bluuebunny/crossref_metadata_2025 huggingface dataset:
Differential Expression of Porins OmpP2A and OmpP2B of
Haemophilus ducreyi
Authors: Prather Derrick T. , Bains Manjeet , Hancock Robert E. W. , Filiatrault Melanie J. , Campagnari Anthony A.
Published: October 2004

Haemophilus ducreyi
is a strict human pathogen and the causative agent of the sexually transmitted disease chancroid. The genome of the human-passaged strain of
H. ducreyi
(35000HP) contains two homologous genes whose protein products have estimated molecular masses of 46 and 43 kDa. A comparative analysis of the deduced amino acid sequences revealed that these proteins share 27 to 33% identity to the outer membrane protein P2 (OmpP2), a major porin of
Haemophilus influenzae
. Therefore, these proteins have been designated OmpP2A and OmpP2B, respectively. The detection of
ompP2A
and
ompP2B
transcripts by reverse transcriptase PCR indicated that these genes were independently transcribed in
H. ducreyi
35000HP. Western blot analysis of outer membrane proteins isolated from a geographically diverse collection of
H. ducreyi
clinical isolates revealed that OmpP2A and OmpP2B were differentially expressed among these strains. Although PCR analysis suggested that
ompP2A
and
ompP2B
were conserved among the strains tested, the differential expression observed was due to nucleotide additions and partial gene deletions. Purified OmpP2A and OmpP2B were isolated under nondenaturing conditions, and subsequent analysis demonstrated that these two proteins exhibited porin activity. OmpP2A and OmpP2B are the first porins described for
H. ducreyi
.
Extracellular Secretion of the
Borrelia burgdorferi
Oms28 Porin and Bgp, a Glycosaminoglycan Binding Protein
Authors: Cluss Robert G. , Silverman Damon A. , Stafford Thomas R.
Published: October 2004

Borrelia burgdorferi
, the Lyme disease pathogen, cycles between its
Ixodes
tick vector and vertebrate hosts, adapting to vastly different biochemical environments. Spirochete gene expression as a function of temperature, pH, growth phase, and host milieu is well studied, and recent work suggests that regulatory networks are involved. Here, we examine the release of
Borrelia burgdorferi
strain B31 proteins into conditioned medium. Spirochetes intrinsically radiolabeled at concentrations ranging from 10
7
to 10
9
cells per ml secreted Oms28, a previously characterized outer membrane porin, into RPMI medium. As determined by immunoblotting, this secretion was not associated with outer membrane blebs or cytoplasmic contamination. A similar profile of secreted proteins was obtained for spirochetes radiolabeled in mixtures of RPMI medium and serum-free Barbour-Stoenner-Kelly (BSK II) medium. Proteomic liquid chromatography-tandem mass spectrometry analysis of tryptic fragments derived from strain B31 culture supernatants confirmed the identity of the 28-kDa species as Oms28 and revealed a 26-kDa protein as 5′-methylthioadenosine/
S
-adenosylhomocysteine nucleosidase (Pfs-2), previously described as Bgp, a glycosaminoglycan-binding protein. The release of Oms28 into the culture medium is more selective when the spirochetes are in logarithmic phase of growth compared to organisms obtained from stationary phase. As determined by immunoblotting, stationary-phase spirochetes released OspA, OspB, and flagellin. Oms28 secreted by strains B31, HB19, and N40 was also recovered by radioimmunoprecipitation. This is the first report of
B. burgdorferi
protein secretion into the extracellular environment. The possible roles of Oms28 and Bgp in the host-pathogen interaction are considered.
Interleukin-10 (IL-10) Counterregulates IL-4-Dependent Effector Mechanisms in Murine Filariasis
Authors: Specht Sabine , Volkmann Lars , Wynn Tom , Hoerauf Achim
Published: October 2004

Interleukin-10 (IL-10) was at first described as a Th2-associated cytokine, although more recent reports have shown that immunosuppression applies to both Th1 and Th2 cell responses, e.g., when produced by T regulatory cells. This concept when applied to human filariasis would argue that high parasite loads are associated with IL-10, while bona fide Th2 responses, mediated by IL-4, IL-5, and IL-13, are associated with parasite containment. To prove this relationship in a causal manner, we investigated the roles of IL-4 and IL-10 in a helminth infection model in which mice genetically deficient for IL-4, IL-10, or IL-4 plus IL-10 were infected with the rodent filariaLitomosoides sigmodontis. Compared to C57BL/6 wild-type and IL-10 knockout (KO) mice, IL-4 KO mice remained susceptible, exhibiting a remarkable number of live adult worms. Interestingly however, when the IL-10 gene was knocked out simultaneously with the IL-4 gene, the susceptibility of IL-4 KO mice was reversed. Although production of IFN-γ was increased in IL-4/IL-10 double-knockout mice, depletion of gamma interferon did not affect worm elimination, so it seems unlikely to be the major factor in mediating resistance in IL-4/IL-10 KO mice. Taken together, the results of this study add proof to the concept that has arisen for human filariasis that IL-10-dependent responses, which are associated with patency, are antagonistic to bona fide Th2 responses, which control parasite loads. The finding that knockout of IL-10 reversed a disease phenotype induced by knockout of IL-4 gives the first causal evidence of an antagonistic activity between IL-4 and IL-10 in an infection in vivo.
Respiratory Hydrogen Use by
Salmonella enterica
Serovar Typhimurium Is Essential for Virulence
Authors: Maier R. J. , Olczak A. , Maier S. , Soni S. , Gunn J.
Published: October 2004

