bluuebunny/crossref_metadata_2025 huggingface dataset:
Plasminogen activator/coagulase gene of Yersinia pestis is responsible for degradation of plasmid-encoded outer membrane proteins
Authors: Sodeinde O A , Sample A K , Brubaker R R , Goguen J D
Published: January 2020

The related family of virulence plasmids found in the three major pathogens of the genus Yersinia all have the ability to encode a set of outer membrane proteins. In Y. enterocolitica and Y. pseudotuberculosis, these proteins are major constituents of the outer membrane when their synthesis is fully induced. In contrast, they have been difficult to detect in Y. pestis. It has recently been established that Y. pestis does synthesize these proteins, but that they are rapidly degraded due to some activity determined by the 9.5-kilobase plasmid commonly found in Y. pestis strains. We show that mutations in the pla gene of this plasmid, which encodes both the plasminogen activator and coagulase activities, blocked this degradation. A cloned 1.4-kilobase DNA fragment carrying pla was also sufficient to cause degradation in the absence of the 9.5-kilobase plasmid.
Inhibition of activated nonresponder C3H/HeJ lymphocytes by lipopolysaccharide endotoxin
Authors: Sultzer B M , Castagna R
Published: January 2020

The B lymphocytes and macrophages of lipopolysaccharide (LPS) nonresponder C3H/HeJ mice were found to respond to certain R types of LPS endotoxin in a fashion resembling that ordinarily seen with the cells from normal responder mice. DNA synthesis, polyclonal antibody synthesis, and interleukin-1 activity were stimulated by Bordetella pertussis LPS and Salmonella minnesota R595 LPS, although to a lesser extent than with responder cells. Mitogenesis stimulated by both LPSs was inhibited by polymyxin B; this finding provided evidence that any trace endotoxin-associated proteins were not responsible for the activity. Of particular interest was the finding that wild-type smooth LPS actually inhibited activation of the C3H/HeJ B cells not only by the LPS but also by mitogenic proteins, including purified protein derivative of tuberculin. The nonspecific nature of this inhibition and the fact that maximal inhibition occurred some 9 to 12 h into the culture period suggested that the proliferation of the B cells was affected by smooth-type LPS in a manner heretofore unrecognized. These findings permit a new approach to the study of how LPS endotoxin affects cells of the immune system.
Lipooligosaccharides from Treponema hyodysenteriae and Treponema innocens
Authors: Halter M R , Joens L A
Published: January 2020

Lipooligosaccharides from Treponema hyodysenteriae serotypes 1 through 7, attenuated T. hyodysenteriae serotypes 1 and 2, and five strains of T. innocens were extracted with hot phenol water. The extracts were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis separation and analyzed by lipopolysaccharide selective silver staining and Western blot (immunoblot) immunodetection. Silver staining revealed the presence of two bands that ranged between 18,000 and 24,000 daltons and that were serotype specific for T. hyodysenteriae. Attenuation of pathogenic strains resulted in the loss of the higher-molecular-weight band. Four of five T. innocens strains also lacked this particular band. T. innocens 421 had six bands between 17,000 and 26,900 daltons. Western blots with hyperimmune rabbit sera and convalescent-phase swine sera revealed antigenic variation among serotypes of T. hyodysenteriae and attenuated serotypes of T. hyodysenteriae. Convalescent-phase swine sera failed to recognize lipopolysaccharides from T. innocens. Differences in results obtained by lipopolysaccharide selective silver staining versus immunoblotting of the lipopolysaccharide preparations probably indicate that these two methods identify separate characteristics of the same molecule.
Inoculation candidiasis in a murine model of severe combined immunodeficiency syndrome
Authors: Mahanty S , Greenfield R A , Joyce W A , Kincade P W
Published: January 2020

To further elucidate the importance of T- and B-lymphocyte-mediated responses in host defense against systemic infection with Candida albicans, we studied this infection in a murine model of severe combined immunodeficiency (SCID). The course of inoculation candidiasis in these mice, which lack functional T and B lymphocytes, was compared with that in immunologically normal BALB/c mice. Mice were inoculated intravenously with 10(5) yeast cells. Quantitative cultures of liver, spleen, and kidneys were performed with necropsy specimens obtained 1, 3, 7, 10, 14, and 21 days after this intravenous inoculation. The differences in the time courses of recovery of organisms from liver and spleen specimens were not significantly different in the SCID mice compared with the BALB/c mice. The recovery of C. albicans from the kidneys was significantly lower in the SCID mice, indicating less persistence of the organism in the kidneys of the SCID mice than in those of the BALB/c mice. These data indicate that defense mechanisms other than T- and B-lymphocyte-mediated mechanisms are primarily responsible for host defense against inoculation candidiasis.
Cloning and expression in Escherichia coli of the perfringolysin O (theta-toxin) gene from Clostridium perfringens and characterization of the gene product
Authors: Tweten R K
Published: January 2020

