bluuebunny/crossref_metadata_2025 huggingface dataset:
Group B Streptococcus type II polysaccharide-tetanus toxoid conjugate vaccine
Authors: Paoletti L C , Wessels M R , Michon F , DiFabio J , Jennings H J , Kasper D L
Published: January 2020

Group B streptococci (GBS) are the most common cause of bacterial sepsis and meningitis in neonates in the United States. Although the capsular polysaccharide of GBS is an important virulence factor, it is variably immunogenic in humans. In this report, we have increased the immunogenicity of GBS type II polysaccharide by coupling it to tetanus toxoid (TT). Like other GBS capsular polysaccharides, the type II polysaccharide has side chains terminating in sialic acid. Controlled periodate oxidation of native II polysaccharide resulted in the conversion of 7% of sialic acid residues to an analog of sialic acid, 5-acetamido-3,5-dideoxy-D-galactosyloctulosonic acid. TT was conjugated to free aldehyde groups created on the oxidized sialic acid residues by reductive amination. Serum from rabbits vaccinated with type II-TT conjugate (II-TT) vaccine contained antibodies specific to type II polysaccharide as well as to TT, whereas rabbits vaccinated with uncoupled native type II polysaccharide failed to produce a type-specific antibody response. Antibodies elicited by II-TT vaccine were serotype specific and mediated phagocytosis and killing in vitro of type II GBS by human peripheral blood leukocytes. Serum from rabbits vaccinated with II-TT vaccine provided 100% protection in a mouse model of GBS type II infection. Antibodies induced by II-TT vaccine were specific for the native but not desialylated type II polysaccharide, suggesting that an important antigenic epitope of II-TT vaccine was dependent on the presence of sialic acid. Therefore, the coupling strategy which selectively modified a portion of the sialic acid residues of types II polysaccharide before coupling the polysaccharide to TT preserved the epitope essential to protective immunity and enhanced the immunogenicity of the polysaccharide.
Identification and characterization of a surface protein-releasing activity in Streptococcus mutans and other pathogenic streptococci
Authors: Lee S F
Published: January 2020

Surface proteins of Streptococcus mutans have been reported to be released into the culture filtrate at concentrations that vary with the growth conditions. The reason for this is not clear. The present study attempts to investigate the mechanism of the protein release. The results showed that whole cells and raffinose-stabilized protoplasts of S. mutans NG8, when incubated in buffers, were capable of releasing their surface proteins in a pH-dependent manner with optimal release at pH 5 to 6. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis revealed that the released proteins were very complex. Two proteins, adhesin P1, which has been previously shown to interact with a human salivary agglutinin, and glucosyltransferase have been identified among the released proteins. The release of adhesin P1 and other proteins was found to be inhibited by heat, Cu2+,Zn2+, and thiol-blocking reagents. The inhibition by heat and Cu2+ was irreversible, whereas that by the thiol-blocking reagents was reversible. EDTA, phenylmethylsulfonyl fluoride, and N-p-tosyl-L-lysyl-chloromethyl ketone had no effect on the release of P1, indicating that the release was probably not due to proteolytic activity. Adhesin P1 from Cu(2+)-inactivated S. mutans NG8 protoplasts could be released by mixing with fresh whole cells and protoplasts, but not the culture filtrate, of a P1-negative mutant of NG8, suggesting that the enzyme is located on the cell surface. This P1-releasing activity was also detected in two other strains of S. mutans and one strain each of S. gordonii, S. agalactiae, S. pneumoniae, and S. pyogenes. The biological role(s) of this enzyme activity remains to be determined. However, owing to its ability to release virulent surface proteins from the cell, it may play an important role in cell surface modulation among the pathogenic streptococci.
Molecular analysis of the Haemophilus ducreyi groE heat shock operon
Authors: Parsons L M , Waring A L , Shayegani M
Published: January 2020

Chancroid is a sexually transmitted genital ulcer disease caused by Haemophilus ducreyi. Previously, we developed diagnostic DNA probes for H. ducreyi (L. M. Parsons, M. Shayegani, A. L. Waring, and L. H. Bopp, J. Clin. Microbiol. 27:1441-1445, 1989). In the present study, DNA sequencing of one of the diagnostic probes revealed two adjacent open reading frames (ORFs). These H. ducreyi ORFs and the encoded proteins show significant homology with the groE genes and GroES and GroEL heat shock proteins from several bacterial pathogens and with conserved eukaryotic 60-kDa heat shock proteins. The first H. ducreyi ORF (groES) is preceded by sequences similar to those of the Escherichia coli consensus heat shock promoters and is 288 nucleotides long and is capable of encoding a protein of 10.3 kDa. The second ORF (groEL) is 1,641 nucleotides long and is capable of encoding a protein of 57.8 kDa. Northern (RNA blot) analysis demonstrated the presence of a high level of groE mRNA in exponential-phase H. ducreyi grown in hemin broth at the organism's optimal growth temperature (33 degrees C), with increased levels seen following heat shock. Heat shock also increased the thermostability of the organisms, since stressed cells were more resistant to the lethal effects of rapid chilling. Electrophoretic analysis and immunoblots demonstrated that the predominant protein produced by exponential-phase H. ducreyi was a heat-inducible, immunoreactive protein of approximately 60 kDa (GroEL). Also, H. ducreyi groE mRNA and GroEL were expressed and inducible by heat in E. coli. This is the first report describing the cloning, sequencing, and expression of H. ducreyi protein-encoding genes.
SCID-Hu mice immunized with a pneumococcal vaccine produce specific human antibodies and show increased resistance to infection
Authors: Aaberge I S , Michaelsen T E , Rolstad A K , Groeng E C , Solberg P , Løvik M
Published: January 2020

