bluuebunny/crossref_metadata_2025 huggingface dataset:
Prairie dog model for antimicrobial agent-induced Clostridium difficile diarrhea
Authors: Muller E L , Pitt H A , George W L
Published: January 2020

We have noted that prairie dogs given cefoxitin develop diarrhea and lose weight yet survive for periods of up to 4 weeks. Therefore, we tested the hypothesis that cefoxitin causes Clostridium difficile cecitis in prairie dogs. Six prairie dogs were given a single intramuscular dose of 100 mg of cefoxitin per kg of body weight, and six control animals received saline; both groups were sacrificed 1 week later. Controls had no diarrhea and lost 2% of their body weight, whereas cefoxitin-treated animals had diarrhea (P less than 0.001) and lost 16% of their body weight (P less than 0.001); one animals died 6 days after cefoxitin challenge. None of the controls yielded C. difficile or had cecal cytotoxin or pseudomembranes detected. Cecal contents from all cefoxitin-treated animals, however, yielded C. difficile (P less than 0.01) and had cecal cytotoxin present (P less than 0.01). Four of five surviving animals also had cecal pseudomembranes present (P less than 0.01). These results demonstrate that in prairie dogs cefoxitin induces C. difficile cecitis. We conclude that the prairie dog is another model for the study of antibiotic-induced diarrhea. The disease in prairie dogs may have a more chronic course than in other animal models of C. difficile-induced diarrhea and may be useful as a model for studying certain aspects of C. difficile-induced diarrhea.
Immunological study of cross-reactive polysaccharide antigens (types a, d, and h) of oral Streptococcus spp. with monoclonal antibodies
Authors: Ota F , Kato H , Fukui K
Published: January 2020

Two monoclonal antibodies against the cross-reactive antigens of S. cricetus (type a) and S. sobrinus (type d) were isolated. Galactose and especially melibiose inhibited the precipitin reaction markedly. Inhibition by melibiose was over 200-fold stronger than that by galactose. This may indicate that galactose-alpha 1, 6-glucose is the predominant antigenic determinant of this cross-reactive antigen. This antigen was also found in S. sobrinus type h strains, but no antigen was found in one type d strain (OMZ176).
Expression of iron-regulated proteins in Yersinia species and their relation to virulence
Authors: Carniel E , Mazigh D , Mollaret H H
Published: January 2020

Under iron-starvation conditions, the different Yersinia species expressed various iron-regulated proteins. Among them, two high-molecular-weight outer membrane proteins were synthesized in high-virulence-phenotype Y. pestis, Y. pseudotuberculosis, and Y. enterocolitica serovars O:8 and O:Tacoma but were present neither in low-virulence phenotype Y. enterocolitica serovars O:3 and O:9 nor in avirulent Y. frederiksenii, Y. kristensenii, Y. intermedia, and Y. enterocolitica serovar O:39. Thus, the degree of virulence correlates with the presence of the two high-molecular-weight proteins in Yersinia species.
Shared epitopes between Mycoplasma pneumoniae major adhesin protein P1 and a 140-kilodalton protein of Mycoplasma genitalium
Authors: Morrison-Plummer J , Lazzell A , Baseman J B
Published: January 2020

Previous serological data have demonstrated cross-reactive antigens between two pathogenic species of mycoplasmas, M. pneumoniae and M. genitalium. Preliminary analysis of sera and monoclonal antibodies (MAbs) to protein antigens of these species showed an immunodominance of adhesin P1 (165 kilodaltons [kDa]) of M. pneumoniae in mice and hamsters and a 140-kDa protein of M. genitalium in mice and experimentally infected chimpanzees. To further characterize these two proteins, we assayed multiple anti-P1 and anti-140-kDa protein MAbs by enzyme-linked immunosorbent assay, immunoblot, and radioimmunoprecipitation techniques. The 140-kDa M. genitalium protein was shown to be surface accessible and insensitive to levels of trypsin which readily degrade protein P1. Peptide mapping was used to identify a unique class of MAbs which bound a cross-reactive molecule common to both the major adhesin protein P1 of M. pneumoniae and the 140-kDa protein of M. genitalium. MAbs generated against both M. pneumoniae and M. genitalium which were reactive with this determinant blocked M. pneumoniae attachment to chicken erythrocytes.
Analysis of T-cell-dependent and -independent antigens of Rickettsia conorii with monoclonal antibodies
Authors: Feng H M , Walker D H , Wang J G
Published: January 2020

