bluuebunny/crossref_metadata_2025 huggingface dataset:
Induction by a Lactic Acid Bacterium of a Population of CD4+T Cells with Low Proliferative Capacity That Produce Transforming Growth Factor β and Interleukin-10
Authors: von der Weid Thierry , Bulliard Christine , Schiffrin Eduardo J.
Published: July 2002

We investigated whether certain strains of lactic acid bacteria (LAB) could antagonize specific T-helper functions in vitro and thus have the potential to prevent inflammatory intestinal immunopathologies. All strains tested induced various levels of both interleukin-12 (IL-12) and IL-10 in murine splenocytes. In particular,Lactobacillus paracasei(strain NCC2461) induced the highest levels of these cytokines. Since IL-12 and IL-10 have the potential to induce and suppress Th1 functions, respectively, we addressed the impact of this bacterium on the outcome of CD4+T-cell differentiation. For this purpose, bacteria were added to mixed lymphocyte cultures where CD4+T-cells from naive BALB/c mice were stimulated weekly in the presence of irradiated allogeneic splenocytes. In these cultures,L. paracaseiNCC2461 strongly inhibited the proliferative activity of CD4+T cells in a dose-dependent fashion. This was accompanied by a marked decrease of both Th1 and Th2 effector cytokines, including gamma interferon, IL-4, and IL-5. In contrast, IL-10 was maintained and transforming growth factor β (TGF-β) was markedly induced in a dose-dependent manner. The bacteria were not cytotoxic, because cell viability was not affected after two rounds of stimulation. Thus, unidentified bacterial components fromL. paracaseiNCC2461 induced the development of a population of CD4+T cells with low proliferative capacity that produced TGF-β and IL-10, reminiscent of previously described subsets of regulatory cells implicated in oral tolerance and gut homeostasis.
Comparison of Serum and Cell-Specific Cytokines in Humans
Authors: Jason Janine , Archibald Lennox K. , Nwanyanwu Okey C. , Byrd Martha G. , Kazembe Peter N. , Dobbie Hamish , Jarvis William R.
Published: July 2002

Cytokines function at the cellular, microenvironmental level, but human cytokine assessment is most commonly done at the macro level, by measuring serum cytokines. The relationships between serum and cellular cytokines, if there are any, are undefined. In a study of hospitalized patients in Malawi, we compared cytometrically assessed, cell-specific cytokine data to serum interleukin 2 (IL-2), IL-4, IL-6, IL-8, IL-10, gamma interferon (IFN-γ), and tumor necrosis factor alpha (TNF-α) levels in 16 children and 71 (IL-2, -4, -6, -10) or 159 (IL-8, IFN-γ, and TNF-α) adults, using Wilcoxon rank sum tests and Pearson's (rp) and Spearman's (rs) rank correlations. For the entire study group, correlations between identical serum and cellular cytokines mainly involved IL-8 and IFN-γ, were few, and were weakly positive (r< 0.40). Blood culture-positive persons had the most and strongest correlations, including those between serum IL-2 levels and the percentages of lymphocytes spontaneously making IL-2 (rs= +0.74), serum IL-8 levels and the percentages of lymphocytes spontaneously making IL-8 (rp= +0.66), and serum IL-10 levels and the percentages of CD8+T cells making TNF-α (rp= +0.89). Human immunodeficiency virus (HIV)-positive persons had the next largest number of correlations, including several serum IL-8 level correlations, correlation of serum IL-10 levels with the percentages of lymphocytes producing induced IL-10 (rs= +0.36), and correlation of serum IFN-γ levels and the percentages of lymphocytes spontaneously making both IL-6 and IFN-γ in the same cell (rp= +0.59). HIV-negative, malaria smear-positive, and pediatric patients had few significant correlations; for the second and third of these subgroups, serum IL-8 level was correlated with the percentage of CD8−T cells producing induced IL-8 (rs= +0.40 andrs= +0.56, respectively). Thus, the strength of associations between serum and cellular cytokines varied with the presence or absence of bloodstream infection, HIV status, and perhaps other factors we did not assess. These results strongly suggest that serum cytokines at best only weakly reflect peripheral blood cell cytokine production and balances.
Antibodies to 60-Kilodalton Heat Shock Protein and Outer Membrane Protein 2 of
Chlamydia pneumoniae
in Patients with Coronary Heart Disease
Authors: Ciervo Alessandra , Visca Paolo , Petrucca Andrea , Biasucci Luigi Maria , Maseri Attilio , Cassone Antonio
Published: July 2002

