bluuebunny/crossref_metadata_2025 huggingface dataset:
A monoclonal antibody to a cell wall component of Candida albicans
Authors: Hopwood V , Poulain D , Fortier B , Evans G , Vernes A
Published: January 2020

A heterologous fusion between mouse myeloma cells and rat lymphocytes resulted in the isolation of a rat immunoglobulin M monoclonal antibody with both agglutinating and precipitating activity. Indirect immunofluorescence and direct agglutination tests showed that the corresponding antigen was present in the cell wall of the three Candida species considered to be the most pathogenic, C. albicans, C. tropicalis, and C. glabrata, and also in the cell wall of C. guilliermondii. The antigen appeared to be predominantly polysaccharide in nature. Precipitation by counterimmunoelectrophoresis suggested that the epitope is shared by at least two separate molecules with different electrophoretic mobilities. Presence of this epitope varied from strain to strain within a given species and may be related to the morphological stage in the cell cycle. Antigen was shown to be present in the cytoplasm, in the periplasmic space, and at the cell surface of C. albicans. Indirect immunofluorescence also suggested that antigen is excreted from the cell.
Polyclonal and monoclonal antibody therapy for experimental Pseudomonas aeruginosa pneumonia
Authors: Pennington J E , Small G J , Lostrom M E , Pier G B
Published: January 2020

A human immunoglobulin G preparation, enriched in antibodies to lipopolysaccharide (LPS) Pseudomonas aeruginosa antigens (PA-IGIV) and murine monoclonal antibodies (MAb) to P. aeruginosa Fisher immunotype-1 (IT-1) LPS antigen and outer membrane protein F (porin), were evaluated for therapeutic efficacy in a guinea pig model of P. aeruginosa pneumonia. The concentration of antibodies to IT-1 LPS was 7.6 micrograms/ml in PA-IGIV and 478 micrograms/ml in the IT-1 MAb preparation. No antibody to IT-1 was detected in MAb to porin. For study, animals were infected by intratracheal instillation of IT-1 P. aeruginosa and then treated 2 h later with intravenous infusions of PA-IGIV, IT-1 MAb, or porin MAb. Control groups received intravenous albumin, and routinely died from pneumonia. Both PA-IGIV (500 mg/kg) and IT-1 MAb (greater than or equal to 2.5 mg/kg) treatment resulted in increased survival (P less than 0.01 to 0.001), and also improved intrapulmonary killing of bacteria. Porin MAb failed to protect from fatal pneumonia. IT-1 MAb treatment produced more survivals than did PA-IGIV treatment but only at dosages of MAb resulting in serum antibody concentrations greater than those achieved with PA-IGIV. PA-IGIV and IT-1 MAb demonstrated in vitro and in vivo (posttreatment guinea pig serum) opsonophagocytic activity for the IT-1 challenge strain. However, the polyclonal preparation required complement, whereas the MAb did not. We conclude that passive immunization with polyclonal hyperimmune P. aeruginosa globulin or with MAb to LPS antigens may be useful in the treatment of acute P. aeruginosa pneumonia. The relative efficacies of such preparations may be limited, however, by their type-specific LPS antibody concentrations.
Ocular sensitization of mice by live (but not irradiated) Chlamydia trachomatis serovar A
Authors: Colley D G , Goodman T G , Barsoum I S
Published: January 2020

Ocular exposure of mice to live elementary bodies of Chlamydia trachomatis serovar A results in immunological sensitization of the mice. This reactivity is manifested by the development of early (5 h) and delayed-type (24 h) dermal reactivity and serovar-specific antibody formation against either live or irradiated (100 kilorads) elementary bodies. Parallel ocular exposure of mice to irradiated elementary bodies does not result in this sensitization. The early and late dermal immune responses induced by ocular exposure to live organisms can be transferred to unexposed mice by serum and lymphoid cell transfers, respectively. It appears that successful murine ocular sensitization by human C. trachomatis serovar A elementary bodies is an ability manifested by live organisms and not by inactivated but antigenic organisms.
Association with HeLa cells of Campylobacter jejuni and Campylobacter coli isolated from human feces
Authors: Fauchere J L , Rosenau A , Veron M , Moyen E N , Richard S , Pfister A
Published: January 2020

We developed a rapid in vitro test for determining the association of Campylobacter jejuni and C. coli with HeLa cells. Association was expressed as a weighted mean of the number of bacteria associated with one cell in an association index (AI). The reproducibility of the AI was checked by repeating the test six times, using four strains chosen at random. Means and standard deviations of the means were 7.3 +/- 1.2, 6.8 +/- 0.9, 1.8 +/- 1.2, and 0.1 +/- 0.2. The experimental conditions for which the results are reliable have been standardized. Among 42 strains from human feces, two groups appeared: for 22 nonassociative strains (52%), AI values ranged from 0.0 to 2.1 (mean +/- SD, 0.5 +/- 0.6); for 20 associative strains (48%), AI values ranged from 3.5 to 8.3 (mean +/- SD, 6.2 +/- 1.4). Of these 42 strains, 17 were clinically documented. Diarrhea occurred more frequently in patients infected with associative strains than in those infected with noninvasive strains (7/7 versus 3/10, P = 0.01). Fever also occurred more frequently in patients infected with associative strains (6/7 versus 2/10, P = 0.03). Transmission electron microscopy and viable counts made after killing of extracellular bacteria by gentamicin support the fact that associated Campylobacter spp. are adherent to the cell membrane and are internalized into cytoplasmic vacuoles. The described test seems to be a convenient and rapid method for estimating the pathogenicity of a given strain.
Immune response of athymic and euthymic germfree mice to Campylobacter spp
Authors: Yrios J W , Balish E
Published: January 2020

