bluuebunny/crossref_metadata_2025 huggingface dataset:
Influence of Synthetic Antiendotoxin Peptides on Lipopolysaccharide (LPS) Recognition and LPS-Induced Proinflammatory Cytokine Responses by Cells Expressing Membrane-Bound CD14
Authors: Iwagaki Akitaka , Porro Massimo , Pollack Matthew
Published: July 2002

Lipopolysaccharides (LPS) are proinflammatory bacterial products implicated in the pathogenesis of gram-negative sepsis and septic shock. Polymyxin B (PMB), a cyclic, cationic peptide antibiotic, inhibits biological activities of LPS through high-affinity binding to the lipid A moiety. Small synthetic peptides have been designed to mimic the primary and secondary structures of PMB to determine structural requirements for binding and detoxification of lipid A and to assess possible therapeutic potential. The purpose of this study was to compare and contrast the endotoxin-neutralizing activities of two synthetic antiendotoxin peptides (SAEP-2 and SAEP-4), PMB, and an LPS core-specific monoclonal antibody (MAb), WN1 222-5, based on their abilities to inhibit CD14-mediated target cell uptake of fluorescein isothiocyanate (FITC)-conjugated LPS, detected by flow cytometry and confocal microscopy, and LPS-induced production of the proinflammatory cytokines, interleukin-6 (IL-6) and tumor necrosis factor alpha (TNF-α), as measured by bioassays. PMB and SAEP-4 produced dose-dependent inhibition of FITC-LPS uptake by CD14-transfected Chinese hamster ovary fibroblasts (CHO-CD14 cells) and by human peripheral blood mononuclear cells. The anti-LPS MAb, WN1 222-5, also blocked LPS uptake by these cells and synergized with PMB and SAEP-4. LPS-induced IL-6 release was inhibited by PMB, SAEP-4, and MAb WN1 222-5, and these inhibitory activities were additive or synergistic. LPS-induced TNF-α release by PBMC was also inhibited by PMB and SAEP-4 alone and in combination with anti-LPS MAb. SAEP-2, in contrast, produced comparatively minor decrements in cellular uptake of LPS and LPS-induced cytokine responses, and did so only in the absence of serum, while a nonsense peptide exerted no discernible inhibitory effect on LPS uptake or LPS-induced cytokine expression in the presence or absence of serum. Thus, PMB and SAEP-4, like the LPS-reactive MAb, WN1 222-5, block proinflammatory activities of LPS in part by preventing LPS recognition by membrane-bound CD14-expressing target cells. Differences in peptide structure, however, like those exemplified by SAEP-2 and SAEP-4, may differentially affect the endotoxin-neutralizing potency of these peptides despite similar binding activity against lipid A, reflecting possible differences in peptide solubility or peptide regulation of intracellular signal transduction.
Antibody-Mediated Elimination of the Obligate Intracellular Bacterial PathogenEhrlichia chaffeensisduring Active Infection
Authors: Winslow Gary M. , Yager Eric , Shilo Konstantin , Volk Erin , Reilly Andrew , Chu Frederick K.
Published: July 2002

It is generally accepted that cellular, but not humoral immunity, plays an important role in host defense against intracellular bacteria. However, studies of some of these pathogens have provided evidence that antibodies can provide immunity if present during the initiation of infection. Here, we examined immunity against infection byEhrlichia chaffeensis, an obligate intracellular bacterium that causes human monocytic ehrlichiosis. Studies with mice have demonstrated that immunocompetent strains are resistant to persistent infection but that SCID mice become persistently and fatally infected. Transfer of immune serum or antibodies obtained from immunocompetent C57BL/6 mice to C57BL/6scidmice provided significant although transient protection from infection. Bacterial clearance was observed when administration occurred at the time of inoculation or well after infection was established. The effect was dose dependent, occurred within 2 days, and persisted for as long as 2 weeks. Weekly serum administration prolonged the survival of susceptible mice. Although cellular immunity is required for complete bacterial clearance, the data show that antibodies can play a significant role in the elimination of this obligate intracellular bacterium during active infection and thus challenge the paradigm that humoral responses are unimportant for immunity to such organisms.
Enterotoxicity and Cytotoxicity of
Vibrio parahaemolyticus
Thermostable Direct Hemolysin in In Vitro Systems
Authors: Raimondi Francesco , Kao Joseph P. Y. , Fiorentini Carla , Fabbri Alessia , Donelli Gianfranco , Gasparini Nicoletta , Rubino Armido , Fasano Alessio
Published: July 2002

Vibrio parahaemolyticus
is a marine bacterium known to be a common cause of seafood gastroenteritis worldwide. The thermostable direct hemolysin (TDH) has been proposed to be a major virulence factor of
V. parahaemolyticus
. TDH causes intestinal fluid secretion as well as cytotoxicity in a variety of cell types. In this study, we investigated the interplay between the hemolysin's enterotoxic and cytotoxic effects by using both human and rat cell monolayers. As revealed by microspectrofluorimetry, the toxin causes a dose-dependent increase in intracellular free calcium in both Caco-2 and IEC-6 cells. This effect was reversible only when low toxin concentrations were tested. The TDH-activated ion influx pathway is not selective for calcium but admits ions such sodium and manganese as well. Furthermore, in the same range of concentration, the hemolysin triggers a calcium-dependent chloride secretion. At high concentrations, TDH induces a dose-dependent but calcium-independent cell death as assessed by functional, biochemical, and morphological assays.
Evidence of Commonality between Canine and Human Extraintestinal PathogenicEscherichia coliStrains That ExpresspapGAllele III
Authors: Johnson James R. , O'Bryan Timothy T. , Low David A. , Ling Gerald , Delavari Parissa , Fasching Claudine , Russo Thomas A. , Carlino Ulrike , Stell Adam L.
Published: July 2002