Based on available annotated gene sequence information, the enteric pathogen salmonella, like other enteric bacteria, contains three putative membrane-associated H
2
-using hydrogenase enzymes. These enzymes split molecular H
2
, releasing low-potential electrons that are used to reduce quinone or heme-containing components of the respiratory chain. Here we show that each of the three distinct membrane-associated hydrogenases of
Salmonella enterica
serovar Typhimurium is coupled to a respiratory pathway that uses oxygen as the terminal electron acceptor. Cells grown in a blood-based medium expressed four times the amount of hydrogenase (H
2
oxidation) activity that cells grown on Luria Bertani medium did. Cells suspended in phosphate-buffered saline consumed 2 mol of H
2
per mol of O
2
used in the H
2
-O
2
respiratory pathway, and the activity was inhibited by the respiration inhibitor cyanide. Molecular hydrogen levels averaging over 40 μM were measured in organs (i.e., livers and spleens) of live mice, and levels within the intestinal tract (the presumed origin of the gas) were four times greater than this. The half-saturation affinity of
S. enterica
serovar Typhimurium for H
2
is only 2.1 μM, so it is expected that H
2
-utilizing hydrogenase enzymes are saturated with the reducing substrate in vivo. All three hydrogenase enzymes contribute to the virulence of the bacterium in a typhoid fever-mouse model, based on results from strains with mutations in each of the three hydrogenase genes. The introduced mutations are nonpolar, and growth of the mutant strains was like that of the parent strain. The combined removal of all three hydrogenases resulted in a strain that is avirulent and (in contrast to the parent strain) one that is unable to invade liver or spleen tissue. The introduction of one of the hydrogenase genes into the triple mutant strain on a low-copy-number plasmid resulted in a strain that was able to both oxidize H
2
and cause morbidity in mice within 11 days of inoculation; therefore, the avirulent phenotype of the triple mutant is not due to an unknown spurious mutation. We conclude that H
2
utilization in a respiratory fashion is required for energy production to permit salmonella growth and subsequent virulence during infection.
Strain-Specific Humoral Response to a Polymorphic Malaria Vaccine
Authors: Flück Christian , Smith Tom , Beck Hans-Peter , Irion Andrea , Betuela Inoni , Alpers Michael P. , Anders Robin , Saul Allan , Genton Blaise , Felger Ingrid
Published: October 2004

The 3D7 form of the merozoite surface protein 2 (MSP2) of
Plasmodium falciparum
was one of three subunits of the malaria vaccine Combination B that were tested in a phase I/IIb double-blind randomized placebo-controlled trial, which was undertaken with 120 Papua New Guinean children of 5 to 9 years of age. Because only one variant of the highly polymorphic MSP2 was used for vaccination, we examined whether the elicited response was directed against conserved or strain-specific epitopes. Postvaccination (week 12) titers of antibody against recombinantly expressed individual domains of MSP2 were measured by enzyme-linked immunosorbent assay and compared to baseline values. We found that vaccination with the 3D7 form of MSP2 induced a significant strain-specific humoral response directed against the repetitive and semiconserved family-specific part. The conserved N- and C-terminal domains were not immunogenic. Titers of antibody against the alternate FC27 family-specific domain showed a tendency to increase in vaccinated children, but there was no increase in antibodies against FC27-type 32-mer repeats. These results indicate that vaccination with one MSP2 variant mainly induced a strain-specific response, which can explain the selective effect of vaccination with combination B on the genotypes of breakthrough parasites. These findings support the inclusion of both family-specific domains (3D7 and FC27) in an improved vaccine formulation.
Mycobacterium marinum
Strains Can Be Divided into Two Distinct Types Based on Genetic Diversity and Virulence
Authors: van der Sar Astrid M. , Abdallah Abdallah M. , Sparrius Marion , Reinders Erik , Vandenbroucke-Grauls Christina M. J. E. , Bitter Wilbert
Published: October 2004

Mycobacterium marinum
causes a systemic tuberculosis-like disease in a large number of poikilothermic animals and is used as a model for mycobacterial pathogenesis. In the present study, we infected zebra fish (
Danio rerio
) with different strains of
M. marinum
to determine the variation in pathogenicity. Depending on the
M. marinum
isolate, the fish developed an acute or chronic disease. Acute disease was characterized by uncontrolled growth of the pathogen and death of all animals within 16 days, whereas chronic disease was characterized by granuloma formation in different organs and survival of the animals for at least 4 to 8 weeks. Genetic analysis of the isolates by amplified fragment length polymorphism showed that
M. marinum
strains could be divided in two clusters. Cluster I contained predominantly strains isolated from humans with fish tank granuloma, whereas the majority of the cluster II strains were isolated from poikilothermic species. Acute disease progression was noted only with strains belonging to cluster I, whereas all chronic-disease-causing isolates belonged to cluster II. This difference in virulence was also observed in vitro: cluster I isolate Mma20 was able to infect and survive more efficiently in the human macrophage THP-1 and the carp leukocyte CLC cell lines than was the cluster II isolate Mma11. We conclude that strain characteristics play an important role in the pathogenicity of
M. marinum
. In addition, the correlation between genetic variation and host origin suggests that cluster I isolates are more pathogenic for humans.
RNA Encoding the MPT83 Antigen Induces Protective Immune Responses againstMycobacterium tuberculosisInfection
Authors: Xue Tian , Stavropoulos Evangelos , Yang Min , Ragno Silvia , Vordermeier Martin , Chambers Mark , Hewinson Glyn , Lowrie Douglas B. , Colston M. Joseph , Tascon Ricardo E.
Published: October 2004

