bluuebunny/crossref_metadata_2025 huggingface dataset:
Immunologic Memory Induced by a Glycoconjugate Vaccine in a Murine Adoptive Lymphocyte Transfer Model
Authors: Guttormsen Hilde-Kari , Wetzler Lee M. , Finberg Robert W. , Kasper Dennis L.
Published: December 2019

We have developed an adoptive cell transfer model in mice to study the ability of a glycoprotein conjugate vaccine to induce immunologic memory for the polysaccharide moiety. We used type III capsular polysaccharide from the clinically relevant pathogen group B streptococci conjugated to tetanus toxoid (GBSIII-TT) as our model vaccine. GBS are a major cause of neonatal infections in humans, and type-specific antibodies to the capsular polysaccharide protect against invasive disease. Adoptive transfer of splenocytes from mice immunized with the GBSIII-TT conjugate vaccine conferred anti-polysaccharide immunologic memory to naive recipient mice. The transfer of memory occurred in a dose-dependent manner. The observed anamnestic immune response was characterized by (i) more rapid kinetics, (ii) isotype switching from immunoglobulin M (IgM) to IgG, and (iii) 10-fold-higher levels of type III-specific IgG antibody than for the primary response in animals with cells transferred from placebo-immunized mice. The adoptive cell transfer model described in this paper can be used for at least two purposes: (i) to evaluate conjugate vaccines with different physicochemical properties for their ability to induce immunologic memory and (ii) to study the cellular interactions required for an immune response to these molecules.
Cloning and Characterization ofCAD1/AAF1, a Gene fromCandida albicansThat Induces Adherence to Endothelial Cells after Expression inSaccharomyces cerevisiae
Authors: Fu Yue , Filler Scott G. , Spellberg Bradley J. , Fonzi William , Ibrahim Ashraf S. , Kanbe Toshio , Ghannoum Mahmoud A. , Edwards John E.
Published: December 2019

Adherence to the endothelial cell lining of the vasculature is probably a critical step in the egress ofCandida albicansfrom the intravascular compartment. To identify potential adhesins that mediate the attachment of this organism to endothelial cells, a genomic library fromC. albicanswas used to transform a nonadherent strain ofSaccharomyces cerevisiae. The population of transformed yeasts was enriched for highly adherent clones by repeated passages over endothelial cells. One clone which exhibited a fivefold increase in endothelial cell adherence, compared withS. cerevisiaetransformed with vector alone, was identified. This organism also flocculated. The candidal DNA fragment within this adherent/flocculent organism was found to contain a single 1.8-kb open reading frame, which was designatedCAD1. It was found to be identical toAAF1. The predicted protein encoded byCAD1/AAF1contained features suggestive of a regulatory factor. Consistent with this finding, immunoelectron microscopy revealed that CAD1/AAF1 localized to the cytoplasm and nucleus but not the cell wall or plasma membrane of the transformed yeasts. Because yeasts transformed withCAD1/AAF1both flocculated and exhibited increased endothelial cell adherence, the relationship between adherence and flocculation was examined.S. cerevisiaeexpressing either of two flocculation phenotypes, Flo1 or NewFlo, adhered to endothelial cells as avidly as did yeasts expressingCAD1/AAF1. Inhibition studies revealed that the flocculation phenotype induced byCAD1/AAF1was similar to Flo1. Thus,CAD1/AAF1probably encodes a regulatory protein that stimulates endothelial cell adherence inS. cerevisiaeby inducing a flocculation phenotype. WhetherCAD1/AAF1contributes to the adherence ofC. albicansto endothelial cells remains to be determined.
Reduced Response to Multiple Vaccines Sharing Common Protein Epitopes That Are Administered Simultaneously to Infants
Authors: Dagan Ron , Eskola Juhani , Leclerc Claude , Leroy Odile
Published: December 2019

The plethora of newly discovered vaccines implies that, in the future, many vaccines will have to be administered simultaneously to infants. We examined the potential interference with the immune response of several coadministered vaccines containing the same protein component, namely, tetanus toxoid (TT). Infants simultaneously receiving a tetravalent pneumococcal vaccine conjugated to TT (PncT) and a diphtheria-tetanus-pertussis–poliovirus–Haemophilus influenzaetype b-tetanus conjugate vaccine showed significantly lower anti-H. influenzaetype b polysaccharide (polyribosylribitol phosphate [PRP]) antibody concentrations than those receiving either a tetravalent pneumococcal vaccine conjugated to diphtheria toxoid or placebo. A dose range study showed that anti-PRP antibody concentrations were inversely related to the TT content of the PncT vaccines administered in infancy. Postimmunization antitetanus antibody concentrations were also affected adversely as the TT content of the coadministered vaccines was increased. This phenomenon, which we believe derives from interference by a common protein carrier, should be taken into account when the introduction of an immunization program including multiple conjugate vaccines is considered.
Expansion of Vγ9Vδ2 T Cells Is Triggered byFrancisella tularensis-Derived Phosphoantigens in Tularemia but Not after Tularemia Vaccination
Authors: Poquet Yannick , Kroca Michal , Halary Franck , Stenmark Stephan , Peyrat Marie-Alix , Bonneville Marc , Fournié Jean Jacques , Sjöstedt Anders
Published: December 2019