Evidence linking
Chlamydia pneumoniae
infection to atherosclerosis and to atherothrombotic events has recently emerged. A primary candidate implicated in these pathogenetic events is the 60-kDa chlamydial heat shock protein (HSP60). Another putative candidate to activate a potential proinflammatory mechanism is the chlamydial outer membrane protein 2 (OMP2). We have generated both HSP60 and OMP2 recombinant antigens in a nondenatured form and shown that (i) the two antigens were highly immunogenic in mice and (ii) murine antisera thus generated recognized the native
C. pneumoniae
proteins. We measured by enzyme linked immunosorbent assay (ELISA) and immunoblot assay antibody titers to the recombinant antigens in samples from 219 patients with coronary heart disease (CHD), 179 patients with unstable angina (UA), 40 patients with acute myocardial infarction (AMI), and 100 age-, sex-, and risk factor-matched healthy controls. We also examined whether anti-HSP60 and/or anti-OMP2 antibodies correlated with anti-
C. pneumoniae
antibodies assessed by a commercial microimmunofluorescence (MIF) assay. Immunoglobulin G (IgG), but neither IgA nor IgM, antibodies against the two recombinant proteins were detected by ELISA. In particular, anti-HSP60 antibodies were detected in >99% of CHD patients versus 0% of the controls, whereas the proportions of anti-OMP2 positive subjects were >70 and 27%, respectively. Nonetheless, among CHD patients, similar frequencies of positive subjects and titers of anti-HSP60 or anti-OMP2 antibodies were present in UA and AMI subjects. The anti-OMP2, but not the anti-HSP60, antibodies showed high specificity. Consistently, high serological correlation was observed between IgG MIF titers and IgG ELISA reactivity to OMP2 but not to HSP60. Overall, the results of this study demonstrate a strong correlation between CHD and anti-HSP60 IgG levels, as measured by our in-house ELISA. They also suggest that recombinant OMP2 ELISA, because of its high specificity and strong correlation with MIF assay, could be a candidate diagnostic marker for
C. pneumoniae
infection, which would be of potential usefulness for its specificity and nonsubjective nature.
Inducible Expression of
Enterococcus faecalis
Aggregation Substance Surface Protein Facilitates Bacterial Internalization by Cultured Enterocytes
Authors: Wells Carol L. , Moore Elizabeth A. , Hoag Julie A. , Hirt Helmut , Dunny Gary M. , Erlandsen Stanley L.
Published: July 2002

Aggregation substance (AS) is an
Enterococcus faecalis
surface protein that may contribute to virulence. Using a recently described system for controlled expression of AS in
E. faecalis
and the heterologous host
Lactococcus lactis
, experiments were designed to assess the effect of AS on bacterial internalization by HT-29 and Caco-2 enterocytes. AS expression was associated with increased internalization of
E. faecalis
by HT-29 enterocytes and of
L. lactis
by HT-29 and Caco-2 enterocytes. Compared to enterocytes cultivated under standard conditions, either cultivation in hypoxia or 1-h pretreatment of enterocytes with calcium-free medium resulted in increased internalization of both
E. faecalis
and
L. lactis
(with and without AS expression). Also, AS expression augmented these increases when
E. faecalis
was incubated with pretreated HT-29 enterocytes and when
L. lactis
was incubated with pretreated Caco-2 and HT-29 enterocytes. These data indicated that AS might facilitate
E. faecalis
internalization by cultured enterocytes.
IncreasedEscherichia coliPhagocytosis in Neutrophils That Have Transmigrated across a Cultured Intestinal Epithelium
Authors: Hofman Paul , Piche Marjorie , Far Dariush Farahi , Le Negrate Gaëlle , Selva Eric , Landraud Luce , Alliana-Schmid Annie , Boquet Patrice , Rossi Bernard
Published: July 2002

The functionality of polymorphonuclear leukocytes (PMNs) once they migrate into the digestive lumen is still ill defined. More specifically, phagocytic function and bactericidal action of PMNs after transepithelial migration have not received much attention. The aim of the present study is to compare PMN behavior before and after transepithelial migration, in particular (i) phagocytosis and bactericidal activity; (ii) expression of surface molecules, particularly those involved in phagocytosis; and (iii) apoptosis. Cultured human intestinal epithelial T84 cell monolayers were used. The effect of transepithelial migration on phagocytosis was evaluated by immunofluorescence and electron microscopy and by flow cytometric assessment of the engulfment of a strain ofEscherichia colitransfected with the green fluorescent protein. Superoxide production by PMNs was investigated by luminol-mediated chemiluminescence. Expression of various surface molecules on PMNs was evaluated by flow cytometry, while PMN apoptosis was assayed by morphologic changes and DNA fragmentation.E. coliphagocytosis by the PMNs was markedly increased after transepithelial migration without modification of superoxide production. CD11b/CD18 and CD47 expression was increased upon PMN transmigration, whereas CD16 expression was decreased and CD29, CD46, CD49e, CD49f, CD55, CD59, CD61, CD95 levels remained unchanged. Apoptosis in transmigrated PMNs was slightly advanced and was observed after 12 h compared to 16 h for nontransmigrated PMNs. In conclusion, the phagocytic capacity of the PMNs is augmented after transepithelial migration, with a dramatic increase in the level of CD11b/CD18 and preservation of the superoxide production. These results suggest a higher bactericidal activity of the PMNs once they have translocated into the digestive lumen.
Acquired, but Not Innate, Immune Responses toStreptococcus pneumoniaeAre Compromised by Neutralization of CD40L
Authors: Hwang Young-il , Nahm Moon H. , Briles David E. , Thomas David , Purkerson Jeffrey M.
Published: July 2002

Streptococcus pneumoniaeis a significant pathogen of young children and the elderly. Systemic infection by pneumococci is a complex process involving several bacterial and host factors. We have investigated the role of CD40L in host defense against pneumococcal infection. Treatment of mice with MR-1 antibody (anti-CD154/CD40L) markedly reduced antibody responses to the pneumococcal protein PspA, elicited by immunization of purified protein or whole bacteria. In mice immunized with whole bacteria, MR-1 treatment reduced antibody responses to capsular polysaccharides but not cell wall polysaccharides. MR-1 did not suppress antibody responses to isolated capsular polysaccharides but did reduce the production of antibody to a capsular polysaccharide-protein conjugate, indicating that when presented in the context of whole bacteria, the humoral response to capsular polysaccharides is partially T-cell dependent. Despite the reduction of the protective humoral responses to pneumococcal infection, administration of MR-1 had no effect on sepsis, lung infection, or nasal carriage in nonimmune mice inoculated with virulent pneumococci. Thus, short-term neutralization of CD40L does not compromise innate host defenses against pneumococcal invasion.
Adherence of Platelets toCandidaSpecies In Vivo
Authors: Robert R. , Nail S. , Marot-Leblond A. , Cottin J. , Miegeville M. , Quenouillere S. , Mahaza C. , Senet J. M.
Published: July 2002

