bluuebunny/crossref_metadata_2025 huggingface dataset:
Purification and properties of a 75-kilodalton major protein, an immunodominant surface antigen, from the oral anaerobe Bacteroides gingivalis
Authors: Yoshimura F , Watanabe K , Takasawa T , Kawanami M , Kato H
Published: January 2020

A 75-kilodalton major protein (75K protein) was purified to homogeneity from the cell lysate fraction and the envelope of Bacteroides gingivalis 381. The 75K protein was originally present in the outer membrane or the outermost part of this organism as a large, stable complex with an apparent molecular weight of about 2,000,000. Heating at 80 degrees C and at higher temperatures in the presence of sodium dodecyl sulfate was needed to completely dissociate it to monomers. Amino acid analysis revealed that the 75K protein had about 50% nonpolar amino acids. Various strains of B. gingivalis but not other bacteria, including oral Bacteroides species tested, contained serologically related 75K proteins when tested in Western blotting (immunoblotting) analysis. The abundance and localization of the 75K protein in this organism suggest that it has the potential to participate in the host-parasite interaction in infection. The 75K protein was, indeed, strongly recognized in patients with adult periodontal diseases. Immunoblotting with sera from patients and with rabbit antisera generated by intravenous inoculations of whole B. gingivalis cells revealed that the 75K protein was an immunodominant antigen on the surface of B. gingivalis.
Structures and sugar compositions of lipopolysaccharides isolated from seven Actinobacillus pleuropneumoniae serotypes
Authors: Byrd W , Kadis S
Published: January 2020

Highly purified lipopolysaccharide (LPS) preparations obtained from seven Actinobacillus pleuropneumoniae strains representative of seven different serotypes were used to determine the structure and monosaccharide composition of the polysaccharide components of each lipopolysaccharide. An indication of the structure of each LPS was obtained by procedures that included sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by silver staining and gel chromatographic fractionation of acetic acid-hydrolyzed LPS. The polysaccharide components of the LPSs were analyzed by gas-liquid chromatography. The LPSs of the strains of serotypes 2, 4, and 7 were of the smooth type, and those of the strains of serotypes 3 and 6 were of the rough type; the LPSs of the strains of serotypes 1 and 5 could be considered semirough. Rhamnose was present only in the O polysaccharide of the smooth-type and semirough-type LPSs, whereas galactose was present only in the O polysaccharide of the smooth-type LPS and in the core oligosaccharides of the rough-type and semirough-type LPSs. Glucoheptose and mannoheptose were present in the core oligosaccharides of all the LPSs except for the strain of serotype 3, in which only mannoheptose was detected. N-Acetylglucosamine was detected only in the O polysaccharides of the strains of serotypes 1 and 5.
Cloning and expression of the Pasteurella multocida toxin gene, toxA, in Escherichia coli
Authors: Petersen S K , Foged N T
Published: January 2020

A chromosomal DNA library of a toxigenic type D strain of Pasteurella multocida subsp. multocida was established in Escherichia coli. From this library two clones, SPE308 and SPE312, were identified by using a monoclonal antibody against the osteoclast-stimulating P. multocida toxin (PMT). Extracts of these clones showed cytopathic activity identical to that of extracts of toxigenic P. multocida. The recombinant plasmids, pSPE308 and pSPE312, directed the synthesis of a protein with an apparent molecular weight of 143,000 which could be specifically detected by anti-PMT antibody. The recombinant toxin, which was located in the cytoplasm of E. coli, was purified by affinity chromatography with immobilized monoclonal antibody and was shown to react in a manner identical to that of PMT in a quantitative structural test using a series of monoclonal antibodies as well as in all quantitative functional tests used, i.e., tests for dermonecrotic activity and mouse lethality and the embryonic bovine lung cell test for cytopathic activity. The gene encoding this toxic activity was named toxA and was found to be present in the chromosome of toxigenic strains only of P. multocida. A probe spanning the toxA gene therefore has potential in the diagnosis and surveillance of progressive atrophic rhinitis in pigs.
Molecular cloning and nucleotide sequence of the alpha-toxin (phospholipase C) of Clostridium perfringens
Authors: Titball R W , Hunter S E , Martin K L , Morris B C , Shuttleworth A D , Rubidge T , Anderson D W , Kelly D C
Published: January 2020

A fragment of DNA containing the gene coding for the phospholipase C (alpha-toxin) of Clostridium perfringens was cloned into Escherichia coli. The cloned DNA appeared to code only for the alpha-toxin and contained both the coding region and its associated gene promoter. The nucleotide sequence of the cloned DNA was determined, and an open reading frame was identified which encoded a protein with a molecular weight of 42,528. By comparison of the gene sequence with the N-terminal amino acid sequence of the protein, a 28-amino-acid signal sequence was identified. The gene promoter showed considerable homology with the E. coli sigma 55 consensus promoter sequences, and this may explain why the gene was expressed by E. coli. The cloned gene product appeared to be virtually identical to the native protein. A 77-amino-acid stretch that was close to the N terminus of the alpha-toxin showed considerable homology with similarly located regions of the Bacillus cereus phosphatidylcholine, preferring phospholipase C and weaker homology with the phospholipase C from Pseudomonas aeruginosa.
T-cell-mediated protection of mice against virulent Mycobacterium tuberculosis
Authors: Leveton C , Barnass S , Champion B , Lucas S , De Souza B , Nicol M , Banerjee D , Rook G
Published: January 2020