Tularemia is a disease caused by the facultative intracellular bacteriumFrancisella tularensis. Here we demonstrate that during the first weeks of infection, a significant increase in levels of Vγ9Vδ2 cells occurred in peripheral blood: in 13 patients analyzed 7 to 18 days after the onset of disease, these lymphocytes represented, on average, 30.5% of CD3+cells and nearly 100% of γδ+T cells. By contrast, after vaccination with the live vaccine strain (LVS) ofF. tularensis, only a minor increase occurred. Eleven days after vaccination, γδ T cells represented an average of 6.7% and Vγ9Vδ2 cells represented an average of 5.3% of T cells, as in control subjects. Since derivatives of nonpeptidic pyrophosphorylated molecules, referred to as phosphoantigens, are powerful stimuli for Vγ9Vδ2 cells, this observation prompted an investigation of phosphoantigens inF. tularensisstrains. TheF. tularensisphosphoantigens triggered in vitro a proliferative response of human Vγ9Vδ2 peripheral blood leukocytes as well as a cytotoxic response and tumor necrosis factor release from a Vγ9Vδ2 T-cell clone. Quantitatively similar phosphoantigenic activity was detected in acellular extracts from two clinical isolates (FSC171 and Schu) and from LVS. Taken together, the chemical nature of the stimulus from the clinical isolates and the significant increase in levels of Vγ9Vδ2 cells in peripheral blood of tularemia patients indicate that phosphoantigens produced by virulent strains ofF. tularensistrigger in vivo expansion of γδ T cells in tularemia.
Generation of Neutralizing Antipeptide Antibodies to the Enzymatic Domain of
Pseudomonas aeruginosa
Exotoxin A
Authors: Elzaim Haissam S. , Chopra Ashok K. , Peterson Johnny W. , Goodheart Rick , Heggers John P.
Published: December 2019

Burn patients suffer a break in the physical barrier (skin), which, when combined with their generalized state of immunodeficiency, creates an open window for opportunistic infections, mainly with
Pseudomonas aeruginosa
. Infection of the burn wound has always been a major factor in retardation of wound healing, and sepsis remains the leading cause of death in burn patients. Because studies have shown that topical treatment with antiexotoxin A (ETA) antibodies significantly increases survival in rats infected with toxin-producing strains of
P. aeruginosa
, we examined 11 synthetic peptides encompassing 12 to 45 amino acid (aa) residues, representing what were predicted by computer analysis to be the most hydrophilic and antigenic regions of ETA. These synthetic peptides were injected into rabbits for antibody production. Different groups of rabbits were immunized with a combination of peptides, with each combination representing one of the three distinct domains of ETA. Animals immunized with various peptide combinations produced peptide-specific antibodies that exhibited cross-reactivity to ETA. Two major epitopes were identified on the ETA molecule by experiments with peptide-specific antibodies in enzyme-linked immunosorbent assay and immunoprecipitation. One of these epitopes was located in the translocation domain (II) (aa 297 to 310), while the other was mapped to the last 13 aa residues at the carboxy-terminal end of the enzymatic domain (III) (aa 626 to 638). Of these two regions, the epitope in the enzymatic domain induced a much higher level of neutralizing antibodies that abrogated the cytotoxic activity of ETA in vitro. Antibodies to this epitope blocked the ADP-ribosyltransferase activity of ETA and appeared to interfere with binding of the substrate elongation factor 2 to the enzymatic active site of the ETA molecule. We conclude that polyclonal, as well as monoclonal, antibodies to short peptides, representing small regions of ETA, may have therapeutic potential in passive immunization or topical treatment of burn patients infected with toxin-producing strains of
P. aeruginosa.
Inactivation of the
gbpA
Gene of
Streptococcus mutans
Increases Virulence and Promotes In Vivo Accumulation of Recombinations between the Glucosyltransferase B and C Genes
Authors: Hazlett Karsten R. O. , Michalek Suzanne M. , Banas Jeffrey A.
Published: December 2019

Glucan-binding protein A (GbpA) of
Streptococcus mutans
has been hypothesized to promote sucrose-dependent adherence and the cohesiveness of plaque and therefore to contribute to caries formation. We have analyzed the adherence properties and virulence of isogenic
gbpA
mutants relative to those of wild-type
S. mutans
. Contrary to expectations, the
gbpA
mutant strains displayed enhanced sucrose-dependent adherence in vitro and enhanced cariogenicity in vivo. In vitro,
S. mutans
was grown in the presence of [
3
H]thymidine and sucrose within glass vials. When grown with constant rotation, significantly higher levels of
gbpA
mutant organisms than of wild type remained adherent to the vial walls. Postgrowth vortexing of rotated cultures significantly decreased adherence of wild-type organisms, whereas the adherence of
gbpA
mutant organisms was unaffected. In the gnotobiotic rat model, the
gbpA
mutant strain was hypercariogenic though the colonization levels were not significantly different from those of the wild type. The
gbpA
mutant strain became enriched in vivo with organisms that had undergone a recombination involving the
gtfB
and
gtfC
genes. The incidence of
gtfBC
recombinant organisms increased as a function of dietary sucrose availability and was inversely correlated with caries development. We propose that the absence of GbpA elevates the cariogenic potential of
S. mutans
by altering the structure of plaque. However, the hypercariogenic plaque generated by
gbpA
mutant organisms may be suboptimal for
S. mutans
, leading to the accumulation of
gtfBC
recombinants whose reduced glucosyltransferase activity restores a less cariogenic plaque structure.
Construction of a Functional Single-Chain Variable Fragment Antibody against Hemagglutinin fromPorphyromonas gingivalis
Authors: Shibata Yasuko , Kurihara Kimiyo , Takiguchi Hisashi , Abiko Yoshimitsu
Published: December 2019

