bluuebunny/crossref_metadata_2025 huggingface dataset:
Experimental Klebsiella pneumoniae burn wound sepsis: role of capsular polysaccharide
Authors: Cryz S J , Fürer F , Germanier R
Published: January 2020

The role of Klebsiella pneumoniae capsular polysaccharide in relation to virulence in a murine burn wound sepsis model was investigated. Burn trauma markedly predisposed mice to lethal K. pneumoniae sepsis. A highly encapsulated variant (KP1-O) derived from K. pneumoniae KP1 was found to be extremely virulent for burned mice (50% lethal dose less than 10 organisms), whereas another variant (KP1-T), which possessed a much smaller capsule, was comparatively nonvirulent (50% lethal dose greater than 10(6) organisms). Production of large quantities of capsular material by KP1-O allowed for its rapid growth in vivo and persistence in the blood and liver. These traits were not demonstrated by KP1-T, which was effectively cleared after challenge.
Reactivity of alkali-soluble, water-soluble cell wall antigen of Coccidioides immitis with anti-Coccidioides immunoglobulin M precipitin antibody
Authors: Cox R A , Huppert M , Starr P , Britt L A
Published: January 2020

The alkali-soluble, water-soluble cell wall antigen of Coccidioides immitis (C-ASWS) mycelia and spherules was shown to react with anti-Coccidioides immunoglobulin M (IgM) precipitin antibody, both in the classical tube precipitin test and in the immunodiffusion assay for tube precipitin antibody (IDTP). The reactions obtained between C-ASWS and reference IgM precipitin antibody were identical to the reaction obtained when reference coccidioidin (CDN) was used. Definitive proof that C-ASWS extracts contain antigenic determinants that are reactive with IgM tube precipitin antibody was obtained by solid-phase immunoadsorption. Elution of reference IDTP antiserum over a column containing mycelium C-ASWS coupled to Sepharose 4B completely adsorbed precipitin antibody; i.e., reactivity in the IDTP was demonstrable in the column eluate but not in the column effluent fraction. The antigenic composition of C-ASWS extracts was evaluated and compared with that of CDN by two-dimensional immunoelectrophoresis against burro anti-CDN. The results established that both mycelium and spherule C-ASWS contain antigenic determinants in common with only one antigen present in CDN. The latter, designated antigen 2, is a large polymer which is predominant among the antigenic components in CDN. On a dry weight comparison, antigen 2 determinants were most concentrated in spherule C-ASWS, followed by mycelium C-ASWS and reference IDTP antigen. The finding that C-ASWS extracts are reactive with IgM tube precipitin antibody and are antigenically identical to antigen 2 in CDN suggests that antigen 2 is the biologically active component of CDN in tube precipitin assays.
Organization and expression of genes involved in the biosynthesis of K99 fimbriae
Authors: de Graaf F K , Krenn B E , Klaasen P
Published: January 2020

Escherichia coli K-12 minicells were used to study the expression of plasmid pFK99 encoding for the production of K99 fimbriae. Plasmid pFK99 is composed of a 6.7-kilobase pair DNA fragment derived from the wild-type K99 plasmid and the vector pBR322. The cloned K99 DNA expressed seven polypeptides with apparent masses of 18.2, 19.0, 21.0, 21.5, 26.5, 33.5, and 76.0 kilodaltons (kd). The 18.2-kd polypeptide was identified as the K99 fimbrial subunit by reaction with specific anti-K99 antibodies. The fimbrial subunit and the 19.0-, 26.5-, 33.5-, and 76.0-kd polypeptides appeared to be synthesized in a precursor form which was ca. 2 kd larger than the mature polypeptide. The location of the structural genes encoding the seven polypeptides on the physical map of pFK99 was established by analyzing a set of deletion derivatives of pFK99. The gene encoding the fimbrial subunit was located at the promoter proximal end of the K99 operon. Only mutants with a deletion in the gene encoding the 33.5- or the 19.0-kd polypeptide or both showed a weak expression of the K99 antigen and a comparably weak agglutination of horse or sheep erythrocytes. None of the deletion mutants was able to adhere to calf intestinal epithelial cells.
Degradation of the human proteinase inhibitors alpha-1-antitrypsin and alpha-2-macroglobulin by Bacteroides gingivalis
Authors: Carlsson J , Herrmann B F , Höfling J F , Sundqvist G K
Published: January 2020

Various strains of black-pigmented Bacteroides species were grown on horse blood agar and suspended in human serum. After various times of incubation the effect of the bacteria on the serum was evaluated by polyacrylamide gel electrophoresis and "rocket" immunoelectrophoresis. The formation of trichloroacetic acid-soluble material in the suspensions and the capacity of the treated sera to inhibit the activity of trypsin were also determined. The two tested strains of Bacteroides gingivalis (W83, H185) degraded most serum proteins, including the plasma proteinase inhibitors alpha-1-antitrypsin and alpha-2-macroglobulin. They did not, however, degrade alpha-1-antichymotrypsin. Bacteroides intermedius NCTC 9336, Bacteroides asaccharolyticus NCTC 9337, and an asaccharolytic oral strain different from B. gingivalis (BN11a-f) did not degrade the plasma proteinase inhibitors. These strains were, however, able to inactivate the capacity of serum to inhibit the activity of trypsin.
Relative potency of virulent versus avirulent Legionella pneumophila for induction of cell-mediated immunity
Authors: Klein T W , Friedman H , Widen R
Published: January 2020