The gene encoding perfringolysin O, the thiol-activated hemolysin from Clostridium perfringens (ATCC 13124), was cloned and expressed in Escherichia coli. A gene library of C. perfringens chromosomal DNA was constructed in bacteriophage lambda EMBL3. A recombinant was identified that produced a hemolysin that was inhibited by cholesterol and was tentatively identified as perfringolysin O. Subcloning experiments localized the perfringolysin O gene (pfo) to a 1.8-kilobase region on the cloned chromosomal fragment. E. coli which carried a plasmid subclone of pfo (pRT1B) expressed perfringolysin O and secreted it into the periplasm. The amino-terminal sequence of the pfo gene product was identical with that determined for perfringolysin O purified from C. perfringens, indicating that E. coli correctly removed the signal peptide during secretion. Purification of the pfo product was accomplished by high-resolution gel filtration and anion-exchange chromatography. Analysis of the pfo product by sodium dodecyl sulfate gel electrophoresis showed that it comigrated with authentic perfringolysin O; both had an estimated molecular weight of 54,000. Two-dimensional tryptic peptide maps of the pfo product and of authentic perfringolysin O purified from C. perfringens were identical. The hemolytic activity of the pfo product was similar to that of authentic perfringolysin O; one hemolytic unit (HU) of the cloned gene product or authentic perfringolysin O corresponded to approximately 1 ng or a hemolytic activity of 10(6) HU per mg.
Nucleotide sequence of the gene for perfringolysin O (theta-toxin) from Clostridium perfringens: significant homology with the genes for streptolysin O and pneumolysin
Authors: Tweten R K
Published: January 2020

The nucleotide sequence was determined for the gene encoding the thiol-activated cytolysin, perfringolysin O (theta-toxin), from Clostridium perfringens. The nucleotide-sequence-derived primary structure of perfringolysin O is 499 residues long and exhibits a 27-amino-acid signal peptide. The calculated molecular weight of the secreted (mature) form of perfringolysin O is 52,469. The deduced amino-terminal sequence of perfringolysin O is identical to that determined for purified perfringolysin O. Hydropathy analysis indicated that, except for the signal peptide, no major stretches of hydrophobic residues are present. Extensive amino acid sequence homology (65%) was detected with the low-molecular-weight form of streptolysin O, and a lesser amount (42%) was detected with pneumolysin. The nucleotide sequence of the perfringolysin O gene (pfo) exhibits approximately 60% homology with the streptolysin O gene (slo) and 48% homology with the pneumolysin gene (ply). All three toxins contain an identical region of 12 amino acids, which includes the essential cysteine of all three toxins. The location of these 12 residues was conserved in all three toxins when the primary sequences were aligned for maximum homology.
Immunological analysis of cell-associated antigens of Bacillus anthracis
Authors: Ezzell J W , Abshire T G
Published: January 2020

Sera from Hartley guinea pigs vaccinated with a veterinary live spore anthrax vaccine were compared with sera from guinea pigs vaccinated with the human anthrax vaccine, which consists of aluminum hydroxide-adsorbed culture proteins of Bacillus anthracis V770-NP-1R. Sera from animals vaccinated with the spore vaccine recognized two major B. anthracis vegetative cell-associated proteins that were either not recognized or poorly recognized by sera from animals that received the human vaccine. These proteins, termed extractable antigens 1 (EA1) and 2 (EA2), have molecular masses of 91 and 62 kilodaltons, respectively. The EA1 protein appeared to be coded by chromosomal DNA, whereas the EA2 protein was only detected in strains that possessed the pXO1 toxin plasmid. Both of the extractable antigen proteins were serologically distinct from the components of anthrax edema toxin and lethal toxin. Following vaccination with the live spore vaccine, the EA1 protein was the predominant antigen recognized, as determined by electrophoretic immunotransblots. Vaccine trials with partially purified EA1 demonstrated that it neither elicits protective antibody against anthrax nor delays time to death in guinea pigs challenged intramuscularly with virulent Ames strain spores. In addition, animals vaccinated with sterile gamma-irradiated cell walls had significant antibody titers to the N-acetylglucosamine-galactose polysaccharide of B. anthracis but were neither protected nor had a delay in time to death following challenge.
Cross-neutralization of staphylococcal and streptococcal pyrogenic toxins by monoclonal and polyclonal antibodies
Authors: Bohach G A , Hovde C J , Handley J P , Schlievert P M
Published: January 2020

We evaluated cross-reactivity of antibodies against staphylococcal and streptococcal pyrogenic toxins. Monoclonal antibodies against staphylococcal enterotoxin (ET) C1 and streptococcal pyrogenic exotoxin (SPE) A were tested for reactivity with homologous and heterologous pyrogenic toxins in vitro. Ten immunoglobulin G1 anti-ET C1 monoclonal antibodies showed little or no cross-reactivity in an enzyme-linked immunosorbent assay, but many of these could neutralize the mitogenic effect of ET B, SPE A, or both. Two immunoglobulin M anti-ET C1 monoclonal antibodies and eight immunoglobulin M anti-SPE A monoclonal antibodies showed extensive cross-reactivity in the enzyme-linked immunosorbent assay and the mitogenicity neutralization assay. No cross-reactivity was observed with SPE C or toxic shock syndrome toxin 1. Rabbits immunized against ET B, ET C1, or SPE A were resistant to challenge with the immunizing toxin. In addition, reciprocal immunity was stimulated by the two ETs, and immunity to SPE A provided protection against ET B but not ET C1. These results show that staphylococcal and streptococcal pyrogenic toxins which share sequence homology have common antigenic determinants which may not be detected in Ouchterlony immunodiffusion assays.
Expression of extracellular acid proteinase by proteolytic Candida spp. during experimental infection of oral mucosa
Authors: Borg M , Rüchel R
Published: January 2020