Although dogs have been proposed as carriers of extraintestinal pathogenicEscherichia coli(ExPEC) with infectious potential for humans, presumed host species-specific differences between canine and human ExPEC strains have cast doubt on this hypothesis. The recent discovery that allele III ofpapG(the P fimbrial adhesin gene) predominates among human cystitis isolates and confers an adherence phenotype resembling that of canine ExPEC prompted the present reevaluation of the canine-human ExPEC connection. Sixteen pairedpap-positive urine and rectalE. coliisolates from dogs with urinary tract infection were studied.papG(adhesin) andpapA(pilin) allele type, agglutination phenotypes, virulence factor genotypes, and randomly amplified polymorphic DNA and pulsed-field gel electrophoresis fingerprints were analyzed and compared with those of human ExPEC controls. The 16 canine strains contained predominantlypapGallele III. Agglutination phenotypes segregated strictly according topapGallele status and were homogeneous among strains with the samepapGallele profile irrespective of their human versus canine origin. Canine and human PapG variant III peptide sequences were highly homologous, without host species-specific differences. The most prevalent caninepapAallele was F48, a novel variant recently identified among human urosepsis isolates. In addition topap, human ExPEC-associated virulence genes detected among the canine strains includedsfa/focDE,sfaS,fyuA,hlyA,cnf1,cdtB,kpsMT-II and -III,rfc,traT,ompT, and a marker for a pathogenicity-associated island from archetypal human ExPEC strain CFT073. Molecular fingerprinting confirmed the fecal origin of all but one canine urine isolate and showed one pair of O6 canine urine and fecal isolates to be extremely similar to an O6 human urosepsis isolate with which they shared all other genotypic and phenotypic characteristics analyzed. These data demonstrate that canine ExPEC strains are similar to, and in some instances essentially indistinguishable from, human ExPEC strains, which implicates dogs and their feces as potential reservoirs ofE. coliwith infectious potential for humans.
Interaction of Human Phagocytes with Pigmentless
Aspergillus
Conidia
Authors: Jahn Bernhard , Boukhallouk Fatima , Lotz Johannes , Langfelder Kim , Wanner Gerhard , Brakhage Axel A.
Published: July 2002

A defect in the
pksP
gene of
Aspergillus fumigatus
is associated with the loss of conidial pigmentation, a profound change of the conidial surface structure, and reduced virulence. The structural change of the conidial surface structure was not observed in similar
A. nidulans wA
mutants. Our data indicate that the pigment of both species is important for scavenging reactive oxygen species and for protection of conidia against oxidative damage.
Identification of Genes Required for Chronic Persistence ofBrucella abortusin Mice
Authors: Hong Priscilla C. , Tsolis Renée M. , Ficht Thomas A.
Published: July 2002

The genetic basis for chronic persistence ofBrucella abortusin lymphoid organs of mice, cows, and humans is currently unknown. We identifiedB. abortusgenes involved in chronic infection, by assessing the ability of 178 signature-tagged mutants to establish and maintain persistent infection in mice. Each mutant was screened for its ability to colonize the spleens of mice at 2 and 8 weeks after inoculation. Comparison of the results from both time points identified two groups of mutants attenuated for chronic infection in mice. The first group was not recovered at either 2 or 8 weeks postinfection and was therefore defective in establishing infection. Mutants in this group carried transposon insertions in genes involved in lipopolysaccharide biosynthesis (wbkA), in aromatic amino acid biosynthesis, and in type IV secretion (virB1andvirB10). The second group, which was recovered at wild-type levels 2 weeks postinfection but not 8 weeks postinfection was able to establish infection but was unable to maintain chronic infection. One mutant in this group carried a transposon insertion in a gene with homology togcvBofMycobacterium tuberculosis, encoding glycine dehydrogenase, an enzyme whose activity is increased during the state of nonreplicating persistence. These results suggest that some mechanisms for long-term persistence may be shared among chronic intracellular pathogens. Furthermore, identification of two groups of genes, those required for initiating infection and those required only for long-term persistence, suggests thatB. abortususes distinct sets of virulence determinants to establish and maintain chronic infection in mice.
Differential Modulation and Subsequent Blockade of Mitogenic Signaling and Cell Cycle Progression byPasteurella multocidaToxin
Authors: Wilson Brenda A. , Aminova Lyaylya R. , Ponferrada Virgilio G. , Ho Mengfei
Published: July 2002

The intracellularly acting protein toxin ofPasteurella multocida(PMT) causes numerous effects in cells, including activation of inositol 1,4,5-trisphosphate (IP3) signaling, Ca2+mobilization, protein phosphorylation, morphological changes, and DNA synthesis. The direct intracellular target of PMT responsible for activation of the IP3pathway is the Gq/11α-protein, which stimulates phospholipase C (PLC) β1. The relationship between PMT-mediated activation of the Gq/11-PLC-IP3pathway and its ability to promote mitogenesis and cellular proliferation is not clear. PMT stimulation of p42/p44 mitogen-activated protein kinase occurs upstream via Gq/11-dependent transactivation of the epidermal growth factor receptor. We have further characterized the effects of PMT on the downstream mitogenic response and cell cycle progression in Swiss 3T3 and Vero cells. PMT treatment caused dramatic morphological changes in both cell lines. In Vero cells, limited multinucleation, nuclear fragmentation, and disruption of cytokinesis were also observed; however, a strong mitogenic response occurred only with Swiss 3T3 cells. Significantly, this mitogenic response was not sustained. Cell cycle analysis revealed that after the initial mitogenic response to PMT, both cell types subsequently arrested primarily in G1and became unresponsive to further PMT treatment. In Swiss 3T3 cells, PMT induced up-regulation of c-Myc; cyclins D1, D2, D3, and E; p21; PCNA; and the Rb proteins, p107 and p130. In Vero cells, PMT failed to up-regulate PCNA and cyclins D3 and E. We also found that the initial PMT-mediated up-regulation of several of these signaling proteins was not sustained, supporting the subsequent cell cycle arrest. The consequences of PMT entry thus depend on the differential regulation of signaling pathways within different cell types.
[Cu,Zn]-Superoxide Dismutase Mutants of the Swine PathogenActinobacillus pleuropneumoniaeAre Unattenuated in Infections of the Natural Host
Authors: Sheehan Brian J. , Langford Paul R. , Rycroft Andrew N. , Kroll J. Simon
Published: July 2002