Guinea pigs were infected with either high-passage, low-virulence Legionella pneumophila or low-passage, high-virulence organisms. On an infectious dosage basis, the high-virulence organisms were much more effective at sensitizing animals for positive skin test responses and splenic lymphocyte proliferation responses to homologous antigens. These results suggest that exposure to low doses of virulent L. pneumophila can effectively prime animals for cell-mediated immune responses.
Ultrastructure of Candida parapsilosis endocarditis
Authors: Marrie T J , Cooper J H , Costerton J W
Published: January 2020

We used light microscopy and scanning and transmission electron microscopy to study the structure of Candida parapsilosis vegetations on five surgically removed porcine bioprosthetic valves. Scanning electron microscopy revealed a "mossy" amorphous surface. In some areas, C. parapsilosis cells could be seen below the surface layer. In areas in which the vegetation had cracked, myriads of healthy C. parapsilosis cells were seen to be loosely adherent to one another. These cells were covered with a flocculent or fibrillar matrix. A compact intercellular matrix was evident by transmission electron microscopy. Hyphal elements were seen invading valvular tissue. The loose matrix that bound large clumps of C. parapsilosis cells explains why large emboli are frequent complications of Candida endocarditis.
Attachment of human and pig (K88) enterotoxigenic Escherichia coli strains to either human or porcine small intestinal cells
Authors: Deneke C F , McGowan K , Larson A D , Gorbach S L
Published: January 2020

Enterotoxigenic Escherichia coli strains pathogenic for humans and enterotoxigenic E. coli strains pathogenic for pigs producing the K88 pili antigen both bound to isolated small intestinal cells from either humans or pigs. Neither the K99 enterotoxigenic E. coli (from lambs and calves) nor the rabbit pathogenic strain RDEC-1 bound to human or pig small intestinal cells under the same conditions.
Detection of free endotoxin in cerebrospinal fluid by the Limulus lysate test
Authors: Munford R S , Hall C L , Grimm L
Published: January 2020

We used a rabbit model of Escherichia coli meningitis to study the basis for positive Limulus lysate tests in infected cerebrospinal fluid. The results indicated that positive Limulus tests are due to endotoxins in cerebrospinal fluid and not to leukocyte proteases or other possible activators of the Limulus clotting system. The results also suggest that bacteria-free endotoxin may be present in localized gram-negative bacterial infections.
Identification and extraction of Pasteurella haemolytica membrane proteins
Authors: Squire P G , Smiley D W , Croskell R B
Published: January 2020

The inner and outer membranes of Pasteurella haemolytica were separated by sucrose density gradient centrifugation after plasmolysis of the cells in 20% sucrose and fragmentation in a French pressure cell. Assays of the two membrane fractions for 2-keto-3-deoxyoctonate, succinate dehydrogenase, and NADH dehydrogenase and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that each of the two membrane fractions was purified fivefold relative to the other. The outer membrane fraction contained two major proteins of molecular weights 30,000 and 42,000 (30K and 42K proteins), and the inner membrane fraction contained five proteins in approximately equal amounts. Intact bacteria as well as membrane fractions were extracted by procedures used by others for vaccine preparation to determine whether the outer membrane proteins were released. Extraction of the isolated membranes with 0.5 M potassium thiocyanate in 0.425 M NaCl with or without EDTA or with M sodium salicylate failed to release more than traces of the outer membrane proteins. Sodium dodecyl sulfate extracted essentially all of the proteins of both membranes, but the products of this procedure were of low solubility and presumably denatured. The inner membrane proteins were extracted with 0.5% Sarkosyl in 0.02 M sodium phosphate buffer (pH 7.5). The 42K outer membrane protein, most of the lipopolysaccharide, and some of the 30K outer membrane protein were extracted with 1% Zwittergent 3-16 in 0.25 M NaCl (pH 8), and the remaining 30K outer membrane protein was extracted with 1% deoxycholate in 0.25% NaCl (pH 8). Extraction of membranes in this sequence yielded partially purified membrane proteins that were soluble in dilute buffers.
T suppressor cells as well as anti-hapten and anti-idiotype B lymphocytes regulate contact sensitivity to oxazolone in mice injected with purified protein derivative from Mycobacterium tuberculosis
Authors: Campa M , Benedettini G , De Libero G , Mori L , Falcone G
Published: January 2020

Purified protein derivative from Mycobacterium tuberculosis inhibits contact sensitivity to oxazolone in mice when given intravenously 24 to 72 h before the antigen. Transfer experiments showed that various types of suppressor cells occurred in the lymph nodes draining the site of sensitization: (i) anti-oxazolone idiotype + B lymphocytes, found at day 3 after sensitization, transferred suppression to syngeneic recipients at the moment of their sensitization; (ii) anti-idiotype B lymphocytes, found at day 3 after sensitization, transferred suppression to syngeneic recipients when sensitization of these animals had been performed 3 days before cell transfer; (iii) T lymphocytes, found only at day 6 after sensitization, inhibited the passive transfer of contact sensitivity, indicating that they were effective on the efferent phase of the immune response. These results indicate that purified protein derivative from M. tuberculosis interferes with contact sensitivity by activating a complex and multiple immunoregulatory circuit.
Immunoelectrophoretic analysis, toxicity, and kinetics of in vitro production of the protective antigen and lethal factor components of Bacillus anthracis toxin
Authors: Ezzell J W , Ivins B E , Leppla S H
Published: January 2020

