bluuebunny/crossref_metadata_2025_split huggingface dataset:
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

ABSTRACT

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

ABSTRACTThe 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

ABSTRACTStreptococcus 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

ABSTRACTThe 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

ABSTRACTThe 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

ABSTRACTSalmonella 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

ABSTRACTDr-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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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

ABSTRACTThis 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

ABSTRACTImmune 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

ABSTRACT

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

ABSTRACTEpidemiologic 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

ABSTRACT

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.