Actinobacillus pleuropneumoniae, the causative agent of porcine pleuropneumonia, contains a periplasmic Cu- and Zn-cofactored superoxide dismutase ([Cu,Zn]-SOD, or SodC) which has the potential, realized in other pathogens, to promote bacterial survival during infection by dismutating host-defense-derived superoxide. Here we describe the construction of a site-specific, [Cu,Zn]-SOD-deficientA. pleuropneumoniaeserotype 1 mutant and show that although the mutant is highly sensitive to the microbicidal action of superoxide in vitro, it remains fully virulent in experimental pulmonary infection in pigs.
RecombinantMycobacterium bovisBCG Expressing Pertussis Toxin Subunit S1 Induces Protection against an Intracerebral Challenge with LiveBordetella pertussisin Mice
Authors: Nascimento Ivan P. , Dias Waldely O. , Mazzantini Rogerio P. , Miyaji Eliane N. , Gamberini Marcia , Quintilio Wagner , Gebara Vera C. , Cardoso Diva F. , Ho Paulo L. , Raw Isaias , Winter Nathalie , Gicquel Brigitte , Rappuoli Rino , Leite Luciana C. C.
Published: July 2002

The recent development of acellular pertussis vaccines has been a significant improvement in the conventional whole-cell diphtheria-pertussis-tetanus toxoid vaccines, but high production costs will limit its widespread use in developing countries. SinceMycobacterium bovisBCG vaccination against tuberculosis is used in most developing countries, a recombinant BCG-pertussis vaccine could be a more viable alternative. We have constructed recombinant BCG (rBCG) strains expressing the genetically detoxified S1 subunit of pertussis toxin 9K/129G (S1PT) in fusion with either the β-lactamase signal sequence or the whole β-lactamase protein, under control of the upregulatedM. fortuitumβ-lactamase promoter, pBlaF*. Expression levels were higher in the fusion with the whole β-lactamase protein, and both were localized to the mycobacterial cell wall. The expression vectors were relatively stable in vivo, since at two months 85% of the BCG recovered from the spleens of vaccinated mice maintained kanamycin resistance. Spleen cells from rBCG-S1PT-vaccinated mice showed elevated gamma interferon (IFN-γ) and low interleukin-4 (IL-4) production, as well as increased proliferation, upon pertussis toxin (PT) stimulation, characterizing a strong antigen-specific Th1-dominant cellular response. The rBCG-S1PT strains induced a low humoral response against PT after 2 months. Mice immunized with rBCG-S1PT strains displayed high-level protection against an intracerebral challenge with liveBordetella pertussis, which correlated with the induction of a PT-specific cellular immune response, reinforcing the importance of cell-mediated immunity in the protection againstB. pertussisinfection. Our results suggest that rBCG-expressing pertussis antigens could constitute an effective, low-cost combined vaccine against tuberculosis and pertussis.
Infection of Fetal Feline Brain Cells in Culture withBartonella henselae
Authors: Muñana Karen R. , Vitek Susanne M. , Hegarty Barbara C. , Kordick Dorsey L. , Breitschwerdt Edward B.
Published: July 2002

Bartonella henselaeis known to cause central nervous system (CNS) disease in humans, and neurological signs have been observed in experimentally infected cats. However, the pathogenesis of CNS disease remains unclear. This study was undertaken to determine whetherB. henselaeinfects feline fetal brain cells in vitro. Microglial-cell- and astrocyte-enriched cultures were inoculated withB. henselae. Giménez staining identified bacterial organisms within microglial cells by day 7 postinoculation. The viability of the intracellular bacteria was demonstrated by incubating cultures with gentamicin and plating cell lysate on agar. Electron microscopy identified intracellular organisms with characteristicBartonellamorphology but identified no ultrastructural abnormalities within infected microglial cells. No evidence of infection was seen inBartonella-inoculated astrocyte cultures. These findings suggest a role for microglia in the pathogenesis ofB. henselae-associated neurological disease.
Identification of Protective Epitopes by Sequencing of the Major Outer Membrane Protein Gene of a Variant Strain ofChlamydia psittaciSerotype 1 (Chlamydophila abortus)
Authors: Vretou Evangelia , Psarrou Evgenia , Kaisar Maria , Vlisidou Isabella , Salti-Montesanto Viviane , Longbottom David
Published: July 2002

Protective monoclonal antibodies (MAbs) to the major outer membrane protein (MOMP) of species of the familyChlamydiaceae, which is the primary vaccine candidate antigen, recognize nonlinear epitopes conferred by the oligomeric conformation of the molecule. Protective MAbs failed to recognize oligomeric MOMP of the variant strain LLG, which bears amino acid substitutions in variable segments (VSs) 1, 2, and 4, and competed with monomer-specific MAbs mapping to these VSs in reference strain 577. The results suggest that multiple sites located in the three VSs contribute to the epitope of protective MAbs.
DNA Immunization withTrypanosoma cruziHSP70 Fused to the KMP11 Protein Elicits a Cytotoxic and Humoral Immune Response against the Antigen and Leads to Protection
Authors: Planelles Lourdes , Thomas M. Carmen , Alonso Carlos , López Manuel C.
Published: July 2002