We sought to protect CBA mice against tuberculosis using in vivo transfer of a T-cell line previously shown to be capable of I-A-restricted recognition of peritoneal macrophages infected in vitro with Mycobacterium tuberculosis. This line induces total bacteriostasis in vitro. In mice that received 500 rads of irradiation 48 h before infection, the T-cell line caused significant prolongation of life when given intravenously with a challenge dose of 5 x 10(6) organisms. Similar experiments with two other T-cell lines showed that these lines offered no protection. Bacterial load at the time of death was inversely related to the time of survival. Thus, death occurred at a lower bacterial load in adoptively protected mice, implying the contribution of an immunopathological component in these animals. The protective T-cell line, which was CD4+ CD8-, had no effect on the rate of growth of strain BCG in CBA nu/nu mice or M. tuberculosis in fully T-cell-deprived mice. This could indicate that CD8+ cells play a role in this system or that there is a need for the recruitment of interleukin 2-producing cells in the recipient. Experiments with monoclonal antibodies to selectively deplete T-cell subsets in normal CBA mice showed that depletion of CD4+ cells strikingly shortened survival, whereas depletion of CD8+ cells did not. However, CD8-depleted mice died with a lower bacterial load than those found in nondepleted controls, and the lesions in CD8-depleted mice were histopathologically distinct. These results suggest that the CD8+ cells either down-regulate bacteriostasis or cause immunopathology in this model and that it is the CD4+ cells that are the major protective subset in long-term protection experiments.
Isolation, structure, and immunogenicity of Pseudomonas aeruginosa immunotype 4 high-molecular-weight polysaccharide
Authors: Pier G B , Pollack M
Published: January 2020

A high-molecular-weight, immunogenic form of the lipopolysaccharide O side chain of Pseudomonas aeruginosa Fisher immunotype 4 (type 4, International Antigenic Typing System 1, Lanyi O:6) was isolated and characterized. Analysis by nuclear magnetic resonance spectroscopy confirmed the structural similarity of this high-molecular-weight polysaccharide and the type 4 O side chain. The polysaccharide was immunogenic in rabbits and mice, eliciting opsonophagocytic killing antibodies. Immunization with the polysaccharide produced significant protection against homologous challenge in both burned and granulocytopenic mice. Naturally acquired opsonic killing antibodies to type 4 polysaccharide were present in sera from unimmunized normal adults at levels comparable to postimmunization levels achieved after immunization with other type-specific polysaccharides. The specificity of the naturally occurring antibodies for the O side chain was documented by immunoblot analysis and inhibition studies. Naturally occurring polysaccharide-specific antibodies were comparable in their protective activity against live challenge in neutropenic animals to immunization-induced murine antibodies with similar specificity. These data suggest that naturally occurring serum antibody to P. aeruginosa type 4 lipopolysaccharide O side chains in most adults is not distinguishable in quantity or quality from immunization-induced antibodies in mice; evaluation of type 4-specific vaccines in humans may be complicated by this finding.
Preliminary evidence that Clostridium perfringens type A enterotoxin is present in a 160,000-Mr complex in mammalian membranes
Authors: Wnek A P , McClane B A
Published: January 2020

Clostridium perfringens type A 125I-enterotoxin (125I-CPE) was bound to rabbit intestinal brush border membranes (BBMs) or Vero cells and then solubilized with 3-[(3-cholamidopropyl)dimethyl-ammonio]-1-propanesulfonate (CHAPS). Solubilized radioactivity was analyzed by gel filtration chromatography on a Sepharose 4B column or by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) without sample boiling and autoradiography. Specifically bound 125I-CPE extracted from either BBMs or Vero cells was primarily associated with a complex of approximately 160,000 Mr. The CPE complex was partially purified by gel filtration or SDS-PAGE without sample boiling. SDS-PAGE analysis with sample boiling of the partially purified 125I-CPE complex from Vero cells or BBMs suggested that CPE complex contains both a 50,000-Mr protein and a 70,000-Mr protein in approximately equimolar amounts. This result is supported by affinity chromatography with CPE immobilized on Sepharose 4B, which showed the specific interaction of similar size proteins with CPE. The simplest explanation for these results is that CPE (Mr 35,000) interacts with 50,000-Mr and 70,000-Mr eucaryotic proteins to form a membrane-dependent complex of approximately 160,000 Mr. These results suggest that the receptor or target site(s) or both for CPE are similar in both BBMs and Vero cells. The significance of these findings in terms of CPE binding, insertion, and biologic action is discussed.
Citrobacter freundii produces an 18-amino-acid heat-stable enterotoxin identical to the 18-amino-acid Escherichia coli heat-stable enterotoxin (ST Ia)
Authors: Guarino A , Giannella R , Thompson M R
Published: January 2020

We purified and sequenced the heat-stable enterotoxin produced by Citrobacter freundii. The toxin was detected during purification by reaction with monoclonal antibody to Escherichia coli heat-stable enterotoxin. The C. freundii toxin amino acid sequence was identical to that of the 18-amino-acid heat-stable enterotoxin (STa) produced by toxigenic E. coli.
Serum antibody responses to the N-acetylneuraminyllactose-binding hemagglutinin of Campylobacter pylori
Authors: Evans D J , Evans D G , Smith K E , Graham D Y
Published: January 2020