We traced an acid proteinase from Candida spp. in the initial stages of the pathogenesis of the mycosis. On infection of human buccal mucosa, proteinase antigens were detected by immuno-scanning electron microscopy on the surface of adhering blastoconidia and invading filamentous cells of C. albicans serotype A. Proteinase antigens were also present on blastoconidia of C. albicans serotype B, but were missing on filamentous cells of this serotype. Proteolytic isolates of C. tropicalis behaved like C. albicans serotype A. An isolate of C. parapsilosis did not express the proteinase antigen under conditions of this study. After infection of mucosa, culture medium of C. albicans or C. tropicalis showed a time-dependent accumulation of acid proteolytic activity, indicating that the visualized antigens represent active proteinase. No such activity was detected in the medium of C. parapsilosis. Preliminary experiments with the proteinase inhibitor pepstatin A revealed an 89% reduction of mucosal adherence of C. albicans (serotype A). These results suggest that Candida proteinase is involved in fungal attachment. The pattern of adherence reflects the differential expression of secretory proteinase by different candidal strains.
Effects of adenylate cyclase toxin from Bordetella pertussis on human neutrophil interactions with Coccidioides immitis and Staphylococcus aureus
Authors: Galgiani J N , Hewlett E L , Friedman R L
Published: January 2020

Bordetella pertussis extract that contained adenylate cyclase toxin produced large increases in human neutrophil cyclic AMP levels and inhibited their oxidative burst, as reflected by luminol-enhanced chemiluminescence and superoxide release. The adenylate cyclase toxin-containing extract blocked neutrophil-mediated inhibition of N-acetylglucosamine incorporation by arthroconidia of Coccidioides immitis in a dose-dependent fashion but had no effect on neutrophil phagocytosis of Candida glabrata and only a slight inhibitory effect on arthroconidial attachment. Neither purified pertussis toxin nor extracts from Bordetella mutants lacking the adenylate cyclase toxin affected neutrophil-mediated inhibition of arthroconidial N-acetylglucosamine incorporation. These studies indicate that adenylate cyclase toxin, alone or in concert with other B. pertussis-elaborated toxins, blocks neutrophil inhibition of arthroconidia, primarily by affecting neutrophil responses other than attachment or phagocytosis.
Use of transformation to construct Neisseria gonorrhoeae strains with altered lipooligosaccharides
Authors: Stein D C , Petricoin E F , Griffiss J M , Schneider H
Published: January 2020

DNA isolated from a nalidixic acid- and rifampin-resistant derivative of Neisseria gonorrhoeae serum-resistant strain 302 (MUG116), a strain that reacts with monoclonal antibody (MAb) 2-1-L8, was used to transform N. gonorrhoeae DOV, a serum-sensitive strain, to antibiotic resistance and/or reactivity with the MAb. MAb 2-1-L8 binds to a 3.6-kilodalton lipooligosaccharide (LOS). Reactivity with MAb 2-1-L8 transformed as a single marker and was unlinked to either of the antibiotic resistance markers. Immunoblot analysis of LOSs separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that new LOSs were synthesized in the transformed cells and that these LOSs corresponded to those of the DNA donor. Although multiple LOS components were made by the transformants, the MAb recognized only one. All transformants that were selected for on the basis of strong reactivity with MAb 2-1-L8 were serum resistant; however, the level of resistance correlated with the apparent loss of recipient LOS components. MAb 2-1-L8-reactive transformants that still produced DOV LOS components remained serum sensitive.
Lipid X protects mice against fatal Escherichia coli infection
Authors: Golenbock D T , Leggett J E , Rasmussen P , Craig W A , Raetz C R , Proctor R A
Published: January 2020

Lipid X, the major monosaccharide precursor of lipid A, is nontoxic and has previously been shown to protect mice and sheep from the harmful effects of endotoxin. To test the hypothesis that lipid X could be therapeutic against infections with gram-negative organisms, neutropenic ICR mice were infected by intramuscular inoculation of Escherichia coli and subsequently treated with lipid X alone or in combination with the antibiotic ticarcillin. Lipid X slightly prolonged survival; treated mice had a significantly improved rate of survival 18 h after intramuscular inoculation as compared with controls (P less than 0.025). By 24 h, however, this difference disappeared. When lipid X was combined with ticarcillin, survival differences were both significant and prolonged. Treatment of mice with one to two doses of lipid X for a total dose of 1 mg intravenously and with 1,200 mg of ticarcillin per kg every 6 h improved survival over a 48-h treatment period from 5 to 23% (P less than 0.0025). Treatment with lipid X and ticarcillin over a broad range of antibiotic dosages in 362 mice demonstrated improved survival of two- to fourfold (P less than 0.0001 at 24 h after inoculation, P less than or equal to 0.0005 at 48 h, and P less than or equal to 0.0001 at 5 days). Lipid X enabled the dose of ticarcillin necessary to protect 50% of mice from death to be reduced by two- to fivefold. Pretreatment with lipid X was not necessary to improve survival: 16 of 17 (94%) infected and visibly ill animals that received lipid X and ticarcillin 6 h after thigh inoculation survived versus 30 of 44 (68%) control animals treated with ticarcillin alone (P less than 0.0001). Lipid X had no antimicrobial activity in vitro. Lipid X is a novel agent that enhances survival in an animal model of severe infection with gram-negative organisms.
The availability of purines influences both the number of parasites and the splenocyte levels of purine-metabolizing enzymes in trypanosome-infected mice
Authors: Albright J W , Albright J F
Published: January 2020

