bluuebunny/crossref_metadata_2025 huggingface dataset:
Characterization of the secreted antigens of Mycobacterium bovis BCG: comparison of the 46-kilodalton dimeric protein with proteins MPB64 and MPB70
Authors: Abou-Zeid C , Harboe M , Rook G A
Published: January 2020

Western blot analysis showed that the 46-kilodalton (kDa) dimeric protein antigen secreted in large amounts by some daughter strains of Mycobacterium bovis BCG corresponded to protein MPB70 present in long-term culture filtrates of the Japanese substrain. The 46/23-kDa antigen is the most abundant protein in supernatant from a 5-day culture but is masked by leaked products in old culture supernatants. No similarities were found between the 46-kDa protein and MPB64, a protein with the same strain distribution, or with the antigen of similar molecular mass recognized by monoclonal antibody SA1.D2D.
Extensive homology between the leukotoxin of Pasteurella haemolytica A1 and the alpha-hemolysin of Escherichia coli
Authors: Strathdee C A , Lo R Y
Published: January 2020

The 19.8- and 101.9-kilodalton leukotoxin proteins of Pasteurella haemolytica (LKTC and LKTA, respectively) share extensive homology with the HLYC and HLYA alpha-hemolysin proteins of Escherichia coli. The leukotoxin LKTA protein cross-reacts with hemolysin-specific antisera in Western blot (immunoblot) analysis, indicating that it shares epitopes with the alpha-hemolysin HLYA protein. Both LKTA and HLYA contain a conserved hydrophobic region, as well as a set of tandemly repeated domains. These features have been implicated in the lytic function of the alpha-hemolysin.
Characterization of genes encoding type 1 fimbriae of Klebsiella pneumoniae, Salmonella typhimurium, and Serratia marcescens
Authors: Clegg S , Purcell B K , Pruckler J
Published: January 2020

With a minicell system, the organization of genes encoding type 1 fimbriae of Salmonella typhimurium, Klebsiella pneumoniae, and Serratia marcescens was determined. In all cases multiple gene products were necessary for the phenotypic expression of fimbriae; thus fimbrial expression in these strains is similar to that in Escherichia coli. The type 1 fimbrial subunit gene was detected by the ability of its product to react with specific antiserum. At least six genes were found to be involved in the expression of type 1 fimbriae by S. typhimurium, and at least four genes constituted the fimbrial gene cluster of K. pneumoniae. In the case of S. marcescens, a minimum of three detectable polypeptides was required for the production of fimbriae. Also, a gene probe consisting in part of nucleotide sequences from the E. coli fimbrial subunit gene hybridized to a discrete DNA fragment derived from the plasmid encoding K. pneumoniae fimbriae. Such a fragment was assumed to contain a gene encoding the structural component of the type 1 fimbriae. Each of the three cloned systems encoded a number of polypeptides which varied in size; thus, the organization and molecular weight of fimbrial accessory proteins of each genus were not identical.
Association and dissociation of Escherichia coli heat-stable enterotoxin from rat brush border membrane receptors
Authors: Cohen M B , Thompson M R , Overmann G J , Giannella R A
Published: January 2020

Escherichia coli heat-stable enterotoxin (ST) binds to receptors on rat intestinal cells and brush border membranes (BBM). We devised experiments to examine the reversibility of ST binding. We found that both 125I-labeled ST and native ST were spontaneously dissociable from the BBM receptor. Radiolabeled ST bound to BBM was also dissociated by the addition of avid goat anti-ST antiserum. Furthermore, using a computer program for analysis of ligand binding, we calculated an apparent Ka of 10(8) liters/mol from competitive inhibition and saturation-binding data. This is significantly lower than the value previously reported by others. Our findings, of a lower Ka and a reversible ST-binding process, suggest that a therapeutic strategy of removing bound ST from its receptor or competing with the enterocyte receptor for unbound ST might be successful in terminating ST-induced secretion.
Expression of staphylococcal enterotoxin C1 in Escherichia coli
Authors: Bohach G A , Schlievert P M
Published: January 2020

The structural gene encoding staphylococcal enterotoxin C1 was cloned into Escherichia coli and localized on a 1.5-kilobase HindIII-ClaI DNA fragment by subcloning. The toxin was partially purified from E. coli clones and shown to be immunologically identical to enterotoxin C1 from Staphylococcus aureus. The cloned toxin also had the same molecular weight (26,000) and charge heterogeneity as staphylococcus-derived enterotoxin. Toxins from both sources were equally biologically active.
A plasmid of enterohemorrhagic Escherichia coli O157:H7 is required for expression of a new fimbrial antigen and for adhesion to epithelial cells
Authors: Karch H , Heesemann J , Laufs R , O'Brien A D , Tacket C O , Levine M M
Published: January 2020

Of 14 strains of Escherichia coli O157:H7 isolated from patients with hemorrhagic colitis or hemolytic uremic syndrome that were examined for fimbriae, the presence of plasmids, and the ability to adhere to intestinal cells, 13 possessed a 60-megadalton plasmid and were fimbriated as assessed by electron microscopy. These strains adhered to Henle 407 intestinal cells but not to HEp-2 cells or erythrocytes. Three strains were cured of the plasmid and thereafter failed to express fimbriae and lost the ability to adhere to intestinal cells. Conversely, E. coli K-12 transformed with the 60-megadalton plasmid from each of the three strains produced fimbriae and was able to adhere to intestinal cells. A single fimbrial subunit of 16 kilodaltons was observed when purified fimbriae from the transformants and from the 60-megadalton plasmid-containing E. coli O157:H7 strains were disaggregated and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Antisera raised against one preparation of the purified fimbriae reacted strongly with 12 of 14 O157:H7 isolates in an agglutination assay and with purified fimbrial preparations from five E. coli O157:H7 strains in an enzyme-linked immunosorbent assay.
Antipyretic activity of a human immunoglobulin preparation for intravenous use in an experimental model of fever in rabbits
Authors: Iwata M , Shimozato T , Tokiwa H , Tsubura E
Published: January 2020

