bluuebunny/crossref_metadata_2025_split huggingface dataset:
Recruitment of CD55 and CD66e Brush Border-Associated Glycosylphosphatidylinositol-Anchored Proteins by Members of the Afa/Dr Diffusely Adhering Family ofEscherichia coliThat Infect the Human Polarized Intestinal Caco-2/TC7 Cells
Authors: Guignot Julie , Peiffer Isabelle , Bernet-Camard Marie-Françoise , Lublin Douglas M. , Carnoy Christophe , Moseley Steve L. , Servin Alain L. 
Published: July 2002

ABSTRACTThe Afa/Dr family of diffusely adheringEscherichia coli(Afa/Dr DAEC) includes bacteria expressing afimbrial adhesins (AFA), Dr hemagglutinin, and fimbrial F1845 adhesin. We show that infection of human intestinal Caco-2/TC7 cells by the Afa/Dr DAEC strains C1845 and IH11128 is followed by clustering of CD55 around adhering bacteria. Mapping of CD55 epitopes involved in CD55 clustering by Afa/Dr DAEC was conducted using CD55 deletion mutants expressed by stable transfection in CHO cells. Deletion in the short consensus repeat 1 (SCR1) domain abolished Afa/Dr DAEC-induced CD55 clustering. In contrast, deletion in the SCR4 domain does not modify Afa/Dr DAEC-induced CD55 clustering. We show that the brush border-associated glycosylphosphatidylinositol (GPI)-anchored protein CD66e (carcinoembryonic antigen) is recruited by the Afa/Dr DAEC strains C1845 and IH11128. This conclusion is based on the observations that (i) infection of Caco-2/TC7 cells by Afa/Dr DAEC strains is followed by clustering of CD66e around adhering bacteria and (ii) Afa/Dr DAEC strains bound efficiently to stably transfected HeLa cells expressing CD66e, accompanied by CD66e clustering around adhering bacteria. Inhibition assay using monoclonal antibodies directed against CD55 SCR domains, and polyclonal anti-CD55 and anti-CD66e antibodies demonstrate that CD55 and CD66e function as a receptors for the C1845 and IH11128 bacteria. Moreover, using structuraldraEgene mutants, we found that a mutant in which cysteine replaced aspartic acid at position 54 displayed conserved binding capacity but failed to induce CD55 and CD66e clustering. Taken together, these data give new insights into the mechanisms by which Afa/Dr DAEC induces adhesin-dependent cross talk in the human polarized intestinal epithelial cells by mobilizing brush border-associated GPI-anchored proteins known to function as transducing molecules.
Expression of AniA, the Major Anaerobically Induced Outer Membrane Protein of
Neisseria gonorrhoeae
, Provides Protection against Killing by Normal Human Sera
Authors: Cardinale J. A. , Clark V. L. 
Published: July 2002

ABSTRACT

Anaerobically grown
Neisseria gonorrhoeae
has previously been shown to have elevated serum resistance in the absence of exogenous CMP-
N
-acetylneuraminic acid or detectable sialylation. We hypothesized that the anaerobically induced gonococcal outer membrane protein AniA might have a role in this phenomenon, as it is the only known gonococcal protein that is absent under aerobic conditions. An
N. gonorrhoeae
F62 derivative, RUG7035, in which
aniA
is under control of the
tac
promoter, was used to examine the effect of AniA expression on serum resistance. In this study, we found that expression of AniA enhanced the serum resistance of
N. gonorrhoeae
and may account for these earlier observations.
CD4+Depletion Selectively Inhibits Eosinophil Recruitment to the Cornea and AbrogatesOnchocerca volvulusKeratitis (River Blindness)
Authors: Hall Laurie R. , Kaifi Jussuf T. , Diaconu Eugenia , Pearlman Eric 
Published: July 2002

ABSTRACTPrevious studies demonstrated that in the murine model ofOnchocerca volvuluskeratitis, neutrophils and eosinophils are recruited into the cornea in a biphasic manner in response to intrastromal injection. To determine if CD4+T cells regulate migration of neutrophils and eosinophils into the cornea, CD4+cells were depleted using monoclonal antibody GK1.5 before intrastromal injection of parasite antigens. Depletion of CD4+cells abrogated corneal opacification at later but not early stages of disease. Consistent with this observation, CD4 depletion significantly impaired recruitment of eosinophils to the cornea but had no effect on neutrophils. These data indicate that CD4+T cells mediate sustainedO. volvuluskeratitis by regulating eosinophil recruitment to the cornea.
High-Affinity, Protective Antibodies to the Binding Domain of Botulinum Neurotoxin Type A
Authors: Pless Dorothy D. , Torres Edna R. , Reinke Emily K. , Bavari Sina 
Published: July 2002