Growth on Trypanosoma musculi in the murine host was limited by the availability of host purines. A portion of the spleen cells of infected mice (many of them granulocytes) displayed high levels of adenosine deaminase (ADA) and purine nucleoside phosphorylase, probably as a compensatory response to extracellular purine deficiency. Injections of adenosine or 2-deoxycoformycin stimulated significant increases in the growth of parasites. 2-Deoxycoformycin treatment also diminished parasite-induced splenomegaly. Treatment of mice with polyethylene glycol-modified ADA, a slowly catabolized form of ADA, had no effect on the course of T. musculi infection, indicating that the parasites can utilize purines other than adenosine. The apparent competition between parasites and host cells for available purines suggests that depletion of extracellular purines should be considered as an approach to treating extracellular trypanosome infections.
Protective role of magnesium in the neutralization by antibodies of Chlamydia trachomatis infectivity
Authors: Peterson E M , Zhong G M , Carlson E , de la Maza L M
Published: January 2020

Neutralization of the infectivity of Chlamydia trachomatis was assessed by using polyclonal antisera and monoclonal antibodies (MAbs). Polyclonal antisera and a species-reactive MAb as well as a subspecies-specific MAb, both of which were directed toward the major outer membrane protein of C. trachomatis, reduced the number of chlamydial inclusion-forming units in an in vitro assay. Neutralization was dependent on the presence of complement. The species-specific MAb reacted with all 15 serovars by a microimmunofluorescence assay and a dot blot enzyme-linked immunosorbent assay with heat-treated elementary bodies. On the other hand, this same MAb reacted with all serovars, except those in the C complex, by the dot blot enzyme-linked immunosorbent assay with viable organisms and neutralized in vitro all 10 serovars tested, except those in the C complex. When neutralization assays were performed in a solution containing Mg2+, neutralization by both polyclonal antisera and MAbs was significantly reduced. A dose response to Mg2+ supplied as MgSO4 revealed that all concentrations tested from 50 to 800 microM had some effect. Concentrations of greater than or equal to 400 microM MgSO4 completely abolished neutralization at the lowest dilution of polyclonal antisera and species-reactive MAb tested. Although Mg2+ also blocked the neutralization effect of the subspecies-specific MAb, this neutralization was not as complete as that observed with the species-reactive MAb. Addition of Mg2+ to the assay over the initial 45 min of incubation of C. trachomatis with MAb and complement showed that the organisms could be rescued to some extent over the first 30 min of incubation, after which time neutralization of infectivity could not be reversed. C. trachomatis treated with Mg2+, the species-reactive MAb, and complement were lethal to mice in an in vivo toxicity and infectivity assay, whereas mice injected with organisms incubated with the same MAb and complement without Mg2+ survived.
Structural and serological relatedness of Haemophilus influenzae type b pili
Authors: LiPuma J J , Gilsdorf J R
Published: January 2020

The structural and serological relatedness of the pilus proteins of several isolates of Haemophilus influenzae type b cultured from patients with invasive disease and from different anatomic sites within the same patient was examined. Epithelial cell-adherent variants of 25 nonadherent parent isolates were obtained by selection for organisms that adhered to human erythrocytes. Outer membrane protein analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed the presence of an additional 24- to 24.5-kilodalton protein among all adherent variants but absent from all nonadherent parent isolates. Polyclonal rabbit antiserum against the intact native pilus protein of H. influenzae M43 cross-reacted with 20 of 25 adherent H. influenzae in both immunodot and slide-agglutination assays. No differences in reactivity among isolates cultured from more than one anatomic site in the same patient were noted. Anti-M43 pilus antiserum had bactericidal activity against both the homologous strain and a heterologous strain that demonstrated serologic identity in the immunodot and slide agglutination assays. The adherence of these strains to human epithelial cells in vitro was inhibited by Fab fragments purified from the antipilus antiserum. These data indicate that a remarkable degree of homogeneity in pilin subunit size exists among the pili of H. influenzae type b and that major antigenic determinants are shared among most of these pili. Also, antibodies directed against H. influenzae pilus proteins may be able to contribute to host defenses through serum bactericidal activity and by blocking the adherence of this bacterium to host epithelial cells.
Inactivation of suppressor T-cell activity by nontoxic monophosphoryl lipid A
Authors: Baker P J , Hiernaux J R , Fauntleroy M B , Prescott B , Cantrell J L , Rudbach J A
Published: January 2020

Treatment with nontoxic monophosphoryl lipid A (MPL), which was derived from a polysaccharide-deficient, heptoseless Re mutant of Salmonella typhimurium, was found to inactivate suppressor T-cell activity, as evidenced by a decrease in the degree of low-dose immunological paralysis expressed and an increase in the magnitude of the antibody response to type III pneumococcal polysaccharide. The effects produced, which could not be attributed to the polyclonal activation of immune B cells by MPL, were dependent upon the dose of MPL used, as well as the time when MPL was given relative to low-dose priming or immunization with type III pneumococcal polysaccharide. Neither amplifier nor helper T-cell activity was decreased by treatment with the same, or larger, doses of MPL. The significance of these findings to the use of MPL as an immunological adjuvant or an immunomodulating agent is discussed.
Effect of neonatal injection with antibodies to Leishmania mexicana on its growth in adult infected mice
Authors: Gorczynski R M
Published: January 2020