We recently reported that Campylobacter pylori possesses a surface-associated fibrillar hemagglutinin which has an affinity for N-acetylneuraminyllactose; this hemagglutinin may function as a colonization factor for attachment to the gastric epithelium. In the present study we examined serum samples obtained from 65 C. pylori-infected individuals with gastric ulcers, duodenal ulcers, or both and from 121 asymptomatic volunteers, including 62 who were infected with C. pylori, for immunoglobulin G (IgG) specific for the C. pylori neuramin-lactose-binding hemagglutinin (NLBH). The NLBH purified from a single isolate was used as the antigen in an enzyme-linked immunosorbent assay. A positive result was obtained with serum from 81.5% (53 of 65) of the individuals with ulcers, 67.7% (42 of 62) of the C. pylori-infected volunteers, and 1.7% (1 of 59) of the C. pylori-negative volunteers. This result confirms that NLBH is produced in vivo. There are several possible explanations for the fact that only 75% of 127 C. pylori-infected individuals were positive for anti-NLBH IgG in serum. We favor the hypothesis that there are probably several antigenically distinct C. pylori NLBHs, i.e., NLBHs which would stimulate antibody that was undetectable by the antigen used in the enzyme-linked immunosorbent assay described here.
Immunohistochemical and electron microscopic study of interaction of Yersinia enterocolitica serotype O8 with intestinal mucosa during experimental enteritis
Authors: Hanski C , Kutschka U , Schmoranzer H P , Naumann M , Stallmach A , Hahn H , Menge H , Riecken E O
Published: January 2020

The experimental infection of mice with Yersinia enterocolitica serotype O8 was investigated in a quantitative and histological study. The course of bacterial penetration and spreading was precisely determined by immunohistochemical staining. After oral administration, the bacteria passed the epithelial barrier of the ileum and spread into the lamina propria. By preference they entered Peyer's patches, which were about 1,000 times more heavily colonized than the surrounding epithelium of a comparable surface area. The bacteria proliferated in the follicles, from which they spread into the lamina propria of the villi. At either site most of the bacteria multiplied extracellularly, with only a small percentage observed to be present within the phagocytes. The bacteria did not appear to be able to pass the intact basement membrane; hence, the integrity of the basement membrane is likely to play a role in determining the route of entry and limit of spread of Y. enterocolitica infection.
Comparison of the biological responses induced by lipopolysaccharide and endotoxin of Treponema hyodysenteriae and Treponema innocens
Authors: Greer J M , Wannemuehler M J
Published: January 2020

The chemical composition and classical biologic activities of lipopolysaccharide (LPS; phenol-water) and endotoxin (butanol-water) preparations from virulent Treponema hyodysenteriae and avirulent Treponema innocens were examined. The LPS and endotoxin preparations from T. hyodysenteriae B204 contained approximately 80.9 and 35.2% hexose, 0.12 and 0.45% thiobarbituric acid-reactive compound, and less than 1 and 11.3% protein, respectively. The LPS and endotoxin preparations of T. innocens B1555a contained approximately 56.3 and 37.8% hexose, 0.45 and 0.4% thiobarbituric acid-reactive compound, and less than 1 and 26% protein, respectively. A silver-stained 7.5 to 15% sodium dodecyl sulfate-polyacrylamide gel showed four bands for the T. hyodysenteriae preparations, while the T. innocens preparations failed to resolve into discrete bands on electrophoresis. We determined by the Limulus amebocyte lysate assay that the treponemal preparations had comparable amounts of endotoxin activity when Escherichia coli LPS was used as a standard. The 50% lethal doses of LPS and endotoxin from T. hyodysenteriae for BALB/cByJ mice were 380 and 80 micrograms, respectively. The treponemal preparations were poor adjuvants, failed to induce a dermal Shwartzman reaction, and were not pyrogenic. The treponemal LPS preparations, unlike the endotoxin preparations, were not mitogenic for murine spleen cells. Differences in virulence between the two treponemal species could not be associated with the biologic activities of the respective LPS or endotoxin moieties, but the endotoxin preparations were consistently more active than the purified LPS preparations.
Dissociation of cell-associated interleukin-1 (IL-1) and IL-1 release induced by lipopolysaccharide and lipid A
Authors: Cavaillon J M , Fitting C , Caroff M , Haeffner-Cavaillon N
Published: January 2020

The capacities of lipopolysaccharide (LPS) and lipid A to trigger mouse BALB/c peritoneal macrophages and to induce the production of cell-associated interleukin-1 (IL-1) and membrane-associated IL-1 and IL-1 release have been compared. Bordetella pertussis lipid A was 1,000 to 10,000 times less efficient than the native LPS to induce IL-1 release by freshly isolated elicited macrophages. When resident macrophages were studied, lipid A, at high concentrations (greater than 2 micrograms/ml), induced significant levels of cell-associated IL-1 but little or no IL-1 release. With synthetic lipid A built up with the Escherichia coli lipid A structure (compound 506), IL-1 activity was present in the supernatants of elicited peritoneal macrophages and to a lesser extent in those of resident macrophages. However, the release of IL-1 induced by synthetic lipid A 506 remained much lower than those induced by rough LPS. Membrane-associated IL-1 could be induced on BALB/c macrophages with LPS and natural or synthetic lipid A, the LPS being the most active. In C3H/HeJ mice, neither natural nor synthetic lipid A could induce detectable cell-associated IL-1, whereas LPS could induce cell-associated and membrane IL-1 activity but no IL-1 release. Our results indicate that fragments of endotoxins may induce the production of IL-1 but the entire structure of the LPS molecule is the most effective to induce intracellular IL-1 production, expression of membrane IL-1, and release of IL-1.
Effect of iron restriction on the outer membrane proteins of Actinobacillus (Haemophilus) pleuropneumoniae
Authors: Deneer H G , Potter A A
Published: January 2020

