MDR-TB, such a therapeutic approach might simplify drug delivery by eliminating injections and might reduce adverse effects through lowering the dose.
Population Pharmacokinetics of Tafenoquine during Malaria Prophylaxis in Healthy Subjects
Authors: Charles Bruce G. , Miller Ann K. , Nasveld Peter E. , Reid Mark G. , Harris Ivor E. , Edstein Michael D.
Published: May 2007

The population pharmacokinetics of tafenoquine were studied in Australian soldiers taking tafenoquine for malarial prophylaxis. The subjects (476 males and 14 females) received a loading dose of 200 mg tafenoquine base daily for 3 days, followed by a weekly dose of 200 mg tafenoquine for 6 months. Blood samples were collected from each subject after the last loading dose and then at weeks 4, 8, and 16. Plasma tafenoquine concentrations were determined by liquid chromatography-tandem mass spectrometry. Population modeling was performed with NONMEM, using a one-compartment model. Typical values of the first-order absorption rate constant (

K
a

), clearance (CL/
F
), and volume of distribution (
V
/
F
) were 0.243 h
−1
, 0.056 liters/h/kg, and 23.7 liters/kg, respectively. The intersubject variability (coefficient of variation) in CL/
F
and
V
/
F
was 18% and 22%, respectively. The interoccasion variability in CL/
F
was 18%, and the mean elimination half-life was 12.7 days. A positive linear association between weight and both CL/
F
and
V
/
F
was found, but this had insufficient impact to warrant dosage adjustments. Model robustness was assessed by a nonparametric bootstrap (200 samples). A degenerate visual predictive check indicated that the raw data mirrored the postdose concentration-time profiles simulated (
n
= 1,000) from the final model. Individual pharmacokinetic estimates for tafenoquine did not predict the prophylactic outcome with the drug for four subjects who relapsed with
Plasmodium vivax
malaria, as they had similar pharmacokinetics to those who were free of malaria infection. No obvious pattern existed between the plasma tafenoquine concentration and the pharmacokinetic parameter values for subjects with and without drug-associated moderate or severe adverse events. This validated population pharmacokinetic model satisfactorily describes the disposition and variability of tafenoquine used for long-term malaria prophylaxis in a large cohort of soldiers on military deployment.
In Vitro Interactions between Apricitabine and Other Deoxycytidine Analogues
Authors: Bethell R. , De Muys J. , Lippens J. , Richard A. , Hamelin B. , Ren C. , Collins P.
Published: May 2007

Apricitabine is a novel deoxycytidine analogue reverse transcriptase inhibitor that is under development for the treatment of human immunodeficiency virus type 1 (HIV-1) infection. Apricitabine is phosphorylated to its active triphosphate by deoxycytidine kinase, which is also responsible for the intracellular phosphorylation of lamivudine (3TC) and emtricitabine (FTC); hence, in vitro studies were performed to investigate possible interactions between apricitabine and these agents. Human peripheral blood mononuclear cells (PBMC) were incubated for 24 h with various concentrations of
3
H-labeled or unlabeled apricitabine, 3TC, or FTC. Intracellular concentrations of parent compounds and their phosphorylated derivatives were measured by high-performance liquid chromatography. In other experiments, viral reverse transcriptase activity was measured in PBMC infected with HIV-1 bearing M184V in the presence of various concentrations of apricitabine and 3TC. [
3
H]apricitabine and [
3
H]3TC were metabolized intracellularly to form mono-, di-, and triphosphates. 3TC and FTC (1 to 10 μM) produced concentration-dependent decreases in apricitabine phosphorylation; in contrast, apricitabine at concentrations of up to 30 μM had no effect on the phosphorylation of 3TC or FTC. The combination of apricitabine and 3TC reduced the antiviral activity of apricitabine against HIV-1: apricitabine concentrations producing 50% inhibition of viral reverse transcriptase were increased two- to fivefold in the presence of 3TC. These findings suggest that nucleoside reverse transcriptase inhibitors with similar modes of action may show biochemical interactions that affect their antiviral efficacy. It is therefore essential that potential interactions between combinations of new and existing agents be thoroughly investigated before such combinations are introduced into clinical practice.
Systematic Derivation of Marker Sets for Staphylococcal Cassette Chromosome
mec
Typing
Authors: Stephens Alex J. , Huygens Flavia , Giffard Philip M.
Published: May 2007