In an effort to elucidate the reason that fever in patients with severe bacterial infections subsided in some cases after the administration of human immunoglobulin preparations for intravenous use (IGIVs), we focused our attention on the antipyretic activity of IGIVs by investigating experimentally produced pyrexia in rabbits with Escherichia coli-derived lipopolysaccharide (LPS). Although little difference in antibody titers against the antigens composing molecules of LPS was found among the IGIVs that were used, IGIVs treated at pH 4 were demonstrated to inhibit a strongly LPS-induced second-phase febrile response, whereas the inhibitory effect of sulfonated and pepsin-treated IGIVs was weak. In vitro experiments on interleukin-1 production by rabbit macrophages stimulated with LPS, silica gel or latex beads and on rosette formation showed that these functions of the cells were also inhibited by IGIVs. The in vivo antipyretic activity and the results of the two in vitro experiments correlated closely. The inhibitory potency decreased in the following order: immunoglobulin G (IgG) treated at pH4, sulfonated IgG, and pepsin-treated IgG. Thus, it is possible that the subsidence of LPS-induced fever by IGIVs was mediated by inhibition of interleukin 1 production by means of binding of IgG to macrophages via an Fc receptor. Results of this study also indicated the importance of the structural integrity of the Fc portion of the IgG contained in the IGIVs to bind with its receptor on the macrophage so as to influence the various functions carried out by the cell.
In vitro effect of synthetic pyocyanine on neutrophil superoxide production
Authors: Miller K M , Dearborn D G , Sorensen R U
Published: January 2020

Pyocyanine, a low-molecular-weight phenazine pigment produced by Pseudomonas aeruginosa, has previously been shown to strongly inhibit human lymphocyte blastogenesis. We now report that synthetic pyocyanine can also affect the generation of superoxide by human peripheral blood polymorphonuclear leukocytes (PMNs) in a dose-dependent manner. Superoxide production by PMNs stimulated with phorbol myristate acetate (PMA) was measured in the presence and absence of pyocyanine, phenazine, and trifluoperazine, a phenothiazine of similar chemical structure to the phenazine pigments. Pyocyanine at 50 microM inhibited superoxide production to 28.9 +/- 2.8% of PMA control values, whereas at the lower concentration of 1 microM, the production of superoxide was significantly enhanced (203 +/- 31.7% of PMA control values). Phenazine, the tricyclic parent compound of pyocyanine, had only a minor effect. Trifluoperazine had a marked inhibitory effect on superoxide generation at concentrations above 1 microM. None of the compounds induced superoxide generation in the absence of PMA. Pyocyanine at all concentrations, unlike phenothiazines, had very little effect on the release of neutrophil granule enzymes. The effect of P. aeruginosa phenazine pigments on polymorphonuclear phagocytes is of significance, since inhibition of host PMN function at sites of infection could result in ineffective bacterial killing, whereas enhanced PMN function could lead to greater tissue damage. These two possibilities are not mutually exclusive and may coexist depending on local pyocyanine concentrations.
Virulence of iron transport mutants of Shigella flexneri and utilization of host iron compounds
Authors: Lawlor K M , Daskaleros P A , Robinson R E , Payne S M
Published: January 2020

Mutants of Shigella flexneri defective in aerobactin-mediated iron transport were assayed for virulence in several model systems. A Tn5 insertion mutant was invasive in HeLa cells, lethal in the chicken embryo, and produced keratoconjunctivitis in the guinea pig, indicating little or no loss of ability to invade and multiply intracellularly. Although the mutant failed to grow in low-iron medium in vitro, growth equivalent to that of the wild type was observed in HeLa cell lysates. Thus, there appears to be sufficient available iron inside the HeLa cell to allow growth in the absence of siderophore synthesis. Possible host iron sources were tested, and both the mutant and wild type utilized hemin or hematin as a sole source of iron. Only the wild-type, aerobactin-producing strain could remove iron from transferrin or lactoferrin. Two deletion mutants were also assayed for virulence and were found to be avirulent for the chicken embryo. These deletions encompass flanking sequences as well as the aerobactin genes; therefore, adjacent genes may be required for virulence.
Keratinolytic activity of cutaneous and oral bacteria
Authors: Mikx F H , de Jong M H
Published: January 2020