Seventy-eight severe combined immunodeficiency (SCID) mice were administered intraperitoneally 1 x 10(7) to 9 x 10(7) human peripheral blood mononuclear cells (PBL) in five experiments. Human immunoglobulin G (IgG) was detected in 70 to 88% of these SCID-PBL-Hu mice after cell transplantation, and all four subclasses were present. The total concentration of human IgG varied from less than 1 to 10.2 g/liter. The SCID-PBL-Hu mice with high concentrations of human IgG regularly had mono- or oligoclonal human IgG bands in serum, as demonstrated by agarose gel electrophoresis. Of the SCID-PBL-Hu mice that were immunized with a 23-valent pneumococcal polysaccharide vaccine, 63 to 78% developed a significant human IgG antipneumococcal antibody response, whereas only very low levels of human IgM and no human IgA antipneumococcal antibodies could be detected. Twelve to twenty-two percent of the SCID-PBL-Hu mice showed signs of leakiness; these mice developed a significant mouse IgM antipneumococcal antibody response and no human antibodies. SCID-PBL-Hu mice were challenged intraperitoneally with 10 50% lethal doses of Streptococcus pneumoniae serotype 4 to study the protective effect of immunization with pneumococcal vaccine. The immunized SCID-PBL-Hu mice showed less bacteremia than did all control groups, and survival was 45 to 60%. None of the unimmunized SCID-PBL-Hu mice survived.
Glycoprotein receptors for a heat-stable enterotoxin (STh) produced by enterotoxigenic Escherichia coli
Authors: Hirayama T , Wada A , Iwata N , Takasaki S , Shimonishi Y , Takeda Y
Published: January 2020

Glycoprotein receptors for heat-stable enterotoxin STh of enterotoxigenic Escherichia coli in the rat intestinal cell membrane were identified and characterized. Incubation of rat intestinal cell membranes with radioiodinated N-5-azidonitrobenzoyl-STh[5-19] (125I-ANB-STh[5-19]) followed by photolysis resulted in specific radiolabeling of two distinct proteins with M(r)s of 200,000 (designated STR-200A and STR-200B). STR-200A was found to be composed of two molecules of a protein with an M(r) of 70,000 (70-kDa protein), whereas STR-200B was composed of two different protein molecules with M(r)s of 53,000 (53-kDa protein) and 77,000 (77-kDa protein). These proteins showed no guanylate cyclase activity. The 70-kDa protein was labeled most with 125I-ANB-STh[5-19], suggesting that STR-200A is the main receptor protein in the rat intestinal cell membrane. The carbohydrate moieties of STR-200A and STR-200B were examined by enzymatic deglycosylation. The 70-kDa protein of STR-200A was found to contain N-linked high-mannose-type and/or hybrid-type oligosaccharides, and results suggested that it possesses at least three N glycosylation sites. The 53-kDa protein of STR-200B was found to have an N-linked complex-type oligosaccharide side chain. The deglycosylated 70-kDa protein retained activity for binding to STh, suggesting that the carbohydrate moieties of these receptor proteins are not important for binding with STh.
Similarity between the group B and A streptococcal C5a peptidase genes
Authors: Cleary P P , Handley J , Suvorov A N , Podbielski A , Ferrieri P
Published: January 2020

Group B streptococci (GBS) and group A streptococci (GAS) are known to have surface-associated peptidase activity which specifically cleaves C5a (C5a-ase), the primary chemotaxin for polymorphonuclear leukocytes. Amplification products were obtained from GBS genomic DNA template by using the polymerase chain reaction and primers corresponding to the C5a peptidase gene of GAS (scpA12). A restriction map of the GBS full-length amplified gene (scpB) was developed. The scpB restriction map was found to be highly similar to that of the analogous gene in GAS. A 50-bp deletion was located near the 5' end of scpB in a region where repeat sequences are found in scpA12. Hybridization experiments confirmed that other serotypes of GBS also carry an scpB-like gene. These results show that GBS contain a gene, scpB, which is very similar to that harbored by M12 GAS. The probability that scpB encodes the C5a-ase protein is discussed.
Depletion of tryptophan is not involved in expression of tryptophanyl-tRNA synthetase mediated by interferon
Authors: Flohr T , Bange F C , von Euch A , Kiekenbeck M , Böttger E C
Published: January 2020

Gamma interferon (IFN-gamma) affects tryptophan metabolism by mediating the expression of indoleamine 2,3-dioxygenase and tryptophanyl-tRNA synthetase. In the present study, we investigated the role of indoleamine 2,3-dioxygenase-mediated tryptophan depletion in the induction of tryptophanyl-tRNA synthetase by IFN-gamma. The addition of excess tryptophan to the culture medium did not affect the induction of tryptophanyl-tRNA synthetase by IFN-gamma, indicating that tryptophan degradation is not directly involved in the IFN-gamma-mediated expression of tryptophanyl-tRNA synthetase.
Molecular characterization of a genomic region associated with virulence in Dichelobacter nodosus
Authors: Katz M E , Strugnell R A , Rood J I
Published: January 2020