Murine immunization withTrypanosoma cruzi KMP11-HSP70fused genes but not theKMP11gene alone elicited both an immunoglobulin G2a long-lasting humoral immune response against KMP11 protein and activation of CD8+cytotoxic T lymphocytes specific for two KMP11 peptides containing A2 motifs. Moreover, protection against the parasite challenge was observed after immunization with the chimeric gene.
Identification of a Group 1-Like Capsular Polysaccharide Operon for
Vibrio vulnificus
Authors: Wright Anita C. , Powell Jan L. , Kaper James B. , Morris J. Glenn
Published: July 2002

Virulence of
Vibrio vulnificus
correlates with changes in colony morphology that are indicative of a reversible phase variation for expression of capsular polysaccharide (CPS). Encapsulated variants are virulent with opaque colonies, whereas phase variants with reduced CPS expression are attenuated and are translucent. Using Tn
phoA
mutagenesis, we identified a
V
.
vulnificus
CPS locus, which included an upstream
ops
element, a
wza
gene (
wza
Vv
), and several open reading frames with homology to CPS biosynthetic genes. This genetic organization is characteristic of group 1 CPS operons. The
wza
gene product is required for transport of CPS to the cell surface in
Escherichia coli
. Polar transposon mutations in
wza
Vv
eliminated expression of downstream biosynthetic genes, confirming operon structure. On the other hand, nonpolar inactivation of
wza
Vv
was specific for CPS transport, did not alter CPS biosynthesis, and could be complemented in
trans
. Southern analysis of CPS phase variants revealed deletions or rearrangements at this locus. A survey of environmental isolates indicated a correlation between deletions in
wza
Vv
and loss of virulent phenotype, suggesting a genetic mechanism for CPS phase variation. Full virulence in mice required surface expression of CPS and supported the essential role of capsule in the pathogenesis of
V
.
vulnificus
.
Reciprocal Protective Immunity againstBordetella pertussisandBordetella parapertussisin a Murine Model of Respiratory Infection
Authors: Watanabe Mineo , Nagai Masaaki
Published: July 2002

The protective immunity induced by infection withBordetella pertussisand withBordetella parapertussiswas examined in a murine model of respiratory infection. Convalescent mice that had been infected by aerosol withB. pertussisor withB. parapertussisexhibited a protective immune response againstB. pertussisand also againstB. parapertussis. Anti-filamentous hemagglutinin (anti-FHA) serum immunoglobulin G (IgG) and anti-FHA lung IgA antibodies were detected in both mice infected withB. pertussisand those infected withB. parapertussis. Antibodies against pertussis toxin (anti-PT) and against killedB. pertussiscells were detected in mice infected withB. pertussis. Pertactin-specific antibodies and antibodies against killedB. parapertussiscells were detected in mice infected withB. parapertussis. Spleen cells from mice infected withB. pertussissecreted interferon-γ (IFN-γ) in response to stimulation by FHA or PT. Spleen cells from mice infected withB. parapertussisalso secreted IFN-γ in response to FHA. Interleukin-4 was not produced in response to any of the antigens tested. The profiles of cytokine secretion in vitro revealed induction of a Th1-biased immune response during convalescence from infection byB. pertussisand byB. parapertussis. It is possible that Th1 and Th2 responses against FHA might be related to the reciprocal protection achieved in our murine model.
Exaggerated Proinflammatory and Th1 Responses in the Absence of γ/δ T Cells after Infection withListeria monocytogenes
Authors: Skeen Marianne J. , Rix Emily P. , Freeman Molly M. , Ziegler H. Kirk
Published: July 2002

While γ/δ T cells are involved in host defense and immunopathology in a variety of infectious diseases, their precise role is not yet clearly defined. In the absence of γ/δ T cells, mice die after infection with a dose ofListeriamonocytogenesthat is not lethal in immunologically intact animals. Morbidity might result from insufficient levels of cytokines normally produced by γ/δ T cells or conversely from an excess of cytokines due to a lack of down-regulation of the inflammatory response in the absence of γ/δ T cells. Consistent with a regulatory role, we found that systemic levels of proinflammatory cytokines (interleukin-6 [IL-6], IL-12, and gamma interferon [IFN-γ]) were significantly higher in the absence of γ/δ T cells during the innate phase of the response. Using combinations of genetically altered and immunodepleted mice, we found evidence for γ/δ T-cell-mediated regulation of IFN-γ production by multiple cell types of both lymphoid and myeloid lineages. The antigen-specific α/β T-cell response that followed the exaggerated innate response was also increased in γ/δ T-cell-deficient mice. These findings are consistent with an emerging picture from a variety of immune response models of a critical role for γ/δ T cells in down-modulation of the immune response.
Interleukin-12- and Gamma Interferon-Dependent Protection against Malaria Conferred by CpG Oligodeoxynucleotide in Mice
Authors: Gramzinski Robert A. , Doolan Denise L. , Sedegah Martha , Davis Heather L. , Krieg Arthur M. , Hoffman Stephen L.
Published: July 2002