Pure cultures of several Campylobacter spp. induced a specific humoral immune response after they colonized and infected gnotobiotic mice; however, Campylobacter-immune mouse serum was not bactericidal (in vitro), manifested a weak agglutination reaction (in vitro), and showed specificity (strain 45100-immune mouse sera) for the homologous (infecting) Campylobacter strain, but was not able to passively protect germfree athymic (nu/nu) BALB/c mice against Campylobacter infection and diarrhea. Active immunization of germfree nu/nu mice with Formalin-killed C. jejuni also did not protect the gnotobiotic mice from Campylobacter infection and diarrhea. It appears from the results of our initial gnotobiotic studies that antibodies in serum against the infecting strain of C. jejuni may not play an essential role in resistance to Campylobacter disease in mice.
Interaction of Clostridium difficile and Escherichia coli with microfloras in continuous-flow cultures and gnotobiotic mice
Authors: Wilson K H , Freter R
Published: January 2020

We studied the interactions between the entire cecal flora of hamsters and the pathogens Clostridium difficile and Escherichia coli in gnotobiotic mice and in a continuous-flow (CF) culture system in which the growth medium consisted of an extract of fecal pellets from germfree mice. CF cultures and germfree mice were colonized first with C. difficile and E. coli and then with the cecal flora of hamsters. Both in vivo and in vitro hamster flora markedly suppressed the potential pathogens. Contents of CF cultures inoculated with hamster flora were introduced into gnotobiotic mice previously colonized with C. difficile and E. coli. These mice were compared with mice given homogenates of hamster ceca. In both groups, the C. difficile population decreased by a factor of more than 10(6) and the E. coli population decreased by a factor of 10(4) to 10(5). CF culture contents also reduced the size of the dilated germfree mouse cecum to normal. When veal infusion broth was used as a medium, contents of CF cultures colonized with hamster flora failed to eliminate C. difficile from mice. Thus, the extract of fecal pellets appeared to contain a substance important for sustained colonization by important components of the cecal flora. We also studied the ability of collections of isolates to suppress the potential pathogens in both gnotobiotic mice and CF cultures. A total of 150 isolates obtained from predominant hamster flora at the ecologic climax stage (C flora) suppressed C. difficile and E. coli to 10 and 1 to 3%, respectively, of the population sizes attained in monoassociated mice. A total of 67 isolates obtained during ecologic succession combined with a C flora consisting of 100 isolates suppressed the potential pathogens to 0.3 and 0.03% of their original levels, respectively. Similar degrees of suppression occurred in CF cultures, further indicating that anaerobic CF cultures are promising models for investigation of the microbial ecology of C. difficile.
Type 2 fimbrial lectin-mediated phagocytosis of oral Actinomyces spp. by polymorphonuclear leukocytes
Authors: Sandberg A L , Mudrick L L , Cisar J O , Brennan M J , Mergenhagen S E , Vatter A E
Published: January 2020

Phagocytosis of Actinomyces viscosus T14V and A. naeslundii WVU45 by human polymorphonuclear leukocytes in the absence of antibody or complement was mediated by the lectin associated with the type 2 fimbriae of these bacteria. This effect was markedly enhanced by exogenous sialidase, an enzyme also secreted by these actinomyces. Since sialidase treatment of the bacteria did not result in increased phagocytosis, this enzyme presumably acts by unmasking receptors for the fimbrial lectin on phagocytic cells. The viability of A. viscosus T14V, which possesses type 1 and type 2 fimbriae (1+ 2+), and A. naeslundii WVU45, which possesses only type 2 fimbriae (2+), was decreased by at least 98% following incubation with polymorphonuclear leukocytes in the presence of sialidase. Entirely analogous findings were obtained with a 1- 2+ mutant of A. viscosus T14V. In contrast, the phagocytosis of 1+ 2- and 1- 2- mutants of A. viscosus T14V and a 2- mutant of A. naeslundii WVU45 was minimal or absent. Lactose and beta-methylgalactoside inhibited the destruction of the bacteria, whereas cellobiose and alpha-methylgalactoside were ineffective. Thus, the type 2 fimbriae of the oral actinomyces recognize galactose-containing receptors on polymorphonuclear leukocytes which have been exposed by the removal of sialic acid, an interaction that is followed by internalization and subsequent killing of the bacteria.
The ability to sensitize host cells for destruction by autologous complement is a general property of lipoteichoic acid
Authors: Weinreb B D , Shockman G D , Beachey E H , Swift A J , Winkelstein J A
Published: January 2020