The aim of this study was to identify optimized sets of genotyping targets for the staphylococcal cassette chromosome
mec
(SCC
mec
). We analyzed the gene contents of 46 SCC
mec
variants in order to identify minimal subsets of targets that provide useful resolution. This was achieved by firstly identifying and characterizing each available SCC
mec
element based on the presence or absence of 34 binary targets. This information was used as input for the software “Minimum SNPs,” which identifies the minimum number of targets required to differentiate a set of genotypes up to a predefined Simpson's index of diversity (
D
) value. It was determined that 22 of the 34 targets were required to genotype the 46 SCC
mec
variants to a
D
of 1. The first 6, 9, 12, and 15 targets were found to define 21, 29, 35, and 39 SCC
mec
variants, respectively. The genotypes defined by these marker subsets were largely consistent with the relationships between SCC
mec
variants and the accepted nomenclature. Consistency was made virtually complete by forcing the computer program to include
ccr1
and
ccr5
in the target set. An alternative target set biased towards discriminating abundant SCC
mec
variants was derived by analyzing an input file in which common SCC
mec
variants were repeated, thus ensuring that markers that discriminate abundant variants had a large effect on
D
. Finally, it was determined that
mecA
single nucleotide polymorphisms (SNPs) can increase the overall genotyping resolution, as different
mecA
alleles were found in otherwise identical SCC
mec
variants.
Experimental Phage Therapy against
Staphylococcus aureus
in Mice
Authors: Capparelli Rosanna , Parlato Marianna , Borriello Giorgia , Salvatore Paola , Iannelli Domenico
Published: May 2007

The present study describes a bacteriophage (M
Sa
) active against
Staphylococcus aureus
, including methicillin-resistant staphylococcal strains. When inoculated into mice simultaneously with
S. aureus
A170 (10
8
CFU/mouse), phage (10
9
PFU) rescued 97% of the mice; when applied to nonlethal (5 × 10
6
CFU/mouse) 10-day infections, the phage also fully cleared the bacteria. The phage M
Sa
, delivered inside macrophages by
S. aureus
, kills the intracellular staphylococci in vivo and in vitro. The phage can also prevent abscess formation and reduce the bacterial load and weight of abscesses. These results suggest a potential use of the phage for the control of both local and systemic human
S. aureus
infections.
Effects of Rifampin and Multidrug Resistance Gene Polymorphism on Concentrations of Moxifloxacin
Authors: Weiner Marc , Burman William , Luo Chi-Cheng , Peloquin Charles A. , Engle Melissa , Goldberg Stefan , Agarwal Vipin , Vernon Andrew
Published: May 2007

Treatment regimens combining moxifloxacin and rifampin for drug-susceptible tuberculosis are being studied intensively. However, rifampin induces enzymes that transport and metabolize moxifloxacin. We evaluated the effect of rifampin and the human multidrug resistance gene (MDR1) C3435T polymorphisms (P-glycoprotein) on moxifloxacin pharmacokinetic parameters. This was a single-center, sequential design study with 16 volunteers in which sampling was performed after four daily oral doses of moxifloxacin (400 mg) and again after 10 days of combined rifampin (600 mg) and moxifloxacin. After daily coadministration of rifampin, the area under the concentration-time curve from 0 to 24 h (AUC
0-24
) for moxifloxacin decreased 27%. Average bioequivalence between moxifloxacin coadministered with rifampin and moxifloxacin alone was not demonstrated: the ratio of geometric means (RGM) of the moxifloxacin AUC
0-24
was 73.3 (90% confidence intervals [CI], 64.3, 83.5) (total
P
value, 0.87 for two one-sided
t
tests). Peak moxifloxacin concentrations, however, were equivalent: the RGM of the maximum concentration of the drug in serum was 93.6 (90% CI, 80.2, 109.3) (total
P
value, 0.049). Concentrations of the sulfate conjugate metabolite of moxifloxacin were increased twofold following rifampin coadministration (AUC
0-24
, 1.29 versus 2.79 μg·h/ml). Concomitant rifampin administration resulted in a 27% decrease in the mean moxifloxacin AUC
0-24
and a marked increase in the AUC
0-24
of the microbiologically inactive M1 metabolite. Additional studies are required to understand the clinical significance of the moxifloxacin-rifampin interaction.
Endogenous Interleukin-18 Improves the Early Antimicrobial Host Response in Severe Melioidosis
Authors: Wiersinga W. Joost , Wieland Catharina W. , van der Windt Gerritje J. W. , de Boer Anita , Florquin Sandrine , Dondorp Arjen , Day Nicholas P. , Peacock Sharon J. , van der Poll Tom
Published: May 2007