A test was developed to measure the keratinolytic activity of cutaneous and oral bacteria. Keratin, labeled with fluorescein isothiocyanate, was used in a phosphate buffer (pH 7.2) with 1 mM dithiothreitol. The degradation of keratin was estimated by measuring the fluorescence of the degradation products in the supernatant of the reaction mixtures in a luminescence spectrometer. Several oral and cutaneous bacteria were investigated: Bacteroides gingivalis, Bacteroides intermedius, Treponema denticola, Actinomyces odontolyticus, Staphylococcus epidermidis, Staphylococcus haemolyticus, Staphylococcus hominis, Staphylococcus saprophyticus, Brevibacterium epidermidis, Brevibacterium lines, Corynebacterium glutamicum, Corynebacterium minutissimum, Corynebacterium ovis, and Rhodococcus equi. The dermatophyte Trichophyton rubrum was used as a control organism. The test offered a simple and quantitative method for the estimation of keratin degradation and enabled us to show keratinolytic activity in Trichophyton rubrum, S. epidermidis, S. haemolyticus, B. linens, B. epidermidis, Bacteroides gingivalis, and Treponema denticola. The keratinolytic activity was cell bound and heat sensitive. The presence of dithiothreitol stimulated the degradation of keratin to mainly high-molecular-weight products.
An immunoreactive, water-soluble conidial wall fraction of Coccidioides immitis
Authors: Cole G T , Kirkland T N , Sun S H
Published: January 2020

Arthroconidia stripped of their outer, hydrophobic wall layer release an immunoreactive, water-soluble fraction (SCWF), the composition of which is examined in this paper. The immunogenicity of SCWF was determined by its reactivity with anti-Coccidioides immitis complement-fixing antibody and tube precipitin antibody in a standardized immunodiffusion assay as well as reactivity in a lymphocyte proliferation assay. SCWF was shown to be more immunoreactive for immune lymphocytes than selected cytosol and culture supernatant fractions of C. immitis. Rabbit antisera raised against SCWF were used in immunoelectron-microscopic and immunofluorescence studies to confirm that the immunoreactive components of SCWF are primarily associated with the inner conidial wall. The antigenic composition of the conidial wall fraction was characterized by advancing-line immunoelectrophoresis with the previously established coccidioidin reference system. Protein composition was characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Chromatographic fractionation of SCWF was performed on a Sephacryl S-300 preparative column. Selected fractions which showed significantly higher immunoreactivity and less complex antigenic composition than whole SCWF were characterized. Two heat-sensitive antigens of Sephacryl fraction 3a were identified. One or both of these antigens may correspond to the complement-fixing antigen, which is potentially important as an immunodiagnostic antigen. Fraction 4 contained a previously described wall-associated antigen (AgCS) which may also be of immunodiagnostic value. We conclude that the conidial envelope is a reservoir of immunoreactive macromolecules which may play significant roles in early stages of infection.
Epidemiological correlation between levels of antibodies promoting merozoite phagocytosis of Plasmodium falciparum and malaria-immune status
Authors: Druilhe P , Khusmith S
Published: January 2020

A phagocytosis assay was used to measure the prevalence of cytophilic antibodies directed against Plasmodium falciparum merozoites in three groups of subjects from Africa: preimmune (individuals aged 2 to 15 years), immune (adults), and postimmune (African adults residing out of the endemic area). Results show that levels of antibodies promoting merozoite phagocytosis (APMP) increase slowly and gradually with age. The production of high levels of APMP requires about 15 years of continuous exposure to malaria and concurs with the ability of exposed individuals to control a high parasitemia and its pathological consequences, such as spleen enlargement. In the absence of antigenic restimulation for more than 1 year (postimmune subjects), APMP titers decrease abruptly. No correlation was found between APMP levels and levels of antimalarial antibodies detected by fluorescence and precipitation assays. Low levels of APMP in subjects susceptible to clinical manifestations of the disease and high levels in subjects in a state of premunition suggest that the results of the merozoite phagocytosis assay more closely reflect clinical immunity than do other markers of antimalarial humoral immunity.
Inhibition of human natural killer cell activity by Pseudomonas aeruginosa alkaline protease and elastase
Authors: Pedersen B K , Kharazmi A
Published: January 2020

The present study was designed to examine the effect of Pseudomonas aeruginosa alkaline protease (AP) and elastase (Ela) on human natural killer (NK) cell activity in vitro. AP and Ela were found to inhibit NK cell function. Addition of alpha interferon and interleukin-2 did not abolish this inhibition of NK cell activity. Adhesion of effector to target cells was studied in a single-cell agarose assay of monocyte-depleted NK-cell-enriched cell populations. AP and Ela were shown to inhibit effector/target cell conjugate formation. Furthermore, AP and Ela inhibited the binding of the monoclonal antibody Leu-11, which reacts with the Fc receptor of NK cells. The inhibition of NK cell binding to the target cell by P. aeruginosa proteases is most likely due to proteolytic cleavage of the surface receptors involved in the binding of the effector cell to the target cell.
Protective immunity in toxoplasmosis: correlation between antibody response, brain cyst formation, T-cell activation, and survival in normal and B-cell-deficient mice bearing the H-2k haplotype
Authors: Brinkmann V , Remington J S , Sharma S D
Published: January 2020