The kinetics of Bacillus anthracis toxin production in culture and its lethal activity in rats, mice, and guinea pigs were investigated. Lethal toxin activity was produced in vitro throughout exponential growth at essentially identical rates in both encapsulated virulent and nonencapsulated avirulent strains. The two toxin proteins which produce lethality when in combination, lethal factor (LF) and protective antigen (PA), could be quantitated directly from culture fluids by rocket immunoelectrophoresis. Using purified preparations of these proteins, we determined that a combination of 8 micrograms of LF and 40 micrograms of PA was required for a maximal rate of killing (39 to 40 min) in Fischer 344 rats (250 to 300 g). Conversely, a minimum of 0.6 microgram of LF and 3 micrograms of PA was required for lethality. The 50% lethal dose for Hartley guinea pigs was 50 micrograms of LF and 250 micrograms of PA, and for Swiss mice it was 2.5 micrograms of LF and 12.5 micrograms of PA. Analyses classically reserved for enzyme kinetic studies were used to study the kinetics of lethal activity in the rat model after intravenous injection of LF-PA mixtures. The amounts of LF and PA which were required to give half the rate of killing (i.e., double the minimum time to death) were 1.2 and 5.8 micrograms, respectively. A theoretical minimum time to death was determined to be 38 min. A third anthrax toxin component, edema factor, was shown to inhibit lethal toxin activity. Edema factor could not be quantitated by rocket immunoelectrophoresis because the protein did not form distinct precipitin bands with available antisera.
Electron microscopic study showing antibody-independent binding of C1q, a subcomponent of the first component of complement, to serum-sensitive salmonellae
Authors: Clas F , Golecki J R , Loos M
Published: January 2020

Effective serum-mediated killing of sensitive gram-negative bacteria requires all the complement components. In the preimmune phase the antibody-independent interaction of the first component of complement, C1, with the bacteria might be especially important. Electron microscopic studies showed that the C1 subcomponent C1q binds only to the serum-sensitive R form of Salmonella minnesota and not to the serum-resistant S form.
Production of macrophage-activating and migration-inhibition factors in vitro by serologically selected and cloned Listeria monocytogenes-specific T cells of the Lyt 1+2- phenotype
Authors: Sperling U , Kaufmann S H , Hahn H
Published: January 2020

Lyt-selected Listeria-immune T lymphocytes from peritoneal exudates and cloned T cells were cocultured with heat-killed listeriae and peritoneal macrophages from nonimmune donors. Supernatants were assayed for: activation of macrophages for tumoristatic and tumoricidal activity via macrophage-activating factors and migration-inhibition factor activity. Peptone-induced peritoneal macrophages were activated by incubation with the supernatants for 24 h. For examination of cytocidal activity, 51Cr-labeled EL4 tumor cells were subsequently added, and 51Cr release was determined. Cytostatic activity was measured by adding unlabeled EL4 tumor cells to the pretreated macrophages and determining [3H]thymidine incorporation 24 h later. Migration-inhibition factor production was examined in an agar microdroplet assay. Only Listeria-specific T cells of the phenotype Lyt 1+2- proved active in these assays, whereas T cells of the phenotype Lyt 1-2+ were not active. When T-cell clones were used, a single clone was capable of inducing macrophage-activating and migration-inhibition factor production at cell concentrations of ca. 10(3)/ml.
Enhancement of Vibrio parahaemolyticus virulence by lysed erythrocyte factor and iron
Authors: Karunasagar I , Joseph S W , Twedt R M , Hada H , Colwell R R
Published: January 2020

The effect of lysed blood on the virulence of Vibrio parahaemolyticus for mice was investigated. A factor present in lysed erythrocytes was found to greatly reduce the 50% lethal dose of V. parahaemolyticus for mice. Similar effects were observed with ferric ammonium citrate and manganous sulfate. V. parahaemolyticus grown in brain heart infusion containing lysed erythrocyte factor appeared to produce a lethal toxin which was either inapparent or absent when the organism was grown in brain heart infusion alone.
Outer membrane proteins from rough strains of four Brucella species
Authors: Santos J M , Verstreate D R , Perera V Y , Winter A J
Published: January 2020

Outer membrane proteins from 15 rough strains of Brucella abortus, B. ovis, B. canis, and B. melitensis were extracted with a dipolar detergent, and outer membrane proteins from selected strains were purified by anion exchange chromatography and gel filtration (Verstreate et al., Infect. Immun. 35:979-989, 1982). Outer membrane proteins produced two types of profiles on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. One type, demonstrated by B. abortus, B. ovis, and B. canis strains, contained the three predominant protein groups present in smooth B. abortus strains (Verstreate et al., Infect. Immun. 35:979-989, 1982): groups 1, 2 (porin [Douglas et al., Infect. Immun. 44:16-21]), and 3. B. melitensis strains demonstrated the second profile type, in which there was an additional band between groups 1 and 2. The relative proportion of porin was considerably lower in B. ovis, B. canis, and B. melitensis than in B. abortus. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis profiles could be used to distinguish B. abortus and B. melitensis from each other and from B. canis and B. ovis. The amino acid compositions of groups 2 and 3 from rough strains of B. abortus, B. canis, and B. melitensis were similar to those of corresponding proteins from smooth B. abortus strains. Zwittergent-soluble fractions from most rough strains contained antigen [b], which cross-reacted with group 2 from smooth B. abortus strains, and antigens [c] and [d], which cross-reacted with group 3 from smooth B. abortus strains. Antigen [a], shared by groups 2 and 3 (D. R. Verstreate and A. J. Winter, Infect. Immun. 46:182-187, 1984), was detected in most rough strains. None of these antigens were related to either rough or smooth lipopolysaccharide.
Interaction of monoclonal antibodies with pertussis toxin and its subunits
Authors: Frank D W , Parker C D
Published: January 2020