We have previously demonstrated that vaccination of mice with plasmid DNA vectors expressing immunodominant mycobacterial genes induced cellular immune responses and significant protection against challenge withMycobacterium tuberculosis. We demonstrate here, using in vitro-synthesized RNA, that vaccination with DNA or RNA constructs expressing theM. tuberculosisMPT83 antigen are capable of inducing specific humoral and T-cell immune responses and confer modest but significant protection againstM. tuberculosischallenge in mice. This is the first report of protective immunity conferred against intracellular bacteria by an RNA vaccine. This novel approach avoids some of the drawbacks of DNA vaccines and illustrates the potential for developing new antimycobacterial immunization strategies.
The Infecting Dose ofChlamydia muridarumModulates the Innate Immune Response and Ascending Infection
Authors: Maxion Heather K. , Liu Wei , Chang Mi-Hyang , Kelly Kathleen A.
Published: October 2004

Murine vaginal infection with the obligate intracellular bacteriumChlamydia muridarumis commonly used as a model for ascendingChlamydiainfections of the human female genital tract. Gamma interferon-producing Th1 cells, in concert with other mononuclear infiltrates, primarily mediate antichlamydial immunity. However, many factors modify this response, including the bacterial load. To investigate the manner in which the inoculating dose ofC. muridarummodulates a genital infection, we measured innate and adaptive cell numbers, CD4+lymphocyte cytokine profile, chemokine expression, course of infection, and pathological sequelae in genital tracts of BALB/c mice infected with doses ofC. muridarumranging from 104to 107inclusion-forming units. We found that the influx of both innate and adaptive immune cells responded similarly in the lower genital tract (cervical-vaginal tissues) and upper genital tract (oviduct tissues) to increasing inoculating doses. However, cells expressing the innate markers Gr-1 and CD11c were affected to a greater degree by increasing dose than lymphocytes of the adaptive immune response (Th1, CD4+, CD8+, CD19+), resulting in a change in the balance of innate and adaptive cell numbers to favor innate cells at higher infecting doses. Surprisingly, we detected greater numbers of viable chlamydiae in the oviducts at lower inoculating doses, and the number of organisms appeared to directly correlate with hydrosalpinx formation after both primary infection and repeat infection. Taken together, these data suggest that innate immune cells contribute to control of ascending infection.
The Type II Heat-Labile Enterotoxins LT-IIa and LT-IIb and Their Respective B Pentamers Differentially Induce and Regulate Cytokine Production in Human Monocytic Cells
Authors: Hajishengallis George , Nawar Hesham , Tapping Richard I. , Russell Michael W. , Connell Terry D.
Published: October 2004

The type II heat-labile enterotoxins, LT-IIa and LT-IIb, exhibit potent adjuvant properties. However, little is known about their immunomodulatory activities upon interaction with innate immune cells, unlike the widely studied type I enterotoxins that include cholera toxin (CT). We therefore investigated interactions of LT-IIa and LT-IIb with human monocytic THP-1 cells. We found that LT-II enterotoxins were inactive in stimulating cytokine release, whereas CT induced low levels of interleukin-1β (IL-1β) and IL-8. However, all three enterotoxins potently regulated cytokine induction in cells activated by bacterial lipopolysaccharide or fimbriae. Induction of proinflammatory (tumor necrosis factor α [TNF-α]) or chemotactic (IL-8) cytokines was downregulated, whereas induction of cytokines with anti-inflammatory (IL-10) or mucosal adjuvant properties (IL-1β) was upregulated by the enterotoxins. These effects appeared to depend on their A subunits, because isolated B-pentameric subunits lacked regulatory activity. Enterotoxin-mediated inhibition of proinflammatory cytokine induction in activated cells was partially attributable to synergism for endogenous production of IL-10 and to an IL-10-independent inhibition of nuclear factor κB (NF-κB) activation. In sharp contrast to the holotoxins, the B pentamers (LT-IIaB and, to a greater extent, LT-IIbB) stimulated cytokine production, suggesting a link between the absence of the A subunit and increased proinflammatory properties. In this regard, the ability of LT-IIbB to activate NF-κB and induce TNF-α and IL-8 was antagonized by the LT-IIb holotoxin. These findings support distinct immunomodulatory roles for the LT-II holotoxins and their respective B pentamers. Moreover, the anti-inflammatory properties of the holotoxins may serve to suppress innate immunity and promote the survival of the pathogen.
Suppression ofPlasmodium chabaudiParasitemia Is Independent of the Action of Reactive Oxygen Intermediates and/or Nitric Oxide
Authors: Gillman Brad M. , Batchelder Joan , Flaherty Patrick , Weidanz William P.
Published: October 2004

The killing of blood-stage malaria parasites in vivo has been attributed to reactive intermediates of oxygen (ROI) and of nitrogen (RNI). However, in the case of the latter, this contention is challenged by recent observations that parasitemia was not exacerbated in nitric oxide synthase (NOS) knockout (KO) (NOS2−/−or NOS3−/−) mice or in mice treated with NOS inhibitors. We now report that the time course shows thatPlasmodium chabaudiparasitemia in NADPH oxidase KO (p47phox−/−) mice also was not exacerbated, suggesting a minimal role for ROI-mediated killing of blood-stage parasites. It is possible that the production of protective antibodies during malaria may mask the function of ROI and/or RNI. However, parasitemia in B-cell-deficient JH−/−× NOS2−/−or JH−/−× p47phox−/−mice was not exacerbated. In contrast, the magnitude of peak parasitemia was significantly enhanced in p47phox−/−mice treated with the xanthine oxidase inhibitor allopurinol, but the duration of patent parasitemia was not prolonged. Whereas the time course of parasitemia in NOS2−/−× p47phox−/−mice was nearly identical to that seen in normal control mice, allopurinol treatment of these double-KO mice also enhanced the magnitude of peak parasitemia. Thus, ROI generated via the xanthine oxidase pathway contribute to the control of ascendingP. chabaudiparasitemia during acute malaria but alone are insufficient to suppress parasitemia to subpatent levels. Together, these results indicate that ROI or RNI can contribute to, but are not essential for, the suppression of parasitemia during blood-stage malaria.
Glucuronoxylomannan, the Major Capsular Polysaccharide of
Cryptococcus neoformans
, Inhibits the Progression of Group B Streptococcal Arthritis
Authors: Tissi Luciana , Puliti Manuela , Bistoni Francesco , Mosci Paolo , Kozel Thomas R. , Vecchiarelli Anna
Published: October 2004