Previous studies have demonstrated that lipoteichoic acid (LTA) from Streptococcus pneumoniae binds to erythrocytes and renders them susceptible to lysis by autologous complement. The present study was performed to determine whether LTA from two other gram-positive bacterial species had the ability to render mammalian cells susceptible to lysis by autologous complement. Human erythrocytes were sensitized with LTA from S. pneumoniae, Streptococcus pyogenes, or Lactobacillus fermentum. Under incubation in normal autologous serum, lysis was observed with each of the LTA-sensitized erythrocyte preparations. When erythrocytes from a C2-deficient patient were sensitized with the LTA preparations and then incubated in autologous, C2-deficient serum, the erythrocytes sensitized with S. pyogenes or L. fermentum LTA demonstrated relatively little lysis, whereas the erythrocytes sensitized with S. pneumoniae LTA yielded near-total lysis. After reconstitution of the C2-deficient serum with purified human C2, lysis was observed with all three LTA preparations. When erythrocytes from an agammaglobulinemic patient were sensitized with either the S. pyogenes or the L. fermentum LTA, they were not lysed in the presence of autologous agammaglobulinemic serum, whereas the erythrocytes sensitized with S. pneumoniae LTA were completely lysed. Serum obtained from the agammaglobulinemic patient after reconstitution with intravenous pooled gamma globulin was able to lyse autologous erythrocytes sensitized with each of the three LTA preparations. These results demonstrate that the ability to render host cells susceptible to lysis by autologous complement is a general property of LTA. Whether activation of the autologous complement occurs by the classical or alternative pathways and whether it is antibody dependent depends on the nature of the bacterial LTA.
Cloning and expression of the major 47-kilodalton surface immunogen of Treponema pallidum in Escherichia coli
Authors: Norgard M V , Chamberlain N R , Swancutt M A , Goldberg M S
Published: January 2020

Monoclonal antibodies directed against the 47-kilodalton (kDa) major outer membrane surface immunogen of virulent Treponema pallidum subsp. pallidum were used to select Escherichia coli recombinant clones expressing the 47-kDa immunogen. The phenotype of the clones was dependent on the presence of recombinant plasmid in the host cell. Southern hybridization revealed that the cloned T. pallidum subsp. pallidum DNA sequence was an accurate representation of the T. pallidum subsp. pallidum genomic DNA arrangement. Purified immunoglobulin G from rabbits experimentally infected with T. pallidum subsp. pallidum and human secondary syphilitic sera specifically reacted with the clones, while normal human serum or immunoglobulin G from normal rabbit serum did not. Results of Southern hybridization indicated that a homologous 47-kDa immunogen gene was absent in at least four species of nonpathogenic treponemes tested, as well as from total rabbit genomic DNA. Rabbit anti-T. phagedenis biotype Reiter (treponemal nonpathogen) antiserum and a monoclonal antibody directed against a common treponemal determinant were unreactive with the clones. Western blotting and radioimmunoprecipitation experiments with specific monoclonal antibodies revealed that the recombinant (E. coli) and native (T. pallidum subsp. pallidum) forms of the antigen had identical electrophoretic mobilities. The availability of recombinant 47-kDa immunogen provides a new opportunity for biochemical analysis of the protein, structure-function studies, examination of its role in microbial pathogenesis, and assessment of its diagnostic and vaccinogenic potentials.
Variation in chemical properties and antigenic determinants among type II heat-labile enterotoxins of Escherichia coli
Authors: Guth B E , Twiddy E M , Trabulsi L R , Holmes R K
Published: January 2020

Type II heat-labile enterotoxin (LT-II) from Escherichia coli 41 was purified and compared with prototype LT-II encoded by genes from E. coli SA53. Both toxins were oligomeric proteins consisting of polypeptides A (Mr, 28,000) and B (Mr, 11,800). The A polypeptides were cleaved by trypsin into fragments A1 (Mr, 21,000) and A2 (Mr, about 7,000). These two toxins were shown to belong to two different subclasses of LT-II. We propose to designate the prototype toxin LT-IIa and the new variant LT-IIb. The pI of LT-IIb was between 5.2 and 5.6, significantly lower than the pI of 6.8 for LT-IIa, and the behavior of LT-IIb during purification differed significantly from that of LT-IIa. The toxic dose of unnicked LT-IIb in the Y1 adrenal-cell assay was 94 pg, but trypsin-treated, nicked LT-IIb was toxic at about 3 pg. In contrast, the toxic dose of LT-IIa was previously shown to be 0.5 to 1 pg for several preparations that varied from unnicked to partially nicked, and treatment with trypsin was not required for full toxicity. The titer of LT-II antiserum in neutralization tests was 100-fold greater against LT-IIa than against LT-IIb. In immunodiffusion tests, LT-IIa and LT-IIb gave a reaction of partial identity. In a radioimmunobinding assay, the titer of LT-IIa antiserum against homologous LT-IIa was approximately 10-fold greater than against LT-IIb. The cholera-E. coli family of heat-labile enterotoxins has been divided into serogroup I, which includes cholera toxin and the antigenic variants of E. coli heat-labile toxin designated LTh-I and LTp-I, and serogroup II, which includes LT-IIa and LT-IIb. The type I and type II toxins do not cross-react in neutralization or immunodiffusion tests. By using very sensitive radioimmunobinding assays, it was possible to demonstrate common antigenic determinants between the type I and type II toxins. However, the titers of antibodies in hyperimmune sera that recognized these common determinants were very low.
Specific attachment of Staphylococcus aureus to immobilized fibronectin
Authors: Maxe I , Rydén C , Wadström T , Rubin K
Published: January 2020

Staphylococcus aureus cells have been shown to possess surface-associated proteins with affinity for soluble fibronectin. We have investigated the ability of these surface proteins to mediate attachment to immobilized fibronectin and collagen. Attachment was quantified by determination of bacterial ATP in a bioluminescence assay. The ability to attach to fibronectin- or collagen-coated plastic surfaces was investigated for four S. aureus strains: Cowan 1, Newman, SA113(83A), and Wood 46. Cells from the different strains varied in their attachment properties, but all cells except those of strain Wood 46 attached readily to substrates coated with fibronectin. Only cells from strain Cowan 1 attached reproducibly to collagen-coated substrates in the absence of fibronectin. The attachment of cells from strain SA113(83A) to fibronectin-coated surfaces was shown to be dependent on time, fibronectin concentration, and bacterial growth phase. Soluble fibronectin or NH2-terminal fibronectin fragment (Mr, 29,000) disturbed the attachment to surfaces coated with fibronectin bound to denatured collagen type I. The attachment process to such substrates was also effectively inhibited by preincubating the substrate with fibronectin-binding proteins isolated from S. aureus Newman and SA113 (83A) and purified with affinity chromatography.
Strain variation in composition and molecular size of the capsular polysaccharide of Cryptococcus neoformans serotype A
Authors: Small J M , Mitchell T G , Wheat R W
Published: January 2020