Hemagglutinin is a major glycoprotein ofPorphyromonas gingivalisvesicles and likely confers the ability to adsorb and penetrate into host tissue cells. To protect this bacterial invasion, murine monoclonal antibody (MAb) Pg-vc, which inhibited the hemagglutinating activity, was prepared by usingP. gingivalisvesicles as an antigen. Western blot analysis revealed that when both MAb Pg-vc and anti-HA-Ag2 antibody raised against theP. gingivalishemagglutinin adhesin (M. Deslauriers and C. Mouton, Infect. Immun. 60:2791–2799, 1992) were allowed to react with protein blots fromP. gingivalisvesicles, a superimposable profile was observed. To obtain a recombinant antibody, cDNAs coding for the variable domains of the L and H chains of MAb Pg-vc were cloned by PCR, and a plasmid specifying a single-chain variable fragment (ScFv) was constructed. Following transformation ofEscherichia colicells, a recombinant ScFv protein was successfully expressed. The immunological properties of this protein were identical to those of the parental murine MAb, specifically recognizing the two proteins (43 and 49 kDa) originating fromP. gingivalisvesicles. In addition, the ScFv antibody inhibited theP. gingivalisvesicle-associated hemagglutinating activity. The amino acid sequences deduced from nucleotide sequencing experiments confirmed that variable heavy-chain and variable light-chain regions belonged to VH1 and Vκ12/13 families, respectively. Since the expression system used in this study can readily provide large quantities of single-chain recombinant antibody, it may be a useful in developing a therapeutic agent for passive immunization in humans.
Essential Role of Gamma Interferon in Survival of Colon Ascendens Stent Peritonitis, a Novel Murine Model of Abdominal Sepsis
Authors: Zantl Niko , Uebe Annette , Neumann Brigitte , Wagner Hermann , Siewert Jörg-Rüdiger , Holzmann Bernhard , Heidecke Claus-Dieter , Pfeffer Klaus
Published: December 2019

Despite considerable progress, peritonitis and sepsis remain life-threatening conditions. To improve the understanding of the pathophysiology encountered in sepsis, a new standardized and highly reproducible murine model of abdominal sepsis termed colon ascendens stent peritonitis (CASP) was developed. In CASP, a stent is inserted into the ascending colon, which generates a septic focus. CASP employing a stent of 14-gauge diameter (14G stent) results in a mortality of 100% within 18 to 48 h after surgery. By inserting stents of small diameters, mortality can be exactly controlled. Thus, CASP surgery with insertion of a 22G or 18G stent (22G or 18G CASP surgery) results in 38 or 68% mortality, respectively. 14G CASP surgery leads to a rapid invasion of bacteria into the peritoneum and the blood. As a consequence, endotoxemia occurs, inflammatory cells are recruited, and a systemic inflammatory response syndrome develops. Interestingly, the most pronounced upregulation of inflammatory cytokines (gamma interferon [IFN-γ], tumor necrosis factor alpha [TNF-α] and interleukin-12) is observed in spleen and lungs. CASP surgery followed by stent removal at specific time intervals revealed that all animals survived if intervention was performed after 3 h, whereas removal of the septic focus after 9 h did not prevent death, suggesting induction of autonomous mechanisms of a lethal inflammatory response syndrome. 18G CASP surgery in IFN-γ receptor-deficient (IFNγR−/−) mice revealed an essential role of IFN-γ in survival of sepsis, whereas TNF receptor p55-deficient (TNFRp55−/−) mice did not show altered survival rates. In summary, this study describes a novel animal model that closely mimics human sepsis and appears to be highly suitable for the study of the pathophysiology of abdominal sepsis. Importantly, this model demonstrates a protective role of IFN-γ in survival of bacterial sepsis.
Differential Early Interactions betweenSalmonella entericaSerovar Typhi and Two Other PathogenicSalmonellaSerovars with Intestinal Epithelial Cells
Authors: Weinstein Debra L. , O’Neill Barbara L. , Hone David M. , Metcalf Eleanor S.
Published: December 2019

Salmonella entericaserovar Typhi (hereafter referred to asS. typhi) is a host-restricted pathogen that adheres to and invades the distal ileum and subsequently disseminates to cause typhoid fever in humans. However,S. typhiappears to be avirulent in small animals. In contrast, other pathogenic salmonellae, such asS. entericaserovars Typhimurium and Dublin (S. typhimuriumandS. dublin, respectively), typically cause localized gastroenteritis in humans but have been used as models for typhoid fever because these organisms cause a disease in susceptible rodents that resembles human typhoid. In vivo,S. typhihas been demonstrated to attach to and invade murine M cells but is rapidly cleared from the Peyer’s patches without destruction of the M cells. In contrast, invasion of M cells byS. typhimuriumis accompanied by destruction of these M cells and subsequently sloughing of the epithelium. These data have furthered our view that the early steps in the pathogenesis of typhoidal and nontyphoidalSalmonellaserovars are distinct. To extend this concept, we have utilized an in vitro model to evaluate three parameters of initial host-pathogen interactions: adherence of threeSalmonellaserovars to human and murine small intestinal epithelial cell (IEC) lines, the capacity of these salmonellae to invade IECs, and the ability of the bacteria to induce interleukin-6 (IL-6) in these cell lines as a measure of host cell activation and the host acute-phase response. The results demonstrate thatS. typhiadheres to and invades human small IECs better than eitherS. typhimuriumorS. dublin. Interestingly,invAandinvEnull mutants ofS. typhiare able neither to adhere to nor to invade IECs, unlikeS. typhimurium invAandinvEmutants, which adhere to but cannot invade IECs.S. typhialso induces significantly greater quantities of IL-6 in human small IEC lines than either of the other twoSalmonellaserovars. These findings suggest that differential host cytokine responses to bacterial pathogens may play an important role in the pathological sequelae that follow infection. Importantly,S. typhimuriumdid not induce IL-6 in murine IECs. SinceS. typhimuriuminfection in mice is often used as a model of typhoid fever, these findings suggest that, at least in this case, the mouse model does not reflect the human disease. Taken together, our studies indicate that (i) marked differences occur in the initial steps ofS. typhi,S. typhimurium, andS. dublinpathogenesis, and (ii) conclusions aboutS. typhipathogenesis that have been drawn from the mouse model of typhoid fever should be interpreted conservatively.
A
Neisseria meningitidis fbpABC
Mutant Is Incapable of Using Nonheme Iron for Growth
Authors: Khun Heng H. , Kirby Shane D. , Lee B. Craig
Published: December 2019