Correlations of Toxoplasma gondii-specific immunoglobulin M (IgM) and IgG production, antigen-specific T-cell activation, and the number of brain cysts were compared in immunocompetent CBA/J (H-2k), C3H/He (H-2k), and B-cell-deficient CBA/N (H-2k) mice. Almost all of the C3H/He mice (94%) survived in comparison to CBA/J (71%) and CBA/N (53%) mice following infection with 20 cysts of Me 49, an avirulent strain of T. gondii. The mortality in susceptible mice was reduced by treatment of the animals with sulfadiazine during the acute stage of infection. Decreased mortality in CBA/J and C3H/He mice as well as in B-cell-deficient mice was paralleled by formation of fewer brain cysts. The Toxoplasma-specific T-cell proliferation was markedly enhanced in all three strains at day 15 postinfection but not at day 45 postinfection when compared to animals not treated with the drug. In contrast, Toxoplasma-specific IgM and IgG levels were lower in CBA/J and CBA/N mice treated with sulfadiazine than in untreated mice of these strains. Although CBA/N mice developed almost no humoral response either with or without drug treatment, they produced fewer brain cysts than normal CBA/J mice. The results indicate a major role of cell-mediated immunity in protection against an acute Toxoplasma infection.
Identification of a Streptococcus agalactiae protein antigen associated with bovine mastitis isolates
Authors: Wanger A R , Dunny G M
Published: January 2020

Immunoblotting was used to analyze the immune response of cows to Streptococcus agalactiae. Antibody from the milk of cows immunized (via the superficial inguinal lymph node) with formalinized S. agalactiae cells or from the milk of cows with S. agalactiae mastitis reacted strongly with a group of high-molecular-weight proteinaceous antigens. The two most predominant antigenic polypeptides in this group had apparent molecular weights of 97,000 and 104,000. Because the data indicated that these two antigens, as well as several minor antigens sometimes observed in the 70- to 100-kilodalton size range, seemed to be different forms of the same protein, we refer to the entire group as Sas97/104. A monoclonal antibody that was reactive with Sas97/104 was derived and was used to purify the antigen by affinity chromatography. Whole-cell and colony blot enzyme-linked immunoassays with either the monoclonal antibody or a polyclonal serum sample raised against the affinity-purified antigen indicated that this antigen (or cross-reactive proteins with higher molecular weights) is present on the S. agalactiae strains that were freshly isolated from mastitic cows. However, the antigen was not detected in S. agalactiae of human origin, bovine strains of S. agalactiae maintained for a prolonged period in the laboratory, or other streptococci. The data are consistent with the notion that Sas97/104 is a surface antigen and does not correspond to previously described type-specific antigens of group B streptococci.
Clonal diversity in Haemophilus pleuropneumoniae
Authors: Musser J M , Rapp V J , Selander R K
Published: January 2020

Genetic diversity among 135 isolates of nine serotypes of Haemophilus pleuropneumoniae recovered from pigs with pleuropneumonia or other invasive diseases in 14 countries was estimated by multilocus enzyme electrophoresis, which detects allelic variation in structural genes. Thirty-two multilocus genotypes (electrophoretic types [ETs]) were distinguished on the basis of allele profiles at 15 enzyme loci, and 36 distinctive combinations of ET and serotype were identified. The recovery of isolates with identical properties in widely separated geographic regions and over a 20-year period indicated that the population structure of H. pleuropneumoniae is clonal. Isolates of the same ET generally shared the same serotype and electrophoretic pattern of the outer membrane proteins, but some ETs were represented by isolates of several different serotypes, outer membrane protein patterns, or both. On average, the genetic diversity among ETs of the same serotype was 56% of the total genetic diversity in the species. Isolates of serotype 1, which are unusually pathogenic, belong to a distinctive group of clones that are closely related to clones marked by serotype 9.
Characterization of a rat salivary sialoglycoprotein complex which agglutinates Streptococcus mutans
Authors: Brack C M , Reynolds E C
Published: January 2020

Rat saliva agglutinated Streptococcus mutans Ingbritt and NCTC 10449 and Streptococcus sanguis NCTC 7864 but not S. mutans NCTC 10921, GS 5, or LM 7, Streptococcus sobrinus 6715-13 or OMZ 65, or Streptococcus cricetus HS 6, as measured turbidometrically. The specificity of agglutination by rat saliva was the same as that by human saliva. Agglutination was associated with a mucin complex (rat salivary agglutinin complex [rS-A]) of sulfated sialoglycoproteins, with a trace of associated lipid and an apparent Mr of 1.6 X 10(6), isolated by gel-filtration Fast Protein Liquid Chromatography. The complex was dissociated in a high-ionic-strength buffer containing 6 M urea and then fractionated by gel filtration and anion-exchange Fast Protein Liquid Chromatography into four sulfated sialoglycoprotein components, designated rS-A-1Q1, rS-A-1Q2, rS-A-1Q3, and rS-A-2, with rS-A-1Q2 being polydisperse through differential glycosylation of the polypeptide backbone. The dissociation destroyed agglutination activity. The polypeptide backbones contained up to 42% serine plus threonine and up to 40% glycine plus alanine plus proline plus valine. The carbohydrate moiety of the rS-A sialoglycoproteins consisted of N-acetylgalactosamine, sialate, galactose, fucose, N-acetylglucosamine, and small amounts of mannose, with the predominant sugar being N-acetylgalactosamine. Agglutination was inhibited by 1 mM EDTA but was restored by 1.5 mM CaCl2. Agglutination was also inhibited by 5 mM CaCl2; nonimmune sera; cationic polymers; and wheat germ, lentil, soybean, and peanut lectins. However, agglutination was not affected by lipoteichoic acid, various anionic proteins, or various sugars. Neuraminidase treatment of rS-A did not affect activity, but tryptic digestion of S. mutans did prevent agglutination. The results are consistent with calcium bridging the negative groups within the rS-A complex and allowing the approach of rS-A to the bacterial cell surface to effect a specific conformational attachment.
Immunoregulatory role of the spleen in antibody responses to pneumococcal polysaccharide antigens
Authors: Cohn D A , Schiffman G
Published: January 2020