Melioidosis is caused by the soil saprophyteBurkholderia pseudomalleiand is endemic in Southeast Asia. The pathogenesis of melioidosis is still largely unknown, although gamma interferon (IFN-γ) seems to play an obligatory role in host defense. Previously, we have shown that IFN-γ production in melioidosis is controlled in part by interleukin-18 (IL-18). The aim of the present study was to determine the role of IL-18 in the immune response toB. pseudomallei. For this the following investigations were performed. (i) Plasma IL-18 and blood monocyte IL-18 mRNA levels were elevated in 34 patients with culture-proven melioidosis compared to the levels in 32 local healthy controls; in addition, IL-18 binding protein levels were markedly elevated in patients, strongly correlating with mortality. (ii) IL-18 gene-deficient (IL-18 knockout [KO]) mice showed accelerated mortality after intranasal infection with a lethal dose ofB. pseudomallei, which was accompanied by enhanced bacterial growth in their lungs, livers, spleens, kidneys, and blood at 24 and 48 h postinfection, compared to wild-type mice. In addition, IL-18 KO mice displayed evidence of enhanced hepatocellular injury and renal insufficiency. Together, these data indicate that the enhanced production of IL-18 in melioidosis is an essential part of a protective immune response to this severe infection.
Heterogeneity of a
Campylobacter jejuni
Protein That Is Secreted through the Flagellar Filament
Authors: Poly Frédéric , Ewing Cheryl , Goon Scarlett , Hickey Thomas E. , Rockabrand David , Majam Gary , Lee Lanfong , Phan Julie , Savarino Nicholas J. , Guerry Patricia
Published: May 2007

Cj0859c, or FspA, is a small, acidic protein of
Campylobacter jejuni
that is expressed by a σ
28
promoter. Analysis of the
fspA
gene in 41 isolates of
C. jejuni
revealed two overall variants of the predicted protein, FspA1 and FspA2. Secretion of FspA occurs in broth-grown bacteria and requires a minimum flagellar structure. The addition of recombinant FspA2, but not FspA1, to INT407 cells in vitro resulted in a rapid induction of apoptosis. These data define a novel
C. jejuni
virulence factor, and the observed heterogeneity among
fspA
alleles suggests alternate virulence potential among different strains.
Surface-Exposed Proteins ofEhrlichia chaffeensis
Authors: Ge Yan , Rikihisa Yasuko
Published: May 2007

The surface proteins ofEhrlichia chaffeensisprovide an important interface for pathogen-host interactions. To investigate the surface proteins ofE. chaffeensis, membrane-impermeable, cleavable Sulfo-NHS-SS-Biotin was used to label intact bacteria. The biotinylated bacterial surface proteins were isolated by streptavidin-agarose affinity purification. The affinity-captured proteins were separated by electrophoresis, and five relatively abundant protein bands containing immunoreactive proteins were subjected to capillary-liquid chromatography-nanospray tandem mass spectrometry analysis. Nineteen out of 22 OMP-1/P28 family proteins, including P28 (which previously was shown to be surface exposed), were detected inE. chaffeensiscultured in human monocytic leukemia THP-1 cells. For the first time, with the exception of P28 and P28-1, 17 OMP-1/P28 family proteins were demonstrated to be expressed at the protein level. The surface exposure of OMP-1A and OMP-1N was verified by immunofluorescence microscopy. OMP-1B was undetectable either by surface biotinylation or by Western blotting of the whole bacterial lysate, suggesting that it is not expressed byE. chaffeensiscultured in THP-1 cells. AdditionalE. chaffeensissurface proteins detected were OMP85, hypothetical protein ECH_0525 (here named Esp73), immunodominant surface protein gp47, and 11 other proteins. The identification ofE. chaffeensissurface-exposed proteins provides novel insights into theE. chaffeensissurface and lays the foundation for rational studies on pathogen-host interactions and vaccine development.
Clostridium difficileToxins A and B Directly Stimulate Human Mast Cells
Authors: Meyer Gesa K. A. , Neetz Anne , Brandes Gudrun , Tsikas Dimitrios , Butterfield Joseph H. , Just Ingo , Gerhard Ralf
Published: May 2007