The neisserial
fbpABC
locus has been proposed to act as an iron-specific ABC transporter system. To confirm this assigned function, we constructed an
fbpABC
mutant in
Neisseria meningitidis
by insertional inactivation of
fbpABC
with a selectable antibiotic marker. The mutant was unable to use iron supplied from human transferrin, human lactoferrin, or iron chelates. However, the use of iron from heme and human hemoglobin was unimpaired. These results support the obligatory participation of
fbpABC
in neisserial periplasmic iron transport and do not indicate a role for this genetic locus in the heme iron pathway.
Construction and Characterization of an Isogenic
slt-ii
Deletion Mutant of Enterohemorrhagic
Escherichia coli
Authors: Gunzer Florian , Bohn Ursula , Fuchs Sibylle , Mühldorfer Inge , Hacker Jörg , Tzipori Saul , Donohue-Rolfe Arthur
Published: December 2019

Enterohemorrhagic
Escherichia coli
(EHEC) produces Shiga-like toxins (SLT), potent protein synthesis inhibitors. To further dissect the role of SLT-II in the course of disease, we have constructed
E. coli
TUV86-2, an isogenic SLT-II-negative mutant of EHEC strain 86-24. The
slt-ii
gene was inactivated by suicide vector mutagenesis. We also isolated derivatives of strain 86-24 that were cured of the phage carrying the toxin genes.
Stable Transfection ofTrypanosoma cruziEpimastigotes with the Trypomastigote-Specific Complement Regulatory Protein cDNA Confers Complement Resistance
Authors: Norris Karen A.
Published: December 2019

Trypanosoma cruziblood stage trypomastigotes are highly resistant to complement-mediated killing in normal serum. A previously described trypomastigote surface glycoprotein was shown to have binding affinity for human complement components C3b and C4b and restrict activation of the complement cascade, thus preventing lysis of the parasites. Insect stage epimastigotes do not produce detectable levels of this 160-kDa complement regulatory protein (CRP) and are highly sensitive to the lytic effects of complement. Epimastigotes were stably transfected with aT. cruziexpression vector carrying the trypomastigote CRP cDNA and produced fully functional recombinant CRP. The recombinant CRP had binding affinity for C3b, and the transfected epimastigotes were protected from complement-mediated lysis. These results demonstrate for the first time that a developmentally regulated gene ofT. cruzitrypomastigotes can be expressed in noninfectious epimastigotes and that production of CRP by epimastigotes is sufficient to confer a virulence-associated trait. Furthermore, these studies demonstrate the critical role that trypomastigote CRP plays in the protection of parasites from the deleterious effects of complement, thus establishing the protein as a virulence factor ofT. cruzi.
Treatment of Mice with Staphylococcal Enterotoxin B Enhances Resolution of an InducedEscherichia coliUrinary Tract Infection and Stimulates Production of Proinflammatory Cytokines
Authors: Morin Michelle D. , Hopkins Walter J.
Published: December 2019

Staphylococcal enterotoxin B (SEB) is a superantigen that causes mass proliferation of murine Vβ8+T cells via major histocompatibility complex (MHC) class II molecules and leads to their apoptosis or anergy. SEB also stimulates other MHC class II-bearing cells to proliferate and secrete cytokines, some of which might enhance early host defenses against urinary tract infections (UTIs). We investigated the effect of SEB administration on the course of an inducedEscherichia coliUTI in mice. Treatment with SEB 3 or 7 days before the infection had no effect on UTI resolution. However, when SEB was administered at the time of infection, bacterial colonization in the bladders was reduced at time points between 6 h and 3 days. This reduction was not due to a physiological effect, such as increased urinary glycosaminoglycans, or altered pH, nor was SEB bactericidal for the inoculum. Cytokine production in the spleens and bladders of SEB-treated and/or infected mice was evaluated by reverse transcription-PCR. SEB treatment resulted in increased levels of interleukin-2 (IL-2), IL-4, IL-6, and IL-10 mRNAs in the spleen and IL-1α, IL-6, granulocyte-macrophage colony-stimulating factor, and tumor necrosis factor alpha transcripts in the bladder. Also, liver cells from SEB-treated mice expressed IL-6 mRNA, which induces the production of acute-phase proteins. These data indicate that SEB treatment in vivo leads to enhanced UTI resolution through a mechanism that may include direct stimulation of effector cells in the bladder, the action of cytokines induced in the spleen, or cytokine-mediated induction of acute-phase proteins.
Evolution of EnterohemorrhagicEscherichia coliHemolysin Plasmids and the Locus for Enterocyte Effacement in Shiga Toxin-ProducingE. coli
Authors: Boerlin Patrick , Chen Shu , Colbourne John K. , Johnson Roger , De Grandis Stephanie , Gyles Carlton
Published: December 2019