The capsule of Cryptococcus neoformans is an important virulence factor. In this investigation capsular polysaccharides (CPSs) were isolated by ethanol precipitation from culture filtrates of C. neoformans serotype A strains 6, 15, 98, 110, and 145. Capsule sizes on India ink examination ranged from barely perceptible (strain 15) to greater than the diameter of the yeast cell (strain 6); the others were intermediate in size. On ion-exchange chromatography on DEAE-cellulose each CPS eluted at 0.2 M NaCl; CPS of strain 15 had two major peaks, designated III and IV. On gel-permeation chromatography CPSs of strains 6, 98, 110, and 145 eluted at the void volume of Sepharose CL-2B in the presence or 0.1 M EDTA, while the CPS of strain 15 eluted in two peaks. Sephacryl S-1000 resolved CPSs of all five strains in the following order, from largest to smallest molecular size: 145 greater than 110 greater than 98 greater than 6 much greater than 15. All five CPSs contained mannose, xylose, and glucuronic acid, while the carboxyl-reduced CPS of strain 110 also contained a large percentage of an inositol-like compound. The CPS of strain 110 contained approximately 30% uronic acid by weight, while the others had 15 to 20%. The composition of peak IV from the CPS of strain 15 resembled those of the other strains; peak III of strain 15 contained a substantial amount of galactose. Each CPS contained less than 0.2% protein by weight. The significant differences in molecular size and sugar composition among CPSs of these strains of C. neoformans serotype A may partially explain strain differences in virulence and biological properties of the organism.
Influence of mouse genotype on passive systemic anaphylaxis by immune complexes
Authors: Portolés A , Rojo J M , Díaz R
Published: January 2020

The influence of mouse genotype on passive systemic anaphylaxis (PSA) by immune complexes was studied. PSA was induced by using Brucella abortus endotoxin as the antigen and rabbit anti-Brucella endotoxin antisera. Experiments using syngeneic mice as well as mice congenic for H-2 showed that the H-2 haplotype influenced the sensitivity of mice to PSA. Among the H-2 haplotypes studied, H-2b was the most sensitive, followed by H-2k and H-2d. Experiments using passive transfer of serum as well as the complement inhibitors suramin and flufenamic acid indicated that variations in complement levels under control of H-2 may be responsible for the effects described. Cyproheptadine, a blocker of serotonin and histamine receptors, and imidazol-alpha-ketoglutarate, an inhibitor of thromboxane synthesis, inhibited PSA, indicating that platelet aggregation, possibly mediated by activated components of the complement cascade, is an important feature in the development of PSA reactions in this system. Differences between strains for protection by cyproheptadine and for the effect of complement inhibitors indicated a role of early components of the classical pathway in this model.
Antigenic variation among Borrelia spp. in relapsing fever
Authors: Kehl K S , Farmer S G , Komorowski R A , Knox K K
Published: January 2020

Seven antigens of Borrelia hermsii, B. parkeri, and B. turicatae with isoelectric points in the range of 4.4 to 5.0 and molecular masses of 40 to 43 kilodaltons played a role in the relapse phenomenon of relapsing fever. Based upon location of the antigens in the outer envelope, the molecular weight, and Western blot analysis, the antigens from each phase of spirochetemia appeared to be a mixture of the serotype-specific antigens of cloned B. hermsii.
Production of immunoglobulin M (IgM) autoantibodies to IgG in rabbits inoculated with Mycoplasma salivarium cells grown in medium supplemented with rabbit serum
Authors: Watanabe T , Shibata K , Hasegawa K
Published: January 2020

Mycoplasma salivarium cells propagated in PPLO broth (Difco Laboratories, Detroit, Mich.) supplemented with rabbit serum (10% [vol/vol]) and harvested by centrifugation were shown to contain rabbit immunoglobulin G (IgG) (approximately 4 to 10% of total cell proteins) and to produce IgM autoantibodies to IgG when inoculated into rabbits.
Fate of Legionella pneumophila Philadelphia-1 strain in resident, elicited, activated, and immune peritoneal macrophages of guinea pigs
Authors: Yoshida S , Mizuguchi Y , Nikaido Y , Mitsuyama M , Nomoto K
Published: January 2020

Legionella pneumophila is known to grow intracellularly in resident peritoneal macrophages of guinea pigs. The present study was done to determine what kinds of macrophage stimulants are able to activate guinea pig macrophages to inhibit intracellular growth of the organism. Peritoneal macrophages were harvested from healthy guinea pigs, from guinea pigs injected intraperitoneally with proteose peptone (PP) or thioglycolate medium, from guinea pigs injected intraperitoneally with live Mycobacterium bovis BCG or killed Propionibacterium acnes (Corynebacterium parvum), and from guinea pigs surviving infection with live L. pneumophila. After in vitro phagocytosis, the L. pneumophila CFU in each well were counted on charcoal-yeast extract agar plates. In the macrophages elicited by PP or thioglycolate medium, the organism grew as well as it did in resident macrophages. In BCG-activated and immune macrophages, growth was inhibited almost completely. In P. acnes-activated macrophages, the initial growth of L. pneumophila was inhibited to some extent, but its growth reached the same level as in the resident and PP-induced macrophages after 3 or 4 days of culture. In the lethal challenge experiments in vivo, the superior protection provided by BCG over P. acnes was ascertained and the importance of macrophages in resistance to L. pneumophila was confirmed. Difference of activation by BCG and P. acnes in relation to the inhibition of intracellular growth of L. pneumophila in guinea pig macrophages is discussed.
Influences of conjugal genetic transfer functions of colicin V plasmids on adhesion of Escherichia coli to murine intestinal tissue
Authors: Darken J , Savage D C
Published: January 2020