Clostridium difficiletoxins A and B (TcdA and TcdB) are the causative agents of antibiotic-associated pseudomembranous colitis. Mucosal mast cells play a crucial role in the inflammatory processes underlying this disease. We studied the direct effects of TcdA and TcdB on the human mast cell line HMC-1 with respect to degranulation, cytokine release, and the activation of proinflammatory signal pathways. TcdA and TcdB inactivate Rho GTPases, the master regulators of the actin cytoskeleton. The inactivation of Rho GTPases induced a reorganization of the actin cytoskeleton accompanied by morphological changes of cells. The TcdB-induced reorganization of the actin cytoskeleton in HMC-1 cells reduced the number of electron-dense mast cell-specific granules. Accordingly, TcdB induced the release of hexosaminidase, a marker for degranulation, in HMC-1 cells. The actin rearrangement was found to be responsible for degranulation since latrunculin B induced a comparable hexosaminidase release. In addition, TcdB as well as latrunculin B induced the activation of p38 mitogen-activated protein kinase (MAPK) and extracellular signal-regulated kinase 1/2 and also resulted in a p38 MAPK-dependent increased formation of prostaglandins D2and E2. The autocrine stimulation of HMC-1 cells by prostaglandins partially contributed to the degranulation. Interestingly, TcdB-treated HMC-1 cells, but not latrunculin B-treated HMC-1 cells, showed a strong p38 MAPK-dependent increase in interleukin-8 release. Differences in the mast cell responses to TcdB and latrunculin B are probably due to the presence of functionally inactive Rho GTPases in toxin-treated cells. Thus, the HMC-1 cell line is a promising model for studying the direct effects ofC. difficiletoxins on mast cells independently of the tissue context.
Plasmodium yoelii
Sporozoites with Simultaneous Deletion of P52 and P36 Are Completely Attenuated and Confer Sterile Immunity against Infection
Authors: Labaied Mehdi , Harupa Anke , Dumpit Ronald F. , Coppens Isabelle , Mikolajczak Sebastian A. , Kappe Stefan H. I.
Published: May 2007

Malaria infection starts when sporozoites are transmitted to the mammalian host during a mosquito bite. Sporozoites enter the blood circulation, reach the liver, and infect hepatocytes. The formation of a parasitophorous vacuole (PV) establishes their intracellular niche. Recently, two members of the 6-Cys domain protein family, P52 and P36, were each shown to play an important albeit nonessential role in
Plasmodium berghei
sporozoite infectivity for the rodent host. Here, we generated
p52/p36
-deficient
Plasmodium yoelii
parasites by the simultaneous deletion of both genes using a single genetic manipulation.
p52/p36
-deficient parasites exhibited normal progression through the life cycle during blood-stage infection, transmission to mosquitoes, mosquito-stage development, and sporozoite infection of the salivary glands.
p52/p36
-deficient sporozoites also showed normal motility and cell traversal activity. However, immunofluorescence analysis and electron microscopic observations revealed that
p52/p36
-deficient parasites did not form a PV within hepatocytes in vitro and in vivo. The
p52/p36
-deficient parasites localized as free entities in the host cell cytoplasm or the host cell nucleoplasm and did not develop as liver stages. Consequently, they did not cause blood-stage infections even at high sporozoite inoculation doses. Mice immunized with
p52/p36
-deficient sporozoites were completely protected against infectious sporozoite challenge. Our results demonstrate for the first time the generation of two-locus gene deletion-attenuated parasites that infect the liver but do not progress to blood-stage infection. The study will critically guide the design of
Plasmodium falciparum
live attenuated malaria vaccines.
Pathogen Specificity and Autoimmunity Are Distinct Features of Antigen-Driven Immune Responses in Neuroborreliosis
Authors: Kuenzle Sandra , von Büdingen Hans-Christian , Meier Mirjam , Harrer Melanie D. , Urich Eduard , Becher Burkhard , Goebels Norbert
Published: May 2007

Neuroborreliosis (NB) is a chronic infectious disease of the central nervous system (CNS) caused by a tick-borne spirochete,
Borrelia burgdorferi
. In addition to direct effects of the causative infectious agent, additional immunity-mediated mechanisms are thought to play a role in the CNS pathology of NB. In order to further understand the involvement of humoral immune mechanisms in NB, we dissected the intrathecal antibody responses down to the single-plasma-cell level. Starting with single-cell reverse transcription-PCR of fluorescence-activated cell sorter-sorted cerebrospinal fluid plasma cells from an NB patient, we identified expanded clones and resurrected the antigen specificity of their secreted antibodies through recombinant expression of the correctly paired immunoglobulin heavy- and light-chain genes as monoclonal antibodies (MAbs). As expected, we found specificity for the causative infectious agent,
B. burgdorferi
, among the clonally expanded plasma cell (cePC)-derived MAbs. However, from an independent cePC of the same patient, we could derive MAbs specific for human CNS myelin, without detectable cross-reactivity with
B. burgdorferi
antigens. While reactivity against
B. burgdorferi
is a known feature of humoral immune responses in NB, we show (i) that immune responses specific for self antigens may be a distinct feature of CNS infections independent of pathogen reactivity and (ii) that humoral autoimmunity in NB (since found in cePC) is the result of a truly antigen-driven immune response. Our findings indicate that in NB mechanisms may be at play that induce distinct immune responses specific for pathogen and self antigens independent from “molecular mimicry.”
Mycolactone-Mediated Inhibition of Tumor Necrosis Factor Production by Macrophages Infected withMycobacterium ulceransHas Implications for the Control of Infection
Authors: Torrado Egídio , Adusumilli Sarojini , Fraga Alexandra G. , Small Pamela L. C. , Castro António G. , Pedrosa Jorge
Published: May 2007