The binding of two monoclonal antibodies to crude and pure preparations of pertussis toxin was examined. Antibody P11B10 reacted with an epitope present on the S2 subunit of pertussis toxin by immunoblot techniques. Radioimmunoprecipitation analysis and immunoaffinity chromatography with P11B10 indicated that subunits S2 and S3 were closely associated. Antibody P7B10 was unreactive in immunoblots and radioimmunoprecipitation but was able to bind and retain toxin subunits during affinity chromatography. The P7B10 epitope may thus be labile to detergent treatment or radioiodination; or the epitope may form as a result of subunit association. Neither antibody alone nor both in combination neutralized the histamine-sensitizing activity of pertussis toxin in passive-transfer experiments. However, toxin isolated by immunoaffinity chromatography with either monoclonal antibody was able to sensitize mice to histamine challenge.
Lack of cross-reactivity of antibodies to ribosomal preparations from Streptococcus mutans with human heart and kidney antigens
Authors: Gregory R L , Shechmeister I L , Brubaker J O , Smedberg C T , Michalek S M , McGhee J R
Published: January 2020

Previous studies have suggested that sera from animals immunized with whole Streptococcus mutans cells may cross-react with human and monkey heart sarcolemmal tissues. In the present study, sera and saliva from rats and rabbits immunized peripherally with ribosomal preparations from S. mutans 6715 (serotype g) or GS-5 (serotype c) were examined for their ability to react with normal human heart sarcolemmal and kidney glomerular tissues by using enzyme-linked immunosorbent and immunofluorescence assays. The results showed that antibodies to serotype g and c ribosomal preparations do not react with either the human heart or renal antigens. Sera from mice immunized with human heart tissue and from a patient with a high anti-streptolysin O titer reacted strongly with human heart sarcolemmal and kidney glomerular tissues. These data indicated that ribosomal preparations from S. mutans lack the putative human heart cross-reactive determinant and suggest that the use of an S. mutans ribosomal vaccine against dental caries may not be pathogenic to human heart or renal tissues.
Atrophic rhinitis in swine: correlation of Pasteurella multocida pathogenicity with membrane protein and lipopolysaccharide patterns
Authors: Lugtenberg B , van Boxtel R , de Jong M
Published: January 2020

Cell envelope proteins and lipopolysaccharides (LPS) of Pasteurella multocida strains associated with atrophic rhinitis in swine were compared by using sodium dodecyl sulfate gel electrophoresis. Among 34 strains, three different types of cell envelope protein patterns, named I (16 strains), II (3 strains), and III (15 strains), could be distinguished. These differences were based on the electrophoretic mobility of the major protein, designated as protein H. Comparison of cell envelope protein type and pathogenicity of the strain, the latter property predicted by the guinea pig skin test, revealed that all type I strains, 6 of 15 type III strains, and none of the type II strains were pathogenic. Although pathogenicity has been correlated with extracellular toxin activity, no protein could be detected in either the cell envelopes or in the extracellular fluid that absolutely correlated with pathogenic strains. Electrophoretic analysis of the LPS revealed that all strains possessed low-molecular-weight LPS, which is inconsistent with the presence of a classical O antigen. The method allowed the detection of at least six types of LPS, which often coincided with a certain cell envelope protein type and with the presence or absence of the pathogenic character of the strain. These results strongly suggest that the sampled swine carry a limited number of P. multocida clones, in each of which the patterns of cell envelope proteins and LPS, as well as the presence or absence of the ability to produce extracellular toxin, are well conserved. Therefore, the possibility is discussed that sodium dodecyl sulfate gel electrophoresis of cell envelope proteins and LPS may be used for the prediction of the pathogenic character of part of the strains. Finally, the typing of strains based on cell envelope protein patterns might contribute to the development of vaccines containing outer membrane proteins as protective antigens.
Immunoglobulin G and M composition of naturally occurring antibody to type III group B streptococci
Authors: Anthony B F , Concepcion N F , Wass C A , Heiner D C
Published: January 2020

Human sera were examined by an enzyme-linked immunosorbent assay for immunoglobulin G (IgG) and IgM antibodies to purified type III polysaccharide of group B streptococci. The antigen-binding capacity of a reference human serum was determined by a radioimmunoassay, and the total antibody content was determined by quantitative precipitation. The serum was then depleted of IgM and IgA to determine the effect on the antigen-binding capacity. Duplicate samples of 81 sera were tested by the enzyme-linked assay in comparison with reference standard serum. Although levels of IgG antibody were greater in subjects who had carried type III streptococci during pregnancy, concentrations of this antibody were generally low. Only 2 of 28 sera (7%) from parturient subjects and 7 of 25 sera (28%) from adult volunteers contained greater than or equal to 1 microgram of IgG antibody per ml; the mean levels were 0.13 and 0.53 micrograms/ml, respectively. In contrast, 19 of 28 maternal sera (68%) and 22 of 25 (88%) volunteer adult sera contained greater than or equal to 1 microgram/ml of IgM antibody; mean levels were 1.33 and 1.54 micrograms/ml, respectively. The cord serum levels of IgG antibody were almost identical to maternal serum concentrations, whereas IgM antibody was essentially undetected.
Immunological evaluation of a component isolated from Mycobacterium bovis BCG with a monoclonal antibody to M. bovis BCG
Authors: Minden P , Kelleher P J , Freed J H , Nielsen L D , Brennan P J , McPheron L , McClatchy J K
Published: January 2020