The in vivo interactions of platelets withCandidaspecies yeast cells were investigated in a murine model. Mice were injected intravenously via the lateral caudal vein, and blood drawn by periorbital puncture was collected in phosphate-buffered saline–formaldehyde to avoid in vitro platelet activation. The study of the clearance of blastoconidia ofCandida albicansandCandida glabratashowed that these cells disappeared quickly from the bloodstream. Microscopic observation of blood samples, stained by Calcofluor white or May Grunwald Giemsa, demonstrated the rapid attachment of platelets to fungal elements of all theCandidaspp. tested. The attachment of murine platelets toC. albicanscells, observed by scanning electron microscopy, revealed morphological changes. The platelets lost their discoid shape, generated pseudopodia, and flattened against the yeast cells. The reversibility of platelet binding toC. albicansby chelating agents suggests a cation-dependent link. In contrast, the fixation ofC. glabrataandCandida tropicaliswas not modified by chelating agents. The mechanisms involved in the in vivo adherence of platelets toCandidacells may therefore differ according to the species ofCandida.
Mucosally Induced Immunoglobulin E-Associated Inflammation in the Respiratory Tract
Authors: Simecka Jerry W. , Jackson Raymond J. , Kiyono Hiroshi , McGhee Jerry R.
Published: July 2002

The purpose of the present study was to determine the immunologic responses, particularly immunopathologic reactions, associated with nasal immunization with the mucosal adjuvant, cholera toxin (CT). BALB/c mice were nasally immunized with tetanus toxoid (TT) combined with CT, and the responses of these mice were determined. After nasal immunization, mice produce a serum antibody response, primarily of the immunoglobulin G (IgG) isotype of predominantly IgG1 subclass, against both TT and CT. Along with the antibody responses, we also found that inflammatory reactions, which could be potentially fatal, developed within the lung. Furthermore, IgE responses were also induced after nasal immunization, and these responses were associated with the detection of interleukin 5 in the serum. Thus, nasal immunization with TT plus CT likely results in the activation of Th2 cells, which may contribute to serious immunopathologic reactions in the lung.
Phase 2 Clinical Trial of AttenuatedSalmonella entericaSerovar Typhi Oral Live Vector Vaccine CVD 908-htrAin U.S. Volunteers
Authors: Tacket Carol O. , Sztein Marcelo B. , Wasserman Steven S. , Losonsky Genevieve , Kotloff Karen L. , Wyant Timothy L. , Nataro James P. , Edelman Robert , Perry Judith , Bedford Philip , Brown David , Chatfield Stephen , Dougan Gordon , Levine Myron M.
Published: July 2002

Salmonella entericaserovar Typhi strain CVD 908-htrAis a live attenuated strain which may be useful as an improved oral typhoid vaccine and as a vector for cloned genes of other pathogens. We conducted a phase 2 trial in which 80 healthy adults received one of two dosage levels of CVD 908-htrAin a double-blind, placebo-controlled, crossover study. There were no differences in the rates of side effects among volunteers who received high-dose vaccine (4.5 × 108CFU), lower-dose vaccine (5 × 107CFU), or placebo in the 21 days after vaccination, although recipients of high-dose vaccine (8%) had more frequent diarrhea than placebo recipients (0%) in the first 7 days. Seventy-seven percent and 46% of recipients of high- and lower-dose vaccines, respectively, briefly excreted vaccine organisms in their stools. All blood cultures were negative. Antibody-secreting cells producing antilipopolysaccharide (LPS) immunoglobulin A (IgA) were detected in 100 and 92% of recipients of high- and lower-dose vaccines, respectively. Almost half the volunteers developed serum anti-LPS IgG. Lymphocyte proliferation and gamma interferon production against serovar Typhi antigens occurred in a significant proportion of vaccinees. This phase 2 study supports the further development of CVD 908-htrAas a single-dose vaccine against typhoid fever and as a possible live vector for oral delivery of other vaccine antigens.
Role of Decay-Accelerating Factor Domains and Anchorage in Internalization of Dr-FimbriatedEscherichia coli
Authors: Selvarangan R. , Goluszko P. , Popov V. , Singhal J. , Pham T. , Lublin D. M. , Nowicki S. , Nowicki B.
Published: July 2002

Dr-fimbriatedEscherichia colicapable of invading epithelial cells recognizes human decay-accelerating factor (DAF) as its cellular receptor. The role of extracellular domains and the glycosylphosphatidylinositol anchor of DAF in the process of internalization of Dr+E. coliwas characterized in a cell-cell interaction model. Binding of Dr+E. colito the short consensus repeat 3 domain of DAF expressed by Chinese hamster ovary cells was critical for internalization to occur. Deletion of short consensus repeat 3 domain or replacement of Ser165by Leu in this domain, or the use of a monoclonal antibody to this region abolished internalization. Replacing the glycosylphosphatidylinositol anchor of DAF with the transmembrane anchor of membrane cofactor protein or HLA-B44 resulted in abolition or reduction of internalization respectively. Cells expressing glycosylphosphatidylinositol-anchored DAF but not the transmembrane-anchored DAF internalized Dr+E. colithrough a glycolipid pathway, since the former cells were more sensitive to inhibition by methyl-β-cyclodextrin, a sterol-chelating agent. Electron microscopic studies revealed that the intracellular vacuoles containing the internalized Dr+E. coliwere morphologically distinct between the anchor variants of DAF. The cells expressing glycosylphosphatidylinositol-anchored DAF contained a single bacterium in tight-fitting vacuoles, while the cells expressing transmembrane-anchored DAF contained multiple (two or three) bacteria in spacious phagosomes. This finding suggests that distinct postendocytic events operate in the cells expressing anchor variants of DAF. We provide direct evidence for the DAF-mediated internalization of Dr+E. coliand demonstrate the significance of the glycosylphosphatidylinositol anchor, which determines the ability and efficiency of the internalization event.
Functional Flexibility of the FimH Adhesin: Insights from a Random Mutant Library
Authors: Schembri Mark A. , Sokurenko Evgeni V. , Klemm Per
Published: July 2002