The major pathogen implicated in footrot, a highly contagious disease of sheep, is the strict anaerobe Dichelobacter nodosus (formerly Bacteroides nodosus). Sequence analysis of a 2,262-bp segment of the D. nodosus genome which is more prevalent in virulent isolates than in other isolates showed the presence of four open reading frames which appeared to have consensus transcriptional and translational start signals. These virulence-associated genes have been designated vapABCD. Two of the three copies of the vap region in the genome of the reference strain D. nodosus A198 were shown to carry all of the vap genes, whereas one copy contained only the vapD gene. The VapD protein was gel purified, shown to contain the predicted amino-terminal sequence, and used to raise rabbit antibodies. Western blots (immunoblots) showed that all of the D. nodosus strains tested that contained the vap region produced the VapD protein. The VapD protein had significant amino acid sequence identity with open reading frame 5 from the cryptic plasmid of Neisseria gonorrhoeae, and the vapBC operon had sequence similarity with the trbH region of the Escherichia coli F plasmid. It is proposed that these gene regions evolved from the integration of a conjugative plasmid from another bacterial species into the D. nodosus chromosome.
Population transcript accumulation of Pseudomonas aeruginosa exotoxin A and elastase in sputa from patients with cystic fibrosis
Authors: Storey D G , Ujack E E , Rabin H R
Published: January 2020

The in vivo regulation of Pseudomonas aeruginosa virulence factors during the chronic lung infections associated with cystic fibrosis is poorly understood. We have developed an approach for the analysis of transcript accumulation of individual virulence factors from the P. aeruginosa populations found in the sputa of patients with cystic fibrosis. This method has been named population transcript accumulation, since we examine the transcript accumulation patterns in RNA extracted from the total bacterial population found in the sputum samples. DNA probes specific for P. aeruginosa elastase (lasB) and exotoxin A (toxA) were used to examine the population transcript accumulation of 21 sputum samples taken from 10 patients. We detected three patterns of population transcript accumulation: lasB and toxA, lasB alone, and neither lasB nor toxA. We also measured the relative levels of elastase and exotoxin A transcript accumulation in 19 of these samples. In the six samples containing both toxA and lasB transcripts, we found that the levels of lasB transcripts were consistently higher than those of toxA. Differences in the stability of the two mRNA species could not completely account for the higher level of lasB population transcript accumulation, since we showed that the mRNA half-life of lasB (11 min) was similar to that of toxA (10 min). Finally, we showed that elastase transcripts could be detected in some samples which contained only mucoid isolates. This finding suggests that both mucoid and nonmucoid populations may be transcribing lasB in the lungs of patients with cystic fibrosis.
Characterization of an amylase-binding component of Streptococcus gordonii G9B
Authors: Scannapieco F A , Haraszthy G G , Cho M I , Levine M J
Published: January 2020

The goal of the present study was to begin characterizing the amylase-binding component(s) on the surface of Streptococcus gordonii G9B. Alkali extracts but not phenol-water extracts of this bacterium inhibited 125I-amylase binding to S. gordonii G9B. To identify the bacterial components involved in amylase binding, the alkali extract was subjected to affinity chromatography on amylase-Sepharose. Immunoblotting with a rabbit antiserum against S. gordonii G9B revealed that a 20-kDa streptococcal component was eluted from the amylase-Sepharose with 1% sodium dodecyl sulfate (SDS), 2 M KSCN, or 0.1 M sodium citrate buffer, pH 4.5. Subsequently, the 20-kDa component was prepared from alkali extracts by electroelution from preparative SDS electrophoresis or by gel filtration chromatography. This component was trypsin sensitive, and an antibody raised against it inhibited the binding of 125I-amylase to S. gordonii G9B. Indirect immunofluorescence microscopy and immunogold electron microscopy demonstrated that both bound amylase and the 20-kDa component were localized to the cell division septum on dividing cells or to polar zones on single cells. In addition, exponentially growing bacteria bound more 125I-amylase than stationary-phase cells did. Collectively, these results suggest that a 20-kDa amylase-binding component is present on the surface of the nascent streptococcal cell wall.
The platelet interactivity phenotype of Streptococcus sanguis influences the course of experimental endocarditis
Authors: Herzberg M C , MacFarlane G D , Gong K , Armstrong N N , Witt A R , Erickson P R , Meyer M W
Published: January 2020

A strain of Streptococcus sanguis that induced rabbit platelets to aggregate in vitro (Agg+ phenotype) was hypothesized to be a more virulent pathogen than an Agg- strain in experimental endocarditis in rabbits. A left ventricular catheter was implanted, and then an Agg+ or Agg- strain was inoculated intravenously. Vegetations formed on the aortic semilunar valves but were unaffected by the duration of implantation of the catheter. Vegetations enlarged by accumulating platelets and their mass increased directly with the duration of endocarditis. Inoculation of the Agg+ strain consistently caused endocarditis with significantly larger vegetations, a more severe clinical course (including febrile episodes, hematological changes, and signs of myocardial ischemia), more gross lesions in major organs, and greater mortality than inoculation with the Agg- strain, saline, or the Agg+ strain pretreated with monospecific rabbit immunoglobulin G or Fab fragments against its platelet aggregation-associated protein (PAAP; class II). In experimental endocarditis, PAAP expressed by Agg+ S. sanguis appeared to be an important virulence factor.
Fusion proteins containing the A2 domain of cholera toxin assemble with B polypeptides of cholera toxin to form immunoreactive and functional holotoxin-like chimeras
Authors: Jobling M G , Holmes R K
Published: January 2020