Mice inoculated with monoclonal antibodies (MAb) directed to Leishmania mexicana antigens were not protected from growth of a subsequent challenge infection; this was the case even when those antibodies were capable of inhibiting parasite growth in vitro. However F(ab')2 fragments of one antibody (1E1) were protective in vivo. When neonatal mice were injected with MAb and subsequently infected as adults, the animals were more susceptible to parasite growth than uninjected controls. This increased susceptibility could be adoptively transferred with Lyt-1+ cells. Separate groups of animals were immunized with different MAb to L. mexicana, and parasite growth in these animals was studied. In no case was parasite growth altered, though these mice did produce specific antibodies directed against the immunizing MAb (anti-idiotypic antibodies). When neonatal mice were injected with these latter reagents, they were found to be more resistant to challenge infection than control animals. This resistance was associated with an enhanced ability of spleen cells from these mice to produce, on stimulation with parasite antigens in vitro, a factor rendering normal macrophages cytocidal for L. mexicana.
Mitogenic activities of synthetic Escherichia coli lipid A and a synthetic partial structure (tripalmitoyl pentapeptide) of E. coli lipoprotein
Authors: Brade L , Bessler W G , Brade H
Published: January 2020

Synthetic Escherichia coli lipid A and synthetic S-[2,3-bis-(palmitoyloxy)propyl]-N-palmitoylpentapeptide (tripalmitoyl pentapeptide [TPP]), representing the mitogenically active principles of bacterial lipopolysaccharide (LPS) and lipoprotein, respectively, were compared for their mitogenic activities on splenocytes of LPS responder (BALB/c) and LPS-low-responder (C3H/HeJ) mice. Whereas lipid A was active only in LPS-responder mice, TPP resulted in mitogenic activation of B lymphocytes from both LPS-responder and LPS-low-responder mice. When the mitogens were added simultaneously, mainly additive effects of both activators were observed. The data suggest that two different B-lymphocyte populations are responding to these two mitogens.
Suppression of T-lymphocyte response by Coccidioides immitis antigen
Authors: Cox R A , Kennell W
Published: January 2020

Intravenous injection of BALB/c mice with coccidioidin or an alkali-soluble cell wall extract of Coccidioides immitis mycelia resulted in the induction of a splenic cell population(s) that suppressed delayed-type hypersensitivity response to coccidioidal antigen. To determine whether the levels of C. immitis antigen produced during the course of active coccidioidal disease might also cause suppression of T-lymphocyte response, BALB/c mice were infected by intranasal instillation of arthroconidia, and 2 weeks later, their sera were evaluated for suppression of T-lymphocyte response in syngeneic recipients. Intravenous transfer of sera, which were shown to contain high levels of coccidioidal antigen by an enzyme-linked immunoadsorbent assay, suppressed the delayed-type hypersensitivity response of recipients to immunization with coccidioidin. Solid-phase immunoadsorption of the sera with goat antibodies to C. immitis antigens removed the suppressive component(s). To determine whether the suppressive effect of circulating coccidioidal antigen(s) was associated with the activation of a splenic suppressor cell(s), as was observed in mice injected intravenously with coccidioidal antigen, spleen cell lysates were prepared from infected donors, and after filtration to remove viable fungi, the lysates were transferred to syngeneic mice. Recipients of lysates from infected but not noninfected donors were suppressed in their response to immunization with coccidioidin. Collectively, these results provide evidence that depressed T-cell responses observed in coccidioidomycosis are associated with, and may be attributable to, the activation of a suppressor cell or factor by circulating C. immitis antigens.
Attempts to characterize the mechanisms involved in mycobacterial growth inhibition by gamma-interferon-activated bone marrow macrophages
Authors: Flesch I E , Kaufmann S H
Published: January 2020

Bone marrow-derived murine macrophages are able to inhibit the growth of Mycobacterium bovis and of some strains of M. tuberculosis after stimulation with either recombinant gamma interferon (rIFN-gamma) or lymphokines from antigen-specific T-cell clones. To elucidate the mechanism(s) involved in antimycobacterial activity, macrophages were infected with M. bovis in the presence of agents thought to influence the antimicrobial effects of phagocytes. Scavengers of toxic oxygen metabolites failed to influence the capacity of IFN-gamma-activated bone marrow macrophages to inhibit the growth of M. bovis. Suramin slightly affected mycobacterial growth in IFN-gamma-activated macrophages, and chloroquine markedly induced growth inhibition of M. bovis in unstimulated macrophages. We conclude that growth inhibition of M. bovis by IFN-gamma-activated macrophages is an oxygen-independent process.
Emergence in gnotobiotic mice of nontoxinogenic clones of Clostridium difficile from a toxinogenic one
Authors: Corthier G , Muller M C
Published: January 2020

In previous studies, we showed that diet composition or Saccharomyces boulardii ingestion could protect gnotobiotic mice against lethal Clostridium difficile infection. Using an original method, we detected nontoxinogenic clones from feces of protected mice challenged with a toxinogenic clone of C. difficile. These clones became established at the same level as the toxinogenic one after about 30 days. In these protected mice bearing nontoxinogenic clones, no enterotoxin production could be detected and cytotoxin titers were highly reduced. These nontoxinogenic clones were genetically stable because nontoxinogenic clones and clones that produce intermediate levels of toxins in vivo did not revert to toxin production, even after repeated culture in vitro. Furthermore, the nontoxinogenic clones were shown to arise from a single toxinogenic clone and were identical to that clone in metabolic patterns and antibiotic sensitivity tests. When mice fed a nonprotective diet were challenged with a nontoxinogenic or intermediate clone, they remained healthy and no toxin production could be detected in their feces. Moreover, these mice were protected against further infections with toxinogenic strains of C. difficile, and a strong antagonism between nontoxinogenic and toxinogenic clones was observed.
Monoclonal antibodies to Staphylococcus aureus laminin-binding proteins cross-react with mammalian cells
Authors: Mota G F , Carneiro C R , Gomes L , Lopes J D
Published: January 2020