Glucuronoxylomannan (GXM), the principal constituent of the
Cryptococcus neoformans
capsule, modulates the inflammatory response of human monocytes in vitro. Here we examine the efficacy of GXM as a novel anti-inflammatory compound for use against experimental septic arthritis. Arthritis was induced in mice by the intravenous injection of 8 × 10
6
CFU of type IV group B streptococcus (GBS). GXM was administered intravenously in different doses (50, 100, or 200 μg/mouse) 1 day before and 1 day after bacterial inoculation. GXM treatment markedly decreased the incidence and severity of articular lesions. Histological findings showed limited periarticular inflammation in the joints of GXM-treated mice, confirming the clinical observations. The amelioration of arthritis was associated with a significant reduction in the local production of interleukin-6 (IL-6), IL-1β, macrophage inflammatory protein 1α (MIP-1α), and MIP-2 and an increase in systemic IL-10 levels. Moreover, peritoneal macrophages derived from GXM-treated mice and stimulated in vitro with heat-inactivated GBS showed a similar pattern of cytokine production. The present study provides evidence for the modulation of the inflammatory response by GXM in vivo and suggests a potential therapeutic use for this compound in pathologies involving inflammatory processes.
Transcriptome of Uropathogenic
Escherichia coli
during Urinary Tract Infection
Authors: Snyder Jennifer A. , Haugen Brian J. , Buckles Eric L. , Lockatell C. Virginia , Johnson David E. , Donnenberg Michael S. , Welch Rodney A. , Mobley Harry L. T.
Published: October 2004

A uropathogenic
Escherichia coli
strain CFT073-specific DNA microarray that includes each open reading frame was used to analyze the transcriptome of CFT073 bacteria isolated directly from the urine of infected CBA/J mice. The in vivo expression profiles were compared to that of
E. coli
CFT073 grown statically to exponential phase in rich medium, revealing the strategies this pathogen uses in vivo for colonization, growth, and survival in the urinary tract environment. The most highly expressed genes overall in vivo encoded translational machinery, indicating that the bacteria were in a rapid growth state despite specific nutrient limitations. Expression of type 1 fimbriae, a virulence factor involved in adherence, was highly upregulated in vivo. Five iron acquisition systems were all highly upregulated during urinary tract infection, as were genes responsible for capsular polysaccharide and lipopolysaccharide synthesis, drug resistance, and microcin secretion. Surprisingly, other fimbrial genes, such as
pap
and
foc/sfa
, and genes involved in motility and chemotaxis were downregulated in vivo.
E. coli
CFT073 grown in human urine resulted in the upregulation of iron acquisition, capsule, and microcin secretion genes, thus partially mimicking growth in vivo. On the basis of gene expression levels, the urinary tract appears to be nitrogen and iron limiting, of high osmolarity, and of moderate oxygenation. This study represents the first assessment of any
E. coli
pathotype's transcriptome in vivo and provides specific insights into the mechanisms necessary for urinary tract pathogenesis.
Listeria monocytogenes-Based Antibiotic Resistance Gene-Free Antigen Delivery System Applicable to Other Bacterial Vectors and DNA Vaccines
Authors: Verch Thorsten , Pan Zhen-Kun , Paterson Yvonne
Published: October 2004

Plasmids represent a powerful tool to rapidly introduce genes into bacteria and help them reach high expression levels. In vaccine development, with live vaccine vectors, this allows greater flexibility and the ability to induce larger antigen amounts through multiple gene copies. However, plasmid retention often requires antibiotic resistance markers, the presence of which has been discouraged in clinical applications by the Food and Drug Administration. Therefore, we developed aListeria monocytogenes-Escherichia colishuttle plasmid that is retained by complementation ofd-alanine racemase-deficient mutant strains both in vitro and in vivo. Our technology potentially allows the production of antibiotic resistance marker-free DNA vaccines as well as bacterial vaccine vectors devoid of engineered antibiotic resistances. As a proof of concept, we applied thed-alanine racemase complementation system to ourListeriacancer vaccine platform. With a transplantable tumor model, we compared the efficacy of the newListeriavector to that of an established vector containing a conventional plasmid carrying a tumor-specific antigen. Both vaccine vector systems resulted in long-term regression of established tumors, with no significant difference between them. Thus, theListeriavaccine vector presented here potentially complies with Food and Drug Administration regulations and could be developed further for clinical use.
Intact Gram-NegativeHelicobacter pylori,Helicobacter felis, andHelicobacter hepaticusBacteria Activate Innate Immunity via Toll-Like Receptor 2 but Not Toll-Like Receptor 4
Authors: Mandell Leisa , Moran Anthony P. , Cocchiarella Andrew , Houghton JeanMarie , Taylor Nancy , Fox James G. , Wang Timothy C. , Kurt-Jones Evelyn A.
Published: October 2004