Cholera enterotoxin (CT) is produced by Vibrio cholerae and excreted into the culture medium as an extracellular protein. CT consists of one A polypeptide and five B polypeptides associated by noncovalent bonds, and CT-B interacts with CT-A primarily via the A2 domain. Treatment of CT with trypsin cleaves CT-A into A1 and A2 fragments that are linked by a disulfide bond. CT-B binds to ganglioside GM1, which functions as the plasma membrane receptor for CT, and the enzymatic activity of A1 causes the toxic effects of CT on target cells. We constructed translational fusions that joined foreign proteins via their carboxyl termini to the A2 domain of CT-A, and we studied the interactions of the fusion proteins with CT-B. The A2 domain was necessary and sufficient to enable bacterial alkaline phosphatase (BAP), maltose-binding protein (MBP) or beta-lactamase (BLA) to associate with CT-B to form stable, immunoreactive, holotoxin-like chimeras. Each holotoxin-like chimera was able to bind to ganglioside GM1. Holotoxin-like chimeras containing the BAP-A2 and BLA-A2 fusion proteins had BAP activity and BLA activity, respectively. We constructed BAP-A2 mutants with altered carboxyl-terminal sequences and tested their ability to assemble into holotoxin-like chimeras. Although the carboxyl-terminal QDEL sequence of the BAP-A2 fusion protein was not required for interaction with CT-B, most BAP-A2 mutants with altered carboxyl termini did not form holotoxin-like chimeras. When holotoxin-like chimeras containing BAP-A2, MBP-A2, or BLA-A2 were synthesized in V. cholerae, they were found predominantly in the periplasm. The toxin secretory apparatus of V. cholerae was not able, therefore, to translocate these holotoxin-like chimeras across the outer membrane.
Lipopolysaccharides of Bacteroides intermedius (Prevotella intermedia) and Bacteroides (Porphyromonas) gingivalis induce interleukin-8 gene expression in human gingival fibroblast cultures
Authors: Tamura M , Tokuda M , Nagaoka S , Takada H
Published: January 2020

Lipopolysaccharides (LPS) prepared from Bacteroides intermedius (Prevotella intermedia) and Bacteroides (Porphyromonas) gingivalis by hot phenol-water extraction induced interleukin-8 (IL-8) mRNA in normal human gingival fibroblast cultures, as demonstrated by Northern (RNA) blot analysis. IL-8 mRNA levels began to increase after a 2-h exposure, reached a maximum after 12 h, and then dropped to the unstimulated level at 48 h. IL-8 mRNA levels were also enhanced in a dose-dependent manner. By contrast, LPS specimens from various Salmonella species with S and R chemotypes and bacterial [corrected] and synthetic lipid A preparations did not increase IL-8 mRNA levels in fibroblasts. Although recombinant human IL-1 alpha induced IL-8 mRNA expression in fibroblast cultures, an antiserum to recombinant human IL-1 alpha did not decrease the IL-8 mRNA accumulation induced by B. intermedius LPS. Fibroblasts primed with natural human gamma interferon (IFN-gamma) expressed higher IL-8 mRNA levels upon stimulation with B. intermedius LPS, but not with Salmonella LPS, compared with nontreated cells. Natural human IFN-beta exhibited a similar priming effect on the fibroblasts, and antiserum to IFN-beta added to the cultures together with B. intermedius LPS decreased the IL-8 mRNA levels. Therefore, endogenous IFN-beta enhanced IL-8 mRNA production in response to B. intermedius LPS in fibroblasts.
Role of the adhesion molecule lymphocyte function associated antigen 1 in toxic shock syndrome toxin 1-induced tumor necrosis factor alpha and interleukin-1 beta secretion by human monocytes
Authors: See R H , Chow A W
Published: January 2020

We previously demonstrated that the induction by staphylococcal toxic shock syndrome toxin 1 (TSST-1) of tumor necrosis factor alpha (TNF-alpha) and interleukin-1 beta (IL-1 beta) secretion by human monocytes requires direct T cell-monocyte contact. In the present study, a role for the adhesion molecule lymphocyte function associated antigen 1 (LFA-1) in TSST-1-induced cytokine secretion by human monocytes among 12 normal healthy donors was investigated. Monoclonal antibodies to the alpha chain (anti-CD11a) and to the beta chain (anti-CD18) of LFA-1 significantly inhibited TSST-1-induced TNF-alpha and IL-1 beta secretion (P < 0.025; Wilcoxon signed-rank test, two tailed), while a control monoclonal antibody directed against the monocyte CD14 antigen had no effect. These results suggest that LFA-1 may play an important role in the secretion of TNF-alpha and IL-1 beta by TSST-1-stimulated human monocytes, likely by promoting cell-cell adhesion between monocytes and lymphocytes.
Characterization of a Borrelia burgdorferi dnaJ homolog
Authors: Anzola J , Luft B J , Gorgone G , Peltz G
Published: January 2020

The gene encoding a Borrelia burgdorferi DnaJ homolog, located immediately 3' of the hsp70 gene, was characterized. Although there is a single copy of the dnaJ gene on the spirochetal chromosome, two distinct dnaJ transcripts are detected in B. burgdorferi RNA. RNA blot analysis indicates that the dnaJ gene can be transcribed alone or as part of a larger transcript containing the hsp70 homolog.
Effect of immunization with Freund's adjuvant and pneumolysin on histologic features of pneumococcal infection in the rat lung in vivo
Authors: Roberts P , Jeffery P K , Mitchell T J , Andrew P W , Boulnois G J , Feldman C , Cole P J , Wilson R
Published: January 2020

Immunization with Freund's adjuvant and pneumolysin and stimulation with Freund's adjuvant alone both reduced the severity of the pneumonia caused by injections of bacteria into the apical lobe bronchi of rats. Neither protocol influenced the incidence of pneumococcal bacteremia. Illness sufficiently severe to require sacrifice was delayed from 2.8 days in nonimmunized animals to 5.7 days in those immunized with Freund's adjuvant and pneumolysin (P < 0.05) and 4.5 days in those stimulated with Freund's adjuvant alone (P, not significant).
Pathogenesis of Providencia alcalifaciens-induced diarrhea
Authors: Albert M J , Alam K , Ansaruzzaman M , Islam M M , Rahman A S , Haider K , Bhuiyan N A , Nahar S , Ryan N , Montanaro J
Published: January 2020