Antibody responses to two structurally different pneumococcal polysaccharides, type 3 (SIII) and type 14 (SXIV), were examined in intact and splenectomized (Sx) A/J mice to determine whether the role of the spleen in immune responses to these antigens varies with respect to the dosage, the antigenic structure, or the interval between immunization and assay. Antibody responses to SIII and SXIV, measured over a 4-week period by radioimmunoassay, differed in antigenic load requirements, kinetics, and extent of dependence upon the spleen. Intact mice given 50 or 100 ng of SIII produced peak antibody responses on day 5, which tapered off by days 14 and 21. Intact mice given SXIV required doses 100 times greater than those of SIII to stimulate high levels of antibody response; antibody responses increased on day 5 and remained elevated through day 28. In Sx mice given 50 or 100 ng of SIII, the peak antibody response on day 5 was obliterated, but extrasplenic sources produced low levels of antibody which peaked by day 14. In Sx mice given SXIV, all anti-SXIV responses were abrogated regardless of the dose or day of assay. Differences between the anti-SIII and anti-SXIV responses in dependence upon the spleen were probably due to structural differences between the two antigens and to the localization of each to different sites in the reticuloendothelial system. These results attest to the importance of the spleen in antibacterial resistance. They show that, even in the presence of extrasplenic antibody synthesis, the spleen is required for early antibody production, the timing of which is critical for the effective clearance of bacteria.
Enhanced resistance of mice to bacterial infection induced by recombinant human interleukin-1a
Authors: Ozaki Y , Ohashi T , Minami A , Nakamura S
Published: January 2020

The effect of recombinant human interleukin-1a on the survival rate of Std-ddY male mice systemically infected with Pseudomonas aeruginosa 12 or Klebsiella pneumoniae P-5709 was evaluated. In P. aeruginosa infection, interleukin-1a given intramuscularly twice, 3 days and 1 day before inoculation of bacteria, most effectively protected animals from death due to infection. The effect was dose dependent, with a maximum survival rate of 92.5% at 10 micrograms per mouse, while only 8.3% of the control group survived until the end of the observation period. The 50% effective dose of interleukin-1a was 0.261 microgram per mouse. In K. pneumoniae infection, interleukin-1a given intramuscularly twice, simultaneously with and 1 day after the inoculation of bacteria, was most effective. The protective effect of interleukin-1a was again dose dependent and was generally more marked than in P. aeruginosa infection. The 50% effective dose was 0.034 microgram per mouse. In both infections, there was no significant increase in the survival rates of animals injected with human albumin or heat-inactivated interleukin-1a. These observations raise the possibility that human interleukin-1a could serve as a therapeutic tool for patients with bacterial infections.
Opsonization of Staphylococcus aureus protects endothelial cells from damage by phagocytosing polymorphonuclear leukocytes
Authors: Vandenbroucke-Grauls C M , Thijssen H M , Verhoef J
Published: January 2020

When phagocytosis of Staphylococcus aureus by human polymorphonuclear leukocytes (PMN) takes place on the surface of cultured human endothelial cells, the endothelial monolayers are damaged by lysosomal enzymes that are released by the PMN. Because PMN can phagocytose opsonized as well as unopsonized staphylococci on an endothelial surface, we studied the role of bacterial opsonization in the damage caused to the endothelium. Phagocytosis of unopsonized S. aureus was accompanied by greater damage (expressed as the percentage of the endothelial cells detached from the culture plates) of the monolayers than was phagocytosis of opsonized S. aureus: 52 +/- 10% and 24 +/- 7%, respectively, after 30 min of phagocytosis and 73 +/- 5% and 50 +/- 6%, respectively, after 60 min of phagocytosis. When correlated to the amount of phagocytosis, this difference was even greater (uptake was 35 +/- 4% for unopsonized S. aureus and 56 +/- 5% for opsonized S. aureus after 30 min and 42 +/- 3% and 60 +/- 5%, respectively, after 60 min). Total release of lysozyme and myeloperoxidase and generation of superoxide anion were the same during phagocytosis of opsonized or unopsonized staphylococci. Adherence of PMN to the endothelial cells was greater during phagocytosis of unopsonized S. aureus: 42 +/- 4% verus 27 +/- 3% during phagocytosis of opsonized staphylococci. Possibly, increased adherence of the PMN resulted in a locally higher concentration of enzymes which induced more damage. We conclude that opsonization of bacteria not only improves bacterial uptake, but also protects bystander cells from damage by the phagocytosing PMN.
Induction of biologically active antibodies by a polyvalent synthetic vaccine constructed without carrier
Authors: Jolivet M E , Audibert F M , Gras-Masse H , Tartar A L , Schlesinger D H , Wirtz R , Chedid L A
Published: January 2020

Four synthetic peptides that copy fragments of two bacterial antigens (Streptococcus pyogenes M protein and diphtheria toxin), one viral antigen (hepatitis B surface antigen), and one parasitic antigen (circumsporozoite protein of Plasmodium knowlesi) were covalently bound within the same construct. This totally synthetic polyvalent administered to mice with Freund complete adjuvant or in saline with murabutide (an adjuvant-active muramyl peptide) elicited high levels of antibodies which, in certain cases, were shown to be biologically active. The results indicated that these antibodies recognized specifically the four peptides. None of the epitopes were immunodominant. It was also demonstrated that the association of several peptides enhanced their respective immunogenicities as compared with those of their homopolymers. Finally, this study shows that a totally synthetic vaccine administered in saline with a synthetic adjuvant can be immunogenic in the absence of a protein carrier.
Characterization of the binding of Pseudomonas aeruginosa alginate to human epithelial cells
Authors: Doig P , Smith N R , Todd T , Irvin R T
Published: January 2020