We and others have previously shown that some microorganisms, including bacteria, express on their surfaces receptors that specifically recognize extracellular matrix proteins, such as laminin, fibronectin, or both. The ability of microorganisms to adhere and to invade might depend on the existence of receptors which could, thus, be correlated with pathogenicity. In the present paper, we report the isolation of five stable cell lines that were producers of monoclonal antibodies to Staphylococcus aureus laminin receptors. One of these antibodies, which was of the immunoglobulin M isotype, blocked the binding of laminin to bacteria before and after fixation and recognized the putative 52-kilodalton laminin-binding protein in whole bacterial extracts. Also, purified receptor was isolated by immunoaffinity chromatography and shown to bind laminin. Furthermore, the same antibodies bound the 67-kilodalton putative receptor from mouse melanoma cells and gave positive immunofluorescence reactions against mammalian tumor cells. These data strongly suggest either the evolutionary conservation of at least some sequences in both procaryotic and eucaryotic laminin-binding proteins or convergent evolution and positive selection of epitopes cross-reacting with laminin. Some of these antibodies to the procaryotic protein could therefore become useful markers for the expression of laminin receptors by cancer cells.
Serological studies of oral Bacteroides intermedius
Authors: Nakazawa F , Zambon J J , Reynolds H S , Genco R J
Published: January 2020

Bacteroides intermedius is a gram negative, anaerobic microorganism associated with certain forms of human periodontal disease, including adult periodontitis and acute necrotizing ulcerative gingivitis. Previous studies have indicated the presence of two DNA homology groups which could be distinguished by analysis of protein patterns on polyacrylamide gel electrophoresis, as well as at least two serogroups within B. intermedius. The present study examined the serology of B. intermedius and determined the distribution of B. intermedius serogroups in clinical isolates and patient plaque samples. Serological reactions with unabsorbed rabbit antisera and antisera immunoabsorbed with B. intermedius strains demonstrated a previously unreported antigenic group within B. intermedius, serogroup C, in both immunodiffusion and immunofluorescence assays. Of 79 B. intermedius isolates from 68 subjects examined with specific antisera, 55% of the isolates and 52% of the subjects were categorized in serogroup C, 40% of the isolates and 46% of the subjects were in serogroup B, and 5% of the isolates and 6% of the subjects were in serogroup A. In 31 samples of subgingival dental plaque from adolescents known to harbor B. intermedius, 81% demonstrated serogroup B, 16% had serogroup A, and 3% had serogroup C.
Identification of Babesia bigemina and Babesia bovis merozoite proteins with isolate- and species-common epitopes recognized by antibodies in bovine immune sera
Authors: McElwain T F , Palmer G H , Goff W L , McGuire T C
Published: January 2020

Immunoprecipitation of radiolabeled antigens with bovine antisera indicated that many Babesia bigemina and Babesia bovis merozoite proteins contain isolate-common epitopes, while at least 16 B. bigemina and 8 B. bovis proteins contain species-cross-reactive epitopes. One immunogenic, isolate-common, and species-specific candidate diagnostic protein from each species was identified.
Binding affinity of serum immunoglobulin G to cardiolipin and other phospholipids in patients with systemic lupus erythematosus and syphilis
Authors: Costello P B , Green F A
Published: January 2020

Qualitative and quantitative assays for human antibodies to cardiolipin and other phospholipids were used in tests for these reactions in sera from patients with systemic lupus erythematosus (SLE) and syphilis. Of 22 SLE serum samples tested by the qualitative assay, 8 showed positive staining to cardiolipin, phosphatidic acid, and/or phosphatidylserine. All 47 syphilitic sera reacted with these three phospholipids. The apparent affinity of anticardiolipin binding was estimated by normalizing absolute binding levels as a function of serum concentration to the maximum percent bound. It was evident that antibody affinity was four- to fivefold lower in the SLE sera than in the syphilitic sera. Twelve serum samples from patients with one or more features of the anti-cardiolipin syndrome demonstrated mean binding values which were not distinguishable from binding in other SLE sera. In sera from patients with active SLE, binding affinity for cardiolipin was somewhat greater than that in samples from patients with inactive disease, but the differences were not statistically significant. The low anticardiolipin binding affinity which was observed in patients with SLE compared with that in patients with syphilis casts doubt on a pathogenic role for these reactions.
Human monoclonal antibodies that protect mice against challenge with Pseudomonas aeruginosa
Authors: Zweerink H J , Gammon M C , Hutchison C F , Jackson J J , Lombardo D , Miner K M , Puckett J M , Sewell T J , Sigal N H
Published: January 2020

Lymphocytes from healthy volunteers and from cystic fibrosis patients were transformed with Epstein-Barr virus and cultured at a limiting dilution to generate lymphoblastoid cell lines that secreted human monoclonal antibodies specific for lipopolysaccharide (LPS) from Pseudomonas aeruginosa. Three cell lines (RM5, FDD7, and 11F9) produced immunoglobulin M (IgM) antibody species that reacted specifically with P. aeruginosa Fisher immunotypes 2, 4, and 5, respectively, and with LPS extracted from these immunotypes. A fourth cell line (9H10) produced a single IgM antibody species that recognized P. aeruginosa immunotypes 3, 6, and 7 and LPS extracted from them. Monoclonal antibodies secreted by cell lines RM5, FDD7, and 11F9 protected neutropenic mice prophylactically against challenge with P. aeruginosa immunotypes 2, 4, and 5, and those secreted by 9H10 protected against P. aeruginosa immunotypes 3 and 6 but did not protect against immunotype 7. In vivo experiments indicated that antibodies protected mice against infection by increasing the rate of bacterial clearance.
Induction of unresponsiveness to gamma interferon in macrophages infected with Mycobacterium leprae
Authors: Sibley L D , Krahenbuhl J L
Published: January 2020