Providencia alcalifaciens is a member of the family Enterobacteriaceae. There are reports that P. alcalifaciens can cause diarrhea, but the mechanism(s) by which it causes diarrhea is known. We studied P. alcalifaciens isolated from a child and two adults with diarrhea for enteropathogenicity. The three isolates did not exhibit any characteristic adherence to cultured HEp-2 cell monolayers, and they did not produce enterotoxins, cytotoxins, or keratoconjunctivitis in the Sereny test. Two isolates invaded cultured HEp-2 cell monolayers, producing localized bacterial clusters and actin condensation. The pattern of actin condensation was different from that produced by enteropathogenic Escherichia coli but similar to that produced by Shigella flexneri. Invasion and actin condensation were poor for the third isolate. Histology of adult rabbit small intestinal loops inoculated with all three isolates revealed bacterial attachment to, penetration of, and microulcer formation on the surface epithelium and hyperemia, edema, and polymorphonuclear cell infiltration of lamina propria. All the isolates produced diarrhea in rabbits with removable intestinal ties, and some of these rabbits developed hindlimb paralysis. Intestinal histology of the rabbits with removable intestinal ties which developed diarrhea showed changes similar to that in adult rabbits on which ileal loop assays had been performed. Transmission electron microscopy of intestinal tissues also confirmed tissue penetration by the isolates. Nerve tissue histology of two rabbits that developed hindlimb paralysis showed focal mononuclear cell infiltration around peripheral nerve sheaths. It is concluded that some strains of P. alcalifaciens are enteropathogenic and that they cause diarrhea by invading the intestinal mucosal epithelium. However, the relevance to human disease of the hindlimb paralysis observed in this animal model is not clear.
Dual roles for class II major histocompatibility complex molecules in staphylococcal enterotoxin-induced cytokine production and in vivo toxicity
Authors: Grossman D , Lamphear J G , Mollick J A , Betley M J , Rich R R
Published: January 2020

The staphylococcal enterotoxins (SE) specifically bind to class II major histocompatibility complex (MHC) proteins, resulting in activation of monocytes and T cells. The SE cause weight loss in mice, which is dependent on T-cell stimulation and tumor necrosis factor alpha (TNF-alpha) production. Here we use a mutant of staphylococcal enterotoxin A that binds class II MHC molecules and activates monocytes but not T cells to evaluate the relative contributions of monocyte- and T-cell-stimulatory activities to in vivo toxicity. The mutant toxin did not cause weight loss in B10. BR mice but did stimulate monocyte TNF-alpha production in vitro, as did the wild-type toxin. Addition of a supernatant from toxin-activated T cells enhanced monocyte-stimulatory activity of both mutant and wild-type toxins fivefold. The effect of the supernatant could be mimicked by recombinant gamma interferon (IFN-gamma) and was inhibited by antibody to IFN-gamma. These results suggest that toxin-induced monocyte TNF-alpha production is upregulated by IFN-gamma, which likely represents the T-cell requirement in SE-mediated weight loss. Our studies thus implicate two distinct class II MHC-dependent signaling pathways for SE, the first involving direct signal transduction through class II MHC molecules mediated by either mutant or wild-type toxin and the second requiring T-cell stimulation by toxin-class II MHC complexes with consequent production of IFN-gamma. We suggest that both pathways are required for optimal monocyte TNF-alpha production in vitro and SE-induced toxicity in vivo.
Intestinal changes associated with expression of immunity to challenge with Eimeria vermiformis
Authors: Rose M E , Millard B J , Hesketh P
Published: January 2020

To provide more information on the mechanisms involved in the immune inhibition of eimeria infections, NIH mice were adoptively immunized against infection with Eimeria vermiformis by the transfer of mesenteric lymph node cells from primed animals and homologously challenged. Subsequent changes in the architecture and cellular composition of the intestine were compared with those observed in similarly challenged susceptible control mice and correlated with the development of the parasite in the two groups. Actively immunized mice were also examined. In adoptively immunized mice, the development of E. vermiformis was inhibited within 3 days of administering the challenge inoculum. Concurrent changes in the intestine included lymphocytic infiltration, crypt hyperplasia, flattening of the crypt epithelium, and a reduction in the number of Paneth cells. Hyperplasia of goblet and pyroninophilic cells in response to challenge, although accelerated and enhanced in adoptively immunized hosts, occurred after the inhibition of the parasites, and mastocytosis was not observed in these animals, findings which suggest that the activities of goblet, pyroninophilic, and mast cells were not instrumental in reducing the numbers of parasites. The intestines of immunized mice contained fewer intraepithelial lymphocytes at the time of inhibition of the parasites than did those of the controls. The protective effects and intestinal changes described above did not differ appreciably from those seen after challenge of mice that had been immunized by infection.
Studies with enteroaggregative Escherichia coli in the gnotobiotic piglet gastroenteritis model
Authors: Tzipori S , Montanaro J , Robins-Browne R M , Vial P , Gibson R , Levine M M
Published: January 2020