Type 1 fimbriae are surface organelles of
Escherichia coli
which mediate
d
-mannose-sensitive binding to different host surfaces. This binding is conferred by the minor fimbrial component FimH. Naturally occurring variants of the FimH protein have been selected in nature for their ability to recognize specific receptor targets. In particular, variants that bind strongly to terminally exposed monomannose residues have been associated with a pathogenicity-adaptive phenotype that enhances
E. coli
colonization of extraintestinal locations such as the urinary bladder. In this study we have used random mutagenesis to specifically identify nonselective mutations in the FimH adhesin which modify its binding phenotype. Isogenic
E. coli
clones expressing FimH variants were tested for their ability to bind yeast cells and model glycoproteins that contain oligosaccharide moieties rich in either terminal monomannose, oligomannose, or nonmannose residues. Both the monomannose- and the oligomannose-binding capacity of type 1 fimbriae could be altered by minor amino acid changes in the FimH protein. The monomannose-binding phenotype was particularly sensitive to changes, with extensive differences in binding being observed in comparison to wild-type FimH levels. Different structural alterations were able to cause similar functional changes in FimH, suggesting a high degree of flexibility to target recognition by this adhesin. Alteration of residue P49 of the mature FimH protein, which occurs within the recently elucidated carbohydrate-binding pocket of FimH, completely abolished its function. Amino acid changes that increased the binding capacity of FimH were located outside receptor-interacting residues, indicating that functional changes relevant to pathogenicity are likely to be due to conformational changes of the adhesin.
Characterization of Heat, Oxidative, and Acid Stress Responses in
Brucella melitensis
Authors: Teixeira-Gomes Ana P. , Cloeckaert Axel , Zygmunt Michel S.
Published: July 2002

Brucella melitensis
is a facultative intracellular pathogen which is able to survive and replicate within phagocytic cells. Therefore, it has to adapt to a range of different hostile environments. In order to understand the mechanisms of intracellular survival employed by virulent
B. melitensis
16M, an initial approach consisting of analysis of the differences in patterns of protein synthesis in response to heat, oxidative, and acid pH stresses by two-dimensional (2-D) polyacrylamide gel electrophoresis was used. Depending on the stress, this involved about 6.4 to 12% of the 676 protein spots detected in 2-D gel electrophoresis. On the basis of N-terminal sequence analysis and database searching, 19 proteins whose level of synthesis was up- or down-regulated by stress conditions were identified. Some of them were previously reported for
Brucella
, such as BvrR, DnaK, GroEL, and Cu-Zn superoxide dismutase (SOD). Eight other proteins closely matched proteins found in other bacteria: AapJ, alpha-ETF, ClpP, Fe and/or Mn SOD, malate dehydrogenase, IalB, 30S ribosomal protein S1, and pyruvate dehydrogenase E1 component beta subunit. Results indicated that
B. melitensis
could bring specific regulatory mechanisms into play in response to stress conditions. For example, the ribosome releasing factor in
B. melitensis
appeared to be a heat shock protein, whereas the ClpP protein, described as a heat shock protein for
Escherichia coli
, was strongly down-regulated in
B. melitensis
in response to heat stress. Some of the identified proteins and their potential specific regulation could be required for the adaptation of
B. melitensis
to environmental stresses encountered in phagocytic cells and possibly for bacterial virulence.
Characterization of Ferrochelatase (
hemH
) Mutations in
Haemophilus influenzae
Authors: Schlör Stefan , Herbert Mark , Rodenburg Michaela , Blass Julia , Reidl Joachim
Published: July 2002

Haemophilus influenzae
lacks most of the biosynthetic enzymes for hemin synthesis. However, the organism has retained ferrochelatase activity, which we identified to be encoded by a
hemH
-homologous gene. In this report we characterize the growth physiology conferred by
hemH
mutations under infection and laboratory conditions.
Infection of Macrophage-Like THP-1 Cells withMycobacterium aviumResults in a Decrease in Their Ability to Phosphorylate Nucleolin
Authors: Garcia Rodolfo C. , Banfi Elena , Pittis Maria G.
Published: July 2002

This study of the phosphorylation ability of macrophage-like cells upon infection withMycobacterium aviumwas undertaken to establish potential targets of the interference with host response mechanisms. Cytosolic and membrane fractions from noninfected and infected cells were incubated with [γ-32P]ATP, in the presence of Mg2+and the absence of Ca2+, and the patterns of phosphoproteins synthesized were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Lower levels of a 110-kDa phosphoprotein were observed in association with cytosolic fractions from mycobacterium-infected cells compared to noninfected cells or cells treated with lipopolysaccharide or having ingestedEscherichia colior killedM. avium. The 110-kDa phosphoprotein was present in the soluble fraction (230,000 ×gsupernatant) after the kinase incubation, from where it was partially purified and identified as phosphonucleolin by amino acid sequencing. The decrease in nucleolin phosphorylation observed was not related to changes in the cytosolic or membrane levels of this protein, and was detected also in the cytosolic fraction of32P-labeled intact cells.
Antigen-Specific Responses to Diphtheria-Tetanus-Acellular Pertussis Vaccine in Human Infants Are Initially Th2 Polarized
Authors: Rowe J. , Macaubas C. , Monger T. M. , Holt B. J. , Harvey J. , Poolman J. T. , Sly P. D. , Holt P. G.
Published: July 2002