The outer membrane protein profile of Actinobacillus (Haemophilus) pleuropneumoniae grown under iron-restricted and iron-replete conditions was studied by polyacrylamide gel electrophoresis and immunoblotting. A virulent serotype 1 isolate synthesized a novel protein with an apparent molecular weight of 105,000 (105K) and increased the synthesis of a 76K protein under iron-restricted conditions. Both proteins were synthesized within 15 min of establishment of iron-restricted conditions. Proteins of equivalent molecular weights could also be induced by iron restriction in serotype 2, 3, 4, 5, and 7 isolates of A. pleuropneumoniae. Convalescent-phase sera from serotype 1-infected pigs contained antibodies which recognized both the 105K and 76K proteins from all six serotypes examined, indicating that these proteins were expressed in vivo and were immunologically conserved. Cells expressing the 105K and 76K proteins also displayed an enhanced ability to bind Congo red and hemin, suggesting that one or both of these proteins functioned to acquire complexed iron during in vivo growth.
Quantitative study of the binding and hemolytic efficiency of Escherichia coli hemolysin
Authors: Eberspächer B , Hugo F , Bhakdi S
Published: January 2020

Mono- and polyclonal antibodies were used to construct a sandwich enzyme-linked immunosorbent assay that permitted quantitation of Escherichia coli hemolysin in soluble and membrane-bound forms. Toxin concentrations of 4 to 14 micrograms/ml were measured in culture supernatants of E. coli LE 2001 at times of peak hemolytic activity. Quantitative studies on the binding of E. coli hemolysin to rabbit erythrocytes were conducted at 0 and 37 degrees C. At 37 degrees C, 85 to 95% of bindable toxin was cell bound after 60 min, and no saturability of binding was observed in the studied range of concentrations, which resulted in deposition of approximately 100 to 50,000 toxin molecules per cell. Binding was slower and less effective at 0 degrees C; however, hemolysis did occur at low temperature. The number of cell-bound toxin molecules required to generate a hemolytic lesion within 60 min was estimated to be approximately 100 molecules per cell at 37 degrees C and 800 to 1,000 molecules per cell at 0 degrees C. Upon prolonged incubation (5 to 20 h, 37 degrees C), the number of molecules evoking a functional lesion decreased to approximately 5 to 20 per cell. These results are compatible with the concept that E. coli hemolysin first adsorbs to the cell surface, with membrane insertion and pore formation following in a second step that may be temporally dissociated from that of binding. The data support the pore concept of toxin action by showing that attachment of a low and finite number of toxin molecules to an erythrocyte will ultimately generate a cytolytic lesion.
DNA and protein sequence homologies between the adhesins of Mycoplasma genitalium and Mycoplasma pneumoniae
Authors: Dallo S F , Chavoya A , Su C J , Baseman J B
Published: January 2020

Mycoplasma genitalium and Mycoplasma pneumoniae are morphologically and serologically related pathogens that colonize the human host. Their successful parasitism appears to be dependent on the product, an adhesin protein, of a gene that is carried by each of these mycoplasmas. Here we describe the cloning and determine the sequence of the structural gene for the putative adhesin of M. genitalium and compare its sequence to the counterpart P1 gene of M. pneumoniae. Regions of homology that were consistent with the observed serological cross-reactivity between these adhesins were detected at both DNA and protein levels. However, the degree of homology between these two genes and their products was much higher than anticipated. Interestingly, the A + T content of the M. genitalium adhesin gene was calculated as 60.1%, which is substantially higher tham that of the P1 gene (46.5%). Comparisons of codon usage between the two organisms revealed that M. genitalium preferentially used A- and T-rich codons. A total of 65% of positions 3 and 56% of positions 1 in M. genitalium codons were either A or T, whereas M. pneumoniae utilized A or T for positions 3 and 1 at a frequency of 40 and 47%, respectively. The biased choice of the A- and T-rich codons in M. genitalium could also account for the preferential use of A- and T-rich codons in conservative amino acid substitutions found in the M. genitalium adhesin. These facts suggest that M. genitalium might have evolved independently of other human mycoplasma species, including M. pneumoniae.
Primary structure of the porin protein of Haemophilus influenzae type b determined by nucleotide sequence analysis
Authors: Hansen E J , Hasemann C , Clausell A , Capra J D , Orth K , Moomaw C R , Slaughter C A , Latimer J L , Miller E E
Published: January 2020

Sequencing techniques for single- and double-stranded DNA were used to determine the nucleotide sequence of the gene encoding P2, the major outer membrane (porin) protein of Haemophilus influenzae type b (Hib). The open reading frame encoding the P2 protein comprised 361 amino acid codons. Comparison of the inferred amino acid sequence with data obtained by amino acid sequencing of the N terminus of the mature or fully processed P2 protein revealed that this protein has a signal peptide composed of 20 amino acids. N-terminal amino acid sequencing of tryptic peptides derived from purified P2 allowed direct identification of 158 of the 341 amino acids in the fully processed P2 protein; there was 100% correlation between these amino acid sequences and that inferred from the nucleotide sequence. The amino acid sequence of Hib P2 protein had 23 to 25% homology with the sequence of the OmpF porin of Escherichia coli and with that of the Neisseria gonorrhoeae porin P.IA. Codon usage in the Hib P2 gene was significantly different from that observed for a gene encoding a porin of E. coli. DNA hybridization studies indicated that there is a single copy of the P2 gene in the Hib chromosome. The availability of the nucleotide and amino acid sequences for the Hib P2 protein will facilitate investigation of the antigenic characteristics and structure-function relationship of this porin.
Effects of cyclosporin A on the cells responsible for the anticryptococcal cell-mediated immune response and its regulation
Authors: Fidel P L , Murphy J W
Published: January 2020