Molecular and genetic studies have demonstrated that members of the Toll-like receptor (TLR) family are critical innate immune receptors. TLRs are recognition receptors for a diverse group of microbial ligands including bacteria, fungi, and viruses. This study demonstrates that distinct TLRs are responsible for the recognition ofHelicobacterlipopolysaccharide (LPS) versus intactHelicobacterbacteria. We show that the cytokine-inducing activity ofHelicobacterLPS was mediated by TLR4; i.e., TLR4-deficient macrophages were unresponsive toHelicobacter pyloriLPS. Surprisingly, the cytokine response to wholeHelicobacterbacteria (H. pylori,H. hepaticus, andH. felis) was mediated not by TLR4 but rather by TLR2. Studies of HEK293 transfectants revealed that expression of human TLR2 was sufficient to confer responsiveness to intactHelicobacterbacteria, but TLR4 transfection was not sufficient. Our studies further suggest thatcagpathogenicity island genes may modulate the TLR2 agonist activity ofH. pyloriascagA+bacteria were more active on a per-cell basis compared tocagAmutant bacteria for interleukin-8 (IL-8) cytokine secretion. Consistent with the transfection studies, analysis of knockout mice demonstrated that TLR2 was required for the cytokine response to intactHelicobacterbacteria. Macrophages from both wild-type and TLR4-deficient mice produced a robust cytokine secretion response (IL-6 and MCP-1) when stimulated with intactHelicobacterbacteria. In contrast, macrophages from TLR2-deficient mice were profoundly unresponsive to intactHelicobacterstimulation, failing to secrete cytokines even at high (100:1) bacterium-to-macrophage ratios. Our studies suggest that TLR2 may be the dominant innate immune receptor for recognition of gastrointestinalHelicobacterspecies.
Role of Protein Tyrosine Kinase p53/56lynin Diminished Lipopolysaccharide Priming of Formylmethionylleucyl- phenylalanine-Induced Superoxide Production in Human Newborn Neutrophils
Authors: Yan Sen Rong , Byers David M. , Bortolussi Robert
Published: October 2004

Human newborns are more susceptible than adults to bacterial infection. With gram-negative bacteria, this may be due to a diminished response of newborn leukocytes to lipopolysaccharide (LPS). Since protein tyrosine kinase inhibition abolishes LPS priming in adult cells, we hypothesized that protein tyrosine kinases may have a critical role in LPS priming of polymorphonuclear neutrophils (PMNs) and that newborn PMNs may have altered protein tyrosine kinase activities. In the present study, we investigated the role ofsrcfamily protein tyrosine kinases in the LPS response of newborn PMNs compared to adult cells. In a respiratory assay, the LPS-primed increase in formylmethionylleucylphenylalanine (fMLP)-triggered O2−release by adult PMNs was greatly decreased by PP1 [4-amino-5-(4-methyphenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine], asrckinase inhibitor, to the level of untreated newborn PMNs, in which LPS failed to prime. LPS activated thesrc-like kinases p59hck(HCK) and p58fgr(FGR) in both adult and newborn PMNs but increased the activation of p53/56lyn(LYN) only in adult cells. In newborn PMNs, LYN was highly phosphorylated independent of LPS. We evaluated subcellular fractions of PMNs and found that the phosphorylated form of LYN was mainly in the Triton-extractable, cytosolic fraction in adult PMNs, while in newborn cells it was located mainly in Triton-insoluble, granule- and membrane-associated fractions. In contrast, the phosphorylated mitogen-activated protein kinases ERK1/2 and p38 were mainly detected in the cytosol in both adult and newborn PMNs. These data indicate a role for LYN in the regulation of LPS priming. The trapping of phosphorylated LYN in the membrane-granule fraction in newborn PMNs may contribute to the deficiency of newborn cells in responding to LPS stimulation.
Application of Mycobacterial Proteomics to Vaccine Design: Improved Protection by
Mycobacterium bovis
BCG Prime-Rv3407 DNA Boost Vaccination against Tuberculosis
Authors: Mollenkopf Hans Joachim , Grode Leander , Mattow Jens , Stein Maik , Mann Peggy , Knapp Bernhard , Ulmer Jeffrey , Kaufmann Stefan H. E.
Published: October 2004

Information from comparative proteome analysis of
Mycobacterium tuberculosis
and
Mycobacterium bovis
bacillus Calmette-Guérin (BCG) principally allows prediction of potential vaccine candidates. Thirty-six
M. tuberculosis
DNA vaccine candidates identified by comparative proteome analysis were evaluated in the mouse model for protection against low-dose aerosol
M. tuberculosis
infection. We identified the DNA vaccine candidate Rv3407 as a protective antigen and analyzed putative major histocompatibility complex class I epitopes by computational predictions and gamma interferon Elispot assays. Importantly, we discovered that the DNA vaccine Rv3407 improved the efficacy of BCG vaccination in a heterologous prime-boost vaccination protocol. Our data demonstrate the rationale of a combination of proteomics, epitope prediction, and broad screening of putative antigens for identification of novel DNA vaccine candidates. Furthermore, our experiments show that heterologous prime-boost vaccination with a defined antigen boost “on top” of a BCG primer provides superior protection against tuberculosis over vaccination with BCG alone.
Combined Vaccine Regimen Based on Parenteral Priming with a DNA Vaccine and Administration of an Oral Booster Consisting of a RecombinantSalmonella entericaSerovar Typhimurium Vaccine Strain for Immunization against Infection with Human-Derived EnterotoxigenicEscherichia coliStrains
Authors: Lásaro Marcio O. , Luiz Wilson B. , Sbrogio-Almeida Maria E. , Nishimura Lucilia S. , Guth Beatriz E. C. , Ferreira Luis C. S.
Published: October 2004