Immune responses to exogenous antigens in infant experimental animals display various degrees of Th2 polarization. Preliminary evidence from small human studies suggest a similar age-dependent response pattern to vaccines, but detailed investigations on vaccine immunity during infancy have not yet been undertaken. We report below the results of a comprehensive prospective study on responses to the tetanus component of the diphtheria, tetanus, acellular pertussis (DTaP) vaccine in a cohort of 55 healthy children, employing peripheral blood mononuclear cells (PBMC) collected at the 2-, 4-, and 6-month vaccinations and at 12 months. Antigen-specific production of interleukin-4 (IL-4), IL-5, IL-6, IL-9, IL-10, IL-13, and gamma interferon (IFN-γ) was determined at each sample point, in parallel with polyclonal (phytohemagglutinin PHA-induced) cytokine responses. Our results indicate early and persistent Th2 responses to the vaccine, in contrast to a more delayed and transient pattern of IFN-γ production. This initial disparity between the Th1 and Th2 components of the vaccine response was mirrored by patterns of polyclonally induced cytokine production, suggesting that the delayed maturation of the Th1 component of the vaccine response during infancy is secondary to developmental processes occurring within the overall Th cell system.
Salmonella enterica
Serovars Gallinarum and Pullorum Expressing
Salmonella enterica
Serovar Typhimurium Type 1 Fimbriae Exhibit Increased Invasiveness for Mammalian Cells
Authors: Wilson Rebecca L. , Elthon Jessica , Clegg Steven , Jones Bradley D.
Published: July 2002

Salmonella enterica
serovars Gallinarum and Pullorum are
S. enterica
biotypes that exhibit host specificity for poultry and aquatic birds and are not normally capable of causing disease in mammalian hosts. During their evolution toward host restriction serovars Gallinarum and Pullorum lost their ability to mediate mannose-sensitive hemagglutination (MSHA), a phenotype correlated with adherence to certain cell types. Because adherence is an essential requirement for invasion of cells by bacterial pathogens, we examined whether MHSA type 1 fimbriae would increase the ability of serovars Pullorum and Gallinarum to invade normally restrictive cells. Serovars Gallinarum and Pullorum expressing
S. enterica
serovar Typhimurium strain LT2 type 1 fimbriae exhibited a 10- to 20-fold increased ability to adhere to and a 20- to 60-fold increased invasion efficiency of the human epithelial HEp-2 cell line. Invasion was accompanied by extensive ruffling of the membranes of the HEp-2 cells. In a murine ligated ileal loop model, a 32% increase in the number of M-cell ruffles was seen when serovar Gallinarum expressed serovar Typhimurium type 1 fimbriae.
Vibrio choleraeO139 Conjugate Vaccines: Synthesis and Immunogenicity ofV. choleraeO139 Capsular Polysaccharide Conjugates with Recombinant Diphtheria Toxin Mutant in Mice
Authors: Kossaczka Zuzana , Shiloach Joseph , Johnson Virginia , Taylor David N. , Finkelstein Richard A. , Robbins John B. , Szu Shousun C.
Published: July 2002

Epidemiologic and experimental data provide evidence that a critical level of serum immunoglobulin G (IgG) antibodies to the surface polysaccharide ofVibrio choleraeO1 (lipopolysaccharide) and ofVibrio choleraeO139 (capsular polysaccharide [CPS]) is associated with immunity to the homologous pathogen. The immunogenicity of polysaccharides, especially in infants, may be enhanced by their covalent attachment to proteins (conjugates). Two synthetic schemes, involving 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and 1-cyano-4-dimethylaminopyridinium tetrafluoroborate (CDAP) as activating agents, were adapted to prepare four conjugates ofV. choleraeO139 CPS with the recombinant diphtheria toxin mutant, CRMH21G. Adipic acid dihydrazide was used as a linker. When injected subcutaneously into young outbred mice by a clinically relevant dose and schedule, these conjugates elicited serum CPS antibodies of the IgG and IgM classes with vibriocidal activity to strains of capsulatedV. choleraeO139. Treatment of these sera with 2-mercaptoethanol (2-ME) reduced, but did not eliminate, their vibriocidal activity. These results indicate that the conjugates elicited IgG with vibriocidal activity. Conjugates also elicited high levels of serum diphtheria toxin IgG. Convalescent sera from 20 cholera patients infected withV. choleraeO139 had vibriocidal titers ranging from 100 to 3,200: absorption with the CPS reduced the vibriocidal titer of all sera to ≤50. Treatment with 2-ME reduced the titers of 17 of 20 patients to ≤50. These data show that, like infection withV. choleraeO1, infection withV. choleraeO139 induces vibriocidal antibodies specific to the surface polysaccharide of this bacterium (CPS) that are mostly of IgM class. Based on these data, clinical trials with theV. choleraeO139 CPS conjugates with recombinant diphtheria toxin are planned.
Inhibition of Adhesion of
Plasmodium falciparum
-Infected Erythrocytes by Structurally Defined Hyaluronic Acid Dodecasaccharides
Authors: Chai Wengang , Beeson James G. , Kogelberg Heide , Brown Graham V. , Lawson Alexander M.
Published: July 2002