Cyclosporin A (CsA), a potent immunosuppressive drug, was used to explore further the induction, expression, and regulation of lymphoid cells involved in the delayed-type hypersensitivity (DTH) response to cryptococcal antigen(s). We found that the induction of the cells responsible for DTH (TDH cells) was not affected by CsA, but their expression was inhibited in CsA-treated mice. The inhibition of expression of the TDH cells could not be attributed to the Cryptococcus neoformans-specific suppressor T (Ts) cells, even though the Ts cells were induced in CsA-treated mice. Instead, the suppressed expression of the TDH cells in CsA-treated mice was a direct effect of CsA or its products. Our studies with CsA also resulted in the first identification of a population of cells that significantly amplify the anticryptococcal DTH response. The amplifier cells were induced in mice that were given a primary immunizing dose of cryptococcal antigen in complete Freund adjuvant, and they amplified the anticryptococcal DTH response in recipient mice when they were transferred at the time of immunization of the recipient. The amplifier cell population was distinct from the TDH cells in that CsA inhibited the production of the amplifying cells but did not affect the induction of TDH cells. Amplification of the DTH response was a cell-mediated event, since cells but not serum from immunized mice mediated the amplified response in recipient mice. Thus, CsA enabled us to characterize anticryptococcal TDH and Ts cells further and to add to the immune cell circuit of the cryptococcal system a distinct population of cells that amplifies the anticryptococcal DTH response.
Bioactivity and immunological characterization of a cholera toxin-cross-reactive cytolytic enterotoxin from Aeromonas hydrophila
Authors: Rose J M , Houston C W , Kurosky A
Published: January 2020

A cytolytic enterotoxin of molecular weight 52,000 was isolated and purified from culture supernatants of a human diarrheal isolate (SSU) of Aeromonas hydrophila. The toxin reacted with cholera antitoxin when tested in an enzyme-linked immunosorbent assay and by Western blot (immunoblot) analysis. The appearance of cytotoxic and hemolytic activities in culture supernatant occurred simultaneously 8 h after the initial inoculation of the culture. Loss of hemolytic activity and cholera toxin cross-reactivity was correlated with heat and pH inactivation. Homologous antibodies neutralized the cytotoxic and hemolytic activities associated with the toxin, but cholera antitoxin did not neutralize these activities. The toxin also possessed enterotoxic activity as demonstrated by fluid accumulation in rabbit ligated intestinal loops. When purified cytolytic enterotoxin was injected intravenously into mice, death occurred within 2 min, whereas mice injected with whole cells or sonicated cell fragments died after several hours or days. Results from 51Cr release experiments demonstrated that the cytolytic enterotoxin had significant membrane-damaging capability. These results indicated that the cytolytic and enterotoxic activities expressed by the described A. hydrophila toxin may contribute significantly to the pathogenesis of disease associated with A. hydrophila.
Bovine lactogenic immunity against cholera toxin-related enterotoxins and Vibrio cholerae outer membranes
Authors: Boesman-Finkelstein M , Walton N E , Finkelstein R A
Published: January 2020

The newly parturient cow secretes large quantities of immunoglobulin G1, a relatively protease- and heat-resistant immunoglobulin, in its colostrum and milk. This study establishes the feasibility of producing protective colostral immunoglobulins by immunizing pregnant cows with cholera toxin (CT), a CT-related enterotoxin from Escherichia coli, and Vibrio cholerae outer membranes (OMs). The OMs were prepared from bacteria grown under iron-replete or iron-deficient (to simulate the in vivo environment) conditions. Immunoglobulins were purified from the colostrum of newly parturient control and immunized cows. The bovine anti-CT and anti-H-LT (CT-related heat-labile enterotoxin produced by diarrheogenic E. coli strains of human origin) antibodies were quantitated by enzyme-linked immunosorbent assays and by neutralization of toxin activity in both Y-1 adrenal cell and infant rabbit assays. The bovine anti-OM antibodies from both high-iron-grown and low-iron-grown vibrios were assessed by bacterial agglutination and by Western blot (immunoblot) analysis of polyacrylamide gel electrophoresis of high-iron-grown and low-iron-grown OMs. To test their protective effect, immunoglobulin preparations were administered orally in infant feeding formula to 6-day-old rabbits. Anti-CT and anti-OM immunoglobulins elicited statistically significant protection against diarrhea in infant rabbits challenged intraintestinally with virulent cholera vibrios.
Immune response to the iron-deprivation-induced proteins of Salmonella typhi in typhoid fever
Authors: Fernandez-Beros M E , Gonzalez C , McIntosh M A , Cabello F C
Published: January 2020

Iron starvation conditions limited the growth of Salmonella typhi, as evidenced by an increase in the lag phase of a culture and a decrease in the number of bacteria reached in the stationary phase. The analysis of the outer membrane of bacteria grown under these conditions identified new protein components with apparent molecular weights of 83,000, 78,000, and 69,000. The extent of induction of these proteins was regulated by increased iron deprivation. Immunoblot analysis showed that the serum of patients with typhoid fever exhibited an immunoglobulin G response to these iron-deprivation-induced proteins. The results of bioassays and DNA-DNA hybridization experiments indicated that pathogenic strains of S. typhi produced enterochelin but not aerobactin. Immunodetection with an anti-FepA antiserum confirmed that one of the induced proteins is the S. typhi analog of the Escherichia coli fepA gene product. These studies suggest a role for iron uptake in the pathogenesis of typhoid fever and confirm the immunogenicity of some of the outer membrane proteins of this pathogen.
Heterogeneity of CD4-positive human T-cell clones which recognize the surface protein antigen of Rickettsia typhi
Authors: Carl M , Vaidya S , Robbins F M , Ching W M , Hartzman R J , Dasch G A
Published: January 2020