Repeated evidence has demonstrated that combined primer-booster immunization regimens can improve both secreted and humoral immune responses to antigens derived from viral, bacterial, and parasitic pathogens. For the present work, we evaluated the synergic serum immunoglobulin G (IgG) and fecal IgA antibody responses elicited in BALB/c mice who were intramuscularly primed with a DNA vaccine, pRECFA, followed by oral boosting with an attenuatedSalmonella entericaserovar Typhimurium vaccine (HG3) strain, with both vaccines encoding the structural subunit (CfaB) of the CFA/I fimbriae produced by human-derived enterotoxigenicEscherichia coli(ETEC) strains. The immunological properties of the vaccine regimen were evaluated according to the order of the administered vaccines, the nature of the oral antigen carrier, the age of the vaccinated animals, the interval between the priming and boosting doses, and the amount of injected DNA. The production of gamma interferon and the IgG2a subclass in serum indicated that mice immunized with the primer-booster regimen developed prevailing type 1 T-cell-dependent immune responses. The synergic effect of the vaccine regimen on the induced antibody responses was also revealed by its ability to block the adhesive properties of CFA/I fimbriae expressed by live bacteria, as shown by the inhibition of Caco-2 cell and human erythrocyte binding. Moreover, DBA2 newborn mice were protected from lethal challenges with a CFA/I+ETEC strain after the incubation of live bacteria with serum samples harvested from mice who were subjected to the primer-booster regimen. We propose, therefore, that the DNA primer-Salmonellabooster regimen represents an alternative for the development of vaccines requiring both mucosal and systemic antibody responses for immunological protection.
Development of Immunity to Serogroup B Meningococci during Carriage of
Neisseria meningitidis
in a Cohort of University Students
Authors: Jordens J. Zoe , Williams Jeannette N. , Jones Graeme R. , Christodoulides Myron , Heckels John E.
Published: October 2004

Understanding the basis of protective immunity is a key requirement for the development of an effective vaccine against infection with
Neisseria meningitidis
of serogroup B. We have conducted a longitudinal study into the dynamics of meningococcal acquisition and carriage in first-year university students. The detection of carriage of serogroup B meningococci correlated with an increase in detection of serum bactericidal activity (SBA) against both colonizing and heterologous serogroup B strains. Once induced, SBA remained high throughout the study. Although students showed increases in antibodies reactive with capsular polysaccharide and lipopolysaccharide (LPS), these antibody responses were transitory, and their decline was not accompanied by a corresponding decline in SBA. In contrast, there was a significant correlation between the presence of antibodies to the PorA outer membrane protein and SBA against both homologous and heterologous strains. SBA induced by a PorA-negative mutant confirmed the contribution of PorA to heterologous activity. Increases in SBA against a range of serogroup B strains were also observed in students in whom no meningococcal carriage was detected. This heterologous protection could not be associated with the presence of antibodies reacting with capsule, LPS, PorA, PorB, Rmp, Opa, Opc, or pilin, demonstrating that other, as yet unidentified, antigens contribute to the development of immunity to serogroup B meningococci. Identification of such antigens with the ability to induce an effective cross-reactive bactericidal response to a range of strains would be a major step in the production of a universally effective vaccine against infections caused by serogroup B meningococci.
Expression of Heterologous Antigens in Commensal
Neisseria
spp.: Preservation of Conformational Epitopes with Vaccine Potential
Authors: O'Dwyer Clíona A. , Reddin Karen , Martin Denis , Taylor Stephen C. , Gorringe Andrew R. , Hudson Michael J. , Brodeur Bernard R. , Langford Paul R. , Kroll J. Simon
Published: October 2004

Commensal neisseriae share with
Neisseria meningitidis
(meningococcus) a tendency towards overproduction of the bacterial outer envelope, leading to the formation and release during growth of outer membrane vesicles (OMVs). OMVs from both meningococci and commensal neisseriae have shown promise as vaccines to protect against meningococcal disease. We report here the successful expression at high levels of heterologous proteins in commensal neisseriae and the display, in its native conformation, of one meningococcal outer membrane protein vaccine candidate, NspA, in OMVs prepared from such a recombinant
Neisseria flavescens
strain. These NspA-containing OMVs conferred protection against otherwise lethal intraperitoneal challenge of mice with
N. meningitidis
serogroup B, and sera raised against them mediated opsonophagocytosis of meningococcal strains expressing this antigen. This development promises to facilitate the design of novel vaccines containing membrane protein antigens that are otherwise difficult to present in native conformation that provide cross-protective efficacy in the prevention of meningococcal disease.
Modeling the Development of Acquired Clinical Immunity to
Plasmodium falciparum
Malaria
Authors: Gatton Michelle L. , Cheng Qin
Published: October 2004

Individuals living in regions where malaria is endemic develop an acquired immunity to malaria which enables them to remain asymptomatic while still carrying parasites. Field studies indicate that cumulative exposure to a variety of diverse
Plasmodium
parasites is required for the transition from symptomatic to asymptomatic malaria. This study used a simulation model of the within-host dynamics of
P. falciparum
to investigate the development of acquired clinical immunity under different transmission conditions and levels of parasite diversity. Antibodies developed to
P. falciparum
erythrocyte membrane protein 1 (PfEMP1), a clonally variant molecule, were assumed to be a key human immunological response to
P. falciparum
infection, along with responses to clonally conserved but polymorphic antigens. The time to the development of clinical immunity was found to be proportional to parasite diversity and inversely proportional to transmission intensity. The effect of early termination of symptomatic infections by chemotherapy was investigated and found not to inhibit the host's ability to develop acquired immunity. However, the time required to achieve this state was approximately double that compared to when no treatment was administered. This study demonstrates that an immune response primarily targeted against PfEMP1 has the ability to reduce clinical symptoms of infections irrespective of whether treatment is administered, supporting its role in the development of acquired clinical immunity. The results also illustrate a novel use for simulation models of
P. falciparum
infections, investigation of the influence of intervention strategies on the development of naturally acquired clinical immunity.
Enhanced Immunogenicity in the Murine Airway Mucosa with an AttenuatedSalmonellaLive Vaccine Expressing OprF-OprI fromPseudomonas aeruginosa
Authors: Arnold Heinz , Bumann Dirk , Felies Melanie , Gewecke Britta , Sörensen Meike , Gessner J. Engelbert , Freihorst Joachim , von Specht Bernd Ulrich , Baumann Ulrich
Published: October 2004