Two strains of enteroaggregative Escherichia coli of human origin fed to gnotobiotic piglets caused diarrhea or death in the majority of them. Histological examination revealed moderate hyperemia of the distal small intestine and cecum, swelling of small intestinal villi, and layers of aggregated bacteria stacked together in a mucus gel-like matrix overlying intact epithelium. These findings confirm that enteroaggregative E. coli strains produce distinctive intestinal lesions different from those caused by other major categories of diarrheagenic E. coli.
Alteration of cytoplasmic Ca2+ in resting and stimulated human neutrophils by short-chain carboxylic acids at neutral pH
Authors: Nakao S , Fujii A , Niederman R
Published: January 2020

The results reported here indicate that the short-chain carboxylic acids acetate and propionate stimulate cytoplasmic calcium mobilization in human polymorphonuclear leukocytes, while butyrate and lactate do not. Together with the results of previous work, this indicates that there are at least three classes of short-chain carboxylic acids: those which can alter only cytoplasmic pH (e.g., lactic acid), those which can alter cytoplasmic pH and actin (e.g., butyric acid), and those which can alter cytoplasmic pH, actin, and calcium (e.g., acetate and propionate).
Similarity of Chlamydia pneumoniae strains in the variable domain IV region of the major outer membrane protein gene
Authors: Gaydos C A , Quinn T C , Bobo L D , Eiden J J
Published: January 2020

DNA was amplified by polymerase chain reaction from the gene encoding the major outer membrane protein (MOMP) of Chlamydia pneumoniae in order to examine the relatedness of strains isolated from diverse geographical regions. Primers for this reaction were chosen to span a 207-bp region comparable to that of the fourth variable segment of the MOMP gene of Chlamydia trachomatis. Among C. trachomatis, sequence heterogeneity is characteristic within variable sequence domain IV (VDIV) and correlates with serovar type. In contrast, sequence analysis of polymerase chain reaction products from 13 C. pneumoniae isolates indicated that all tested strains were identical in this segment of the MOMP gene. The predicted amino acid sequences from the C. pneumoniae VDIV gene products shared only 13.3 to 30% homology with published VDIV regions from serovars of C. trachomatis. Homology of these VDIV amino acid sequences with sequences from strains of C. psittaci ranged from 45.7 to 60%. The sequence conservation of the VDIV region of the MOMP gene indicates that C. pneumoniae strains may be more genetically homogeneous than C. trachomatis or Chlamydia psittaci strains. Future investigations of antigenic diversity among C. pneumoniae strains should be aimed at the evaluation of variation in other regions of the C. pneumoniae genome.
Involvement of Pf155/RESA and cross-reactive antigens in Plasmodium falciparum merozoite invasion in vitro
Authors: Wåhlin B , Sjölander A , Ahlborg N , Udomsangpetch R , Scherf A , Mattei D , Berzins K , Perlmann P
Published: January 2020

Lines of Plasmodium falciparum FCR3 either expressing or not expressing the blood-stage antigen Pf155/RESA were used to analyze the possible involvement of this antigen in the merozoite invasion process in vitro. Antibodies from human sera, affinity purified on synthetic peptides corresponding to C-terminal repeated sequences in Pf155/RESA, were shown to inhibit merozoite invasion of both types of parasites with similar efficiency. Reversal of the invasion inhibition by fusion proteins containing repeated sequences of Pf155/RESA but not of the cross-reactive antigens Ag332 and Pf11.1 indicated that the inhibitory antibodies had similar target antigens in both Pf155/RESA+ and Pf155/RESA- parasites that involved cross-reacting epitopes present in Pf155/RESA. Rabbit antibodies specific for Pf155/RESA repeats inhibited merozoite invasion of Pf155/RESA expressing parasites efficiently but had no or very small effect on the invasion of Pf155/RESA-deficient parasites. In contrast, rabbit antibodies specific for Ag332 repeats as well as human antibodies affinity purified on synthetic Ag332 peptides inhibited merozoite invasion of both types of parasites with high efficiency. A similar inhibition pattern was seen with the human monoclonal antibody 33G2, which has specificity for Ag332 but also cross-reacts with Pf155/RESA and Pf11.1. Taken together, our data suggest that Pf155/RESA and related cross-reactive antigens as well as Ag332 are involved in the merozoite invasion process and may constitute targets for invasion inhibitory antibodies.
Molecular chimerization of Pasteurella haemolytica leukotoxin to interleukin-2: effects on cytokine and antigen function
Authors: Hughes H P , Campos M , Potter A A , Babiuk L A
Published: January 2020

A chimeric recombinant protein composed of the lktA gene product from Pasteurella haemolytica fused to bovine interleukin-2 (IL-2) was made. The LKT-IL-2 chimera was compared with recombinant bovine IL-2 with regard to the ability to induce proliferative responses and LAK cell activity in bovine peripheral blood mononuclear cells in vitro. In both instances, chimerization had no effect on IL-2 activity. Similarly, the LKT component was unaffected in its ability to induce an effective immune response after immunization. The adjuvant properties of IL-2 have been established in a number of models, and this effect was tested by using the chimera. A multiple-injection protocol of LKT-IL-2 was compared with single-dose administration of LKT. The results obtained indicate that while there was no increase in specific antibody production, the IL-2 component of the chimera may be able to affect antigen-specific proliferation, as assessed by limiting-dilution analysis. Use of cytokine-antigen chimeras may provide a valuable antigen-adjuvant formulation that is simple to produce and purify and thus have economic advantages over conventional preparations. Furthermore, chimerization will also ensure that the adjuvant acts at the same site as the antigen, thus optimizing immunostimulatory activity.
Helicobacter mustelae isolation from feces of ferrets: evidence to support fecal-oral transmission of a gastric Helicobacter
Authors: Fox J G , Paster B J , Dewhirst F E , Taylor N S , Yan L L , Macuch P J , Chmura L M
Published: January 2020