We recently reported that
Plasmodium falciparum
-infected erythrocytes (IRBCs) can adhere to hyaluronic acid (HA), which appears to be a receptor, in addition to chondroitin sulfate A (CSA), for parasite sequestration in the placenta. Further investigations of the nature and specificity of this interaction indicate that HA oligosaccharide fragments competitively inhibit parasite adhesion to immobilized purified HA in a size-dependent manner, with dodecasaccharides being the minimum size for maximum inhibition. Rigorously purified and structurally defined HA dodecasaccharides, free of contamination by CSA or other glycosaminoglycans, effectively inhibited IRBC adhesion to HA but not CSA, providing compelling evidence of a specific interaction between IRBCs and HA.
Role of
Mycobacterium tuberculosis
Copper-Zinc Superoxide Dismutase
Authors: Dussurget Olivier , Stewart Graham , Neyrolles Olivier , Pescher Pascale , Young Douglas , Marchal Gilles
Published: July 2002

Superoxide dismutases (SODs) play an important role in protection against oxidative stress and have been shown to contribute to the pathogenicity of many bacterial species. To determine the function of the mycobacterial copper and zinc-cofactored SOD (CuZnSOD), we constructed and characterized
Mycobacterium tuberculosis
and
Mycobacterium bovis
BCG CuZnSOD null mutants. Both strains were more sensitive to superoxides and hydrogen peroxide than were their respective parental strains. The survival of
M. bovis
BCG in unstimulated as well as activated mouse bone marrow-derived macrophages was not affected by the loss of CuZnSOD. The survival of CuZnSOD deficient-
M. tuberculosis
in guinea pig tissues was comparable to that of its parental strain. These results indicate that the mycobacterial CuZnSOD is not essential for intracellular growth within macrophages and does not detectably contribute to the pathogenicity of
M. tuberculosis
.
Proteins PblA and PblB of
Streptococcus mitis
, Which Promote Binding to Human Platelets, Are Encoded within a Lysogenic Bacteriophage
Authors: Bensing Barbara A. , Siboo Ian R. , Sullam Paul M.
Published: July 2002

The binding of platelets by bacteria is a proposed central mechanism in the pathogenesis of infective endocarditis. Platelet binding by
Streptococcus mitis
strain SF100 (an endocarditis isolate) was recently shown to be mediated in part by the surface proteins PblA and PblB. The genes encoding PblA and PblB are clustered with genes nearly identical to those of streptococcal phages r1t, 01205, and Dp-1, suggesting that
pblA
and
pblB
might reside within a prophage. To address this possibility, cultures of SF100 were exposed to either mitomycin C or UV light, both of which are known to induce the lytic cycle of many temperate phages. Both treatments caused a significant increase in the transcription of
pblA
. Treatment with mitomycin C or UV light also caused a substantial increase in the expression of PblA and PblB, as detected by Western blot analysis of proteins in the SF100 cell wall. By electron microscopy, phage particles were readily visible in the supernatants from induced cultures of SF100. The phage, designated SM1, had a double-stranded DNA genome of approximately 35 kb. Southern blot analysis of phage DNA indicated that
pblA
and
pblB
were contained within the SM1 genome. Furthermore, Western blot analysis of phage proteins revealed that both PblA and PblB were present in the phage particles. These findings indicate that PblA and PblB are encoded by a lysogenic bacteriophage, which could facilitate the dissemination of these potential virulence determinants to other bacterial pathogens.
Targeting of Nasal Mucosa-Associated Antigen-Presenting Cells In Vivo with an Outer Membrane Protein A Derived fromKlebsiellapneumoniae
Authors: Goetsch Liliane , Gonzalez Alexandra , Plotnicky-Gilquin Hélène , Haeuw Jean François , Aubry Jean Pierre , Beck Alain , Bonnefoy Jean Yves , Corvaı̈a Nathalie
Published: July 2002

Administration of vaccines by the nasal route has recently proven to be one of the most efficient ways for inducing both mucosal and systemic antibody responses in experimental animals. Our results demonstrate that P40, a well-defined outer membrane protein A fromKlebsiellapneumoniae, is indeed a carrier molecule suitable for nasal immunization. Using fragments from the respiratory syncytial virus subgroup A (RSV-A) G protein as antigen models, it has been shown that P40 is able to induce both systemic and mucosal immunity when fused or coupled to a protein or a peptide and administered intranasally (i.n.) to naive orK.pneumoniae-primed mice. Confocal analyses of nasal mucosa-associated lymphoid tissue after i.n. instillation of P40 showed that this molecule is able to cross the nasal epithelium and target CD11c-positive cells likely to be murine dendritic cells or macrophages. More importantly, this targeting of antigen-presenting cells following i.n. immunization with a subunit of the RSV-A molecule in the absence of any mucosal adjuvant results in both upper and lower respiratory tract protection against RSV-A infection.
Association of Mitogen-Activated Protein Kinase Pathways with Gingival Epithelial Cell Responses toPorphyromonas gingivalisInfection
Authors: Watanabe Kiyoko , Yilmaz Özlem , Nakhjiri Simin F. , Belton Carol M. , Lamont Richard J.
Published: July 2002