We constructed an oral live vaccine based on the attenuatedaroAmutantSalmonella entericaserovar Typhimurium strain SL3261 expressing outer membrane proteins F and I (OprF-OprI) fromPseudomonas aeruginosaand investigated it in a mouse model. Strains with in vivo inducible protein expression with thePpacCpromoter showed good infection rates and immunogenicity but failed to engender detectable antibodies in the lung. However, a systemic booster vaccination following an oral primary immunization yielded high immunoglobulin A (IgA) and IgG antibody levels in both upper and lower airways superior to conventional systemic or mucosal booster vaccination alone. In addition, the proportion of IgG1 and IgG2a antibodies suggested that the systemic booster does not alter the more TH1-like type of response induced by the oralSalmonellaprimary vaccination. We conclude that an oral primary systemic booster vaccination strategy with an appropriate mucosal vector may be advantageous in diseases with the risk ofP. aeruginosaairway infection, such as cystic fibrosis.
Effect of Dosage on Immunogenicity of a Vi Conjugate Vaccine Injected Twice into 2- to 5-Year-Old Vietnamese Children
Authors: Canh Do Gia , Lin Feng-ying (Kimi) , Thiem Vu Dinh , Trach Dang Duc , Trong Nguyen Dinh , Mao Nguyen Duc , Hunt Steven , Schneerson Rachel , Robbins John B. , Chu Chiayung , Shiloach Joseph , Bryla Dolores A. , Bonnet Marie-Claude , Schulz Dominique , Szu Shousun C.
Published: October 2004

In a double-blind, randomized, and placebo-controlled previous trial, the efficacy of Vi-
r
EPA for typhoid fever in 2- to 5-year-olds was 89.0% for 46 months. Vi-
r
EPA contained 25 μg of Vi and induced a greater-than-eightfold rise in immunoglobulin G (IgG) anti-Vi in all of the vaccinees tested. In this investigation, we conducted a dosage-immunogenicity study of 5, 12.5, and 25 μg of Vi-
r
EPA in this age group. Two doses of Vi-
r
EPA were injected 6 weeks apart. Blood samples were taken before and at 10 weeks (4 weeks after the second injection) and 1 year later. All postimmunization geometric mean (GM) levels were higher than the preimmune levels (
P
< 0.0001). At 10 weeks, the GM IgG anti-Vi level elicited by 25 μg (102 EU/ml) was higher than those elicited by 12.5 μg (74.7 EU/ml) and 5 μg (43 EU/ml) (
P
< 0.004): all of the children had ≥3.52 EU/ml (estimated minimum protective level). One year later, the levels declined about sevenfold (13.3 and 11.3 versus 6.43 EU/ml,
P
< 0.0001) but remained significantly higher than the preimmune levels (
P
< 0.0001), and >96% of the children had a greater-than-eightfold rise. This study also confirmed the safety and consistent immunogenicity of the four lots of Vi-
r
EPA used in this and previous trials.
Quorum Sensing: a Transcriptional Regulatory System Involved in the Pathogenicity of
Burkholderia mallei
Authors: Ulrich Ricky L. , DeShazer David , Hines Harry B. , Jeddeloh Jeffrey A.
Published: October 2004

Numerous gram-negative bacterial pathogens regulate virulence factor expression by using a cell density mechanism termed quorum sensing (QS). An in silico analysis of the
Burkholderia mallei
ATCC 23344 genome revealed that it encodes at least two
luxI
and four
luxR
homologues. Using mass spectrometry, we showed that wild-type
B. mallei
produces the signaling molecules
N
-octanoyl-homoserine lactone and
N
-decanoyl-homoserine lactone. To determine if QS is involved in the virulence of
B. mallei
, we generated mutations in each putative
luxIR
homologue and tested the pathogenicities of the derivative strains in aerosol BALB/c mouse and intraperitoneal hamster models. Disruption of the
B. mallei
QS alleles, especially in RJ16 (
bmaII
) and RJ17 (
bmaI3
), which are
luxI
mutants, significantly reduced virulence, as indicated by the survival of mice who were aerosolized with 10
4
CFU (10 50% lethal doses [LD
50
s]). For the
B. mallei
transcriptional regulator mutants (
luxR
homologues), mutation of the
bmaR5
allele resulted in the most pronounced decrease in virulence, with 100% of the challenged animals surviving a dose of 10 LD
50
s. Using a Syrian hamster intraperitoneal model of infection, we determined the LD
50
s for wild-type
B. mallei
and each QS mutant. An increase in the relative LD
50
was found for RJ16 (
bmaI1
) (>967 CFU), RJ17 (
bmaI3
) (115 CFU), and RJ20 (
bmaR5
) (151 CFU) compared to wild-type
B. mallei
(<13 CFU). These findings demonstrate that
B. mallei
carries multiple
luxIR
homologues that either directly or indirectly regulate the biosynthesis of an essential virulence factor(s) that contributes to the pathogenicity of
B. mallei
in vivo.
Differences in Cell Activation byChlamydophila pneumoniaeandChlamydia trachomatisInfection in Human Endothelial Cells
Authors: Krüll M. , Kramp J. , Petrov T. , Klucken A. C. , Hocke A. C. , Walter C. , Schmeck B. , Seybold J. , Maass M. , Ludwig S. , Kuipers Jens G. , Suttorp N. , Hippenstiel S.
Published: October 2004