Four monoclonal antibodies from euthymic mice and two monoclonal antibodies from athymic mice were directed against antigens of Rickettsia conorii, as shown by both indirect immunofluorescence and an enzyme immunoassay. There was extensive cross-reactivity with other spotted fever group rickettsiae. Euthymic monoclonal antibodies 3-2 and 9-2 (immunoglobulin G2a [IgG2a]) and 27-10 (IgG1) distinctly outlined the acetone-fixed rickettsial surface, as determined by indirect immunofluorescence; only monoclonal antibody 3-2 reacted with the intact rickettsial surface, as determined by colloidal gold-protein A negative-stain electron microscopy. Athymic monoclonal antibodies 32-2 and 35-3 (IgM) and euthymic monoclonal antibody 31-15 (IgG3) all demonstrated an irregular, extrarickettsial morphology, as determined by immunofluorescence, and ultrastructural cell wall blebs that were readily shed from the rickettsial surface. Monoclonal antibody 3-2, the only antibody to confer protection in lethally challenged mice, reacted with a high-molecular-weight protein in Western immunoblots. Monoclonal antibodies 31-15, 32-2, and 35-3 reacted with a "ladder" of proteinase K-resistant, lipopolysaccharidelike antigens. None of the monoclonal antibodies stabilized the ultrastructural rickettsial slime layer, but both athymic and euthymic polyclonal antibodies to R. conorii did. This is, to the best of our knowledge, the first report of the production of monoclonal antibodies to R. conorii and their use for antigenic analysis.
Physiology of the potentiation of lethal endotoxin shock by streptococcal pyrogenic exotoxin in rabbits
Authors: Murai T , Ogawa Y , Kawasaki H , Kanoh S
Published: January 2020

Streptococcal pyrogenic exotoxin (SPE) dramatically potentiates the lethal shock induced by gram-negative bacterial endotoxin. To provide further understanding of the mechanism underlying the potentiating effect, the physiological basis for the toxic synergism of the two toxins was investigated. Pretreatment of rabbits with an intravenous (i.v.) dose (10 micrograms/kg of body weight) of SPE greatly enhanced the endotoxin lethality and reduced the 50% lethal dose to less than 5 micrograms of endotoxin per kg. The SPE pretreatment dose caused severe pathophysiological changes in combination with a small i.v. dose of endotoxin (1 microgram/kg). These changes included transient hyperglycemia followed by profound hypoglycemia, elevation of the blood lipoperoxide level, and an acute increase in plasma beta-glucuronidase activity. These changes were comparable with those in animals given a large i.v. dose of endotoxin (100 micrograms/kg) alone. An injection of SPE alone did not alter any of the parameters described above. These results suggest that SPE renders rabbits more sensitive to extensive pathophysiologic effects of endotoxin, and the potentiating effect on endotoxin lethality may thus involve a general potentiation of physiologic failures. The SPE pretreatment depressed the vascular clearance of a large dose of endotoxin (100 micrograms/kg) but failed to affect that of a small dose of endotoxin (1 microgram/kg). The data suggest that the potentiating effect is not readily explained solely on the basis of the decreased clearance of endotoxin.
Pathogenic capacity of proteases from Serratia marcescens and Pseudomonas aeruginosa and their suppression by chicken egg white ovomacroglobulin
Authors: Molla A , Matsumura Y , Yamamoto T , Okamura R , Maeda H
Published: January 2020

The pathogenicities of three proteases from Serratia marcescens, two proteases from Pseudomonas aeruginosa, and one thermolysin from Bacillus stearothermophilus were examined. All proteases tested caused acute liquefactive necrosis of the cornea and descemetocele formation in guinea pig eyes after intrastromal injection, with the exception of the 60-kilodalton protease from S. marcescens, which produced only an opaque lesion. When injected into guinea pig skin, the protease also enhanced vascular permeability, which was followed by edema formation. The permeability-enhancing activity of the proteases increased in parallel with the concentration of the enzymes. When tested in vitro for its effect on these bacterial proteases, chicken egg white ovomacroglobulin (ovoM) inhibited the enzymatic activity of all the proteases after a short incubation period at an enzyme/inhibitor ratio (molar) of 1:1 to 1:4 or at a lower concentration after a longer incubation period. Such treatment of the proteases with chicken egg white ovoM before injection intrastromally into the eyes or intradermally into the clipped flanks of guinea pigs protected the cornea from destruction or completely prevented the permeability reaction and edema formation. No inhibitory effects of plasma protease inhibitors against these bacterial proteases were noted. Since the proteases are critical in the pathogenic processes caused by the bacteria, these results suggest a beneficial effect of ovoM against bacterial infections.
Intergeneric bacterial coaggregations involving mutans streptococci and oral actinomyces
Authors: Crowley P J , Fischlschweiger W , Coleman S E , Bleiweis A S
Published: January 2020