This study assessed the diversity of the enterohemorrhagicEscherichia coli(EHEC) hemolysin gene (ehxA) in a variety of Shiga toxin-producingE. coli(STEC) serotypes and the relationship betweenehxAtypes and virulence markers on the locus for enterocyte effacement (LEE). Restriction fragment length polymorphism of theehxAgene and flanking sequences and of theE. coliattaching and effacing (eae) gene was determined for 79 EHEC hemolysin-positive STEC isolates of 37 serotypes. Two main groups of EHEC hemolysin sequences and associated plasmids, which corresponded to theeae-positive and theeae-negative isolates, were delineated. Comparisons of theehxAgene sequences of representative isolates of each group showed that this gene and the rest of the EHEC hemolysin operon are highly conserved. Digestion of anehxAPCR product with the restriction endonucleaseTaqI showed a unique restriction pattern foreae-negative isolates and another one for isolates of serotypes O157:H7 and O157:NM. A conserved fragment of 5.6 kb with four potential open reading frames was identified on the EHEC hemolysin plasmid ofeae-positive STEC. Phylogenetic analysis of a subset of 27 STEC isolates, one enteropathogenicE. coliisolate, and a K-12 reference isolate showed thateae-positive STEC isolates all belong to a single evolutionary lineage and that the EHEC hemolysin plasmid and theehxAgene evolved within this lineage without recent horizontal transfer. However, theeaegene and the LEE appear to have been transferred horizontally within this STEC lineage on several occasions. The reasons for the lack of transfer or maintenance of the LEE in other STEC lineages are not clear and require further study.
Conformational Dependence ofAnaplasma marginaleMajor Surface Protein 5 Surface-Exposed B-Cell Epitopes
Authors: Munodzana Devere , McElwain Terry F. , Knowles Donald P. , Palmer Guy H.
Published: December 2019

TheAnaplasma marginaleouter membrane is composed of immunogenic major surface proteins (MSPs) linked both covalently and noncovalently in multimeric complexes (M. C. Vidotto, T. C. McGuire, T. F. McElwain, G. H. Palmer, and D. P. Knowles, Infect. Immun. 62:2940–2946). Consequently, effective induction of antibody against surface-exposed MSP epitopes has been postulated to require maintenance of MSP secondary through quatenary structures. Using MSP5 as a model and the approach of epitope mapping with recombinant expressed full-length and truncated proteins, we demonstrated that the immunodominant surface epitope bound by monoclonal antibody (MAb) ANAF16C1 required disparate amino- and carboxy-terminal regions of MSP5, indicating the conformational dependence of this epitope. The required amino-terminal MSP5 region included the cysteines involved in intramolecular disulfide bonding. The dependence of the immunodominant epitope on disulfide bonding was confirmed by loss of MAb ANAF16C1 binding to MSP5 following disulfide bond reduction and covalent modification of the reduced sulfhydryl groups. The recognition of the MSP5 immunodominant epitope by antibody induced by protective immunization withA. marginaleouter membranes was also conformationally dependent, as shown by the loss of epitope binding following serum adsorption with native but not reduced and denaturedA. marginale. Importantly, the antibody response to all immunodominant MSP5 surface epitopes was restricted to conformationally dependent epitopes, since the binding of polyclonal anti-MSP5 antibody to theA. marginalesurface could be blocked by adsorption with native but not denatured and reduced MSP5. These results confirm the importance of the secondary and tertiary structures of MSP epitopes as immune system targets and support the testing of immunogens which maintain the required conformation.
Antibody-Secreting Cells in the Stomachs of Symptomatic and AsymptomaticHelicobacter pylori-Infected Subjects
Authors: Mattsson A. , Quiding-Järbrink M. , Lönroth H. , Hamlet A. , Ahlstedt I. , Svennerholm A.-M.
Published: December 2019

In this study we analyzed whether infection withHelicobacter pylorigives rise to specific B-cell responses against a number of putative virulence factors ofH. pylori, e.g., urease, flagellin, and different bacterial surface antigens, locally in the gastric mucosa. This was studied in antrum and corpus biopsies collected from 11H. pylori-infected patients with duodenal ulcers, 11 asymptomaticH. pyloricarriers, and 13 noninfected, healthy controls. Mononuclear cells were isolated from the biopsies and assayed for frequencies of total andH. pylori-specific antibody-secreting cells (ASCs) by means of the enzyme-linked immunospot technique. TheH. pylori-infected subjects had remarkably higher frequencies of total immunoglobulin A (IgA)- and IgM-secreting cells than the noninfected subjects, while the frequencies of IgG-secreting cells were virtually the same in the different groups. In addition, most of the infected subjects had IgA ASCs reacting withH. pylorimembrane proteins, flagellin, and urease, while none of the noninfected subjects had any detectableH. pylori-reactive ASCs. Furthermore, half of the infected subjects also had ASCs reacting with aHelicobacter-specific 26-kDa protein, while only a few of them had ASCs reacting with neutrophil-activating protein, the neuraminyllactose-binding hemagglutinin HpaA, or lipopolysaccharides purified from differentH. pyloristrains. The frequencies ofH. pylori-specific ASCs in the antrum and corpus were almost identical, and no differences in either antigen specificity or magnitude of the B-cell response in the stomach could be detected between the ulcer patients and the asymptomaticH. pyloricarriers. This study demonstrates thatH. pyloriinfection induces strong antibody responses in the human gastric mucosa, both in asymptomatic carriers and in duodenal ulcer patients. However, the outcome of infection could not be explained by differences in the local B-cell response to any of the antigens used in this study.
Strain-Dependent Activation of Monocytes and Inflammatory Macrophages by Lipopolysaccharide ofPorphyromonas gingivalis
Authors: Shapira Lior , Champagne Catherine , Van Dyke Thomas E. , Amar Salomon
Published: December 2019