A component of Mycobacterium bovis BCG referred to as BCG-a was isolated through the combined use of monoclonal antibody directed to BCG and affinity chromatography. Analysis of BCG-a by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a single prominent band with a molecular weight of ca. 10,000. Structural characterization of BCG-a consisting of amino acid composition and amino-terminal sequence determination was carried out. The intact BCG-a antigen was bound by neither the lectin from common lentils nor concanavalin A, implying that BCG-a does not carry any asparagine-linked oligosaccharides. Immunoprecipitation of 125I-labeled BCG-a with polyclonal and monoclonal antibodies directed against BCG resulted in bands having the same mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis as did free 125I-BCG-a. In radioimmunoassays 125I-BCG-a was bound by the monoclonal antibody and by polyclonal antibodies from rabbits that had been immunized to BCG and to Mycobacterium tuberculosis H37Rv. Antibodies to nontuberculous and to nonacid-fast bacteria bound BCG-a poorly or not at all. The binding of 125I-BCG-a by the monoclonal antibody was readily inhibited by extracts of BCG and H37Rv, but it was not as readily inhibited by extracts of nontuberculous mycobacteria and was not at all inhibited by extracts of nonacid-fast bacteria. Considerable inhibition was similarly observed by surface antigens of nonviable, intact BCG organisms. Delayed cutaneous hypersensitivity reactions to small concentrations of BCG-a were elicited in guinea pigs that had been immunized with BCG or H37Rv antigens, but such reactions were not elicited in unimmunized animals.
Solubilization and partial characterization of the intestinal receptor for Escherichia coli heat-stable enterotoxin
Authors: Dreyfus L A , Robertson D C
Published: January 2020

Binding of Escherichia coli strain 431 heat-stable enterotoxin (STa) and activation of intestinal particulate guanylate cyclase by E. coli STa were studied with rat intestinal epithelial cells and brush border membranes (BBMs). The rates of guanylate cyclase stimulation by 431 STa in cells and BBMs were rapid, with maximal levels of cyclic GMP observed within 5 min. Specific binding of 125I-labeled STa from E. coli 431 (431 125I-STa) and activation of guanylate cyclase by unlabeled 431 STa were observed with intestinal BBMs; however, neither was detected with membranes from nonintestinal tissues. The STa receptor was solubilized with 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate, a nondenaturing dipolar ionic detergent, in yields of approximately 50%. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the detergent-solubilized receptor-431 125I-STa complex, followed by autoradiography, showed that 431 125I-STa bound to a single BBM component with a molecular weight of about 100,000. Binding of 431 STa to its solubilized receptor was saturable, specific, and essentially irreversible. Pretreatment of the soluble receptor with trypsin and pronase but not chymotrypsin decreased binding of 431 125I-STa. The 431 STa-receptor complex was dissociated by boiling in the presence of 1% sodium dodecyl sulfate, incubation with 0.5 M acetic acid, or reduction with dithiothreitol. In contrast to the residual particulate guanylate cyclase activity of detergent-treated membranes, solubilized guanylate cyclase was not stimulated by STa. Membrane structure appears to play an important role in the coordination of STa binding and stimulation of guanylate cyclase activity.
In vivo analysis of impaired macrophage bactericidal capacity during experimental African trypanosomiasis
Authors: Glick D L , Jones J F
Published: January 2020

Since innate resistance of mice to Salmonella typhimurium depends on an intact macrophage system, we have used this bacterium to investigate the effect of Trypanosoma brucei subsp. rhodesiense infection on macrophage phagocytic and cytolytic function. CBA/CaJ mice infected with T. brucei subsp. rhodesiense have decreased resistance to S. typhimurium, since doubly infected mice rapidly succumb to sublethal doses of S. typhimurium. Although trypanosomiasis is known to suppress antibody formation, such a suppression of antibody does not seem to play a role in trypanosome-induced sensitivity to S. typhimurium. A trypanosome-induced blockade of the reticuloendothelial system also does not occur, since parasitized and control mice clear S. typhimurium from the blood equally well. Early killing (0 to 48 h) of S. typhimurium in the liver and spleen is mainly macrophage mediated, and mice infected with trypanosomes kill S. typhimurium in the liver and spleen very poorly. Apparently trypanosomiasis inhibits macrophage bactericidal activity, but has no effect on phagocytosis.
Mouse monoclonal antibodies reactive with J5 lipopolysaccharide exhibit extensive serological cross-reactivity with a variety of gram-negative bacteria
Authors: Nelles M J , Niswander C A
Published: January 2020

We describe two mouse monoclonal antibodies reactive with lipopolysaccharide derived from the J5 mutant of Escherichia coli O111:B4. These antibodies react with purified lipopolysaccharide derived from rough mutants of E. coli and Salmonella typhimurium and also with lipopolysaccharide associated with both smooth- and rough-phenotype, gram-negative bacteria. Both antibodies appear to bind determinants present in the lipopolysaccharide core region, and this reactivity is inhibited in the presence of polymyxin B. Although their patterns of reactivity differ, both antibodies exhibit extensive serological cross-reactivity with a variety of gram-negative bacteria. Reagents of this type should prove useful in animal models to delineate the requisite affinity, epitope specificity, immunoglobulin class, etc., needed for the prevention and treatment of gram-negative bacteremia.
Survival and growth of Yersinia pestis within macrophages and an effect of the loss of the 47-megadalton plasmid on growth in macrophages
Authors: Charnetzky W T , Shuford W W
Published: January 2020