The pathogenicity ofMycobacterium ulcerans, the agent of Buruli ulcer, depends on the cytotoxic exotoxin mycolactone. Little is known about the immune response to this pathogen. Following the demonstration of an intracellular growth phase in the life cycle ofM. ulcerans, we investigated the production of tumor necrosis factor (TNF) induced by intramacrophage bacilli of diverse toxigenesis/virulence, as well as the biological relevance of TNF duringM. ulceransexperimental infections. Our data show that murine bone marrow-derived macrophages infected with mycolactone-negative strains ofM. ulcerans(nonvirulent) produce high amounts of TNF, while macrophages infected with mycolactone-positive strains of intermediate or high virulence produce intermediate or low amounts of TNF, respectively. These results are in accordance with the finding that TNF receptor P55-deficient (TNF-P55 KO) mice are not more susceptible than wild-type mice to infection by the highly virulent strains but are more susceptible to nonvirulent and intermediately virulent strains, demonstrating that TNF is required to control the proliferation of these strains in animals experimentally infected byM. ulcerans. We also show that mycolactone produced by intramacrophageM. ulceransbacilli inhibits, in a dose-dependent manner, but does not abrogate, the production of macrophage inflammatory protein 2, which is consistent with the persistent inflammatory responses observed in experimentally infected mice.
The Large Clostridial Toxins from
Clostridium sordellii
and
C. difficile
Repress Glucocorticoid Receptor Activity
Authors: Tait A. Sasha , Dalton Monique , Geny Blandine , D'Agnillo Felice , Popoff Michel R. , Sternberg Esther M.
Published: May 2007

We have previously shown that
Bacillus anthracis
lethal toxin represses glucocorticoid receptor (GR) transactivation. We now report that repression of GR activity also occurs with the large clostridial toxins produced by
Clostridium sordellii
and
C. difficile
. This was demonstrated using a transient transfection assay system for GR transactivation. We also report that
C. sordellii
lethal toxin inhibited GR function in an ex vivo assay, where toxin reduced the dexamethasone suppression of the proinflammatory cytokine tumor necrosis factor alpha (TNF-α). Furthermore, the glucocorticoid antagonist RU-486 in combination with
C. sordellii
lethal toxin additively prevented glucocorticoid suppression of TNF-α. These findings corroborate the fact that GR is a target for the toxin and suggest a physiological role for toxin-associated GR repression in inflammation. Finally, we show that this repression is associated with toxins that inactivate p38 mitogen-activated protein kinase (MAPK).
Multiple Consecutive Lavage Samplings Reveal Greater Burden of Disease and Provide Direct Access to the Nontypeable
Haemophilus influenzae
Biofilm in Experimental Otitis Media
Authors: Leroy Magali , Cabral Howard , Figueira Marisol , Bouchet Valérie , Huot Heather , Ram Sanjay , Pelton Stephen I. , Goldstein Richard
Published: May 2007

The typically recovered quantity of nontypeable
Haemophilus influenzae
(NTHi) bacteria in an ex vivo middle ear (ME) aspirate from the chinchilla model of experimental otitis media is insufficient for direct analysis of gene expression by microarray or of lipopolysaccharide glycoforms by mass spectrometry. This prompted us to investigate a strategy of multiple consecutive lavage samplings to increase ex vivo bacterial recovery. As multiple consecutive lavage samples significantly increased the total number of bacterial CFU collected during nasopharyngeal colonization or ME infection, this led us to evaluate whether bacteria sequentially acquired from consecutive lavages were similar. Comparative observation of complete ex vivo sample series by microscopy initially revealed ME inflammatory fluid consisting solely of planktonic-phase NTHi. In contrast, subsequent lavage samplings of the same infected ear revealed the existence of bacteria in two additional growth states, filamentous and biofilm encased. Gene expression analysis of such ex vivo samples was in accord with different bacterial growth phases in sequential lavage specimens. The existence of morphologically distinct NTHi subpopulations with varying levels of gene expression indicates that the pooling of specimens requires caution until methods for their separation are developed. This study based on multiple consecutive lavages is consistent with prior reports that NTHi forms a biofilm in vivo, describes the means to directly acquire ex vivo biofilm samples without sacrificing the animal, and has broad applicability for a study of mucosal infections. Moreover, this approach revealed that the actual burden of bacteria in experimental otitis media is significantly greater than was previously reported. Such findings may have direct implications for antibiotic treatment and vaccine development against NTHi.
Uropathogenic
Escherichia coli
Outer Membrane Antigens Expressed during Urinary Tract Infection
Authors: Hagan Erin C. , Mobley Harry L. T.
Published: May 2007