We have previously demonstrated that Mycobacterium leprae-burdened granuloma macrophages isolated from infected nude mice are refractory to activation by gamma interferon (IFN-gamma). To explore further both the afferent and efferent functional capacity of M. leprae-infected macrophages, we examined the IFN-gamma-mediated activation of resident mouse peritoneal macrophages infected in vitro with live or dead M. leprae. When IFN-gamma was administered within 24 h of M. leprae infection, macrophages were fully activated. However, defective activation was evident at 3 to 5 days postinfection in macrophages that were heavily burdened with viable M. leprae. This defect was evident by four parameters of activation in which IFN-gamma failed to stimulate the enhancement of microbicidal activity, cytotoxicity for tumor target cells, O2- production, and surface Ia antigen expression. The development of defective activation closely followed an increase in macrophage production of prostaglandin E2. Defective activation of M. leprae-burdened macrophages was reversible by indomethacin, and a similar block in IFN-gamma activation was observed in three of these four parameters in normal macrophages treated with exogenous prostaglandin E2. Thus, infection of mouse macrophages with M. leprae appears to restrict IFN-gamma-mediated activation at least in part by induction of inhibitory levels of prostaglandin E2.
T-cell subsets in delayed-type hypersensitivity, protection, and granuloma formation in primary and secondary Listeria infection in mice: superior role of Lyt-2+ cells in acquired immunity
Authors: Mielke M E , Ehlers S , Hahn H
Published: January 2020

Immunity to Listeria monocytogenes was studied in mice treated with rat monoclonal antibodies (MAbs) specific for the Thy-1.2, L3T4, and Lyt-2 T-cell markers. Three characteristic T-cell-mediated phenomena were investigated. Delayed-type hypersensitivity (DTH) to listerial antigen was totally abolished in mice treated with anti-Thy-1.2 or anti-L3T4 MAbs, whereas anti-Lyt-2 MAb treatment had no effect, regardless of whether the MAb was given during the induction or the expression of DTH. On the other hand, the elimination of bacteria from the spleens of infected animals was inhibited only by the application of either anti-Thy-1.2 MAb or anti-Lyt-2 MAb. This could be shown most impressively during the secondary infection of immune mice with a normally lethal dose of listeriae. In this situation, treatment with anti-Lyt-2 MAb sufficed to completely abolish immunologic memory, whereas anti-L3T4 MAb had only a marginal effect on antibacterial protection. However, the accelerated development of mononuclear cell foci in the livers of immune mice was inhibited by the application of both anti-L3T4 MAb and anti-Lyt-2 MAb. It is concluded that in murine listeriosis, DTH and acquired immunity to reinfection are dissociable phenomena. Although DTH is a function of L3T4+ T lymphocytes, Lyt-2+ T cells are necessary and sufficient for the expression of acquired resistance to L. monocytogenes. The roles of the different T-cell subsets in granuloma formation warrant further investigation.
Cloning of the gene encoding streptococcal immunoglobulin A protease and its expression in Escherichia coli
Authors: Gilbert J V , Plaut A G , Fishman Y , Wright A
Published: January 2020

We have identified and cloned a 6-kilobase-pair segment of chromosomal DNA from Streptococcus sanguis ATCC 10556 that encodes immunoglobulin A (IgA) protease activity when cloned into Escherichia coli. The enzyme specified by the iga gene in plasmid pJG1 accumulates in the periplasm of E. coli MM294 cells and has a substrate specificity for human IgA1 identical to that of native S. sanguis protease. Hybridization experiments with probes from within the encoding DNA showed no detectable homology at the nucleotide sequence level with chromosomal DNA of gram-negative bacteria that excrete IgA protease. Moreover, the S. sanguis iga gene probes showed no detectable hybridization with chromosomal DNA of S. pneumoniae, although the IgA proteases of these two streptococcal species cleaved the identical peptide bond in the human IgA1 heavy-chain hinge region.
Molecular cloning and expression of a Streptococcus mutans major surface protein antigen, P1 (I/II), in Escherichia coli
Authors: Lee S F , Progulske-Fox A , Bleiweis A S
Published: January 2020

Antigen P1, also called I/II, is one of the most abundant cell wall proteins of the mutans streptococci. It has been suggested that P1 may be involved in cell adherence to tooth surfaces and in sucrose-induced cell aggregation. As a first step toward fully understanding its biological functions, the P1 gene, which has been designated spaP1, from Streptococcus mutans NG5 (serotype c) has been cloned into Escherichia coli JM109 by a shotgun procedure with pUC18 as the vector. The recombinant strain expressing P1 carries a 5.2-kilobase DNA insert whose restriction map has been determined. This map is completely different from that of spaA of Streptococcus sobrinus (serotype g), even though P1 and SpaA are antigenically related. Southern hybridization revealed that DNA sequences closely homologous to spaP1 were present in serotypes c, e, and f, and similar sequences also existed in strains of serotypes a and d. The expression of the cloned spaP1 was found to be independent of the lac inducer and the orientation of the DNA insert, suggesting that it carries its own promoter. Western blotting (immunoblotting) revealed at least 20 bands reacting with a mixture of three anti-P1 monoclonal antibodies. The highest-molecular-weight reactive band was comparable in size to the parent P1 (185 kilodaltons [kDa]); however, the major reactive bands were smaller (approximately 160 kDa). Expression of cloned P1 in E. coli LC137 (htpR lonR9) resulted in the increased prominence of the 185-kDa protein reactive band. Ouchterlony immunodiffusion showed partial identity between the parent and cloned P1. In E. coli, P1 was detected primarily in the periplasm and extracellular fluid.
Conservation of protective and nonprotective epitopes in M proteins of group A streptococci
Authors: Miller L , Burdett V , Poirier T P , Gray L D , Beachey E H , Kehoe M A
Published: January 2020