Seroepidemiological studies and demonstration of viable bacteria in atherosclerotic plaques have linkedChlamydophila pneumoniaeinfection to the development of chronic vascular lesions and coronary heart disease. In this study, we characterizedC. pneumoniae-mediated effects on human endothelial cells and demonstrated enhanced phosphorylation and activation of the endothelial mitogen-activated protein kinase (MAPK) family members extracellular receptor kinase (ERK1/2), p38-MAPK, and c-Jun-NH2kinase (JNK). Subsequent interleukin-8 (IL-8) expression was dependent on p38-MAPK and ERK1/2 activation as demonstrated by preincubation of endothelial cells with specific inhibitors for the p38-MAPK (SB202190) or ERK (U0126) pathway. Inhibition of either MAPK had almost no effect on intercellular cell adhesion molecule 1 (ICAM-1) expression. WhileChlamydia trachomatiswas also able to infect endothelial cells, it did not induce the expression of endothelial IL-8 or ICAM-1. These effects were specific for a direct stimulation with viableC. pneumoniaeand independent of paracrine release of endothelial cell-derived mediators like platelet-activating factor, NO, prostaglandins, or leukotrienes. Thus,C. pneumoniaetriggers an early signal transduction cascade in target cells that could lead to endothelial cell activation, inflammation, and thrombosis, which in turn may result in or promote atherosclerosis.
The Protective Effect of the
Mycobacterium bovis
BCG Vaccine Is Increased by Coadministration with the
Mycobacterium tuberculosis
72-Kilodalton Fusion Polyprotein Mtb72F in
M. tuberculosis
-Infected Guinea Pigs
Authors: Brandt Lise , Skeiky Yasir A. W. , Alderson Mark R. , Lobet Yves , Dalemans Wilfried , Turner Oliver C. , Basaraba Randall J. , Izzo Angelo A. , Lasco Todd M. , Chapman Philip L. , Reed Steven G. , Orme Ian M.
Published: October 2004

A tuberculosis vaccine candidate consisting of a 72-kDa polyprotein or fusion protein based upon the Mtb32 and Mtb39 antigens of
Mycobacterium tuberculosis
and designated Mtb72F was tested for its protective capacity as a potential adjunct to the
Mycobacterium bovis
BCG vaccine in the mouse and guinea pig models of this disease. Formulation of recombinant Mtb72F (rMtb72F) in an AS02A adjuvant enhanced the Th1 response to BCG in mice but did not further reduce the bacterial load in the lungs after aerosol challenge infection. In the more stringent guinea pig disease model, rMtb72F delivered by coadministration with BCG vaccination significantly improved the survival of these animals compared to BCG alone, with some animals still alive and healthy in their appearance at >100 weeks post-aerosol challenge. A similar trend was observed with guinea pigs in which BCG vaccination was boosted by DNA vaccination, although this increase was not statistically significant due to excellent protection conferred by BCG alone. Histological examination of the lungs of test animals indicated that while BCG controls eventually died from overwhelming lung consolidation, the majority of guinea pigs receiving BCG mixed with rMtb72F or boosted twice with Mtb72F DNA had mostly clear lungs with minimal granulomatous lesions. Lesions were still prominent in guinea pigs receiving BCG and the Mtb72F DNA boost, but there was considerable evidence of lesion healing and airway remodeling and reestablishment. These data support the hypothesis that the coadministration or boosting of BCG vaccination with Mtb72F may limit the lung consolidation seen with BCG alone and may promote lesion resolution and healing. Collectively, these data suggest that enhancing BCG is a valid vaccination strategy for tuberculosis that is worthy of clinical evaluation.
The Hyphal and Yeast Forms ofCandida albicansBind the Complement Regulator C4b-Binding Protein
Authors: Meri T. , Blom A. M. , Hartmann A. , Lenk D. , Meri S. , Zipfel P. F.
Published: October 2004

Candida albicans, an important pathogenic yeast, activates all three pathways of the complement system. To understand how this yeast evades the effects of the activated system, we have analyzed the binding of the classical pathway inhibitor C4b-binding protein (C4BP) byC. albicans. Purified native as well as recombinant C4BP bound dose dependently to the yeast and hyphal forms, as shown by multiple methods, such as confocal microscopy, flow cytometry, a novel enzyme-linked immunosorbent assay, absorption from human serum, and direct binding assays with purified proteins. A prominent binding site was identified at the tip of the germ tube, a structure that is considered important for tissue penetration and pathogenesis. The binding site in C4BP was localized to the two N-terminal complement control protein domains by using recombinant deletion constructs and site-specific monoclonal antibodies. As the alternative pathway inhibitors factor H and FHL-1 also bind toC. albicans, the binding of all three plasma proteins was compared. Simultaneous binding of the classical regulator C4BP and the alternative pathway regulator factor H was demonstrated by confocal microscopy. In addition, FHL-1 competed for binding with C4BP, suggesting that these two related complement regulators bind to the same structures on the yeast surface. The surface-attached C4BP maintains its complement regulatory activities and inactivates C4b. The surface-attached human C4BP serves multiple functions relevant for immune evasion and likely pathogenicity. It inhibits complement activation at the yeast surface and, in addition, mediates adhesion ofC. albicansto host endothelial cells.