Porphyromonas gingivalisis one of the pathogens associated with periodontal diseases, and its lipopolysaccharide (LPS) has been suggested as a possible virulence factor, acting by stimulation of host cells to secrete proinflammatory mediators. However, recent studies have shown thatP. gingivalisLPS inhibited some components of the inflammatory response. The present study was designed to test the hypothesis that there are strain-dependent variations in the ability ofP. gingivalisLPS to elicit the host inflammatory response. By using LPS preparations from two strains ofP. gingivalis, W50 and A7346, the responses of mouse macrophages and human monocytes were evaluated by measuring the secretion of nitric oxide (NO) and tumor necrosis factor alpha (TNF-α). Both direct and indirect (priming) effects were investigated. LPS fromSalmonella typhosawas used as a reference LPS.P. gingivalisA7436 LPS induced lower secreted levels of NO from the tested cells thanS. typhosaLPS but induced similar levels of TNF-α. In contrast, LPS fromP. gingivalisW50 did not induce NO or TNF-α secretion. Preincubation of macrophages with LPS fromS. typhosaorP. gingivalisA7436 prior to stimulation withS. typhosaLPS upregulated NO secretion and downregulated TNF-α secretion, while preincubation withP. gingivalisW50 LPS enhanced both TNF-α and NO secretory responses. These results demonstrate that LPSs derived from different strains ofP. gingivalisvary in their biological activities in vitro. The findings may have an impact on our understanding of the range ofP. gingivalisvirulence in vivo.
Multiple Fimbrial Adhesins Are Required for Full Virulence of
Salmonella typhimurium
in Mice
Authors: van der Velden Adrianus W. M. , Bäumler Andreas J. , Tsolis Renée M. , Heffron Fred
Published: December 2019

Adhesion is an important initial step during bacterial colonization of the intestinal mucosa. However, mutations in the
Salmonella typhimurium
fimbrial operons
lpf
,
pef
, or
fim
only moderately alter mouse virulence. The respective adhesins may thus play only a minor role during infection or
S. typhimurium
may encode alternative virulence factors that can functionally compensate for their loss. To address this question, we constructed mutations in all four known fimbrial operons of
S. typhimurium
:
fim
,
lpf
,
pef
, and
agf
. A mutation in the
agfB
gene resulted in a threefold increase in the oral 50% lethal dose (LD
50
) of
S. typhimurium
for mice. In contrast, an
S. typhimurium
strain carrying mutations in all four fimbrial operons (quadruple mutant) had a 26-fold increased oral LD
50
. The quadruple mutant, but not the
agfB
mutant, was recovered in reduced numbers from murine fecal pellets, suggesting that a reduced ability to colonize the intestinal lumen contributed to its attenuation. These data are evidence for a synergistic action of fimbrial operons during colonization of the mouse intestine and the development of murine typhoid fever.
A Low Concentration of Ethanol Reduces the Chemiluminescence of Human Granulocytes and Monocytes but Not the Tumor Necrosis Factor Alpha Production by Monocytes after Endotoxin Stimulation
Authors: Parlesak Alexandr , Diedrich Jens P. , Schäfer Christian , Bode Christiane
Published: December 2019

The ability of polymorphonuclear neutrophils (PMNs) and monocytes (Mφ) to produce reactive oxygen species (ROS) has been related closely to their potential in the killing of microorganisms. Ethanol has been shown to impair the generation of ROS in these phagocytes after stimulation with some immunogens and to increase the susceptibility of alcohol abusers to infectious diseases. As endotoxemia is common in alcohol abusers, we investigated the effect of ethanol (21.7 mmol/liter) on the luminol-amplified chemiluminescence of PMNs and Mφ after endotoxin stimulation and the release of tumor necrosis factor alpha (TNF-α) from Mφ. Further, the efficiency of ethanol to inactivate chemically generated ROS was tested. Significant stimulation of ROS release occurred at endotoxin concentrations of 1 ng/ml or higher in both PMNs and Mφ. Ethanol significantly suppressed the formation of ROS in both cell types, the decrease being more pronounced in Mφ (−73.8%) than in PMNs (−45.7%). The correlations between endotoxin concentration and the amount of released ROS showed a dose-dependent, sigmoidal course. Concentrations of endotoxin necessary for half-maximum stimulation were nearly identical (6 to 8 ng/ml) in both PMNs and Mφ, independent of the presence of ethanol. In contrast to ROS formation, ethanol had no effect on the amount of TNF-α produced by endotoxin-stimulated Mφ. Ethanol was shown to be unable to decrease the levels of chemically generated ROS under physiological conditions. Therefore, ethanol cannot be assumed to be an “antioxidative” compound but rather seems to modify processes of endotoxin recognition, intracellular signal transduction, or metabolism.
Humoral Immunity toBorrelia burgdorferiN40 Decorin Binding Proteins during Infection of Laboratory Mice
Authors: Feng Sunlian , Hodzic Emir , Stevenson Brian , Barthold Stephen W.
Published: December 2019