The alginate produced by Pseudomonas aeruginosa has been reported to play a role in the adhesion of this bacterium to epithelial cell surfaces, although some controversy concerning this role exists. To clarify this controversy, we investigated the ability of alginate to bind to human buccal epithelial cells (BECs) and human tracheal epithelial cells (TECs). Alginate from P. aeruginosa 492c bound to both BECs and TECs. Alginate from strain 492c was found to be multivalent and thus capable of agglutinating both BECs and TECs. The multivalency of alginate complicated the determination of the number of alginate-specific receptors on the BEC and the apparent association constant (Ka). By using the analysis of Hogg and Winzor (Biochim. Biophys. Acta 843:159-163, 1985), an average valency of 2.6 BEC binding domains per alginate molecule was determined, and the maximum binding capacity per BEC was calculated to be 5.8 X 10(-4) micrograms, with a Ka of 4.1 X 10(-2) ml/micrograms. The binding of alginate to immobilized BECs (where only 50% of the BEC surface is exposed) yielded values of 2.52 X 10(-4) micrograms of alginate per BEC for the maximum binding capacity per BEC and a Ka of 3.30 X 10(-2) ml/micrograms. The alginate-specific site on the BEC surface was trypsin sensitive. Alginate from P. aeruginosa 492a did not bind to BECs, differing substantially from that of strain 492c. The data presented here demonstrate that alginate purified from some strains of P. aeruginosa may bind to TECs and BECs in a defined, specific manner, whereas alginate from other strains does not, reflecting structural diversity in P. aeruginosa alginates.
Antimicrobial binding of a radiolabeled cationic neutrophil granule protein
Authors: Farley M M , Shafer W M , Spitznagel J K
Published: January 2020

A 57,000-dalton protein (CAP57) purified from human polymorphonuclear leukocytes has antimicrobial activity against a number of gram-negative bacteria. We developed a procedure using solid-phase Iodo-gen to radiolabel CAP57 without destroying its antibacterial activity. Iodinated and native CAP57 were electrophoretically identical. Autoradiographs of sodium dodecyl sulfate-polyacrylamide gels revealed greater than 95% of the 125I in a single heavy band in the 57,000-molecular-weight region. The quantity of [125I]CAP57 bound to bacterial test strains was directly proportional to the sensitivity to CAP57.
Two-dimensional gel electrophoresis and immunoblotting of Campylobacter outer membrane proteins
Authors: Dunn B E , Blaser M J , Snyder E L
Published: January 2020

We characterized outer membrane proteins (OMPs) from selected Campylobacter jejuni, C. coli, and C. fetus strains by two-dimensional gel electrophoresis (2DGE), using isoelectric focusing and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and by immunoblotting with immune rabbit serum. The flagellar band with a molecular mass of 63 kilodaltons (kDa) demonstrated previously by one-dimensional SDS-PAGE was shown by 2DGE to consist of one or several charge trains, depending upon the species, strain, and type of preparation studied; each of the individual peptides was found to be antigenic by immunoblotting. In contrast, in all of the strains studied, the major OMP (43 to 44 kDa) of C.jejuni and C. coli consisted of a single isomeric form which was weakly immunogenic. Several minor proteins (29 to 31 kDa) were found to be strongly immunogenic by immunoblotting. C. fetus strains possessed two major OMPs of 45 to 47 kDa, each of which consisted of either a single isomer or a major isomer comprising at least 90% of the major OMP. Serum-resistant strains of C. fetus possessed an acid-labile 100-kDa glycoprotein (pI, 4.1) which was markedly diminished or absent in serum-sensitive strains. These 2DGE analyses provide information that is useful in taxonomic and epidemiologic studies and for the purification of surface antigens for the development of campylobacter vaccines and may also facilitate the identification of specific virulence factors.
Effects of immunosuppression on avian coccidiosis: cyclosporin A but not hormonal bursectomy abrogates host protective immunity
Authors: Lillehoj H S
Published: January 2020

The effects of cyclosporin A (CsA) treatment and hormonal bursectomy on Eimeria tenella infection of chickens were investigated to evaluate the role of humoral antibody and cell-mediated immunity (CMI) in the host protective immunity to an intestinal protozoan disease, coccidiosis. Hormonal bursectomy had no significant effect on the host response to E. tenella. CsA treatment had a differential effect on the course of disease depending on how CsA was given relative to infection. Daily administration of CsA for 7 days beginning 1 day before primary infection with E. tenella enhanced disease resistance, whereas a single dose of CsA given before primary infection enhanced disease susceptibility compared with that of untreated controls. Chickens treated with CsA during the primary infection were resistant to reinfection at 5 weeks post-primary infection. Treatment of chickens immune to E. tenella with CsA at the time of secondary infection abrogated their resistance to reinfection despite the presence of high levels of coccidia-specific secretory immunoglobulin A and serum immunoglobulin G. Splenic lymphocytes obtained after CsA treatment demonstrated a substantially depressed concanavalin A response, but not a depressed lipopolysaccharide response. Because CsA was not directly toxic to parasites in vivo when administered during the secondary infection, these results suggest that CsA interacts with the immune system to allow priming during the primary infection, while interfering with the effector function of CMI during the secondary infection. Taken together, present findings indicate that CMI plays a major role in host protective immunity to E. tenella.
Purification, characterization, and toxicity of the sulfhydryl-activated hemolysin listeriolysin O from Listeria monocytogenes
Authors: Geoffroy C , Gaillard J L , Alouf J E , Berche P
Published: January 2020