The survival and growth of Yersinia pestis cells within mouse peritoneal cavities and within mouse peritoneal macrophages maintained in vitro was examined. Two strains were used which differed only in that one (KIM) contained the 47-megadalton plasmid associated with virulence and the second (KIM1) lacked this plasmid. The KIM cells, but not the KIM1 cells, acquired some resistance to phagocytosis during growth at 37 degrees C which was not evident when cells were grown at 26 degrees C. Whether previously grown at 26 or 37 degrees C, however, a substantial portion of the cells of either strain which were phagocytized were apparently killed after phagocytosis in vivo, although this was not observed in vitro. KIM cells which survived phagocytosis proliferated within macrophages in vivo, but no increase in viable cells was seen with the KIM1 cells. Growth of the KIM1 cells within macrophages in vitro required that a complex supportive medium be used in which the bacteria could have grown if extracellular. This was not the case for the KIM cells which proliferated within macrophages supported in medium not permissive to bacterial growth. After phagocytosis of cells of either strain by macrophages maintained in vitro, phagolysosome formation occurred normally, as shown by the acridine orange dye staining technique. KIM and KIM1 cells were equally sensitive to hydrogen peroxide and superoxide anion, although the sensitivity in each case varied with growth temperature. The oxidative burst, as determined by the luminol chemiluminescence assay, was low when compared with that seen after phagocytosis of Escherichia coli cells. Chemiluminescence after phagocytosis of yeast cells by macrophages which had engulfed KIM or KIM1 was also low. We conclude that survival within macrophages is substantially independent of the 47-megadalton plasmid and may be a consequence, as least in part, of blockage of the oxidative burst or rapid removal of the oxidizing compounds formed. The 47-megadalton plasmid is apparently required for subsequent proliferation within the macrophage.
Enhancement of experimental bacteremia and endocarditis caused by dysgonic fermenter (DF-2) bacterium after treatment with methylprednisolone and after splenectomy
Authors: Butler T , Johnston K H , Gutierrez Y , Aikawa M , Cardaman R
Published: January 2020

The dysgonic fermenter-2 bacterium is a newly recognized fastidious gram-negative bacillus that causes bacteremia and sometimes endocarditis in immunocompromised persons after they are bitten by dogs. To develop an experimental model of this infection, we placed polyethylene catheters across the aortic valves of New Zealand white rabbits, which were inoculated intravenously the next day with dysgonic fermenter-2 bacteria. After 1 week, the rabbits were killed and the endocardial vegetations were homogenized for quantitative culture. Large inocula (1.3 X 10(10) to 2.1 X 10(10) viable bacteria) were required to produce infected vegetations. All infected rabbits had negative blood cultures at the time of autopsy and most developed serum agglutinins against dysgonic fermenter-2 bacteria. Three daily injections of methylprednisolone (30 mg/kg), starting the day before inoculation, significantly increased the incidence of endocarditis and the number of bacteria per gram of infected vegetation (P less than 0.05). Treatment with methylprednisolone prolonged the initial bacteremia and caused significant increases in the numbers of bacteria per gram of blood, spleen, and liver compared with those of untreated controls (P less than 0.05). Rabbits that had previously undergone splenectomy showed prolongation of the initial bacteremia but no significant increase in the incidence of infected vegetations. These results showed that the dysgonic fermenter-2 bacterium is a pathogen that causes endocarditis in rabbits but that it requires a large inoculum and produces blood culture-negative infections. Treatment with methylprednisolone enhances infection by prolonging the initial bacteremia and probably by diminishing bactericidal activity in the vegetations.
Cloning and characterization of genes involved in production of mannose-resistant, neuraminidase-susceptible (X) fimbriae from a uropathogenic O6:K15:H31 Escherichia coli strain
Authors: Hacker J , Schmidt G , Hughes C , Knapp S , Marget M , Goebel W
Published: January 2020

The uropathogenic Escherichia coli strain 536 (O6:K15:H31) exhibits a mannose-resistant hemagglutination phenotype (Mrh) with bovine erythrocytes and delayed Mrh with human and guinea pig erythrocytes. Neuraminidase treatment of the erythrocytes abolishes mannose resistant hemagglutination, which is typical for X fimbriae. E. coli strain 536 synthesizes two different fimbriae (Fim phenotype) protein subunits, 16.5 and 22 kilodaltons in size. In addition the strain shows mannose-sensitive hemagglutination and common type I (F1) fimbriae. The cosmid clone E. coli K-12(pANN801) and another nine independently isolated Mrh+ cosmid clones derived from a cosmid gene bank of strain 536 express the 16.5-kilodalton protein band, but not the 22-kilodalton protein, indicating an association of the Mrh+ property with the "16.5-kilodalton fimbriae." All cosmid clones were fimbriated, and they reacted with antiserum produced against Mrh+ fimbriae of the E. coli strain HB101(pANN801) and lacked mannose-sensitive hemagglutination (F1) fimbriae. From the Mrh fim cosmid DNA pANN801, several subclones coding for hemagglutination and X fimbriae were constructed. Subclones that express both hemagglutination and fimbriae and subclones that only code for the hemagglutination antigen were isolated; subclones that only produce fimbriae were not detected. By transposon Tn5 mutagenesis we demonstrated that about 6.5 kilobases of DNA is required for the Mrh+ Fim+ phenotype, and the 1.5- to 2-kilobase DNA region coding for the structural protein of the fimbriae has been mapped adjacent to the region responsible for the Mrh+ phenotype. Two different regions can thus be distinguished in the adhesion determinant, one coding for hemagglutination and the other coding for fimbria formation. Transformation of plasmid DNA from these subclones into a Mrh- Fim- mutant of E. coli 536 and into a galE (rough) strain of Salmonella typhimurium yielded transformants that expressed both hemagglutination and fimbria production.
Relation of capsular materials and colony opacity to virulence of Vibrio vulnificus
Authors: Yoshida S , Ogawa M , Mizuguchi Y
Published: January 2020