Cells of strains of Escherichia coli K-12 291 bearing one of three colicin V plasmids, pF54, pH247, or pF70, were tested in comparison with cells of strain 291 for their ability to adhere to murine intestinal tissue in vitro. The plasmids were either repressed or derepressed in conjugal genetic transfer functions. The strains bearing pF54 and pH247 repressed in transfer functions (pColVF54 luminal diameter r and pColVH247 luminal diameter r) adhered in higher numbers to the murine tissue than did the host strain lacking the plasmid or the strains containing the plasmids with active transfer functions (pColVF54drd and pColVH247drd). The number of cells (CFU) of strain 291(pColVF54 luminal diameter r) adherent to the tissue was related directly to the time of incubation (up to 30 min) and to the number of cells (CFU) to which the tissues were exposed. As indicated by tests for sensitivity to F factor (F)-pilus-specific bacteriophages, the cells of strains bearing the plasmids derepressed for conjugal functions had F pili on their surfaces, while such structures were missing from cells of the parental strain (291) and the strains containing the plasmids in repressed form. This finding was supported by transmission electron microscopy of cells of strain 291, 291(pColVF54 luminal diameter r), and 291(pColVF54drd). F pili could be seen on cells of the latter strain but not on those of the parental strain or the strain bearing pColVF54 luminal diameter r. Pili other than F pili were not seen on cells of the strains bearing pF54 in either form. Strains 291(pColVF70drd) and 291(pColVF70 luminal diameter r) adhered to the tissues in numbers comparable to those of strain 291. Nevertheless, these findings are further evidence that for certain colicin V plasmids (A. M. Nilius and D. C. Savage, Infect. Immun. 43:947-953, 1984), conjugal genetic transfer functions influence properties that may be important in the pathogenesis of invasive E. coli strains.
Use of monoclonal antibodies to protein F of Pseudomonas aeruginosa as opsonins for phagocytosis by macrophages
Authors: Battershill J L , Speert D P , Hancock R E
Published: January 2020

Five protein F-specific monoclonal antibodies were found to opsonize Pseudomonas aeruginosa for complement-independent phagocytosis by unelicited mouse peritoneal macrophages, mouse macrophage cell line P388D1, and human monocyte-derived macrophages. Immunoglobulin G1 antibodies seemed to be a preferred isotype.
Activities of complete and truncated forms of pertussis toxin subunits S1 and S2 synthesized by Escherichia coli
Authors: Locht C , Cieplak W , Marchitto K S , Sato H , Keith J M
Published: January 2020

The genes encoding the S1 and S2 subunits of pertussis toxin were expressed in Escherichia coli under lac operon transcription and translation control with pUC8 and pUC18 as the expression vectors. Various versions of the subunits were detected with anti-S1 or anti-S2 monoclonal antibodies. Recombinant S1, but not S2, subunit contained the enzymatic NAD-glycohydrolase and NAD:Gi ADP-ribosyltransferase activities. Both activities were also expressed by a truncated version of the S1 subunit in which the 48 carboxy-terminal amino acid residues, including a predicted Rossman structure and one of the two cysteines, had been deleted. The epitope for an anti-S2 monoclonal antibody was localized to the N-terminal 40-amino-acid region of the S2 subunit. Both the S1 and S2 subunits expressed in E. coli reacted with human hyperimmune serum. The full length and the truncated recombinant S1 subunit also reacted in Western blots with a neutralizing and protective monoclonal anti-S1 antibody. The different versions of S1 and S2 subunits expressed in E. coli are useful for mapping active sites, epitopes, and regions that interact with receptors or the other subunits in the holotoxin. These recombinant subunits will also facilitate the development of a safer, new-generation vaccine against whooping cough.
Antigenic identity of biologically active antigens in coccidioidin and spherulin
Authors: Cox R A , Britt L A
Published: January 2020

We recently reported the isolation of three clinically relevant antigens from coccidioidin; viz., the antigen that is reactive in the immunodiffusion (ID) assay for detecting tube precipitin (TP) antibody (designated IDTP); the antigen that is reactive in the ID assay for detecting complement-fixing antibody (designated IDCF); and the heat-stable (HS) antigen which, when demonstrated in soluble extracts of fungal cultures by using the IDHS assay, establishes the mycologic identification of Coccidioides immitis. This investigation was undertaken to determine whether the IDTP, IDCF, and IDHS antigens isolated from coccidioidin are of antigenic identity with components in spherulin. By employing the purified coccidioidin antigens in line-immunoelectrophoresis with spherulin and by assaying coccidioidin and spherulin by tandem two-dimensional immunoelectrophoresis against antisera produced to the purified antigens, we report that these three coccidioidin-derived antigens are antigenically identical to precipitinogens in spherulin.
Enhancement of non-Candida antibody responses by Candida albicans cell wall glycoprotein
Authors: Domer J E , Elkins K L , Ennist D L , Stashak P W , Garner R E , Baker P J
Published: January 2020