We purified and characterized an extracellular hemolysin produced by Listeria monocytogenes. Hemolysin production was greatly enhanced by growing bacteria in resin (Chelex)-treated medium. This hemolysin was separated as a homogeneous protein of 60,000 daltons by using thiol-disulfide exchange affinity chromatography. This protein was a sulfhydryl-activated toxin, termed listeriolysin O, which shared the classical properties of other bacterial sulfhydryl-activated toxins: inhibition by very low amounts of cholesterol; activation by reducing agents and suppression of the lytic activity by oxidation; antigenic cross-reactivity with streptolysin O. However, listeriolysin O differed remarkably from the other sulfhydryl-activated toxins in that its cytolytic activity towards erythrocytes from various animal species was maximum at low pH (approximately 5.5) and was undetectable at pH 7.0. This suggests that the lytic activity of the toxin in host tissues might be better expressed in the acidic microenvironment, including macrophage phagosomes where bacteria presumably replicate. Listeriolysin O was lethal to mice (50% lethal dose of ca. 0.8 microgram) and induced a rapid inflammatory reaction when injected intradermally. These results favor the view that listeriolysin O might play a major role during intracellular replication of L. monocytogenes, ultimately promoting death of infected macrophages.
Discrepancy in assessment of phagosome-lysosome fusion with two lysosomal markers in murine macrophages infected with Candida albicans
Authors: Mor N , Goren M B
Published: January 2020

Phagosome-lysosome fusion (P-LF) was studied in cultured mouse resident peritoneal macrophages after phagocytosis of Candida albicans. The macrophages were labeled with acridine orange (AO), the electronopaque colloidal Thorotrast, or both markers. After phagocytosis of heat-killed C. albicans, both markers were delivered to more than 95% of phagosomes. After ingestion of viable C. albicans by labeled cells, delivery of AO to phagosomes was highly suppressed (90%), and yet Thorotrast delivery was almost universal. After phagocytosis and 60 min of incubation, about 10 to 20% of the yeasts were killed, and a similar fraction of phagosomes was stained by the fluorescent marker. The evidence from Thorotrast transfer and assessment of yeast viability indicates that C. albicans largely resists intracellular killing by resident macrophages in the face of entirely uninhibited P-LF. We infer that AO must transfer to nearly all of the phagosomes but that it is evidently recognizable only in those in which the yeasts have been killed or possibly severely injured. This conclusion constitutes yet another limitation in the usefulness of AO for studying P-LF.
Immunosuppressive effect of cyclosporin A on Mycobacterium bovis BCG infections in mice
Authors: Takashima T , Collins F M
Published: January 2020

The effect of increasing doses of cyclosporin A (CsA) given to mice infected intravenously with Mycobacterium bovis BCG was investigated. Development of both tuberculin hypersensitivity and acquired antituberculous resistance was suppressed in a dose-responsive manner. Daily dosages at 100 mg/kg of body weight prevented any reduction in the BCG counts within the lungs, liver, or spleen. This effect was associated with lowered nonspecific resistance to a Listeria monocytogenes challenge and a decline in specific protective immunity adoptively transferred to naive recipients. CsA treatment had no effect on antilisterial activity by activated macrophages or on the antituberculous immunity expressed by specific memory T cells. CsA treatment inhibited the ability of BCG-vaccinated mice to produce gamma interferon (IFN-gamma) after a secondary stimulation with live BCG or with lipopolysaccharide. Spleen cells from BCG-infected mice which were exposed to daily treatment with CsA showed reduced IFN-gamma production in response to purified protein derivative or concanavalin A stimulation, suggesting that the immunosuppressive effect of CsA on BCG-infected mice was expressed by inhibiting the development of effector T cells responsible for the production of IFN-gamma.
Isolation and characterization of circulating immune complexes from patients with pneumococcal pneumonia
Authors: Mellencamp M A , Preheim L C , McDonald T L
Published: January 2020

Circulating immune complexes (CIC) were isolated from serum samples from patients with bacteremic and nonbacteremic pneumococcal pneumonia. Overall, 63% (26 of 41) of patients with pneumococcal pneumonia had elevated levels of immunoglobulin G (IgG)-containing CIC. IgM-containing CIC were identified in samples from only three patients. Serum samples from nonbacteremic patients contained significantly higher levels of IgG-containing CIC (96.6 +/- 111.7 micrograms/ml) than did samples from bacteremic patients (31.7 +/- 26.9 micrograms/ml) during week 1 in hospital (P less than 0.05). Immune complexes levels did not correlate with IgG concentrations in serum or anticapsular antibody levels. Immune complexes from nonbacteremic patients had sedimentation coefficients of greater than 19s by density gradient ultracentrifugation. In contrast, CIC from bacteremic patients had smaller coefficients, of between 9s and 14s. Pneumococcal capsular antigens were identified in concentrated dissociated CIC from both patient groups by counterimmunoelectrophoresis.
The etiologic agents of leprosy and tuberculosis share an immunoreactive protein antigen with the vaccine strain Mycobacterium bovis BCG
Authors: Shinnick T M , Sweetser D , Thole J , van Embden J , Young R A
Published: January 2020