Colonies which varied in opacity were isolated from the four strains of Vibrio vulnificus. Opaque and translucent colonial types of the strains were distinguished from the corresponding parent strains. Variation in the opacity of colonies formed by each strain was accompanied by variation of capsular material formation, which was clarified by electron microscopy of the organisms stained with ruthenium red. The opaque-type colonies of the strains had capsular materials. On the other hand, three translucent-type colonies had no observable capsular materials, and one had incomplete capsular materials, in contrast to the corresponding opaque type. The corresponding opaque and translucent types of the strains were compared for points of virulence in mice and guinea pigs. By having capsular materials, the bacterial strains acquired resistance to serum bactericidal action, antiphagocytic activity, high lethality for mice, and strong invasiveness in the subcutaneous tissue of guinea pigs. Capsular materials of V. vulnificus were considered to be important for the expression of virulence.
Effect of immune complexes from mastitic milk on blocking of Fc receptors and phagocytosis
Authors: Targowski S P , Klucinski W
Published: January 2020

Fc receptors on the surface of milk leukocytes from normal glands, bronchial leukocytes, mastocytoma P-815 cells, and murine leukemia L1210 cells were blocked significantly (P less than 0.01) by cavian and bovine milk collected from inflamed glands (mastitic milk), their wheys, and in vitro-prepared immune complexes composed of the whey from normal milk and serum. Blocking of Fc receptors indicated the presence of immune complexes in the mastitic milk and was detected by inhibition of rosette formation with sensitized erythrocytes or attachment of the aggregated immunoglobulin G. The binding of immune complexes to these cells was also determined by staining with fluorescein isothiocyanate-labeled protein A. As the mastitis subsided, the blocking effect of the mastitic milk also declined markedly. There was no significant difference in blocking capacity between mastitic milk and its whey. The blocking capacity of normal cavian or bovine milk and their wheys was insignificant. Whey from mastitic milk also inhibited phagocytosis of opsonized staphylococci by alveolar macrophages. We suggest that the blocking of Fc receptors on phagocytic cells adversely affects phagocytosis.
Lactate dehydrogenase from Streptococcus mutans: purification, characterization, and crossed antigenicity with lactate dehydrogenases from Lactobacillus casei, Actinomyces viscosus, and Streptococcus sanguis
Authors: Sommer P , Klein J P , Schöller M , Frank R M
Published: January 2020

A cytoplasmic fructose-1,6-diphosphate-dependent lactate dehydrogenase (LDH; EC 1.1.1.27) from Streptococcus mutans OMZ175 was purified to homogeneity as judged by sodium dodecyl sulfate-gel electrophoresis. The purification consisted of ammonium sulfate precipitation of the cytoplasmic fraction, DEAE-Sephacel and Blue-Sepharose CL.6B chromatography, and Sephacryl S200 gel permeation. The catalytic activity of the purified enzyme required the presence of fructose-1,6-diphosphate with a broad optimum between pH 5 and 6.2. The concentration of fructose-1,6-diphosphate required for half-maximal velocity was around 0.02 mM and was affected by the pyruvate concentration. The enzyme seemed to have at least two binding sites for the activator which interact in a cooperative manner. Increasing concentrations of fructose-1,6-diphosphate up to 2 mM enhanced the relative affinity of the enzyme for pyruvate and modified the pyruvate saturation curve from sigmoidal to hyperbolic. The enzyme activity showed also a sigmoidal response to NADH, exhibiting two binding sites for the cofactor with a Hill coefficient of about 1.9. The molecular weight of the native enzyme was 150,000 as determined by gel permeation on Sephacryl S200. Monomers (38,000 daltons) and dimers (85,000 daltons) were observed by sodium dodecyl sulfate-gel electrophoresis; the latter form was dissociated after reduction with 2-mercaptoethanol, and the enzyme could be considered a tetramer. Antibodies obtained against the purified S. mutans OMZ175 LDH cross-reacted with the sodium dodecyl sulfate-dissociated forms of LDHs from different S. mutans serotypes, Streptococcus sanguis OMZ9, Lactobacillus casei ATCC 4646, and Actinomyces viscosus NY 1. A competitive enzyme-linked immunosorbent assay allowed us to detect a very close relationship between the native states of L-LDHs from S. mutans serotypes and S. sanguis. Cross-reactions were also observed with the LDHs from A. viscosus and L. casei, the latter being the least related. A very weak immunological relationship was obtained between the L-LDH from S. mutans OMZ175 and the D-LDH from Lactobacillus leichmannii, whereas no cross-reaction could be detected with mammal LDHs.
Temperature-inducible surface fibrillae associated with the virulence plasmid of Yersinia enterocolitica and Yersinia pseudotuberculosis
Authors: Kapperud G , Namork E , Skarpeid H J
Published: January 2020