Unmethylated CpG dinucleotides in bacterial DNA or synthetic oligodeoxynucleotides (ODNs) cause B-cell proliferation and immunoglobulin secretion, monocyte cytokine secretion, and activation of natural killer (NK) cell lytic activity and gamma interferon (IFN-γ) secretion in vivo and in vitro. The potent Th1-like immune activation by CpG ODNs suggests a possible utility for enhancing innate immunity against infectious pathogens. We therefore investigated whether the innate immune response could protect against malaria. Treatment of mice with CpG ODN 1826 (TCCATGACGTTCCTGACGTT, with the CpG dinucleotides underlined) or 1585 (ggGGTCAACGTTGAgggggG, with g representing diester linkages and phosphorothioate linkages being to the right of lowercase letters) in the absence of antigen 1 to 2 days prior to challenge withPlasmodium yoeliisporozoites conferred sterile protection against infection. A higher level of protection was consistently induced by CpG ODN 1826 compared with CpG ODN 1585. The protective effects of both CpG ODNs were dependent on interleukin-12, as well as IFN-γ. Moreover, CD8+T cells (but not CD4+T cells), NK cells, and nitric oxide were implicated in the CpG ODN 1585-induced protection. These data establish that the protective mechanism induced by administration of CpG ODN 1585 in the absence of parasite antigen is similar in nature to the mechanism induced by immunization with radiation-attenuatedP. yoeliisporozoites or with plasmid DNA encoding preerythrocytic-stageP. yoeliiantigens. We were unable to confirm whether CD8+T cells, NK cells, or nitric oxide were required for the CpG ODN 1826-induced protection, but this may reflect differences in the potency of the ODNs rather than a real difference in the mechanism of action of the two ODNs. This is the first report that stimulation of the innate immune system by CpG immunostimulatory motifs can confer sterile protection against malaria.
Experimental Model of Human Body Louse Infection Using Green Fluorescent Protein-Expressing
Bartonella quintana
Authors: Fournier Pierre-Edouard , Minnick Michael F. , Lepidi Hubert , Salvo Eric , Raoult Didier
Published: July 2002

A laboratory colony of human body lice was experimentally infected by feeding on rabbits made artificially bacteremic with a green fluorescent protein-expressing
Bartonella quintana. B. quintana
was detected in the gut and feces until death but not in the eggs. The life span of the lice was not modified. The rabbit model should provide valuable clues to the role of lice in the transmission of
B. quintana
.
Antibody against Surface-Bound C5a Peptidase Is Opsonic and Initiates Macrophage Killing of Group B Streptococci
Authors: Cheng Qi , Carlson Brian , Pillai Sub , Eby Ron , Edwards Lorri , Olmsted Stephen B. , Cleary Patrick
Published: July 2002

The capsular polysaccharides of group B streptococci (GBS) are a primary focus of vaccine development. Immunogenicity and long-lasting protection are best achieved by conjugating polysaccharides to a T-cell-dependent protein antigen. Streptococcal C5a peptidase (SCPB) is a conserved surface protein that is expressed by all streptococcal serotypes tested to date, and it is a possible carrier protein that could itself induce a protective immune response. Clearance of GBS from lungs, mucosal surfaces, or blood probably depends on the opsonophagocytic response of tissue-specific macrophages and polymorphonuclear leukocytes (PMNs). In this study, we examined the potential of antibody directed against SCPB from a serotype II strain to enhance the capacity of mouse bone marrow macrophages (from primary cultures) and human PMNs in whole blood to kill GBS in vitro. Our experiments demonstrated thatStreptococcusserotypes Ia, Ib, II, III, and V, preopsonized with anti-SCPB antibody, were killed more rapidly by cultured macrophages and PMNs in whole blood than were nonopsonized GBS. The increased rate of killing was accompanied by an increased macrophage oxidative burst. Furthermore, opsonization was serotype transparent. Immunization with SCPB conjugated to capsular polysaccharide type III produced polysaccharide-specific antibodies. It is interesting that this antiserum promoted serotype-independent killing of streptococci. These data support the use of SCPB in a GBS polysaccharide conjugate vaccine. SCPB not only enhanced the immunogenicity of polysaccharide components of the vaccine, but it might also induce additional serotype-independent protective antibodies.
Stimulation of Dendritic Cells via CD40 Enhances Immune Responses toMycobacterium tuberculosisInfection
Authors: Demangel Caroline , Palendira Umaimainthan , Feng Carl G. , Heath Andrew W. , Bean Andrew G. D. , Britton Warwick J.
Published: July 2002

The resolution of pulmonary tuberculosis (TB) critically depends on the development of the Th1 type of immune responses, as exemplified by the exacerbation of TB in IL-12-deficient mice. Therefore, vaccination strategies optimizing IL-12 production by antigen-presenting cells (APC) in response to mycobacteria may have enhanced protective efficacy. Since dendritic cells (DC) are the critical APC for activation of CD4+and CD8+T cells, we examined whether stimulation ofMycobacterium bovisbacillus Calmette Guérin (BCG)-infected DC via CD40 increased their ability to generate Th1-oriented cellular immune responses. Incubation of DC with an agonistic anti-CD40 antibody activated CD40 signaling in DC, as shown by increased expression of major histocompatibility complex class II and costimulatory molecules, mRNA production for proinflammatory cytokines and interleukin 12 (IL-12) p40. This activation pattern was maintained when DC were stimulated with anti-CD40 antibody and infected with BCG. Importantly, CD40-stimulated BCG-infected DC displayed increased capacity to release bioactive IL-12 and to activate gamma interferon (IFN-γ) producing T cells in vitro. Moreover, when C57BL/6 mice were immunized with these DC and challenged with aerosolMycobacterium tuberculosis, increased levels of mRNA for IL-12 p40, IL-18, and IFN-γ were present in the draining mediastinal lymph nodes. However, the mycobacterial burden in the lungs was not reduced compared to that in mice immunized with BCG-infected non-CD40-stimulated DC. Therefore, although the manipulation of DC via CD40 is effective for enhancing immune responses to mycobacteria in vivo, additional strategies are required to increase protection against virulentM. tuberculosisinfection.
Whole-Cell but Not Acellular Pertussis Vaccines Induce Convulsive Activity in Mice: Evidence of a Role for Toxin-Induced Interleukin-1β in a New Murine Model for Analysis of Neuronal Side Effects of Vaccination
Authors: Donnelly Sheila , Loscher Christine E. , Lynch Marina A. , Mills Kingston H. G.
Published: July 2002