Immunity to the typhus group of rickettsiae is largely dependent on the effector function of several classes of T lymphocytes, including those which produce gamma interferon. Since the surface protein antigen (SPA) derived from typhus group rickettsiae has been shown to be an effective immunogen in animal models, human T-cell clones specific for the SPA of Rickettsia typhi were isolated and tested for their antigenic specificity, as well as for their ability to produce gamma interferon. Eighteen CD4-positive clones specific for the SPA of R. typhi exhibited considerable diversity in their response to the SPAs derived from two strains of Rickettsia prowazekii and from Rickettsia canada. The vast majority of clones also recognized the SPAs from R. prowazekii but not from R. canada. Two heteroclitic clones demonstrated significantly higher proliferative responses to the SPAs derived from one or both of the R. prowazekii strains than to the SPA of R. typhi, and one clone demonstrated a significantly higher response to the SPA of R. typhi than to the other SPAs. All 18 clones produced gamma interferon in response to SPA stimulation. We conclude that the SPAs from typhus group rickettsiae can elicit both a diverse T-cell response in humans and the efficient stimulation of gamma interferon-mediated immunity.
Use of mitochondrial and ribosomal DNA polymorphisms to classify clinical and soil isolates of Histoplasma capsulatum
Authors: Spitzer E D , Lasker B A , Travis S J , Kobayashi G S , Medoff G
Published: January 2020

We have developed an improved scheme for the classification of environmental and clinical isolates of Histoplasma capsulatum that is based on analysis of mitochondrial DNA (mtDNA) and ribosomal DNA (rDNA). Strains were initially divided into mtDNA groups according to restriction digests of whole-cell DNA and Southern hybridization with cloned mtDNA probes. Strains within a mtDNA class could be further grouped by polymorphisms in rDNA. The majority of soil and clinical isolates from the United States had identical mtDNA patterns; however, rDNA polymorphisms were common in both types of isolates. The combination of mtDNA and rDNA typing described in this report will be useful in resolving questions concerning the epidemiology of H. capsulatum infections.
Nucleotide sequence of the plasminogen activator gene of Yersinia pestis: relationship to ompT of Escherichia coli and gene E of Salmonella typhimurium
Authors: Sodeinde O A , Goguen J D
Published: January 2020

We have determined the nucleotide sequence of the 1.4-kilobase DNA fragment containing the plasminogen activator gene (pla) of Yersinia pestis, which determines both plasminogen activator and coagulase activities of the species. The sequence revealed the presence of a 936-base-pair open reading frame that constitutes the pla gene. This reading frame encodes a 312-amino-acid protein of 34.6 kilodaltons and containing a putative 20-amino-acid signal sequence. The presence of a single large open reading frame is consistent with our previous conclusion that the two Pla proteins which appear in the outer membrane of pla+ Y. pestis are derived from a common precursor. The deduced amino acid sequence of Pla revealed that it possesses a high degree of homology to the products of gene E of Salmonella typhimurium and ompT of Escherichia coli but does not possess significant homology to other plasminogen activators of known sequence. We also identified a transcription unit that resides on the complimentary strand and overlaps the pla gene.
Properties of the major antigens of rat and human Pneumocystis carinii
Authors: Linke M J , Cushion M T , Walzer P D
Published: January 2020

The major rat and human Pneumocystis carinii antigens were analyzed for their susceptibility to treatment with enzymes and other procedures by immunoblotting, immunofluorescence, and light microscopy. Carbohydrate residues were further analyzed by lectin-binding experiments. The 116-kilodalton (kDa) band of rat P. carinii was susceptible to proteolytic (e.g., trypsin) and glycolytic (e.g., Zymolyase) treatments but not to a variety of other procedures (e.g., lipase). This moiety reacted strongly with concanavalin A and wheat germ agglutinin, indicating the presence of mannosyl or glucosyl and N-acetylglucosamine residues. Immunofluorescence staining and surface labeling suggested that the 116-kDa antigen was located on the P. carinii cell wall. The 45- and 50-kDa bands were as sensitive as the 116-kDa band to degradative treatments when studied after immobilization onto nitrocellulose but were more resistant to proteolytic enzymes when studied in situ on whole organisms. These moieties exhibited poor binding to lectins and reactivity by surface-labeling procedures. The 116-kDa band of human P. carinii appeared to be a glycoprotein with characteristics similar to those of its counterpart in rats, whereas the human P. carinii 40-kDa band exhibited protein and carbohydrate properties more closely related to those of the 45- and 50-kDa rat-derived antigens. We conclude that P. carinii antigens are complex glycoproteins and that this information will be helpful in developing strategies for their isolation and purification and study of their function.
Protection of mice against Listeria monocytogenes infection by recombinant human tumor necrosis factor alpha
Authors: Desiderio J V , Kiener P A , Lin P F , Warr G A
Published: January 2020