ABorrelia burgdorferiN40 genomic expression library was screened with serum from actively infected mice to identify gene products that elicit protective immunity. A clone that contained a putative bicistronic operon containing two genes that encoded 20- and 22-kDa lipoproteins was identified and sequenced. These genes showed homology with the genes encoding decorin binding proteins DbpB and DbpA, respectively, ofB. burgdorferi297 and B31.N40-dbpADNA hybridized withB. burgdorferiN40 DNA on a single 48-kb linear plasmid. Homologous genes could be amplified under various degrees of stringency by PCR or hybridized by Southern blotting fromB. burgdorferisensu stricto N40 and B31, and fromB. burgdorferisensu lato PBi and 25015, but not PKo. Recombinant N40-DbpB and N40-DbpA were reactive with antibody in serum from infected mice, and serum was more reactive against N40-DbpA than againstB. burgdorferiN40 recombinant P39, OspC, or OspA. Sera from mice infected withB. burgdorferisensu lato strains PKo and PBi were weakly reactive against N40-DbpB and N40-DbpA, and sera from mice infected with 25015 were moderately reactive, compared to sera from mice infected withB. burgdorferiN40. Hyperimmunization of mice with N40-DbpA, but not N40-DbpB, induced protective immunity against syringe challenge with culturedB. burgdorferiN40. DbpA may therefore be one of the antigens responsible for eliciting protective antibody known to exist in serum from infected mice. DNA amplification and serology suggest that DbpB and DbpA are likely to have homologs throughout theB. burgdorferisensu lato family, but they are likely to be heterogeneous.
Liposomes Containing Lipid A Serve as an Adjuvant for Induction of Antibody and Cytotoxic T-Cell Responses against RTS,S Malaria Antigen
Authors: Richards Roberta L. , Rao Mangala , Wassef Nabila M. , Glenn Gregory M. , Rothwell Stephen W. , Alving Carl R.
Published: December 2019

Encapsulation of soluble protein antigens in liposomes was previously shown to result in processing of antigen via the major histocompatibility complex class I pathway, as evidenced by costaining of thetrans-Golgi region of murine bone marrow-derived macrophages (BMs) by fluorophore-labeled liposomal antigen and by atrans-Golgi-specific fluorescent lipid. Evidence is presented here that free or liposome-encapsulated RTS,S, a particulate malaria antigen consisting of hepatitis B particles coexpressed with epitopes from thePlasmodium falciparumcircumsporozoite protein, also was localized in thetrans-Golgi after incubation with BMs, suggesting processing by the class I pathway. An in vivo cytotoxic T-lymphocyte (CTL) response was detected, however, only after immunization with RTS,S encapsulated in liposomes containing lipid A and not after immunization with free RTS,S or with RTS,S encapsulated in liposomes lacking lipid A. Therefore, intracellular delivery of antigen containing CTL epitopes to the Golgi of BMs does not necessarily result in a CTL response in vivo unless an additional adjuvant, such as liposomes containing lipid A, is utilized. Encapsulation of RTS,S in liposomes containing monophosphoryl lipid A (MPL) resulted in a dose-dependent enhancement of the NANP-specific immunoglobulin G (IgG) antibody response compared to that of free RTS,S. The IgG1 and IgG2a subclasses predominated after immunization with RTS,S encapsulated in liposomes containing MPL. These results demonstrate that encapsulation of a lipid-containing particulate antigen, such as RTS,S, in liposomes containing lipid A can enhance both humoral and cellular immune responses.
Signal Transduction duringLegionella pneumophilaEntry into Human Monocytes
Authors: Coxon Patricia Y. , Summersgill James T. , Ramirez Julio A. , Miller Richard D.
Published: December 2019

Legionella pneumophilacauses Legionnaires’ disease by replication in alveolar macrophages and monocytes. The bacteria are internalized most efficiently by opsonin-dependent, CR3-mediated phagocytosis. This investigation focused on determining the role of actin polymerization and phosphorylation signals in this uptake mechanism. Uptake inhibition assays and confocal microscopic analysis indicated that entry ofL. pneumophilaactivated tyrosine kinase (TK) and protein kinase C (PKC) and induced actin polymerization at the site of bacterial entry. UponL. pneumophilaentry, six major cellular proteins (75, 71, 59, 56, 53, and 52 kDa) were TK phosphorylated in soluble fractions of monocytes, and three of these proteins (52, 53, and 56 kDa) were consistently found in insoluble (i.e., cytoskeletal) fractions of monocytes as well. Tyrosine phosphorylation was suppressed when cells were pretreated with the kinase inhibitor genistein, tyrphostin, or staurosporine. A similar tyrosine-phosphorylated protein pattern was observed with CR3-mediated entry of avirulentL. pneumophila,Escherichia coli, or zymosan into monocytes. This study has shown that PKC and TK signals which activate actin polymerization during the process of phagocytosis are induced uponL. pneumophilaentry. In addition, CR3 receptor-mediated phagocytosis into monocytes may involve tyrosine phosphorylation of similar proteins, regardless of the particle being phagocytosed. Therefore, the tyrosine-induced phosphorylation observed during opsonizedL. pneumophilaentry is not a virulence-associated event.
Goldfish,
Carassius auratus
, a Novel Animal Model for the Study of
Mycobacterium marinum
Pathogenesis
Authors: Talaat Adel M. , Reimschuessel Renate , Wasserman Steven S. , Trucksis Michele
Published: December 2019

We have developed an animal model for studying mycobacterial pathogenesis using
Mycobacterium marinum
and the goldfish,
Carassius auratus
. Goldfish are injected intraperitoneally with doses between 10
2
and 10
9
CFU of
M. marinum
organisms. Depending on the dose of
M. marinum
organisms administered, an acute or chronic disease is produced. The acute disease is characterized by systemic mycobacterial infection, severe peritonitis, tissue necrosis, and a short median survival time. The chronic disease is characterized by granuloma formation in all organs and survival of animals to the end point of the experiment (56 days). Colony counts in organ homogenates showed recovery of mycobacteria from a high percentage of inoculated animals. We believe this well-characterized animal model will be useful for studying mycobacterial pathogenesis.
Secreted Aspartyl Proteinases and Interactions of
Candida albicans
with Human Endothelial Cells
Authors: Ibrahim Ashraf S. , Filler Scott G. , Sanglard Dominique , Edwards John E. , Hube Bernhard
Published: December 2019