Immunization with the whole-cell pertussis vaccine (Pw), while effective at preventing whooping cough in infants, has been associated with local, systemic, and neuronal reactions, including fevers and convulsions in children. In contrast, the new acellular pertussis vaccines (Pa) have a considerably improved safety profile. The lack of an appropriate animal model has restricted investigations into the mechanisms by which neurological reactions are induced by vaccination. Here we describe a novel murine model wherein seizure-like behavioral changes are induced following parenteral administration of Pw. The proinflammatory cytokine interleukin-β (IL-1β), production of which has been associated with many neurodegenerative conditions, was significantly increased in the hippocampus and hypothalamus of vaccinated animals. Accompanying this change was a decrease in release of the inhibitory neurotransmitters γ-aminobutyric acid and adenosine in the hippocampus. Seizure-like behavioral changes were significantly reduced following inhibition of IL-1β production by the administration of an inhibitor of IL-1β-converting enzyme and were almost completely abrogated in IL-1 receptor type I knockout mice. These results suggest a causal relationship between IL-1β induction and convulsive behavior following Pw vaccination. Significantly, Pa neither increased IL-1β nor induced behavioral changes in mice, but did induce the anti-inflammatory cytokine IL-10. In contrast, administration of active pertussis toxin and lipopolysaccharide, residual in Pw but absent from Pa, also induced convulsive activity. Our findings provide the first direct evidence of an immunological basis for pertussis vaccine reactogenicity and suggest that active bacterial toxins are responsible for the neurologic disturbances observed in children immunized with Pw.
Cloning, Expression, and Characterization of a Neuraminidase Gene fromArcanobacterium pyogenes
Authors: Jost B. H. , Songer J. G. , Billington S. J.
Published: July 2002

Arcanobacterium pyogenesis an opportunistic pathogen, associated with suppurative infections in domestic animals. In addition to pyolysin, a pore-forming, cholesterol-binding toxin,A. pyogenesexpresses a number of putative virulence factors, including several proteases and neuraminidase activity. A 3,009-bp gene,nanH, was cloned and sequenced and conferred neuraminidase activity on anEscherichia colihost strain. The predicted 107-kDa NanH protein displayed similarity to a number of bacterial neuraminidases and contained the RIP/RLP motif and five copies of the Asp box motif found in all bacterial neuraminidases. Recombinant His-tagged NanH was found to have pH and temperature optima of 5.5 to 6.0 and 55°C, respectively. Insertional deletion of thenanHgene resulted in the reduction, but not absence, of neuraminidase activity, indicating the presence of a second neuraminidase gene inA. pyogenes. NanH was localized to theA. pyogenescell wall.A. pyogenesadhered to HeLa, CHO, and MDBK cells in a washing-resistant manner. However, thenanHmutant was not defective for adherence to epithelial cells. The role of NanH in host epithelial cell adherence may be masked by the presence of a second neuraminidase inA. pyogenes.
Expression ofAnaplasma marginaleMajor Surface Protein 2 Variants in Persistently Infected Ticks
Authors: de la Fuente José , Kocan Katherine M.
Published: July 2002

Anaplasma marginale, an intraerythrocytic ehrlichial pathogen of cattle, establishes persistent infections in both vertebrate (cattle) and invertebrate (tick) hosts. The ability ofA. marginaleto persist in cattle has been shown to be due, in part, to major surface protein 2 (MSP2) variants which are hypothesized to emerge in response to the bovine immune response. MSP2 antigenic variation has not been studied in persistently infected ticks. In this study we analyzed MSP2 inA. marginalepopulations from the salivary glands of maleDermacentor variabilispersistently infected withA. marginaleafter feeding successively on one susceptible bovine and three sheep. New MSP2 variants appeared in eachA. marginalepopulation, and sequence alignment of the MSP2 variants revealed multiple amino acid substitutions, insertions, and deletions. These results suggest that selection pressure on MSP2 occurred in tick salivary glands independent of the bovine immune response.
Evidence for the Emergence of Non-O1 and Non-O139
Vibrio cholerae
Strains with Pathogenic Potential by Exchange of O-Antigen Biosynthesis Regions
Authors: Li Manrong , Shimada Toshio , Morris J. Glenn , Sulakvelidze Alexander , Sozhamannan Shanmuga
Published: July 2002

The novel epidemic strain
Vibrio cholerae
O139 Bengal originated from a seventh-pandemic O1 El Tor strain by antigenic shift resulting from homologous recombination-mediated exchange of O-antigen biosynthesis (
wb*
) clusters. Conservation of the genetic organization of
wb*
regions seen in other serogroups raised the possibility of the existence of pathogenic non-O1 and non-O139
V. cholerae
strains that emerged by similar events. To test this hypothesis, 300
V. cholerae
isolates of non-O1 and non-O139 serogroups were screened for the presence of virulence genes and an epidemic genetic background by DNA dot blotting, IS
1004
fingerprinting, and restriction fragment length polymorphism (RFLP) analysis. We found four non-O1 strains (serogroups O27, O37, O53, and O65) with an O1 genetic backbone suggesting exchange of
wb*
clusters. DNA sequence analysis of the O37
wb*
region revealed that a novel ∼23.4-kb gene cluster had replaced all but the ∼4.2-kb right junction of the 22-kb O1
wbe
region. In sharp contrast to the backbones, the virulence regions of the four strains were quite heterogeneous; the O53 and O65 strains had the El Tor vibrio pathogenicity island (VPI) cluster, the O37 strain had the classical VPI cluster, and the O27 strain had a novel VPI cluster. Two of the four strains carried CTXφ; the O27 strain possessed a CTXφ with a recently reported immune specificity (
rstR-4
** allele) and a novel
ctxB
allele, and the O37 strain had an El Tor CTXφ (

rstR
ET

allele) and novel
ctxAB
alleles. Although the O53 and O65 strains lacked the
ctxAB
genes, they carried a pre-CTXφ (i.e.,

rstR
cla

). Identification of non-O1 and non-O139 serogroups with pathogenic potential in epidemic genetic backgrounds means that attention should be paid to possible future epidemics caused by these serogroups and to the need for new, rapid vaccine development strategies.
A Lipid Core Peptide Construct Containing a Conserved Region Determinant of the Group A Streptococcal M Protein Elicits Heterologous Opsonic Antibodies
Authors: Olive Colleen , Batzloff Michael R. , Horváth Anikó , Wong Allan , Clair Timothy , Yarwood Penny , Toth Istvan , Good Michael F.
Published: July 2002