Two cell wall glycoprotein extracts from Candida albicans (glycoprotein [GP] and peptidoglucomannan [PGM]) were tested for their influence on antibody responses to type III pneumococcal polysaccharide and sheep erythrocytes. GP was isolated from lipid-extracted cell walls with ethylenediamine, whereas PGM was extracted with dilute sodium hydroxide. Both glycoproteins increased the number of antibody-producing plaque-forming cells in the spleens of mice immunized with type III polysaccharide or sheep erythrocytes, although PGM appeared to be about 10 times more effective. PGM could be administered up to 3 days prior to immunization with sheep erythrocytes to elicit enhancement; it did not have to be administered by the same route as the immunogen to cause significant enhancement. Enhancement did not appear to be the result of a direct mitogenic effect of GP and PGM on lymphocytes, nor did these glycoproteins appear to stimulate the production of B-cell growth factors or interleukin 2.
Cloning of genes for production of Escherichia coli Shiga-like toxin type II
Authors: Newland J W , Strockbine N A , Neill R J
Published: January 2020

Genes controlling production of Shiga-like toxin type II (SLT-II) in Escherichia coli were cloned from the SLT-II-converting bacteriophage 933W and compared with the Shiga-like toxin type I (SLT-I) genes previously isolated and described from phage 933J. Subcloning analysis identified a region within the 4.9-kilobase EcoRI fragment of phage 933W that was associated with SLT-II production. Experiments with E. coli minicells containing these subclones demonstrated that the 4.9-kilobase EcoRI fragment encodes the structural genes for SLT-II. These experiments additionally showed the genetic organization of the SLT-II genes to be the same as that of the SLT-I genes, with the coding sequence for the large A subunit adjacent to that for the smaller B subunit. The mobilities of the SLT-II subunits in sodium dodecyl sulfate-polyacrylamide gels were slightly greater than those determined for the SLT-I subunits. Although apparent processing of the SLT-I subunits was observed with polymyxin B treatment of the labeled minicells, no processing of the SLT-II subunits was detected. Southern blot hybridization studies suggested that the DNA fragment carrying the SLT-II structural genes shares approximately 50 to 60% homology with the DNA of the SLT-I structural genes.
Serological relationships between Escherichia coli and Salmonella smooth- and rough-mutant lipopolysaccharides as revealed by enzyme-linked immunosorbent assay for human immunoglobulin G antiendotoxin antibodies
Authors: Barclay G R , Scott B B
Published: January 2020

Reactions of sera from healthy blood donors to smooth and rough mutant lipopolysaccharides (LPS) from Escherichia coli O111:B4 and Salmonella minnesota which had been complexed with polymyxin B were determined by enzyme-linked immunosorbent assay. Relative enzyme-linked immunosorbent assay reactivities were examined for prima facie evidence of cross-reactivity by analyses of correlation and relative scatter. Patterns of reactivity were interpreted in relation to published structures. Evidence was obtained for two dominant epitopes associated with the lipid A-2-keto-3-deoxyoctulosonic acid and heptose regions of the LPS core. Sera demonstrated apparent exclusive antibody homogeneity in that given sera showed only one of the two possible specificities, which occurred with equal frequency. Cross-reactivity was found in the lipid A-2-keto-3-deoxyoctulosonic acid region for all LPS. Cross-reactivity was found in the heptose region for larger rough mutant S. minnesota LPS and smooth S. minnesota LPS and for E. coli O111:B4 LPS but not for E. coli rough mutant J5 LPS, whose heptose region appears to be different from those of the other organisms.
Cytosolic calcium changes in individual neutrophils stimulated by opsonized and unopsonized Candida albicans hyphae
Authors: Levitz S M , Lyman C A , Murata T , Sullivan J A , Mandell G L , Diamond R D
Published: January 2020

Previous experiments suggest the critical central role of the neutrophil (PMN) respiratory burst in the prevention and containment of disseminated candidiasis. A rise in cytosolic free calcium concentrations ([Ca2+]i) has been documented as an early event after PMN stimulation which is involved in the subsequent genesis of microbicidal and inflammatory respiratory burst products. [Ca2+]i were therefore determined in individual PMN, loaded with the fluorescent calcium probe fura-2 as they attached to and spread over serum-opsonized or unopsonized Candida albicans hyphae, particles that are too big to be completely ingested. After contact between hyphae and PMN, the PMN rapidly spread over hyphal surfaces. Although both opsonized and unopsonized hyphae stimulated similar magnitudes of peak median increases in PMN [Ca2+]i, the kinetics of responses differed; median [Ca2+]i peaked within 1 min after contact with opsonized hyphae versus 4 min after contact with unopsonized hyphae. Moreover, a detectable calcium transient did not invariably follow contact and spreading of each individual PMN over a hyphal surface. In contrast to patterns seen after stimulation of PMN with opsonized zymosan, in which [Ca2+]i is greatest in the periphagosomal region, there was a more uniform distribution throughout the cytoplasm in PMN stimulated with the noningestable hyphae. These alterations in the early patterns and timing of PMN stimulation may reflect analogous differences in subsequent events which control the efficiency and specificity of microbicidal responses to uningestible hyphae and which also determine whether host tissues are damaged by the generation of toxic PMN activation products.
Protein variability among strains of Mycoplasma pulmonis
Authors: Watson H L , Davidson M K , Cox N R , Davis J K , Dybvig K , Cassell G H
Published: January 2020