The endothelial cell interactions of homozygous null mutants of
Candida albicans
that were deficient in secreted aspartyl proteinase 1 (Sap1), Sap2, or Sap3 were investigated. Only Sap2 was found to contribute to the ability of
C. albicans
to damage endothelial cells and stimulate them to express E-selectin. None of the Saps studied appears to play a role in
C. albicans
adherence to endothelial cells.
Clearance ofShigella flexneriInfection Occurs through a Nitric Oxide-Independent Mechanism
Authors: Way Sing Sing , Goldberg Marcia B.
Published: December 2019

Nitric oxide (NO) generated by gamma interferon (IFN-γ) activation of macrophages mediates the killing of many intracellular pathogens. IFN-γ is essential to innate resistance toShigella flexneriinfection. We demonstrate that NO is produced followingS. flexneriinfection both in mice and in activated cells in vitro and that while it is able to killS. flexneriin a cell-free system, it is not required for clearance ofS. flexneriin either infected mice or in activated cells in vitro.
Heterogeneous Expression and Release of CD14 by Human Gingival Fibroblasts: Characterization and CD14-Mediated Interleukin-8 Secretion in Response to Lipopolysaccharide
Authors: Sugawara Shunji , Sugiyama Akiko , Nemoto Eiji , Rikiishi Hidemi , Takada Haruhiko
Published: December 2019

To identify the role in periodontal inflammatory diseases of human gingival fibroblasts (HGF), the major constituents of gingival tissue, the expression of CD14, a possible lipopolysaccharide (LPS) receptor, and the release of soluble CD14 (sCD14) by HGF were examined. Among the HGF samples from the nine donors tested, more than 50% of the HGF from five donors expressed CD14 but less than 20% of HGF from the other four donors did so, as determined by flow cytometric analysis. The CD14 expression on the cell surface was correlated with the expression of CD14 mRNA. The HGF and skin and lung fibroblasts tested expressed no CD18, which indicates that fibroblasts do not possess other LPS receptors, such as CD11b/CD18 and CD11c/CD18. The CD14 expression by the HGF was decreased after subculturing and was highest at the confluent stage of culture. The treatment of high-CD14-expressing (CD14high) HGF with phosphatidylinositol-phospholipase C reduced CD14 expression; this result and the increase in a 55-kDa CD14 indicate that the membrane CD14 (mCD14) on the HGF may be a 55-kDa glycosylphosphatidylinositol-anchored protein. CD14highHGF spontaneously released 48- and 57-kDa sCD14. The total release of sCD14 by the HGF was augmented by gamma interferon andEscherichia coliLPS in accordance with the increased expression of mCD14. The CD14highHGF secreted interleukin-8 in response to LPS, and the secretion was completely inhibited by anti-CD14 antibody. These results suggest that (i) HGF consist of populations that are heterogeneous on the basis of different levels of expression of CD14 and (ii) CD14highHGF secrete inflammatory cytokines in response to LPS via CD14.
Role of Alveolar Macrophages in Initiation and Regulation of Inflammation inPseudomonas aeruginosaPneumonia
Authors: Kooguchi Kunihiko , Hashimoto Satoru , Kobayashi Atsuko , Kitamura Yoshihiro , Kudoh Ichidai , Wiener-Kronish Jeanine , Sawa Teiji
Published: December 2019

To evaluate the role of alveolar macrophages (AMs) in acutePseudomonas aeruginosapneumonia in mice, AMs were depleted by aerosol inhalation of liposomes containing clodronate disodium. AM-depleted mice were then intratracheally infected with 5 × 105CFU ofP. aeruginosa. In addition to monitoring neutrophil recruitment and chemokine releases, lung injury was evaluated soon after infection (8 h) and at a later time (48 h). At 8 h, depletion of AMs reduced neutrophil recruitment, chemokine release, and lung injury. At 48 h, however, depletion of AMs decreased bacterial clearance and resulted in delayed movement of neutrophils from the site of inflammation with aggravated lung injury. With instillation of 5 × 107CFU of bacteria, AM-depleted mice showed low mortality within 24 h of infection but high mortality at a later time, in contrast to non-AM-depleted mice. These results demonstrate that depletion of AMs has beneficial early effects but deleterious late effects on lung injury and survival in cases ofP. aeruginosapneumonia.
Intestinal Immune Responses to an Inactivated Oral Enterotoxigenic
Escherichia coli
Vaccine and Associated Immunoglobulin A Responses in Blood
Authors: Åhrén Christina , Jertborn Marianne , Svennerholm Ann-Mari
Published: December 2019

An inactivated oral enterotoxigenic
Escherichia coli
(ETEC) vaccine against ETEC diarrhea was given to 25 adult Swedish volunteers. The vaccine consisted of formalin-killed
E. coli
bacteria expressing the most common colonization factor antigens (CFAs), i.e., CFA/I, -II, and -IV, and recombinantly produced cholera B subunit (CTB). Immunoglobulin A (IgA) antibody responses in intestinal lavage fluid to CTB and CFAs were determined and compared with corresponding responses in stool extracts and serum as well as with IgA antibody-secreting cell (ASC) responses in peripheral blood. Two doses of vaccine induced significant IgA responses to the different CFAs in lavage fluid in 61 to 87% of the vaccinees and in stool in 38 to 81% of them. The most frequent responses were seen against CFA/I. The magnitudes of the antibody responses against CTB and CFA/I in stool correlated significantly (CTB,
P
< 0.01; CFA/I,
P
< 0.05) with those in intestinal lavage. Intestinal lavage responses against CFAs were best reflected by the ASC responses, with the sensitivity of the ASC assay being 80 to 85%, followed by stool (sensitivity of 50 to 88%) and serum antibody (sensitivity of 7 to 65%) analyses. CTB-specific immune responses were seen in >90% of the vaccinees in all assays.