The study reported here investigated the immunogenicity and protective potential of a lipid core peptide (LCP) construct containing a conserved region determinant of M protein, defined as peptide J8. Parenteral immunization of mice with LCP-J8 led to the induction of high-titer serum immunoglobulin G J8-specific antibodies when the construct was coadministered with complete Freund's adjuvant (CFA) or administered alone. LCP-J8 in CFA had significantly enhanced immunogenicity compared with the monomeric peptide J8 given in CFA. Moreover, LCP-J8/CFA and LCP-J8 antisera opsonized four different group A streptococcal (GAS) strains, and the antisera did not cross-react with human heart tissue proteins. These data indicate the potential of an LCP-based M protein conserved region GAS vaccine in the induction of broadly protective immune responses in the absence of a conventional adjuvant.
Identification of Novel Adhesins from Group B Streptococci by Use of Phage Display Reveals that C5a Peptidase Mediates Fibronectin Binding
Authors: Beckmann Christiane , Waggoner Joshua D. , Harris Theresa O. , Tamura Glen S. , Rubens Craig E.
Published: July 2002

Group B streptococci (GBS) are a major cause of pneumonia, sepsis, and meningitis in newborns and infants. GBS initiate infection of the lung by colonizing mucosal surfaces of the respiratory tract; adherence of the bacteria to host cells is presumed to be the initial step in and prerequisite for successful colonization (G. S. Tamura, J. M. Kuypers, S. Smith, H. Raff, and C. E. Rubens, Infect. Immun. 62:2450-2458, 1994). We have performed a genome-wide screen to identify novel genes of GBS that mediate adherence to fibronectin. A shotgun phage display library was constructed from chromosomal DNA of a serotype Ia GBS strain and affinity selected on immobilized fibronectin. DNA sequence analysis of different clones identified 19 genes with homology to known bacterial adhesin genes, virulence genes, genes involved in transport or metabolic processes, and genes with yet-unknown function. One of the isolated phagemid clones showed significant homology to the gene (scpB) for the GBS C5a peptidase, a surface-associated serine protease that specifically cleaves the complement component C5a, a chemotaxin for polymorphonuclear leukocytes. In this work we have demonstrated that affinity-purified recombinant ScpB and a peptide ScpB fragment (ScpB-PDF), similar to the peptide identified in the phagemid, bound fibronectin in a concentration-dependent manner. Adherence assays to fibronectin were performed, comparing an isogenicscpBmutant to the wild-type strain. Approximately 50% less binding was observed with the mutant than with the wild-type strain. The mutant phenotype could be fully restored by intranscomplementation of the mutant with the cloned wild-typescpBgene, providing further evidence for the role of ScpB in fibronectin adherence. Our results suggest that C5a peptidase is a bifunctional protein, which enzymatically cleaves C5a and mediates adherence to fibronectin. Since binding of fibronectin has been implicated in attachment and invasion of eukaryotic cells by streptococci, our results may imply a second important role for this surface protein in the pathogenesis of GBS infections.
Dissemination of the Phage-Associated Novel Superantigen Gene
speL
in Recent Invasive and Noninvasive
Streptococcus pyogenes
M3/T3 Isolates in Japan
Authors: Ikebe Tadayoshi , Wada Akihito , Inagaki Yoshishige , Sugama Kumiko , Suzuki Rieko , Tanaka Daisuke , Tamaru Aki , Fujinaga Yoshihiro , Abe Yoshiaki , Shimizu Yoshikata , Watanabe Haruo ,   the Working Group for Group A Streptococci in Japan
Published: July 2002

In Japan, more than 10% of streptococcal toxic shock-like syndrome (TSLS) cases have been caused by
Streptococcus pyogenes
M3/T3 isolates since the first reported TSLS case in 1992. Most M3/T3 isolates from TSLS or severe invasive infection cases during 1992 to 2001 and those from noninvasive cases during this period are indistinguishable in pulsed-field gel electropherograms. The longest fragments of these recent isolates were 300 kb in size, whereas those of isolates recovered during or before 1973 were 260 kb in size. These 260- and 300-kb fragments hybridized to each other, suggesting the acquisition of an about 40-kb fragment by the recent isolates. The whole part of the acquired fragment was cloned from the first Japanese TSLS isolate, NIH1, and its nucleotide sequence was determined. The 41,796-bp fragment is temperate phage φNIH1.1, containing a new superantigen gene
speL
near its right attachment site. The C-terminal part of the deduced amino acid sequence of
speL
has 48 and 46% similarity with well-characterized erythrogenic toxin SpeC and the most potent superantigen, SmeZ-2, respectively. None of 10 T3 isolates recovered during or before 1973 has
speL
, whereas all of 18 M3/T3 isolates recovered during or after 1992 and, surprisingly,
Streptococcus equi
subsp.
equi
ATCC 9527 do have this gene. Though plaques could not be obtained from φNIH1.1, its DNA became detectable from the phage particle fraction upon mitomycin C induction, showing that this phage is not defective. A horizontal transfer of the phage carrying
speL
may explain the observed change in M3/T3
S. pyogenes
isolates in Japan.
Overproduction of Type 8 Capsular Polysaccharide AugmentsStaphylococcus aureusVirulence
Authors: Luong Thanh T. , Lee Chia Y.
Published: July 2002