Mitogen-activated protein (MAP) kinase pathways are key factors in host signaling events and can also play important roles in the internalization of pathogenic bacteria by host cells.Porphyromonas gingivalis, a periodontal pathogen,can efficiently invade human gingival epithelial cells (GECs). In this study, we examined the activation of MAP kinase pathways in GECs infected withP. gingivalis. c-Jun N-terminal kinase (JNK) was activated after 5 min of infection withP. gingivalis,whereas noninvasiveStreptococcus gordoniidid not have a significant effect on JNK activation. In contrast, extracellular signal-regulated kinase (ERK) 1/2 was downregulated in a dose-dependent manner byP. gingivalis, but not byS. gordonii, after a 15-min exposure. Nonmetabolically activeP. gingivaliscells were unable to modulate MAP kinase activity. U0126, a specific inhibitor of MEK1/2 (ERK1/2 kinase), and toxin B, a specific inhibitor of Rho family GTPases, had no effect onP. gingivalisinvasion. Genistein, a tyrosine protein kinase inhibitor, blocked uptake ofP. gingivalis. The transcriptional regulator NF-κB was not activated byP. gingivalis. These results suggest thatP. gingivaliscan selectively target components of the MAP kinase pathways. ERK1/2, while not involved inP. gingivalisinvasion of GECs, may be downregulated by internalizedP. gingivalis. Activation of JNK is associated with the invasive process ofP. gingivalis.
Koala Biovar of
Chlamydia pneumoniae
Infects Human and Koala Monocytes and Induces Increased Uptake of Lipids In Vitro
Authors: Coles Katie A. , Timms Peter , Smith David W.
Published: July 2002

We examined the ability of the koala biovar of
Chlamydia pneumoniae
to infect both Hep-2 cells and human monocytes and the effect of infection on the formation of foam cells. The koala biovar produced large inclusions in both human and koala monocytes and in Hep-2 cells. Koala
C. pneumoniae
induced foam cell formation with and without added low-density lipoprotein, in contrast to TW183, which produced increased foam cell formation only in the presence of low-density lipoprotein.
Neural Route of CerebralListeria monocytogenesMurine Infection: Role of Immune Response Mechanisms in Controling Bacterial Neuroinvasion
Authors: Jin Yuxuan , Dons Lone , Kristensson Krister , Rottenberg Martı́n E.
Published: July 2002

The pathologic features of cerebralListeria monocytogenesinfection strongly suggest that besides hematogenous spread, bacteria might also spread via a neural route. We propose that after snout infection of recombination activating gene 1 (RAG-1)-deficient mice,L. monocytogenesspreads to the brain via a neural route. The neural route of invasion is suggested by (i) the immunostaining ofL. monocytogenesin the trigeminal ganglia (TG) and brain stem but not in other areas of the brain; (ii) the kinetics of bacterial loads in snout, TG, and brain; and (iii) the increased resistance of mice infected with aplcBbacterial mutant (unable to spread from cell to cell). Gamma interferon (IFN-γ) plays a protective role in neuroinvasion; inducible nitric oxide synthase (iNOS) accounts only partially for the protection, as shown by a comparison of the susceptibilities of IFN-γ receptor (IFN-γR)-deficient, iNOS-deficient, and wild-type mice to snout infection withL. monocytogenes. The dramatically enhanced susceptibility ofRAG-1-deficient, IFN-γR gene-deficient mice indicated the overall importance of innate immune cells in the release of protective levels of IFN-γ. The source of IFN-γ appeared to be NK cells, as shown by use ofRAG-1-deficient, γ-chain receptor gene-deficient mice; NK cells played a relevant protective role in neuroinvasion through a perforin-independent mechanism. In vitro evidence indicated that IFN-γ can directly induce bacteriostatic mechanisms in neural tissue.
T Cells That React to the ImmunodominantLeishmania majorLACK Antigen Prevent Early Dissemination of the Parasite in Susceptible BALB/c Mice
Authors: Schilling Sabrina , Glaichenhaus Nicolas
Published: July 2002

Susceptibility of BALB/c mice toLeishmania majordepends on the early production of IL-4 by CD4+T cells which react to the parasite LACK antigen. Here, we show that LACK-specific cells are rapidly recruited to the site of infection and favor the early dissemination ofL. majorto the internal organs.
Restoration of Mga Function to aStreptococcus pyogenesStrain (M Type 50) That Is Virulent in Mice
Authors: Limbago Brandi , McIver Kevin S. , Penumalli Vikram , Weinrick Brian , Scott June R.
Published: July 2002

The Mga protein in B514Sm, aStreptococcus pyogenesstrain isolated as a mouse pathogen, contains amino acid substitutions at conserved sites that render the protein defective. Replacement ofmga50with the functional homologmga4.1restored full expression of Mga-regulated proteins. Restoration of Mga function did not affect fibrinogen binding, nor did it affect virulence in several mouse models of group A streptococcus infection.
Commercial Preparations of Lipoteichoic Acid Contain Endotoxin That Contributes to Activation of Mouse Macrophages In Vitro
Authors: Gao Jian Jun , Xue Qiao , Zuvanich Eleanor G. , Haghi Kevin R. , Morrison David C.
Published: July 2002

Lipoteichoic acids (LTA), cell wall components of gram-positive bacteria, have been reported to induce various inflammatory mediators and to play a key role in gram-positive-microbe-mediated septic shock. In a large number of these studies, investigators used commercially available LTA purified from a variety of gram-positive bacteria, including
Staphylococcus aureus
,
Bacillus subtilis
, and
Streptococcus sanguis
. We report here that, although these commercially available LTA could be readily shown to stimulate production of nitric oxide (NO) in RAW 264.7 mouse macrophages, the activity was dramatically inhibited by polymyxin B, a relatively specific inhibitor of endotoxin biological activity. One-step purification of the commercially available
S. aureus
LTA using hydrophobic interaction chromatography resulted in two well-separated peak fractions, one highly enriched for LTA and a second highly enriched for endotoxin. The LTA-enriched fractions did not induce production of NO in RAW 264.7 macrophages, although they caused a dose-dependent induction of NO in the presence of low concentrations of gamma interferon (IFN-γ) (which by itself induced little NO), regardless of the presence of polymyxin B. In contrast, the endotoxin-enriched fractions by themselves inhibited in high levels of NO in RAW 264.7 macrophages but activity was almost completely inhibited in the presence of polymyxin B. Consistent with these findings, our data also indicate that commercial LTA preparations from
S. aureus
,
B. subtilis
, and
S. sanguis
were not able to induce NO from lipopolysaccharide-hyporesponsive C3H/HeJ mouse peritoneal macrophages, but in the presence of IFN-γ, these LTA preparations were able to induce relatively high levels of NO from C3H/HeJ macrophages. These results indicate that commercially available LTA can contain contaminating and potentially significant levels of endotoxin that can be expected to contribute to the putative macrophage-stimulating effects of LTA as assessed by NO production. The fact that the purified LTA, by itself, was not able to induce significant levels of NO secretion in RAW 264.7 macrophages supports the conclusion that caution in attributing high-level biological activity to this microbial cell wall constituent should be exercised.
Pulmonary Inflammation Disrupts Surfactant Function duringPneumocystis cariniiPneumonia
Authors: Wright Terry W. , Notter Robert H. , Wang Zhengdong , Harmsen Allen G. , Gigliotti Francis
Published: July 2002