The amino acid sequences of the 65-kilodalton antigens of Mycobacterium leprae, Mycobacterium tuberculosis, and Mycobacterium bovis BCG display greater than 95% homology.
Trichomonas vaginalis NYH286 phenotypic variation may be coordinated for a repertoire of trichomonad surface immunogens
Authors: Alderete J F
Published: January 2020

Trichomonas vaginalis isolate NYH286 was fractionated with immunoglobulin G of sera from patients with trichomoniasis. Subpopulations of trichomonads with phenotypes of either patient serum-immunoglobulin G reactive (PS+) or nonreactive (PS-) were obtained. Flow cytofluorometry of PS+ and PS- subpopulations with a monoclonal antibody called C20A3 which reacts with a high-molecular-weight immunogen of T. vaginalis gave corresponding fluorescent (positive) and nonfluorescent (negative) phenotypes. No relationship was seen between PS+ and PS- phenotypes and binding of soybean agglutinin, wheat germ agglutinin, and concanavalin A, showing that PS- organisms still possessed carbohydrate moieties on their surfaces based on lectin binding. Phenotypic variation among the PS+ and PS- trichomonads was observed during in vitro growth. A positive-to-negative phenotype shift was also recorded for parasites obtained from lesions of mice subcutaneously infected with PS+ trichomonads. The involvement of surface proteins in the differential PS+ and PS- reactions was supported by soluble antigen and whole cell radioimmunoprecipitation assays. Finally, enhanced parasitism and killing of HeLa cells in monolayer cultures were observed for PS- subpopulations as compared with PS+ counterparts. The data support the idea that phenotypic variation for T. vaginalis may be coordinated for a repertoire of trichomonad immunogens and that such membrane dynamics influence expression of virulence determinants for these sexually transmitted disease agents.
In vivo killing and degradation of Mycobacterium aurum within mouse peritoneal macrophages
Authors: Silva M T , Appelberg R , Silva M N , Macedo P M
Published: January 2020

We studied the in vivo killing and degradation of Mycobacterium aurum, a nonpathogenic, acid-fast bacillus, within macrophages after inoculation into the peritoneal cavity of CD-1 mice. The degradative process could be divided in five successive steps that were characterized on ultrastructural and cytochemical grounds and the relative contributions of which were determined by quantitative electron microscopy of samples taken at different times. The main ultrastructural alterations observed during the degradative process were ribosome disaggregation, coagulation of the cytoplasmic matrix, and change in the membrane profile from asymmetric to symmetric, with loss of the polysaccharide components from the outer layer, followed by membrane solubilization and intracellular clearing, followed by digestion of the innermost (peptidoglycan) layer of the cell wall, and at the end of the process, disorganization and collapse of the remaining layers of the cell wall. The correlation between viability and morphology indicated that the first ultrastructural signs of viability loss are cytoplasmic coagulation, change in the membrane geometry, and disappearance of ribosomes. The labeling of lysosomes of peritoneal macrophages with ferritin or by the cytochemical demonstration of inorganic trimetaphosphatase showed that fusion of lysosomes with phagosomes containing mycobacteria occurs in the phagocytes in the mouse peritoneal cavity and is already extensive as soon as 1 h after the inoculation of the bacilli.
Marker exchange mutagenesis of the aerolysin determinant in Aeromonas hydrophila demonstrates the role of aerolysin in A. hydrophila-associated systemic infections
Authors: Chakraborty T , Huhle B , Hof H , Bergbauer H , Goebel W
Published: January 2020

We report here on the isolation of isogenic strains of Aeromonas hydrophila AB3 deleted for a segment of the aerolysin gene. All aer mutants obtained lacked the 49-kilodalton aerolysin gene product and were neither hemolytic for blood erythrocytes nor cytotoxic for Chinese hamster ovary tissue culture cells. One such mutant, AB3-5, was used in a mouse toxicity model to evaluate the role of aerolysin in the pathogenesis of A. hydrophila infections. The strain had a 50% lethal dose (LD50) of greater than 10(9) as compared with the parental strain which had an LD50 of 5 X 10(7). Reintegration of the deleted segment into AB3-5 resulted in an LD50 of 6 X 10(7) cells for this revertant. Furthermore, all mice injected with a sublethal dose of the parental strains developed necrotic lesions; this was never obtained with the aerolysin-deficient strain AB3-5. More importantly, specific neutralizing antibody to aerolysin was detected in mice surviving A. hydrophila infection, demonstrating that aerolysin is produced during the course of systemic A. hydrophila infections.
Release of lactoferrin by polymorphonuclear leukocytes after aerosol challenge with Escherichia coli
Authors: LaForce F M , Boose D S
Published: January 2020

Mice with cyclophosphamide-induced granulocytopenia were challenged with aerosolized Escherichia coli, their lungs were lavaged at 1 and 4 h, and total cell counts, differential counts, and levels of lactoferrin, transferrin, and albumin were measured in the lung lavage fluid. Lung lavage fluid from cyclophosphamide-treated mice had few neutrophils and no increase in lactoferrin levels, whereas control mice had significant increases in both. Transferrin levels did not change in either group. Neutrophils are the source of increased lactoferrin levels in lung lavage fluid after aerosol challenge.