Mutans streptococci (MS) representing eight different serotypes were tested for their ability to coaggregate in vitro with oral actinomyces and other streptococcal species. Of the mutans streptococci tested, only strains of S. cricetus (formerly S. mutans serotype a) displayed pronounced coaggregations and only with certain strains of actinomyces. S. cricetus coaggregated, by lactose nonreversible mechanisms, with serotype 4 Actinomyces naeslundii WVU963 and WVU924 and with serotype 2 Actinomyces odontolyticus WVU758. The first pair was disaggregated by protein denaturants (e.g., sodium dodecyl sulfate and urea) and EDTA. This coaggregation was inhibited when the streptococcal, but not the actinomyces, partner was pretreated with either heat or protease, suggesting the presence of a protein mediator on only the streptococcal cell surface. The S. cricetus-A. odontolyticus coaggregation appeared to involve protein components on each cell, as shown by the lack of coaggregation after pretreatment of either cell type with heat or proteases. This coaggregation was also reversed by sodium dodecyl sulfate and urea, as well as by sodium deoxycholate, but not by EDTA. The data indicate that different mechanisms may be involved in each of these coaggregations.
Isolation and characterization of monoclonal antibodies specific for antigen P1, a major surface protein of mutans streptococci
Authors: Ayakawa G Y , Boushell L W , Crowley P J , Erdos G W , McArthur W P , Bleiweis A S
Published: January 2020

A panel of 15 murine monoclonal antibodies (MAbs; 14 immunoglobulin G1, 1 immunoglobulin G2a) directed against antigen P1, a major surface protein of mutans streptococci, was prepared. All of these MAbs reacted by the enzyme-linked immunosorbent assay with solubilized wall material from Streptococcus mutans Ingbritt 175 (a serotype c strain which retains significant amounts of P1 in its cell wall), culture supernatant fluid from Ingbritt 162 (a strain which excretes large amounts of P1 into the culture medium), and purified P1. By Western immunoblotting, these MAbs were observed to react with a high-molecular-weight polypeptide which comigrated with antigen P1. None of these MAbs cross-reacted with human heart tissue or with various eucaryotic proteins. When whole cells of various strains of mutans streptococci were screened against the panel of MAbs, the strongest reactivities were noted with strains of serotype c and e S. mutans, while a serotype f strain of S. mutans, along with S. sobrinus and S. cricetus strains, reacted somewhat more weakly. S. rattus strains were completely negative. Results obtained with bacterial culture supernatants were qualitatively similar. The surface localization of antigen P1 was confirmed by electron microscopy with an indirect immunogold technique. In sectioned S. mutans cells, labeling appeared to be associated with a fibrillar "fuzzy coat" layer, which was far more prominent on cells of Ingbritt 175 than on those of Ingbritt 162.
Genetic control of immune response to staphylococcal exfoliative toxin A in mice
Authors: Machida K , Sakurai S , Kondo I , Ikawa S
Published: January 2020