Helicobacter mustelae has been isolated from stomachs of ferrets with chronic gastritis and ulcers. When H. mustelae is inoculated orally into H. mustelae-negative ferrets, the animals become colonized and develop gastritis, a significant immune response, and a transient hypochlorhydria. All of these features mimic Helicobacter pylori-induced gastric disease in humans. Because the epidemiology of H. pylori infection is poorly understood and its route of transmission is unknown, the feces of weanling and adult ferrets were cultured for the presence of H. mustelae. H. mustelae was isolated from the feces of 11 of 36 ferrets by using standard helicobacter isolation techniques. H. mustelae was identified by biochemical tests, ultrastructural morphology, reactivity with specific DNA probes, and 16S rRNA sequencing. H. mustelae was not recovered from 20-week-old ferrets which had been H. mustelae positive as weanlings, nor was H. mustelae recovered from 1-year-old ferrets. Isolation of H. mustelae from feces may correspond to periods of transient hypochlorhydria, or H. mustelae may be shed in feces intermittently. The H. mustelae-colonized ferret provides an ideal model for studying the pathogenesis and transmission of H. pylori-induced gastric disease.
Identification of the protective 44-kilodalton recombinant antigen of Ehrlichia risticii
Authors: Shankarappa B , Dutta S K , Mattingly-Napier B
Published: January 2020

Protective studies were conducted with mice by using recombinantly produced antigens, polyacrylamide gel electrophoresis-fractionated antigens, and a monoclonal antibody specific to the 28-kDa antigen of Ehrlichia risticii. Analysis of E. risticii-infected cell culture used as the challenge inoculum indicated an inverse relationship between the progression of cell culture infection and the infective capability of E. risticii for mice. A recombinant 44-kDa antigen was found to protect mice considerably against challenge infection, while the monoclonal antibody and fractionated antigens were not protective. A potentiation of protection was observed when the recombinant 44-kDa antigen was combined with the recombinant 70-kDa antigen and used for mouse immunization.
Insertion sequence IS1016 and absence of Haemophilus capsulation genes in the Brazilian purpuric fever clone of Haemophilus influenzae biogroup aegyptius
Authors: Dobson S R , Kroll J S , Moxon E R
Published: January 2020

Brazilian purpuric fever (BPF) strains of Haemophilus influenzae biogroup aegyptius form a clone of organisms distinct from more innocuous, conjunctivitis-associated isolates. There has been controversy over whether the virulence of BPF strains might derive from the presence of a polysaccharide capsule analogous to that found in conventional invasive H. influenzae, a controversy fuelled by the observation (G. M. Carlone, L. Gorelkin, L. L. Gheesling, A. L. Erwin, S. K. Hoiseth, M. H. O. Mulks, S. P. Connor, R. S. Weyant, J. Myrick, L. Rubin, R. S. Mumford III, E. H. White, R. J. Arko, B. Swaminathan, L. M. Graves, L. W. Mayer, M. K. Robinson, S. P. Caudill, and the Brazilian Purpuric Fever Study Group, J. Clin, Microbiol. 27:609-614, 1989) that a capsulation DNA probe from H. influenzae type b hybridized uniquely to BPF strains. In this work, the basis for this hybridization has been established as the possession by BPF strains, but not by non-BPF strains, of the Haemophilus insertion element IS1016. Although IS1016 is associated with the capsulation locus in some Haemophilus spp., a Southern hybridization study suggests that in BPF strains there are no capsulation genes.
Binding of cultured mammalian cells to immobilized bacteria
Authors: Leong J M , Moitoso de Vargas L , Isberg R R
Published: January 2020

The invasin protein of Yersinia pseudotuberculosis binds to integrin receptors on mammalian cells and promotes cellular penetration. We demonstrate here that the cell attachment activity of invasin can be detected in bacterial colonies that have been immobilized on filter membranes. Invasin expressed in either Escherichia coli K-12 or Y. pseudotuberculosis mediated binding to membranes, and as few as 10(5) Y. pseudotuberculosis resulted in detectable attachment of cultured epithelial cells. A similar binding activity was detected in clinical isolates of the related pathogen Y. enterocolitica but not in environmental isolates. Although there exist multiple mechanisms for the binding of wild-type organisms to host cells, efficient mammalian cell binding to immobilized Y. pseudotuberculosis required expression of a functional invasin protein. Several pathogens that are known to bind or penetrate mammalian cells were also tested, and only one of these bound cultured mammalian cells efficiently.
CD8+ cells and natural cytotoxic activity among spleen, blood, and heart lymphocytes during the acute phase of Trypanosoma cruzi infection in rats
Authors: Sato M N , Yamashiro-Kanashiro E H , Tanji M M , Kaneno R , Higuchi M L , Duarte A J
Published: January 2020

The infection developed by Wistar Furth rats inoculated with the Y strain of Trypanosoma cruzi was the experimental model used in our study. The results showed that this infection altered considerably the CD4/CD8 lymphocyte subset ratio and the natural cytotoxic activity of mononuclear cells in the spleen, blood, and myocardial tissue. Concomitantly, an expansion of the number of cells expressing major histocompatibility complex (MHC) class II antigens was observed, as well as spontaneous development of high levels of blast cells, mainly in the spleen. The inflammatory infiltration of the myocardium, made up essentially of CD8+ cells (cytotoxic/suppressor T cells, natural killer cells), was initially found at 9 days postinfection, spread continuously, and was observed until the death of the animals at about 18 days postinfection. T. cruzi infection also enhanced the natural killer activity of mononuclear cells in the blood, spleen, and myocardium. Sorting these cells by affinity columns showed that the natural killer function was performed exclusively by the CD8+ population, which did not express MHC class II antigens. It was shown that the polyclonal T-lymphocyte activation induced by T. cruzi infection results in a wide distribution of CD8+ cells with enhanced natural cytotoxic activity in the spleen, blood, and cardiac tissue.
Longitudinal study of Plasmodium falciparum polymorphic antigens in a malaria-endemic population
Authors: Conway D J , Greenwood B M , McBride J S
Published: January 2020