Recombinant human tumor necrosis factor alpha (rHuTNF-alpha) administered intravenously to mice resulted in enhanced resistance to a lethal challenge infection of Listeria monocytogenes given 24 h later. The observed protection was lost following treatment of the rHuTNF-alpha preparations with rabbit polyclonal antibody rHuTNF-alpha but not with normal rabbit immunoglobulin G.
Human milk immunoglobulin A antibodies to Shigella virulence determinants
Authors: Cleary T G , Winsor D K , Reich D , Ruiz-Palacios G , Calva J J
Published: January 2020

Because human milk is thought to protect infants from shigellosis, we evaluated milk for immunoglobulin A to Shigella virulence determinants. Milk was preincubated to remove antibodies unrelated to each locus of interest, using defined Shigella and E. coli hybrids containing known Shigella genetic segments prior to immunoblotting. The milk could not be shown to contain antibodies to chromosomally encoded virulence loci except for the expected antibodies to the products of the histidine locus. However, all the milk samples contained antibodies to antigens encoded by the large virulence plasmid. The finding of these antibodies suggests a possible mechanism by which human milk might protect infants.
Roles of interferon and cellular adhesion molecules in bacterial activation of human natural killer cells
Authors: Lindemann R A
Published: January 2020

Interaction of lipopolysaccharide (LPS) from enteric and oral bacteria with natural killer (NK) cells enhanced cytotoxicity against NK-sensitive and NK-resistant targets. This activation occurred without expansion of the NK cell population or without changes in the leukocyte function-associated antigen family of cellular adhesion molecule (CAM) expression on NK cells. Significant interferon (IFN) titers were measured in LPS-lymphocyte supernatants, and antibody to IFN-alpha blocked LPS activation. LPS-induced NK cytotoxicity was inhibited by antibodies to individual alpha chains of CAM and, more profoundly, by antibody to the beta chain of CAM. However, LPS, when preincubated with NK cells, did not compete with subsequent anti-CAM antibody binding as detected by flow cytometry. Anti-CAM antibodies had no effect on NK activation by IFN, but antibodies to either CD11a or CD11c abrogated IFN production induced by LPS. These findings suggest that LPS binds NK cells at non-CAM sites, resulting in the release of IFN. IFN then acts in an autocrine manner independent of CAM to enhance NK cytotoxicity. Interaction of anti-CAM antibodies with CAM may provide a negative signal in regulating LPS-induced IFN production.
Cloning and temperature-dependent expression in Escherichia coli of a Legionella pneumophila gene coding for a genus-common 60-kilodalton antigen
Authors: Hoffman P S , Butler C A , Quinn F D
Published: January 2020

All Legionella species express a 60-kilodalton (kDa) protein which contains a genus-specific epitope recognized by murine monoclonal antibody GW2X4B8B2H6. A genomic cosmid library of Legionella pneumophila chromosomal DNA was constructed in pHC79 and screened for 60-kDa antigen-expressing clones with the monoclonal antibody. A 3.2-kilobase EcoRI fragment from cosmid 14B11 expressing a 60-kDa protein was subcloned into pUC19 (pSH16), and deletion of a 1.2-kilobase HindIII fragment (pSH16A) generated a 33-kDa truncated polypeptide no longer reactive with the monoclonal antibody. Southern blot analysis of chromosomal DNA from selected Legionella species restricted with EcoRI and probed with the 1.2-kilobase fragment coding for the carboxyl region of the protein revealed DNA homology which was not observed with DNA from Escherichia coli. Maxicell analysis of pSH16 identified a second polypeptide of approximately 15 kDa expressed from a gene (htpA) upstream of the gene coding the 60-kDa protein (htpB). Both proteins were preferentially synthesized by L. pneumophila following heat shock (temperature shift from 25 to 42 degrees C), and under steady-state growth conditions the relative level of 60-kDa protein was unaffected by temperature. In E. coli, expression of a 60-kDa protein from pSH16 also increased following heat shock (25 to 42 degrees C), but under steady-state conditions expression was temperature dependent. Temperature-dependent expression from pSH16 was not observed in an rpoH (htpR) mutant strain of E. coli. The Legionella 60-kDa protein appears to be a heat shock protein which shares cross-reactive epitopes with the GroEL homolog of E. coli. In addition, a region of htpB encoding the 27-kDa carboxyl portion of the protein containing the monoclonal antibody-reactive epitope also contains DNA sequences unique to and conserved within the genus.
Characterization of a suppressor factor that regulates phagocytosis by macrophages in murine cryptococcosis
Authors: Blackstock R , Hall N K , Hernandez N C
Published: January 2020

A T-suppressor factor which inhibits the phagocytic activity of a macrophage subset has been further characterized. This suppressor factor was first described for a murine model of cryptococcosis but was later found to be common to models of immunologic unresponsiveness. The suppressor factor was produced when suppressor cells were cultured in the presence of specific cryptococcal antigen. It could not be extracted from spleen cells and was not induced by antigen in cultures of lymph node cells. The suppressor factor was filtered through Amicon filters of 100-kilodalton (kDa) exclusion limit but was retained by filters excluding molecules of less than 50 kDa. By Sephadex G-100 chromatography, the factor eluted just ahead of bovine serum albumin (68 kDa). The activity of the suppressor factor could not be inhibited by anticryptococcal antibody, but it was inhibited by anti-I-J alloantiserum of the same genotype as the lymphocyte which produced the factor. Absorption with an encapsulated strain of Cryptococcus neoformans removed the suppressor factor from culture supernatants, while absorption with a nonencapsulated mutant or an unrelated yeast cell had not effect. On the basis of these observations, it was apparent that the suppressor factor was idiotypic in nature and that I-J and/or the I-J-interactive molecule played a role in the function of the suppressor factor. The requirement for antigenic stimulation for the production of suppressor factor in vitro distinguished it from the T-suppressor factor 3 described by others which regulates delayed-type hypersensitivity in cryptococcosis.
Protective effect of recombinant murine granulocyte-macrophage colony-stimulating factor against Pseudomonas aeruginosa infection in leukocytopenic mice
Authors: Tanaka T , Okamura S , Okada K , Suga A , Shimono N , Ohhara N , Hirota Y , Sawae Y , Niho Y
Published: January 2020