Uncomplicated urinary tract infection (UTI) caused by uropathogenic
Escherichia coli
(UPEC) represents a prevalent and potentially severe infectious disease. In this study, we describe the application of an immunoproteomics approach to vaccine development that has been used successfully to identify vaccine targets in other pathogenic bacteria. Outer membranes were isolated from pyelonephritis strain
E. coli
CFT073 cultured under conditions that mimic the urinary tract environment, including iron limitation, osmotic stress, human urine, and exposure to uroepithelial cells. To identify antigens that elicit a humoral response during experimental UTI, outer membrane proteins were separated by two-dimensional gel electrophoresis and probed using pooled antisera from 20 CBA/J mice chronically infected with
E. coli
CFT073. In total, 23 outer membrane antigens, including a novel iron compound receptor, reacted with the antisera and were identified by mass spectrometry. These antigens also included proteins with known roles in UPEC pathogenesis, such as ChuA, IroN, IreA, Iha, IutA, and FliC. These data demonstrate that an antibody response is directed against these virulence-associated factors during UTI. We also show that the genes encoding ChuA, IroN, hypothetical protein c2482, and IutA are significantly more prevalent (
P
< 0.01) among UPEC strains than among fecal-commensal
E. coli
isolates. Thus, we suggest that the conserved outer membrane antigens identified in this study could be rational candidates for a UTI vaccine designed to elicit protective immunity against UPEC infection.
Exogenous Farnesol Interferes with the Normal Progression of Cytokine Expression during Candidiasis in a Mouse Model
Authors: Navarathna Dhammika H. M. L. P. , Nickerson Kenneth W. , Duhamel Gerald E. , Jerrels Thomas R. , Petro Thomas M.
Published: May 2007

Candida albicans, a dimorphic fungus composed of yeast and mycelial forms, is the most common human fungal pathogen. Th1 cytokines such as interleukin-2 (IL-2), gamma interferon (IFN-γ), and tumor necrosis factor alpha (TNF-α), which are induced by macrophage IL-12, are critical to resistance against systemic candidiasis, while Th2 cytokines such as IL-4 and IL-5 are less critical. Farnesol is a quorum-sensing molecule produced byC. albicansthat controls the formation of mycelia but is also a virulence factor. To determine whether farnesol enhances the virulence ofC. albicansby modulating the production of Th1 and Th2 cytokines, mice were pretreated with farnesol prior to intravenous infection with a sublethal dose of farnesol-producingC. albicans. Production of IL-2, IL-4, IL-5, TNF-α, IFN-γ, and IL-12 was evaluated by bead-array flow cytometry and enzyme-linked immunosorbent assay. Mice exhibited an elevation in serum TNF-α levels at 48 h and an elevation in IFN-γ and IL-12 levels at 6 to 12 h after infection withC. albicans. Pretreatment with farnesol significantly reduced the elevation of both IFN-γ and IL-12 but not TNF-α. In contrast, mice pretreated with farnesol exhibited an unexpected elevation in IL-5 levels. To determine whether farnesol has a direct effect on macrophage production of IL-12, peritoneal macrophages were pretreated with farnesol prior to stimulation with IFN-γ plus lipopolysaccharide (LPS). Farnesol inhibited production of both IL-12 p40 and p70 from IFN-γ/LPS-stimulated macrophages. Therefore, the role of farnesol in systemic candidiasis is likely due to its ability to inhibit the critical Th1 cytokines IFN-γ and IL-12 and perhaps to enhance a Th2 cytokine, IL-5.
Identification of a LolC Homologue inBurkholderia pseudomallei, a Novel Protective Antigen for Melioidosis
Authors: Harland David N. , Chu Karen , Haque Ashraful , Nelson Michelle , Walker Nicola J. , Sarkar-Tyson Mitali , Atkins Timothy P. , Moore Benjamin , Brown Katherine A. , Bancroft Gregory , Titball Richard W. , Atkins Helen S.
Published: May 2007