Plasmodium falciparum merozoite surface antigens MSP1 and MSP2 and an exported antigen, Exp-1, exhibit allelic polymorphism in natural populations. To explain this, one hypothesis is that antigen polymorphisms are maintained by frequency-dependent immune selection. An expectation of the hypothesis is that rare variants have an advantage over common variants because of a lower level of acquired immunity against them and thus increase in frequency until an equilibrium is attained. To test this hypothesis, the frequencies of polymorphic epitopes of MSP1, MSP2, and Exp-1 were determined among isolates from malaria patients in an urban area of The Gambia, during different periods of one transmission season (1988) and in different years (1982, 1983, 1988, and 1989). The frequencies remained very stable throughout the period of study, alternative epitope variants remaining either rare or common, without shifts in relative frequencies. These results are discussed with reference to the immune-selection hypothesis, with the conclusion that frequencies of the major dimorphic serological classes of MSP1 are probably not maintained by immune selection.
Partial characterization and staphylocidal activity of thrombin-induced platelet microbicidal protein
Authors: Yeaman M R , Puentes S M , Norman D C , Bayer A S
Published: January 2020

Thrombin-induced platelet microbicidal protein (PMP) is considered to play an important role in preventing an important role in preventing streptococcal endocarditis. However, the structural features and functions of PMPs have not been well characterized, and their antibacterial spectra against other common endocarditis pathogens, such as the staphylococci, are not known. Thrombin stimulation of washed rabbit platelets (10(8)/ml) yielded a PMP-rich preparation with a specific activity of approximately 25 U/mg of protein as determined by Bacillus subtilis bioassay. Twenty-eight clinical and laboratory Staphylococcus aureus isolates, exposed to a standardized PMP preparation (100 U/ml for 2 h at 37 degrees C), exhibited a Poisson-distributed heterogeneity to the bactericidal action of PMP, with approximately one-third designated as PMP resistant. Gel filtration chromatography (Sephadex G-50) identified the bioactive moiety within PMP preparations to be in the major protein elution peak; sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) presumptively identified PMP as a low-molecular-weight (MW) (8,500) protein present only in such bioactive protein peaks. Both the bioactivity of PMP preparations and the low-MW protein band were removable by specific anionic membranes (e.g., cellulose-acetate/nitrate), as well as by a variety of anionic resins, further corroborating the suspected cationic charge of PMP. In addition, both PMP bioactivity and the low-MW protein band were recoverable by 1.5 M NaCl elution of the anionic membrane filters post-PMP adsorptive removal. Adsorption of bioactive PMP preparations by highly PMP-susceptible B. subtilis (10(8) CFU/ml, 30 min) resulted in a near-complete loss of residual bioactivity; in contrast, adsorption of bioactive PMP preparations with less PMP-susceptible S. aureus strains failed to reduce bioactivity. Significant lysozyme contamination of PMP-rich preparations was ruled out by determination of differences between bioactive PMP preparations and exogenous lysozyme as regards (i) relative heat stabilities; (ii) differential bactericidal activity versus B. subtilis and Micrococcus luteus; and (iii) SDS-PAGE protein profiles. These data show that the bioactive PMP protein moiety is of low MW, is heat stable, is probably cationic (similar to leukocyte-derived defensins), and possesses potent bactericidal activity against a significant percentage of S. aureus isolates.
Characterization of a cryptic gene pair from Neisseria gonorrhoeae that is common to pathogenic Neisseria species
Authors: Seifert H S , Wilson D
Published: January 2020

A pair of genes, each of which produces in Escherichia coli a 20-kDa, periplasmically localized protein that cross-reacts with anti-rpoN monoclonal antibody, was isolated from Neisseria gonorrhoeae. Homologs of the two genes were detected in pathogenic Neisseria species but not in commensal species. These genes are designated cnp1 and cnp2 (cryptic neisserial protein).
Regional and generalized changes in cytosolic free calcium in monocytes during phagocytosis
Authors: Kim E , Enelow R I , Sullivan G W , Mandell G L
Published: January 2020

We measured and visualized cytosolic free calcium ([Ca2+]i) in individual human peripheral blood monocytes during phagocytosis by using the fluorescent indicator fura-2. Monocytes exhibit a rapid rise in [Ca2+]i from a basal level of 75 +/- 11 nM to a peak level of 676 +/- 78 nM (means +/- standard errors of the means; P less than 0.001) by 34 +/- 5 s after contact with opsonized zymosan particles, and a thin rim of high [Ca2+]i was observed surrounding the ingested particle.
Generation of human monoclonal antibodies that confer protection against pertussis toxin
Authors: Zaccolo M , Roggero S , Armellini D , Pegoraro L , Rappuoli R , Malavasi F
Published: January 2020

A panel of human monoclonal antibodies reactive with pertussis toxin has been generated by means of Epstein-Barr virus infection. One of these, the 3F11 monoclonal antibody, showed the ability to neutralize in vitro and in vivo the toxic effects of the toxin. Western blot (immunoblot) analysis located the 3F11 epitope on the S3 subunit.