ABSTRACT

Monoclonal antibodies (MAbs) were prepared against the putative binding domain of botulinum neurotoxin A (BoNT/A), a nontoxic 50-kDa fragment. Initially, all fusion products were screened against the holotoxin BoNT/A and against the binding fragment, BoNT/A H
C
. Eleven neutralizing hybridomas were cloned, and their specific binding to BoNT/A H
C
was demonstrated by surface plasmon resonance, with dissociation constants ranging from 0.9 to <0.06 nM. Epitope mapping by real-time surface plasmon resonance showed that the antibodies bound to at least two distinct regions of the BoNT/A H
C
fragment. These MAbs will be useful tools for studying BoNT/A interactions with its receptor, and they have potential diagnostic and therapeutic applications.
Generation and Surface Localization of Intact M Protein inStreptococcus pyogenesAre Dependent onsagA
Authors: Biswas Indranil , Germon Pierre , McDade Kathleen , Scott June R. 
Published: July 2002

ABSTRACTThe M protein is an important surface-located virulence factor ofStreptococcuspyogenes, the group A streptococcus (GAS). Expression of M protein is primarily controlled by Mga, a transcriptional activator protein. A recent report suggested that thesaglocus, which includes nine genes necessary and sufficient for production of streptolysin S, another GAS virulence factor, is also needed for transcription ofemm, encoding the M protein (Z. Li, D. D. Sledjeski, B. Kreikemeyer, A. Podbielski, and M. D. Boyle, J. Bacteriol. 181:6019–6027, 1999). To investigate this in more detail, we constructed an insertion-deletion mutation insagA, the first gene in thesaglocus, in the M6 strain JRS4. The resulting strain, JRS470, produced no detectable streptolysin S and showed a drastic reduction in cell surface-associated M protein, as measured by cell aggregation and Western blot analysis. However, transcription of theemmgene was unaffected by thesagAmutation. Detailed analysis with monoclonal antibodies and an antipeptide antibody showed that the M protein in thesagAmutant strain was truncated so that it lacks the C-repeat region and the C-terminal domain required for anchoring it to the cell surface. This truncated M protein was largely found, as expected, in the culture supernatant. Lack of surface-located M protein made thesagAmutant strain susceptible to phagocytosis. Thus, althoughsagAdoes not affect transcription of the M6 protein gene, it is needed for the surface localization of this important virulence factor.
Codon Optimization of Gene Fragments Encoding
Plasmodium falciparum
Merzoite Proteins Enhances DNA Vaccine Protein Expression and Immunogenicity in Mice
Authors: Narum David L. , Kumar Sanjai , Rogers William O. , Fuhrmann Steven R. , Liang Hong , Oakley Miranda , Taye Alem , Sim B. Kim Lee , Hoffman Stephen L. 
Published: July 2002

ABSTRACT

In contrast to conventional vaccines, DNA and other subunit vaccines exclusively utilize host cell molecules for transcription and translation of proteins. The adenine plus thymine content of
Plasmodium falciparum
gene sequences (∼80%) is much greater than that of
Homo sapiens
(∼59%); consequently, codon usage is markedly different. We hypothesized that modifying codon usage of
P. falciparum
genes encoded by DNA vaccines from that used by the parasite to those resembling mammalian codon usage would lead to increased
P. falciparum
protein expression in vitro in mouse cells and increased antibody responses in DNA-vaccinated mice. We synthesized gene fragments encoding the receptor-binding domain of the 175-kDa
P. falciparum
erythrocyte-binding protein (EBA-175 region II) and the 42-kDa C-terminal processed fragment of the
P. falciparum
merozoite surface protein 1 (MSP-1
42
) using the most frequently occurring codon in mammals to code for each amino acid, and inserted the synthetic genes in DNA vaccine plasmids. In in vitro transient-expression assays, plasmids containing codon-optimized synthetic gene fragments (pS plasmids) showed greater than fourfold increased protein expression in mouse cells compared to those containing native gene fragments (pN plasmids). In mice immunized with 0.5, 5.0, or 50 μg of the DNA plasmids, the dose of DNA required to induce equivalent antibody titers was 10- to 100-fold lower for pS than for pN plasmids. These data demonstrate that optimizing codon usage in DNA vaccines can improve protein expression and consequently the immunogenicity of gene fragments in DNA vaccines for organisms whose codon usage differs substantially from that of mammals.
Virulence ofLeishmania infantumIs Expressed as a Clonal and Dominant Phenotype in Experimental Infections
Authors: Garin Yves Jean-François , Sulahian Annie , Pratlong Francine , Meneceur Pascale , Gangneux Jean-Pierre , Prina Eric , Dedet Jean-Pierre , Derouin Francis 
Published: July 2002