Different inbred and congenic resistant strains of mice were immunized with staphylococcal exfoliative toxin A (ETA). In antibody responses measured in sera of mice by a passive hemagglutination technique, A/J, DBA/2, BALB/c, B10A, B10D2, B10S, and A.SW were high responders. C57BL/10 (B10), A.BY, and DBA/1 were low responders. The congenic C3H/HeJ and C3H.SW mice were, respectively, high and low responders. The observation that the immune responses of the mice to ETA were closely linked with the haplotypes of their H-2 complexes suggests the existence of an H-2-linked immune response (Ir) gene coding for the production of humoral antibodies to ETA. Four B10A recombinants were used to map this gene within the H-2 complex. The finding that B10A(2R) and B10A(4R) were high responders, whereas B10A(3R) and B10A(5R) were low responders, indicates that the gene controlling antibody response to ETA is located in the I-A subregion or the H-2K end within the H-2 complex. We wish to propose the name Ir-ETA for this gene. The function of Ir-ETA seems to be at least related to antigen recognition at the T-lymphocyte level. Neonatal mice are generally susceptible to ETA regardless of their H-2 haplotypes. However, the neonatal mice born to a high-responder mother immunized with ETA were resistant to the subcutaneous challenge of ETA, but those born to an immunized low-responder mother were susceptible to the challenge. This result suggests that if the mother is a high responder to ETA and is effectively immunized with ETA, the maternal immunity makes it possible to neutralize this toxin in neonatal mice.
Transposon Tn5 mutagenesis of Brucella abortus
Authors: Smith L D , Heffron F
Published: January 2020

We demonstrate that transposon-mediated mutagenesis can be used to construct mutations in the pathogen Brucella abortus. We have used both a plasmid and a bacteriophage to introduce either Tn5 or Tn5 lac into the Brucella chromosome. B. abortus is naturally sensitive to kanamycin. We have selected 22 independent kanamycin-resistant colonies in strain US-19 and 19 colonies in strain 2308. We have demonstrated by Southern hybridization that Tn5 was inserted into the chromosome.
Gamma interferon reverses inhibition of leukocyte bactericidal activity by a 25-kilodalton fraction from Mycobacterium tuberculosis
Authors: Wadee A A , Cohen J D , Rabson A R
Published: January 2020

In this study we examined the effects of Mycobacterium tuberculosis cell extracts on the phagocytic activity of polymorphonuclear leukocytes and cultured peripheral blood monocytes. M. tuberculosis cell extracts were fractionated on Sephacryl S-200 columns, and a 25-kilodalton glycolipoprotein was shown to inhibit the intracellular killing ability of these leukocytes but had no effect on their phagocytic potential. This same fraction inhibited fusion of phagosomes with lysosomes, as assessed by noting the transfer of acridine orange from lysosomes to phagosomes. This fraction was shown to have a maximal inhibitory effect when it was in the form of an intact carbohydrate-lipid-protein complex. Gamma interferon (IFN-gamma), but not IFN-alpha, reversed the inhibitory effect of the mycobacterial component on bactericidal activity and on fusion of phagosomes and lysosomes. Thus, this 25-kilodalton fraction of M. tuberculosis cell extract may be important in protecting organisms against phagocytic degradation, an effect which can be reversed by IFN-gamma.
Characterization of a native polysaccharide hapten from Brucella melitensis
Authors: Moreno E , Mayer H , Moriyon I
Published: January 2020

The 13C nuclear magnetic resonance spectrum of Brucella melitensis native polysaccharide hapten proved to be very similar to that generated by the O-specific chain (PS) isolated from B. melitensis lipopolysaccharide; that is, to a linear polymer in which the repeating unit is composed of five N-formylperosaminyl residues, one of them being substituted at position C-3 and the other four at position C-2. The serological analysis suggests that the so-called A determinant is present solely in Brucella abortus PS, the M determinant is only in B. melitensis PS, and the extensive cross-reaction observed is due to a determinant shared by both polysaccharides.
Production of monoclonal antibodies against serotype strains of Pseudomonas aeruginosa
Authors: Lam J S , MacDonald L A , Lam M Y
Published: January 2020

A panel of 22 monoclonal antibodies against 8 of the 17 International Antigenic Typing Scheme (IATS) serotypes of Pseudomonas aeruginosa was produced. The antibodies were characterized for cross-reactivities, isotypes, titers, and epitope specificities. The results complemented those of our previous study and marked the completion of a set of monoclonal antibodies for serotyping P. aeruginosa.
Purification and characterization of levanase from Actinomyces viscosus ATCC 19246
Authors: Igarashi T , Takahashi M , Yamamoto A , Etoh Y , Takamori K
Published: January 2020