Melioidosis is an emerging disease of humans in Southeast Asia and tropical Australia. The bacterium causing this disease,Burkholderia pseudomallei, is also considered a bioterrorism agent, and as yet there is no licensed vaccine for preventingB. pseudomalleiinfection. In this study, we evaluated selected proteins (LolC, PotF, and OppA) of the ATP-binding cassette systems ofB. pseudomalleias candidate vaccine antigens. Nonmembrane regions of theB. pseudomalleiproteins were expressed and purified fromEscherichia coliand then evaluated as vaccine candidates in an established mouse model ofB. pseudomalleiinfection. When delivered with the monophosphoryl lipid A-trehalose dicorynomycolate adjuvant, the proteins stimulated antigen-specific humoral and cellular immune responses. Immunization with LolC or PotF protein domains afforded significant protection against a subsequent challenge withB. pseudomallei. The most promising vaccine candidate, LolC, provided a greater level of protection when it was administered with immune-stimulating complexes complexed with CpG oligodeoxynucleotide 10103. Immunization with LolC also protected against a subsequent challenge with a heterologous strain ofB. pseudomallei, demonstrating the potential utility of this protein as a vaccine antigen for melioidosis.
Periodontal Bacterial DNA Suppresses the Immune Response to Mutans Streptococcal Glucosyltransferase
Authors: Taubman Martin A. , Han Xiaozhe , LaRosa Karen B. , Socransky Sigmund S. , Smith Daniel J.
Published: May 2007

Certain CpG motifs found in bacterial DNA enhance immune responses through Toll-like receptor 9 (TLR-9) and may also demonstrate adjuvant properties. Our objective was to determine if DNA from bacteria associated with periodontal disease could affect the immune response to other bacterial antigens in the oral cavity.Streptococcus sobrinusglucosyltransferase (GTF), an enzyme involved in dental caries pathogenesis, was used as a test antigen. Rowett rats were injected with aluminum hydroxide (alum) with buffer, alum-GTF, or alum-GTF together with eitherEscherichia coliDNA,Fusobacterium nucleatumDNA, orPorphyromonas gingivalisDNA. Contrary to expectation, animals receiving alum-GTF plus bacterial DNA (P. gingivalisin particular) demonstrated significantly reduced serum immunoglobulin G (IgG) antibody, salivary IgA antibody, and T-cell proliferation to GTF compared to animals immunized with alum-GTF alone. A diminished antibody response was also observed after administration of alum-GTF with theP. gingivalisDNA either together or separately, indicating that physical complexing of antigen and DNA was not responsible for the reduction in antibody. Since TLR triggering by DNA induces synthesis of prospective suppressive factors (e.g., suppressor of cytokine signaling [SOCS]), the effects ofP. gingivalisDNA and GTF exposure on rat splenocyte production of SOCS family molecules and inflammatory cytokines were investigated in vitro.P. gingivalisDNA significantly up-regulated SOCS1 and SOCS5 expression and down-regulated interleukin-10 expression by cultured splenocytes. These results suggested that DNA from periodontal disease-associated bacteria did not enhance, but in fact suppressed, the immune response to a protein antigen from cariogenic streptococci, potentially through suppressive SOCS components triggered by innate mechanisms.
Extracytoplasmic-Stress-Responsive Pathways Modulate Type III Secretion in
Yersinia pseudotuberculosis
Authors: Carlsson Katrin E. , Liu Junfa , Edqvist Petra J. , Francis Matthew S.
Published: May 2007