ABSTRACTHumanLeishmania infantuminfection results in a spectrum of clinical expressions ranging from cutaneous to either asymptomatic or fatal visceral disease. In this context, characterization of parasite virulence appears to be relevant as a biological marker of intrinsic parasitic factors that can affect the pathology of leishmaniasis. Since parasite populations in naturally infected hosts are likely to be composed of multiclonal associations, we first explored the biodiversity of parasite virulence at the intrastrain level in vitro and in vivo by using 11 clones isolated from three strains previously known to express different virulence phenotypes in mice. Subsequently, we studied the course of infection in mice inoculated simultaneously or successively with strains or clones showing various virulence phenotypes. Analysis of in vitro growth characteristics showed no differences among clones from the different parental strains. By contrast, in vivo experiments evidenced a marked intrastrain heterogeneity of virulence to mice. One out of five clones obtained from a virulent strain showed a typical virulence phenotype, while the remaining four clones had low-virulence profiles, as did the six clones isolated from two low-virulence strains. In mixed multiclonal infections, the virulence phenotype was expressed as a dominant character over the associated low-virulence clones. After a challenge with either a homologous or a heterologous strain or clone, virulence phenotypes were conserved and expressed as in naive mice independently from the preexisting population. These results strongly suggest that parasite virulence inL. infantumvisceral leishmaniasis is clonal and dominant in nature.
Differential Changes in Heat Shock Protein-, Lipoarabinomannan-, and Purified Protein Derivative-Specific Immunoglobulin G1 and G2 Isotype Responses during BovineMycobacterium aviumsubsp.paratuberculosisInfection
Authors: Koets Ad P. , Rutten Victor P. M. G. , de Boer Masja , Bakker Douwe , Valentin-Weigand Peter , van Eden Willem 
Published: July 2002

ABSTRACTBovine paratuberculosis is caused by infection of young calves withMycobacterium aviumsubsp.paratuberculosis. In some of the chronically infected cows the long asymptomatic stage (2 to 4 years) is followed by a rapid progression to a clinical stage due to protein-losing enteropathy, which will ultimately be fatal. The current dogma is that in early stages of disease the cell-mediated responses predominate, whereas in the clinical stage of the disease the humoral responses prevail, possibly signaling a switch in immune reactivity related to disease progression. We developed immunoglobulin M (IgM)-, IgA-, and IgG1- and IgG2-isotype-specific enzyme-linked immunosorbent assays forM. aviumsubsp.paratuberculosis-derived antigens (heat shock proteins of 70 kDa [Hsp70] and 65 kDa [Hsp65], lipoarabinomannan [LAM], andM. aviumsubsp.paratuberculosispurified protein derivative PPD [PPDP]). The serological responses of cows in different stages of paratuberculosis were used to evaluate the putative shift in immune responsiveness. In the clinical stage the PPDP-specific IgG1 responses were increased compared to those in the asymptomatic stage. However, total IgG1 and IgG2 and the Hsp70-, Hsp65-, and LAM-specific isotype responses were decreased in the clinical stage were decreased compared to those in the asymptomatic stage of disease. Thus, the classical pattern was found only for PPDP antigens and the IgG1 isotype. For other antigens and isotypes and the total IgG levels, the response pattern is different and indicates that there is no uniform association with increased antibody responses during the progression from the asymptomatic stage to the clinical stage of bovine paratuberculosis.
Expression of C-Reactive Protein in the Human Respiratory Tract
Authors: Gould Jane M. , Weiser Jeffrey N. 
Published: July 2002