When cultivated at 37 degrees C in static broth, human clinical isolates of Yersinia enterocolitica (serogroups O:3, O:8, and O:9) and Yersinia pseudotuberculosis (serogroup O:III) produced numerous nonflagellar surface appendages, which appeared as a lawn of fine fibrillae, each having a diameter of 1.5 to 2.0 nm and a length of 50 to 70 nm. Cultivation at 22 degrees C resulted in complete disappearance of the fibrillae. The phenotypic expression of these appendages was correlated with the presence of the 40- to 48-megadalton virulence plasmid and was strongly affected by the growth medium. Evidence is presented which suggests that these plasmid-mediated, temperature-inducible surface fibrillae are responsible for autoagglutination and are related to production of one prominent, Sarkosyl-insoluble polypeptide of ca. 180 kilodaltons in the bacterial outer membrane.
Depressing hepatic macrophage complement receptor function causes increased susceptibility to endotoxemia and infection
Authors: Loegering D J , Blumenstock F A
Published: January 2020

Previous work has demonstrated that in vivo hepatic macrophage complement receptor clearance function is depressed after thermal injury. To determine whether impairment of complement receptor function is important in host defense, the present study evaluated the effect of the depression of complement receptor function in uninjured animals on susceptibility to endotoxin shock and bacterial infection. Hepatic complement receptor clearance function was evaluated by measuring the hepatic uptake of a test dose (2.9 X 10(8)/100 g) of rat erythrocytes coated with anti-erythrocyte immunoglobulin M (EIgM) or EIgG in rats. Depression of hepatic complement receptor function was induced by the injection of EIgG. The hepatic uptake of the test dose of EIgM or EIgG was depressed after the injection of 8.7 X 10(8) EIgG per 100 g and 17.4 X 10(8) EIgG per 100 g but not after the injection of 2.9 X 10(8) EIgG per 100 g. This effect was shown not to be due to a decrease in hepatic blood flow or a depletion of serum C3 and was, therefore, due to a depression in hepatic macrophage complement receptor clearance function. Susceptibility to endotoxin shock was increased with the dose of 8.7 X 10(8) EIgG per 100 g, and susceptibility to infection with Pseudomonas aeruginosa was increased with the dose of 17.4 X 10(8) EIgG per 100 g. Therefore, depression of hepatic macrophage complement receptor clearance function with EIgG is associated with depressed host defense.
Effect of piliation on interactions of Haemophilus influenzae type b with human polymorphonuclear leukocytes
Authors: Tosi M F , Anderson D C , Barrish J , Mason E O , Kaplan S L
Published: January 2020

Piliated, adherent (P+) and nonpiliated, nonadherent (P-) strains of Haemophilus influenzae type b (Hib) were compared with respect to their ability to induce polymorphonuclear leukocyte (PMN) chemiluminescence (CL) and superoxide (O2-) generation and their susceptibility to phagocytosis by PMNs. P+ strains opsonized in normal human serum (NHS) induced significantly greater CL than did P- strains (500 X 10(5) +/- 112 X 10(5) versus 242 X 10(5) +/- 65 X 10(5) total counts per 60 min; P less than 0.001) when reacted with normal PMNs. Contributions of immunoglobulin and complement to CL activity in these mixtures were shown by findings of lower overall levels of CL when hypogammaglobulinemic serum or heat-inactivated NHS was used to opsonize either P+ or P- organisms. Results obtained with mixtures of hypogammaglobulinemic plus adsorbed heat-inactivated NHS (with P+ or P- organisms) suggested a role for an antipilus antibody in the enhancement of CL by these strains. NHS-opsonized P+ strains also induced significantly greater (P less than 0.002) O2- generation than did P- strains (2.83 +/- 0.08 versus 1.94 +/- 0.14 nmol of ferricytochrome c reduced per 10 min/10(6) PMN). Comparable ingestion of P+ or P- strains opsonized in NHS by PMNs was demonstrated by a radiolabeled uptake technique and transmission electron microscopy, and primary granule release (beta-glucuronidase) was comparable during ingestion of P+ or P- strains. The basis for the observed enhanced capacity of P+ Hib to stimulate PMN oxidative metabolism as compared with P- organisms is uncertain. Possible clinical implications of these findings deserve further study.
Production of Listeria-specific rat T-cell clones and role of interleukin-2 receptors in regulation of Listeria-dependent T-cell clone growth in vitro
Authors: Stolpmann R M , Näher H , Osawa H , Herrmann T , Hahn H , Diamantstein T
Published: January 2020

Splenic T lymphocytes from rats immunized with the facultative intracellular bacterium Listeria monocytogenes were cloned by the limiting-dilution technique in the presence of accessory cells, heat-killed L. monocytogenes as antigen, and conditioned medium containing interleukin-2. The cloned rat T-cells were Listeria-specific cells, and their proliferation depended on class II-restricted antigen presentation by accessory cells. As demonstrated by their reactivity to the monoclonal antibody W3/25, the clones were of helper cell phenotype. Cloned-cell proliferation depended on repeated (or continuous) exposure to antigen. When antigen was omitted from the system, cell growth subsided over time, and cells finally ceased to grow. By the use of the monoclonal antibody ART-18, which recognizes the interleukin-2 receptor, it was shown that cessation of growth was accompanied by the disappearance of interleukin-2 receptors from the cell surface. The addition of antigen to the culture resulted in the reexpression of interleukin-2 receptors and concomitant resumption of proliferation.