Type 8 capsular polysaccharide (CP8) is the most prevalent capsule type in clinical isolates ofStaphylococcus aureus. However, its role in virulence has not been clearly defined. CP8 strains such as strain Becker produce a small amount of capsule on their surface in vitro. In contrast, CP1 strains such as strain M produce a large amount of capsule, which has been shown to be an important antiphagocytic virulence factor. Thecap8andcap1operons, required for the synthesis of CP8 and CP1, respectively, have been cloned and sequenced. To test whether CP8 contributes to the pathogenesis ofS. aureus, we replaced the weak native promoter of thecap8operon in strain Becker with the strong constitutive promoter of thecap1operon of strain M. The resultant strain, CYL770, synthesizedcap8-specific mRNA at a level about sevenfold higher than that in the parent strain. Remarkably, the CYL770 strain produced about 80-fold more CP8. In a mouse infection model of bacteremia, the CP8-overproducing strain persisted longer in the bloodstream, the liver, and the spleen in mice than the parent strain. In addition, strain CYL770 was more resistant to ospsonophagocytosis in vitro by human polymorphonuclear leukocytes. These results indicate that CP8 is an antiphagocytic virulence factor ofS. aureus.
Contribution of Interleukin-11 and Prostaglandin(s) in Lipopolysaccharide-Induced Bone Resorption In Vivo
Authors: Li Li , Khansari Alireza , Shapira Lior , Graves Dana T. , Amar Salomon
Published: July 2002

We previously demonstrated that interleukin-1 (IL-1) and tumor necrosis factor (TNF) activities only partially account for calvarial bone resorption induced by local application of lipopolysaccharide (LPS) in mice. The present study was undertaken to determine the role and relative contribution of IL-11 and prostaglandin(s) (PG[s]) in LPS-induced bone resorption in vivo. A one-time dose of LPS was injected into the subcutaneous tissue overlying calvaria of mice lacking IL-1 receptor type I (IL-1RI−/−), mice lacking TNF receptor p55 and IL-1RI (TNFRp55−/−-IL-1RI−/−), and wild-type mice. Mice were then treated with injections of anti-IL-11 monoclonal antibody (MAb), indomethacin, or phosphate-buffered saline (PBS) and sacrificed 5 days later. Histological sections stained for tartrate-resistant acid phosphatase (TRAP) were quantified by histomorphometric analysis. At low doses of LPS (100 μg/mouse), the percentages of bone surface covered by osteoclasts were found to be similar in three strains of mice. The increase was reduced by 37% with anti-IL-11 MAb and by 46% with indomethacin. At higher doses of LPS (500 μg/mouse), we found an eightfold increase in these percentages in wild-type mice and a fivefold increase in these percentages in IL-1RI−/−and TNFRp55−/−−IL-1RI−/−mice after normalizing with the value from the saline-PBS control group in the same strain of mice. The increase was reduced by 55 and 69% in wild-type mice and by 50 and 57% in IL-1RI−/−and TNFRp55−/−−IL-1RI−/−mice treated with anti-IL-11 MAb or indomethacin, respectively. Our findings suggest that in vivo, at low doses of LPS (100 μg/mouse), LPS-induced bone resorption is mediated by IL-11 and PGs, while at high doses of LPS (500 μg/mouse), it is mediated by IL-11, PGs, IL-1, and TNF signaling. IL-11 and PGs mediate LPS-induced bone resorption by enhancing osteoclastogenesis independently of the IL-1 or TNF signaling.
Characterization of a
Sinorhizobium meliloti
ATP-Binding Cassette Histidine Transporter Also Involved in Betaine and Proline Uptake
Authors: Boncompagni Eric , Dupont Laurence , Mignot Tam , Østeräs Magne , Lambert Annie , Poggi Marie-Christine , Le Rudulier Daniel
Published: July 2002

The symbiotic soil bacterium
Sinorhizobium meliloti
uses the compatible solutes glycine betaine and proline betaine for both protection against osmotic stress and, at low osmolarities, as an energy source. A PCR strategy based on conserved domains in components of the glycine betaine uptake systems from
Escherichia coli
(ProU) and
Bacillus subtilis
(OpuA and OpuC) allowed us to identify a highly homologous ATP-binding cassette (ABC) binding protein-dependent transporter in
S. meliloti
. This system was encoded by three genes (
hutXWV
) of an operon which also contained a fourth gene (
hutH2
) encoding a putative histidase, which is an enzyme involved in the first step of histidine catabolism. Site-directed mutagenesis of the gene encoding the periplasmic binding protein (
hutX
) and of the gene encoding the cytoplasmic ATPase (
hutV
) was done to study the substrate specificity of this transporter and its contribution in betaine uptake. These mutants showed a 50% reduction in high-affinity uptake of histidine, proline, and proline betaine and about a 30% reduction in low-affinity glycine betaine transport. When histidine was used as a nitrogen source, a 30% inhibition of growth was observed in
hut
mutants (
hutX
and
hutH2
). Expression analysis of the
hut
operon determined using a
hutX-lacZ
fusion revealed induction by histidine, but not by salt stress, suggesting this uptake system has a catabolic role rather than being involved in osmoprotection. To our knowledge, Hut is the first characterized histidine ABC transporter also involved in proline and betaine uptake.
Isolation and Characterization of Canthaxanthin Biosynthesis Genes from the Photosynthetic Bacterium
Bradyrhizobium
sp. Strain ORS278
Authors: Hannibal Laure , Lorquin Jean , D'Ortoli Nicolas Angles , Garcia Nelly , Chaintreuil Clemence , Masson-Boivin Catherine , Dreyfus Bernard , Giraud Eric
Published: July 2002

A carotenoid biosynthesis gene cluster involved in canthaxanthin production was isolated from the photosynthetic
Bradyrhizobium
sp. strain ORS278. This cluster includes five genes identified as
crtE
,
crtY
,
crtI
,
crtB
, and
crtW
that are organized in at least two operons. The functional assignment of each open reading frame was confirmed by complementation studies.