ABSTRACTC-reactive protein (CRP) is a normal constituent of human sera synthesized by hepatocytes and induced by proinflammatory cytokines. The function of this acute-phase reactant includes activation of complement and enhancement of opsonophagocytosis. CRP binds to phosphorylcholine (ChoP), a constituent of eukaryotic membranes that is also found on the cell surface of major bacterial pathogens of the human respiratory tract, includingStreptococcus pneumoniaeandHaemophilus influenzae. The presence of CRP on mucosal surfaces and role in innate immunity in the human respiratory tract where ChoP-containing organisms reside have not been previously studied. We have shown using a monoclonal antibody to CRP that CRP is present in inflamed (0.17 to 42 μg/ml) and uninflamed (<0.05 to 0.88 μg/ml) secretions from the human respiratory tract in sufficient quantities for an antimicrobial effect. In addition, the CRP gene was expressed in epithelial cells of the human respiratory tract using in situ hybridization on nasal polyps and reverse transcriptase PCR of pharyngeal cells in culture. The complement-dependent bactericidal activity of normal nasal airway surface fluid and sputum against ChoP-expressingH. influenzaewas abolished when the secretions were pretreated to remove CRP. In summary, the results indicate that CRP is present in secretions of the human respiratory tract, that human respiratory epithelial cells are capable of CRP expression, and that this protein may contribute to bacterial clearance in the human respiratory tract.
Upregulation of Toll-Like Receptor 2 Gene Expression in Macrophage Response to Peptidoglycan and High Concentration of Lipopolysaccharide Is Involved in NF-κB Activation
Authors: Liu Yan , Wang Yin , Yamakuchi Munekazu , Isowaki Sumikazu , Nagata Etsuro , Kanmura Yuichi , Kitajima Isao , Maruyama Ikuro 
Published: July 2002

ABSTRACTToll-like receptors 2 and 4 (TLR2 and TLR4) have been found to transduce signals of peptidoglycan (PGN) and lipopolysaccharide (LPS), respectively, for NF-κB activation. However, little is known about the expression and regulation of the TLR2 gene in monocytes/macrophages in response to the two typical bacterial products. We show in the present study that both PGN and a high concentration of LPS increase TLR2 gene expression in macrophage-like cells, 1α,25-dihydroxyvitamin D3-differentiated human HL60 and mouse RAW264.7 cells, and human monocytes in a dose- and time-dependent manner. Actinomycin D and pyrrolidine dithiocarbamate inhibition of gene transcription and NF-κB activation, respectively, blocks LPS- and PGN-elevated TLR2 mRNA in monocytic cells. The LPS-induced increase in TLR2 mRNA in monocytic cells is abolished by polymyxin B pretreatment and is observed in peripheral blood mononuclear cells from pigs subjected to endotoxic shock. Further, high concentrations of LPS and synthetic lipid A increase TLR2 mRNA expression in peritoneal macrophages from both TLR4-deficient C3H/HeJ mice and normal C3H/HeN mice, a process that constitutes induction of TLR4-independent TLR2 expression. These findings demonstrate that TLR2 gene expression is upregulated in macrophage responses to PGN and to high concentrations of LPS in vitro and in vivo and correlates with NF-κB activation.
CD4+T Helper 1 Cells Facilitate Regression of Murine Lyme Carditis
Authors: Bockenstedt Linda K. , Kang Insoo , Chang Christopher , Persing David , Hayday Adrian , Barthold Stephen W. 
Published: July 2002

ABSTRACTMurine Lyme borreliosis, caused by infection with the spirocheteBorrelia burgdorferi, results in acute arthritis and carditis that regress as a result ofB. burgdorferi-specific immune responses.B. burgdorferi-specific antibodies can attenuate arthritis in mice deficient in both B cells and T cells but have no effect on carditis. Because macrophages comprise the principal immune cell in carditis, T-cell responses that augment cell-mediated immunity may be important for carditis regression. To investigate this hypothesis, we examined the course of Lyme carditis in mice selectively deficient in B cells or αβ T cells. Our results show that carditis regresses in B-cell-deficient B10.Akmice but not in αβ T-cell-deficient mice, independently of the mouse strain background. Despite prominent macrophage infiltrates, hearts fromB. burgdorferi-infected αβ T-cell-deficient mice had less mRNA for tumor necrosis factor alpha as measured by reverse transcription-PCR compared to infected control mice. Anti-inflammatory cytokine mRNA levels were equivalent. Adoptive transfer of gamma interferon-secreting CD4+T cells into infected αβ T-cell-deficient mice promoted carditis resolution. These results show that αβ T cells can promote resolution of murine Lyme carditis and are the first demonstration of a beneficial role for CD4+T helper 1 cells in this disease.
Cloning, Sequencing, and Expression of the Leukotoxin Gene fromFusobacterium necrophorum
Authors: Narayanan Sanjeev Kumar , Nagaraja T. G. , Chengappa M. M. , Stewart George C. 
Published: July 2002