The proteins of Mycoplasma pulmonis were examined by two-dimensional polyacrylamide gel electrophoresis and immunoblotting. Comparison of profiles from silver-stained two-dimensional polyacrylamide gel electrophoresis of 18 strains of M. pulmonis allowed identification of five proteins that were strain variable. These variable proteins were not dependent on the medium used to grow the organisms and were shown by reaction with serum samples from naturally infected rats and mice to be produced in vivo and not to be medium components. Identification of strain-variable proteins may lead to an explanation of the differences in properties found among M. pulmonis strains.
Quantitative analysis of the binding and oligomerization of staphylococcal alpha-toxin in target erythrocyte membranes
Authors: Reichwein J , Hugo F , Roth M , Sinner A , Bhakdi S
Published: January 2020

The binding of staphylococcal alpha-toxin to rabbit and human erythrocytes was quantitated over a wide range of toxin concentrations (3 x 10(-11) to 3 x 10(-6) M) with the use of an enzyme-linked immunosorbent assay that permitted simultaneous quantitation of monomeric and oligomeric toxin forms. Three basic observations were made. First, in no range of concentrations did the binding of alpha-toxin to rabbit erythrocytes display characteristics of a receptor-ligand interaction. Net binding to rabbit cells was nil at sublytic concentrations (10(-10) M or 3 ng/ml). The onset of binding occurred at around 10 ng/ml and remained fairly constant and ineffective (5 to 8% of toxin offered) over a wide concentration range (up to 10 micrograms/ml). Second, hemolysis of rabbit and human erythrocytes at 37 degrees C was always accompanied by the formation of toxin oligomers in the membrane. Third, overall toxin binding at 0 degree C followed a pattern similar to that at 37 degrees C. However, oligomer formation and cell lysis were retarded (but not totally inhibited) at 0 degree C. When rabbit erythrocytes were incubated with low levels of toxin at 0 degree C (0.5 microgram/ml) for 30 min, the toxin became bound exclusively in monomer form, and no lysis occurred. When cells thus treated were washed and suspended at 37 degrees C, lysis rapidly ensued, and native monomeric toxin was replaced by oligomeric toxin. The collective results directly support the oligomer pore concept of toxin action and also indicate that toxin oligomers form by lateral aggregation of bound monomers in the bilayer. They speak against the existence of specific binding sites for alpha-toxin on rabbit erythrocytes.
Structural studies of the serotype-f polysaccharide antigen from Streptococcus mutans OMZ175
Authors: Linzer R , Reddy M S , Levine M J
Published: January 2020

The serotype f antigen of Streptococcus mutans has been described as a rhamnose-glucose polysaccharide associated with the bacterial cell wall. In this study, the structure of serotype f polysaccharide was examined by analyses of the methylated derivatives of the antigen and the periodate-oxidized antigen. Methylated derivatives were characterized with a gas chromatograph-mass spectrometer. The polysaccharide appeared to have a backbone of alternating 1,3- and 1,2,3-linked rhamnose units. Branching occurred at the 3-position of the 1,2,3-linked rhamnose. Side chains were composed of terminal alpha-linked glucose units. A small proportion of longer side chains containing 1,2- and 1,6-linked glucose units were noted in some preparations; however, these determinants were not reactive with serotype f antisera.
Characterization by affinity electrophoresis of an alpha-1,6-glucan-binding protein from Streptococcus sobrinus
Authors: Landale E C , McCabe M M
Published: January 2020

Glucan-binding protein 1 (GBP1), the most abundant glucan-binding protein isolated from culture supernatants of Streptococcus sobrinus 6715-49, has been purified by affinity chromatography on Sephadex G-50 followed by gel permeation chromatography with Bio-Gel P-10. The specificity and affinity of GBP1 for glucans were assessed by affinity electrophoresis. GBP1 did not detectably bind to glucans lacking linear arrays of alpha-1,6 linkages. The association constant for the linear alpha-1,6-glucan Dextran T2000 was 3 x 10(7) M-1. Providing small isomaltosaccharide ligands to compete with this dextran indicated that the binding site maximally accommodated isomaltosaccharides with a degree of polymerization of 8. When glucans produced by purified S. sobrinus glucosyltransferases were tested, GBP1 displayed the highest affinity for the glucan from the soluble-product, primer-independent glucosyltransferase.
Passive transfer of acquired resistance to Listeria monocytogenes infection is independent of mononuclear cell granuloma formation
Authors: Roberts E C , Demartini J C , Orme I M
Published: January 2020

This study documents the formation of leukocyte foci in the livers of mice infused with either normal or immune T cells and then challenged intravenously with Listeria monocytogenes. The results show that the transfer of antilisterial resistance occurred before mononuclear cell granuloma formation and was associated instead with the appearance of foci of infiltrating lymphocytes and neutrophils. Numbers of these foci remained low in mice which received immune cells but increased progressively until death in mice which received normal cells. These findings do not support the previous hypothesis that a major component of acquired resistance against Listeria infection involves the rapid generation of mononuclear cell granuloma formation under the control of immune T cells.
Nucleotide sequence of the streptolysin O (SLO) gene: structural homologies between SLO and other membrane-damaging, thiol-activated toxins
Authors: Kehoe M A , Miller L , Walker J A , Boulnois G J
Published: January 2020

The complete nucleotide sequence of a cloned streptolysin O (SLO) gene and the amino acid sequence of SLO, predicted from the DNA sequence, are reported. SLO contains a single cysteine residue located close to the C terminus of the molecule and shares extensive structural homologies with other thiol-activated toxins, which allow us to predict functionally important features.
Proline requirement for glucose utilization by Peptostreptococcus anaerobius ATCC 27337
Authors: Curtis M A , Wittenberger C L , Thompson J
Published: January 2020