Carefully controlled hybridization experiments with probes from a cloned serotype 5 M protein (M5) gene (smp5) were performed with DNA isolated from heterologous M types of group A streptococci, and the homologies detected by hybridization were compared with the ability of anti-pepM5 serum to cross-opsonize heterologous M types. As previously reported (J.R. Scott, S.K. Hollingshead, and V.A. Fischetti, Infect. Immun. 52:609-612, 1986), extensive structural homologies exist among the 3' ends of heterologous M protein genes, but there appears to be an increase in sequence variation as one moves towards the 5' ends. However, a clear, predictive correlation between the hybridization patterns and cross-opsonization was not observed. Antibodies raised to a synthetic peptide corresponding to central, conserved sequences adjacent to the C-terminal sides of the pepsin cleavage sites in M5, serotype 6 M protein, and serotype 24 M protein cross-reacted with heterologous acid-extracted M antigens but were not protective and did not bind to intact streptococcal cells, indicating that these epitopes are inaccessible on the intact cell surface. Removal of the N-terminal half of M5, serotype 6 M protein, or serotype 24 M protein by pepsin exposed the conserved epitope on the cell surface. These results suggest that immunoaccessible protective epitopes are confined to the highly variable N-terminal halves of M proteins and that a single, broadly conserved protective M protein epitope does not exist.
Protective immunity in murine histoplasmosis: functional comparison of adoptively transferred T-cell clones and splenic T cells
Authors: Deepe G S
Published: January 2020

In this study, I examined whether a murine T-cell line and three clones that recognize Histoplasma capsulatum antigens in vitro could confer protection in vivo against a challenge of Histoplasma yeasts. C57BL/6 mice were each inoculated with 5 X 10(4) yeasts intravenously; 1 h later, 5 X 10(6) or 2 X 10(7) resting T cells were inoculated intravenously. At week 1 of infection, the T-cell line and all clones failed to reduce the number of H. capsulatum CFU in the spleens of mice compared with numbers in infected controls. Administration of recombinant interleukin 2 or cyclophosphamide to infected mice did not potentiate the functional activity in vivo of either the T-cell line or the clones. In contrast, inoculation with 2 X 10(7) CD4+ but not CD8+ cells isolated from the spleens of mice immunized with 10(6) viable yeast cells sharply diminished the number of CFU in the spleens of infected animals. Moreover, splenic CD4+ cells from immune mice transferred a delayed-type hypersensitivity response, whereas the T-cell line and clones did not. Injection Injection of an equal number of cloned T cells and CD8+ splenocytes from immune mice did not transfer resistance to infected mice. Additional studies were undertaken to determine if the ineffectiveness of cloned T cells was associated with a failure to migrate to and survive within spleens of infected mice. B6.PL Thy-1a/Cy mice, which are genetically identical to C57BL/6 mice except that T cells of the former bear Thy-1.1 rather than Thy-1.2, were inoculated with Histoplasma yeasts and then injected with immune CD4+ splenocytes or a T-cell clone. At days 1 and 7 of infection, virtually no Thy-1.2+ cells were detected in the spleens of infected mice given cloned T cells. However, the spleens of animals inoculated with immune CD4+ cells contained a small but significant (P less than 0.01) proportion of Thy-1.2+ cells at both day 1 and day 7 postinoculation of H. capsulatum. Thus, the failure of T-cell clones to transfer protection against H. capsulatum may be explained by defective trafficking or poor survival in vivo or both.
Identification of Babesia bovis merozoite surface antigens by using immune bovine sera and monoclonal antibodies
Authors: Goff W L , Davis W C , Palmer G H , McElwain T F , Johnson W C , Bailey J F , McGuire T C
Published: January 2020

Three Babesia bovis merozoite surface proteins with relative molecular weights of 37,000, 42,000, and 60,000 were identified by indirect immunofluorescence of live merozoites and by immunoprecipitation of 125I-surface-labeled merozoite proteins with immune bovine sera and monoclonal antibodies. These proteins were clearly of parasite origin, as evidenced by immunoprecipitation of metabolically labeled [( 35S]methionine) merozoites from cultures with specific antimerozoite monoclonal antibodies. In addition, two other proteins were identified with these methods. An 85-kilodalton protein was considered to be of parasite origin based on fluorescence reactivity with a monoclonal antibody. However, this protein was not detected after immunoprecipitation of metabolically labeled parasites, and thus, the exact nature of its origin is equivocal. A fifth protein of 145 kilodaltons was detected by immunoprecipitation after metabolic labeling but was not directly apparent on the surfaces of live merozoites. Since merozoite surface proteins may be important in the induction of protective immunity, those identified here are candidates for vaccine studies.