ABSTRACTFusobacterium necrophorumis a gram-negative, rod-shaped, anaerobic bacterium that is a primary or secondary etiological agent in a variety of necrotic purulent infections in animals and humans. Included are diseases of cattle such as liver abscesses and foot rot, which have economically important consequences for the cattle industry. The major virulence factor of this bacterium is leukotoxin, a secreted protein of high molecular weight active against leukocytes from ruminants. The screening of a genomic DNA library with polyclonal antisera raised against native affinity-purified leukotoxin and further extension of the sequence using inverse PCR led to the cloning of the entire leukotoxin gene. The leukotoxin gene open reading frame (ORF;lktA) consists of 9,726 bp and encodes a protein of 3,241 amino acids with an overall molecular weight of 335,956. The leukotoxin does not have sequence similarity with any other bacterial leukotoxin. Five truncated overlapping polypeptides covering the wholelktAORF were used to immunize rabbits. In Western blot assays, polyclonal antisera raised against all five truncated polypeptides recognized affinity-purified leukotoxin fromF. necrophorumculture supernatant in a Western blot assay. Antisera directed against two of the five polypeptides had neutralizing activity against the toxin. The entire leukotoxin ORF was expressed inEscherichia coli. Flow-cytometric analysis showed that the recombinant leukotoxin was active against bovine polymorphonuclear leukocytes and was inhibited with antiserum raised against theF. necrophorumleukotoxin. Southern blot hybridization analysis revealed different patterns oflktAhybridizing bands between isolates of the two subspecies ofF. necrophorum.
DNA Vaccine Combinations Expressing Either Tissue Plasminogen Activator Signal Sequence Fusion Proteins or Ubiquitin-Conjugated Antigens Induce Sustained Protective Immunity in a Mouse Model of Pulmonary Tuberculosis
Authors: Delogu Giovanni , Li Amy , Repique Charlene , Collins Frank , Morris Sheldon L. 
Published: July 2002

ABSTRACTDNA vaccination has emerged as a powerful approach in the search for a more efficacious vaccine against tuberculosis. In this study, we evaluated the effectiveness of immunizing with combinations of 10 different tuberculosis DNA vaccines that expressed mycobacterial proteins fused at the N terminus to eukaryotic intracellular targeting sequences. In one vaccine combination, the genes were fused to the tissue plasminogen activator signal sequence (TPA), while in a second combination the same 10 genes were expressed as ubiquitin (Ub)-conjugated proteins. In ex vivo studies in which the secretion of gamma interferon was measured, cellular immune responses were detected in mice vaccinated with either the TPA DNA vaccine combination or the Ub DNA vaccine combination at 7 and 14 days following a low-doseMycobacterium tuberculosischallenge. Moreover, mice vaccinated with the TPA combination, the Ub combination, andMycobacterium bovisBCG were able to limit the growth of tubercle bacilli in the lung and spleen after a virulent tuberculous aerosol challenge. Histopathological analyses also showed that mice immunized with the DNA vaccine combinations had substantially improved postinfection lung pathology relative to the naïve controls. Finally, in three different long-term experiments, the survival periods following aerogenic challenge were extended as much as sevenfold for vaccinated mice compared to naïve controls. Interestingly, in all three experiments, no significant differences were detected in the mean times to death for mice immunized with the TPA combination or the Ub combination relative to the BCG controls. In conclusion, these studies demonstrate the effectiveness of immunization with DNA vaccine combinations against tuberculosis and suggest that further testing of these plasmid cocktails is warranted.
Failure of theMycobacterium bovisBCG Vaccine: Some Species of Environmental Mycobacteria Block Multiplication of BCG and Induction of Protective Immunity to Tuberculosis
Authors: Brandt Lise , Feino Cunha Joana , Weinreich Olsen Anja , Chilima Ben , Hirsch Penny , Appelberg Rui , Andersen Peter 
Published: July 2002

ABSTRACTThe efficacy ofMycobacterium bovisbacillus Calmette-Guérin (BCG) vaccine against pulmonary tuberculosis (TB) varies enormously in different populations. The prevailing hypothesis attributes this variation to interactions between the vaccine and mycobacteria common in the environment, but the precise mechanism has so far not been clarified. Our study demonstrates that prior exposure to live environmental mycobacteria can result in a broad immune response that is recalled rapidly after BCG vaccination and controls the multiplication of the vaccine. In these sensitized mice, BCG elicits only a transient immune response with a low frequency of mycobacterium-specific cells and no protective immunity against TB. In contrast, the efficacy of TB subunit vaccines was unaffected by prior exposure to environmental mycobacteria. Six different isolates from soil and sputum samples from Karonga district in Northern Malawi (a region in which BCG vaccination has no effect against pulmonary TB) were investigated in the mouse model, and two strains of theMycobacterium aviumcomplex were found to block BCG activity completely.