DuringPneumocystis cariniipneumonia (PCP) in mice, the degree of pulmonary inflammation correlates directly with the severity of lung function deficits. Therefore, studies were undertaken to determine whether the host inflammatory response contributes to PCP-related respiratory impairment, at least in part, by disrupting the pulmonary surfactant system. Protein and phospholipid content and surfactant activity were measured in the lavage fluid of infected mice in either the absence or presence of an inflammatory response. At 9 weeks postinfection withP. carinii, nonreconstituted SCID mice exhibited no signs of pulmonary inflammation, respiratory impairment, or surfactant dysfunction. Lavage fluid obtained from these mice had protein/phospholipid (Pr/PL) ratios (64% ± 4.7%) and minimum surface tension values (4.0 ± 0.9 mN/m) similar to those ofP. carinii-free control mice. However, when infected SCID mice were immunologically reconstituted, an intense inflammatory response ensued. Pr/PL ratios (218% ± 42%) and minimum surface tension values (27.2 ± 2.7 mN/m) of the lavage fluid were significantly elevated compared to those of the lavage fluid from infected, nonreconstituted mice (P< 0.05). To examine the specific role of CD8+T-cell-mediated inflammation in surfactant dysfunction during PCP, mice with defined T-cell populations were studied.P. carinii-infected, CD4+-depleted mice had elevated lavage fluid Pr/PL ratios (126% ± 20%) and elevated minimum surface tension values (16.3 ± 1.0 mN/m) compared to normal mice (P< 0.05). However, when infected mice were additionally depleted of CD8+cells, Pr/PL ratios were normal and surfactant activity was improved. These findings demonstrate that the surfactant pathology associated with PCP is related to the inflammatory process rather than being a direct effect ofP. carinii. Moreover, CD8+lymphocytes are involved in the mechanism leading to surfactant dysfunction.
Variant-Specific Surface Protein Switching inGiardia lamblia
Authors: Nash T. E. , Luján H. T. , Mowatt M. R. , Conrad J. T.
Published: July 2002

Surface antigen switching inGiardia lambliawas analyzed using monoclonal antibodies specific for two variant-specific surface proteins (VSPs). Two VSPs were detected on the surface of single trophozoites. Dual expression persisted for 13 h but disappeared at 36 h, as in other parasites that undergo surface antigenic variation.
Pseudomonas aeruginosa-Induced Apoptosis Involves Mitochondria and Stress-Activated Protein Kinases
Authors: Jendrossek Verena , Grassmé Heike , Mueller Ilka , Lang Florian , Gulbins Erich
Published: July 2002

Pseudomonas aeruginosa, a gram-negative facultative pathogen, causes severe infections in immunocompromised and cystic fibrosis patients. However, the molecular details of the interaction betweenP. aeruginosaand mammalian cells are still largely unknown. Here we demonstrate that infection of human conjunctiva epithelial Chang cells with the well-characterizedP. aeruginosastrain PAO-I results in rapid induction of apoptosis. Apoptosis was mediated by mitochondrial alterations, in particular mitochondrial depolarization, synthesis of reactive oxygen intermediates, and release of cytochromec, as well as an activation of Jun N-terminal kinases (JNK). Stimulation of these events was dependent on upregulation of CD95 on infected cells, and a deficiency of CD95 or the CD95 ligand prevented mitochondrial changes, JNK activation, and apoptosis upon infection. Further, efficient apoptosis of Chang epithelial cells required infection with liveP. aeruginosa, adhesion but not invasion of the bacteria, and expression of the type III secretion system in PAO-I. The data indicate a type III secretion system-dependent, sequential activation of several signaling pathways byP. aeruginosaPAO-I, resulting in apoptosis of the infected cell.
Expression ofpls, a Gene Closely Associated with themecAGene of Methicillin-ResistantStaphylococcus aureus, Prevents Bacterial Adhesion In Vitro
Authors: Savolainen K. , Paulin L. , Westerlund-Wikström B. , Foster T. J. , Korhonen T. K. , Kuusela P.
Published: July 2002

Theplsgene, coding for a large surface protein of methicillin-resistantStaphylococcus aureus, was cloned from a strain which adheres poorly to several mammalian proteins. The structure ofplsrevealed three distinct repeat regions, one of which was a serine-aspartate repeat characteristic of the Clf-Sdr family of surface proteins in staphylococci. The lengths of the repeat regions varied in different clinical strains and could be used as epidemiological markers.plswas found to be closely associated with themecAgene by pulsed-field gel electrophoresis analysis ofSmaI-digested DNA. Aplsmutant constructed by allele replacement adhered well to immobilized fibronectin and immunoglobulin G, in contrast to the parental strain, suggesting that Pls could have a role in preventing adhesion at some stages during an infection.