Resting cells of Peptostreptococcus anaerobius maintained under anaerobic conditions were unable to metabolize either glucose or alanine. The addition of proline to the appropriate suspension, however, resulted in the immediate utilization of both compounds. Fermentation of alanine by the cells required that stoichiometric concentrations of proline be present in the medium; and during the oxidation of alanine, proline was simultaneously reduced to the ring cleavage product delta-aminovaleric acid. Although proline was required to initiate glucose transport, stoichiometric amounts of the imino acid were not necessary for glucose fermentation. Proline also stimulated the uptake and concomitant phosphorylation of the nonmetabolizable glucose analog 2-deoxy-D-glucose. The proline requirement for glucose transport by P. anaerobius could be replaced by adding ferricyanide or simply by aerating the cell suspension. The initiation of sugar uptake by proline, ferricyanide, and O2 was attributed to the capacity of these compounds to function as electron acceptors, which permitted reoxidation of the (reduced) intracellular nucleotide pool and the formation (from an endogenous reserve) of the high-energy donor(s) required for the vectorial transport and phosphorylation of sugar.
Adherence to human colonocytes of an Escherichia coli strain isolated from severe infantile enteritis: molecular and ultrastructural studies of a fibrillar adhesin
Authors: Hinson G , Knutton S , Lam-Po-Tang M K , McNeish A S , Williams P H
Published: January 2020

Escherichia coli 469-3 (O21:H-) was isolated from a child with severe enteritis. Ultrastructural analysis of the surface of the strain indicated the presence of very fine fimbriae which mediated mannose-resistant hemagglutination of human blood and caused the bacteria to adhere to human epithelial cell lines and to brush borders of isolated human colonic, but not duodenal, enterocytes. A cosmid library of total DNA of the strain, expressed in laboratory strains of E. coli, was screened by a rapid hemadsorption method, and a number of positive clones were identified. Restriction endonuclease fragments specifying mannose-resistant adherence were subcloned from the cosmid DNA of a strongly hemagglutinating clone in a plasmid vector. The identity of the adhesin was confirmed by biochemical, electron-microscopic, and immunological comparisons with the adhesin synthesized by the clinical isolate. It comprised a high-molecular-weight aggregate of a 14,000-dalton subunit protein which bound antiserum raised against the mannose-resistant adhesin of strain 469-3. The adhesin was synthesized by both the clone and the parental strain at growth temperatures above 18 degrees C but by only a fraction of the cells in a pure culture, although all the bacteria which adhered to human cells expressed the protein.
Purification and characterization of a protease from Bacteroides gingivalis 381
Authors: Tsutsui H , Kinouchi T , Wakano Y , Ohnishi Y
Published: January 2020

An intracellular membrane-free, trypsinlike protease was isolated from cells of Bacteroides gingivalis 381. The protease was extracted from the cells by ultrasonic treatment and was purified about 250-fold with a recovery of 2% by sequential procedures. The properties of the protease were as follows: its optimal pH was 8.5; its activity was almost completely lost on incubation at 50 degrees C for 15 min; its activity was inhibited by diisopropylfluorophosphate, p-toluenesulfonyl-L-lysine chloromethyl ketone hydrochloride, leupeptin, Mn2+, Cu2+, and Zn2+; it hydrolyzed casein, azocasein, N-alpha-benzoyl-DL-arginine-p-nitroanilide (BAPNA), bovine serum albumin, azocoll, and gelatin, but not N-alpha-benzoyl-DL-lysine-p-nitroanilide or human serum immunoglobulin A; its molecular weight was estimated as 45,000 by gel filtration and 50,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis; and its Km values for azocasein and BAPNA were 1.11% and 0.19 mM, respectively.
Suppression of the delayed-type hypersensitivity and cell-mediated immune responses to Listeria monocytogenes induced by Pseudomonas aeruginosa
Authors: Blackwood L L , Lin T , Rowe J I
Published: January 2020

Pseudomonas aeruginosa-mediated suppression of the immune response to Listeria monocytogenes was investigated in mice. Because delayed-type hypersensitivity (DTH) footpad swelling to L. monocytogenes was suppressed equally in lipopolysaccharide-responsive and -hyporesponsive mouse strains, the lipopolysaccharide component of P. aeruginosa could not have been the suppressive agent. Mucoid P. aeruginosa cells were no more suppressive than their nonmucoid revertants; therefore, mucoid coating was not an additional immunosuppressive element. Interleukin-1 and macrophage inhibitory factor production to L. monocytogenes and clearance of L. monocytogenes from mouse spleens were all decreased by prior Pseudomonas infection, indicating that cell-mediated immunity, as well as DTH, was decreased to a sublethal Listeria dose. The timing of Pseudomonas exposure relative to Listeria sensitization was varied. P. aeruginosa injected 24 or 6 h before or at the same time as L. monocytogenes depressed DTH to Listeria challenge 7 days later. Animals treated in this way could not respond to reinfection with L. monocytogenes at 13 days. P. aeruginosa administered to L. monocytogenes-sensitized mice at the time of footpad challenge was suppressive, but these mice responded normally upon reinfection. It appears that P. aeruginosa induced two types of suppression to L. monocytogenes: a transient suppression, affecting DTH challenge but not resensitization, and a longer lasting suppression that did not permit mice exposed to P. aeruginosa at the time of Listeria sensitization to respond to subsequent Listeria exposure.