Three signal transduction pathways, the two-component systems CpxRA and BaeSR and the alternative sigma factor σ
E
, respond to extracytoplasmic stress that facilitates bacterial adaptation to changing environments. At least the CpxRA and σ
E
pathways control the production of protein-folding and degradation factors that counter the effects of protein misfolding in the periplasm. This function also influences the biogenesis of multicomponent extracellular appendages that span the bacterial envelope, such as various forms of pili. Herein, we investigated whether any of these regulatory pathways in the enteropathogen
Yersinia pseudotuberculosis
affect the functionality of the Ysc-Yop type III secretion system. This is a multicomponent molecular syringe spanning the bacterial envelope used to inject effector proteins directly into eukaryotic cells. Disruption of individual components revealed that the Cpx and σ
E
pathways are important for
Y. pseudotuberculosis
type III secretion of Yops (
Yersinia
outer proteins). In particular, a loss of CpxA, a sensor kinase, reduced levels of structural Ysc (
Yersinia
secretion) components in bacterial membranes, suggesting that these mutant bacteria are less able to assemble a functional secretion apparatus. Moreover, these bacteria were no longer capable of localizing Yops into the eukaryotic cell interior. In addition, a
cpxA lcrQ
double mutant engineered to overproduce and secrete Yops was still impaired in intoxicating cells. Thus, the Cpx pathway might mediate multiple influences on bacterium-target cell contact that modulate
Yersinia
type III secretion-dependent host cell cytotoxicity.
Opa Proteins of Pathogenic Neisseriae Initiate Src Kinase-Dependent or Lipid Raft-Mediated Uptake via Distinct Human Carcinoembryonic Antigen-Related Cell Adhesion Molecule Isoforms
Authors: Schmitter Tim , Pils Stefan , Weibel Stephanie , Agerer Franziska , Peterson Lisa , Buntru Alexander , Kopp Kathrin , Hauck Christof R.
Published: May 2007

Several pathogenic bacteria exploit human carcinoembryonic antigen-related cell adhesion molecules (CEACAMs) for adhesion to and invasion into their host cells. CEACAM isoforms have characteristic expression patterns on epithelial, endothelial, or hematopoietic cells, providing bacteria with distinct sets of receptors on particular tissues. For example, while CEACAM1 and CEACAM6 have a wide tissue distribution, CEACAM3, CEACAM4, and CEACAM8 are uniquely expressed on primary human granulocytes, whereas CEA and CEACAM7 are limited to epithelia. By reconstitution of a CEACAM-deficient cell line with individual CEACAMs, we have analyzed the requirements for CEACAM-mediated internalization ofNeisseria gonorrhoeae. Our results point to two mechanistically different uptake pathways triggered by either epithelial CEACAMs (CEACAM1, CEA, and CEACAM6) or the granulocyte-specific CEACAM3. In particular, CEACAM3-mediated uptake critically depends on Src family protein tyrosine kinase (PTK) activity, and CEACAM3 associates with the SH2 domains of several Src PTKs. In contrast, epithelial CEACAMs require the integrity of cholesterol-rich membrane microdomains and are affected by cholesterol depletion, whereas CEACAM3-mediated uptake by transfected cells or the opsonin-independent phagocytosis by human granulocytes is not altered in the presence of cholesterol chelators. These results allow the subdivision of all human CEACAMs known to be utilized as pathogen receptors into functional groups and point to important consequences for bacterial engagement of distinct CEACAM isoforms.
Toll-Like Receptor 2-Mediated Signaling Requirements forFrancisella tularensisLive Vaccine Strain Infection of Murine Macrophages
Authors: Cole Leah E. , Shirey Kari Ann , Barry Eileen , Santiago Araceli , Rallabhandi Prasad , Elkins Karen L. , Puche Adam C. , Michalek Suzanne M. , Vogel Stefanie N.
Published: May 2007

Francisella tularensis, an aerobic, non-spore-forming, gram-negative coccobacillus, is the causative agent of tularemia. We reported previously thatF. tularensislive vaccine strain (LVS) elicited strong, dose-dependent NF-κB reporter activity in Toll-like receptor 2 (TLR2)-expressing HEK293T cells and proinflammatory gene expression in primary murine macrophages. Herein, we report thatF. tularensisLVS-induced murine macrophage proinflammatory cytokine gene and protein expression are overwhelmingly TLR2 dependent, as evidenced by the abrogated responses of TLR2−/−macrophages.F. tularensisLVS infection also increased expression of TLR2 both in vitro, in mouse macrophages, and in vivo, in livers fromF. tularensisLVS-infected mice. Colocalization of intracellularF. tularensisLVS, TLR2, and MyD88 was visualized by confocal microscopy. Signaling was abrogated if theF. tularensisLVS organisms were heat or formalin killed or treated with chloramphenicol, indicating that the TLR2 agonist activity is dependent on new bacterial protein synthesis.F. tularensisLVS replicates in macrophages; however, bacterial replication was not required for TLR2 signaling because LVSΔguaA, anF. tularensisLVSguanine auxotroph that fails to replicate in the absence of exogenous guanine, activated NF-κB in TLR2-transfected HEK293T cells and induced cytokine expression in wild-type macrophages comparably to wild-typeF. tularensisLVS. Collectively, these data indicate that the primary macrophage response toF. tularensisLVS is overwhelmingly TLR2 dependent, requires de novo bacterial protein synthesis, and is independent of intracellularF. tularensisreplication.