The effects of recombinant murine granulocyte-macrophage colony-stimulating factor (rmGM-CSF) against Pseudomonas aeruginosa infection in ICR mice were investigated. Mice were treated with cyclophosphamide (CPA) and were then injected intraperitoneally with rmGM-CSF three times daily, beginning on the day after CPA treatment, for 7 days. The number of peripheral blood leukocytes in both CPA- and rmGM-CSF-treated mice and control CPA-treated mice reached a nadir on day 4, when P. aeruginosa was injected intraperitoneally. The administration of rmGM-CSF significantly increased the proportion of survivors among mice infected with a lethal dose of P. aeruginosa. This effect was further analyzed by monitoring sequential changes in leukocyte count and bacterial growth in various organs. The number of bacteria in the peritoneal cavities, peripheral blood samples, and livers of GM-CSF-treated mice decreased to an undetectable level after a transient increase, and the number was significantly lower than that in control mice. In GM-CSF-treated mice, the neutrophil levels in peripheral blood started to increase 5 days after CPA administration and were consistently higher than those in controls. Furthermore, the neutrophils in GM-CSF-treated mice were more mature morphologically. Thus, the prophylactic effect of rmGM-CSF against P. aeruginosa infection may result from a rapid recovery of myelopoiesis and a partial enhancement of mature neutrophil function.
Cell surface molecules involved in early events in T-cell mitogenic stimulation by staphylococcal enterotoxins
Authors: Vroegop S M , Buxser S E
Published: January 2020

We tested the mitogenic response to staphylococcal enterotoxin (SE) type A and SE type B in spleen cells from five strains of mice and found consistent and significant differences among the strains. We chose to study the mitogenic responses of two of these strains, C58BL/6J and BALB/cJ, in greater detail. We investigated the effects of specific monoclonal antibodies to cell surface determinants on SE-induced mitogenesis. Monoclonal antibodies against Ia (class II major histocompatibility complex) determinants blocked SE-induced mitogenesis. Both I-A and I-E molecules can participate in the stimulation, and in BALB/cJ mice which express both types of class II molecules both must be blocked to prevent mitogenesis. Mitogenesis was not inhibited by monoclonal antibodies specific for class I major histocompatibility complex antigens or monoclonal antibodies specific for Mac-1, Lyt-1, or Lyt-2 cell surface proteins. Monoclonal antibodies specific for the T-cell surface antigens L3T4 and T3 also substantially inhibited SE-induced mitogenesis. This implicates participation of the T-cell antigen receptor complex in stimulation induced by the SEs. Elimination of L3T4+ helper-inducer T cells abolished the mitogenic response of spleen cells to SE. Reconstitution of L3T4-depleted spleen cells with L3T4+ T cells showed that the level of the mitogenic response was directly proportional to the number of L3T4+ cells added. Elimination of Lyt-2+ cells resulted in a 50% decrease in the response to SEs. These results indicate that L3T4+ T cells are required for the mitogenic response to SE, but both L3T4+ and Lyt 2+ T cells participate in SE-induced mitogenesis. Our results suggest that both Ia and the T-cell antigenic receptor complex are involved in SE-induced mitogenesis.
Role of adherence in pathogenesis of Enterococcus faecalis urinary tract infection and endocarditis
Authors: Guzmàn C A , Pruzzo C , LiPira G , Calegari L
Published: January 2020

Enterococcus faecalis strains isolated from urinary tract infections (UTIs) and endocarditis were analyzed for their ability to adhere to urinary tract epithelial cells (ECs) and Girardi heart (GH) and human embryonic kidney (HEK) cell cultures. UTI isolates adhered to urinary tract ECs more efficiently than to the cultured cells, at the same time showing the least affinity for GH cells. In contrast, endocarditis isolates adhered to GH cell cultures more readily than to urinary tract ECs. Moreover, although strains isolated from endocarditis adhered to GH cells more efficiently than those derived from UTI, the latter strains adhered to urinary tract cells better than the former. Studies of the ability of GH and HEK cells to internalize E. faecalis showed that for UTI isolates, 9 to 74% of adhered bacteria were internalized, while for endocarditis isolates, the percentage varied from 76 to 82%. All strains were able to associate with human neutrophils; endocarditis strains, however, associated less efficiently than UTI isolates. Growth in serum raised the adherence of all tested strains by at least 1.5- to 3-fold, with the greatest increase being observed in UTI strain adherence to GH cells (8-fold). In contrast, the association of serum-grown cells with polymorphonuclear leukocytes was reduced by two- to fivefold. In both cases, the observed serum-dependent alterations were cancelled by a few subcultures in brain heart infusion broth. These results indicate that adhesive properties are important virulence factors in the pathogenesis of UTI and endocarditis and also suggest that UTI strains showing the highest invasion and adhesive potential invade the kidneys, cause bacteremia, and, after having expressed the serum-dependent surface modification, colonize the heart.