The extracellular levanase of Actinomyces viscosus ATCC 19246 was purified about 3,701-fold in 11% yield from the bacterial culture supernatant by means of ammonium sulfate precipitation, followed by DE52 column chromatography, Sephadex G-100 gel filtration, hydroxylapatite column chromatography, and Bio-Gel A 1.5m gel filtration. The molecular weight of the enzyme was estimated to be 89,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme was optimally reactive at pH 6.0 and at 45 degrees C. The activity was inhibited by MnCl2, BaCl2, FeCl3, ZnCl2, HgCl2, and EDTA at a final concentration of 1 mM. The inhibition by EDTA was recovered by adding CaCl2 or MgCl2. The enzyme specifically hydrolyzed levan, but not sucrose, raffinose, melezitose, inulin, and dextran. These results indicate that the purified enzyme is specific for fructan (i.e., levan), which mainly consists of beta-(2,6) linkages.
Adherence of group B streptococci to adult and neonatal epithelial cells mediated by lipoteichoic acid
Authors: Teti G , Tomasello F , Chiofalo M S , Orefici G , Mastroeni P
Published: January 2020

We have investigated the role of lipoteichoic acid in mediating the adherence of different serotypes of group B streptococci to human adult and neonatal epithelial cells. Pretreatment of neonatal buccal and vaginal epithelial cells with lipoteichoic acid, but not with deacylated lipoteichoic acid, induced a marked inhibition in the adherence of all strains tested. Pretreatment of bacteria with substances known to bind lipoteichoic acid, such as monoclonal and polyclonal antipolyglycerophosphate antibodies and albumin, also resulted in adherence inhibition. Group B streptococci adhered in 6- to 10-fold-higher numbers to buccal epithelial cells from neonates older than 3 days than to those from neonates less than 1 day old. This increase in receptiveness for group B streptococci was paralleled by an increased ability of epithelial cells from older neonates to bind group B streptococcal lipoteichoic acid. These data suggest a role for the lipid portion of lipoteichoic acid in the adherence of different serotypes of group B streptococci to vaginal and neonatal epithelial cells.
Prior exposure to subimmunogenic amounts of some bacterial lipopolysaccharides induces specific immunological unresponsiveness
Authors: Elkins K L , Stashak P W , Baker P J
Published: January 2020

Pretreatment (priming) of BALB/c mice with a low (subimmunogenic) dose of Escherichia coli O113 lipopolysaccharide (LPS) generates immunological memory 7 to 30 days later; the direct (immunoglobulin M) plaque-forming cell (PFC) responses produced after subsequent immunization with an optimal dose are 4 to 20 times greater than those of unprimed mice. By contrast, priming with a low dose of E. coli O55 LPS, followed by immunization with an optimally immunogenic dose 2 to 30 days later, resulted in a significantly reduced antibody response. Similar results were obtained with Serratia marcescens LPS. Dose-response studies indicated that such unresponsiveness is antigen specific and could be induced with subimmunogenic amounts of LPS. Priming reduced the magnitude of the PFC response to all immunizing doses of LPS tested. Unresponsiveness is not due to (i) an alteration in the time course of the PFC response or to (ii) a change in the isotype of the anti-LPS antibody produced after priming and immunization.
Mechanisms of specific immunological unresponsiveness to bacterial lipopolysaccharides
Authors: Elkins K L , Stashak P W , Baker P J
Published: January 2020

Low-dose priming of mice with Escherichia coli O113 lipopolysaccharide (LPS) results in the development of immunological memory, whereas low-dose priming with E. coli O55 LPS or Serratia marcescens LPS induces significant antigen-specific unresponsiveness. All three preparations of LPS induced proliferation of mouse splenocytes with similar time course and [3H]thymidine uptake. There was no correlation between the small amounts of serum antibody detected by enzyme-linked immunosorbent assay after low-dose priming and the subsequent generation of either memory or unresponsiveness. Further, the passive transfer of small amounts of LPS-specific antibody had no significant effect on the magnitude of the plaque-forming cell (PFC) response elicited after subsequent immunization. Reduction of the PFC response to E. coli O55 LPS occurred after low-dose priming of nu/nu (as well as nu/+) mice; however, unresponsiveness could not be generated in nu/nu mice by low-dose priming with S. marcescens LPS. Thus, although the development of low-dose unresponsiveness to S. marcescens LPS appears to involve T cells, the response of E. coli O55 LPS does not. Enhancement of the primary PFC response to S. marcescens LPS could be transferred with low-dose primed spleen cells depleted of Lyt-2+ T cells; this suggests that the magnitude of the PFC response to this preparation of LPS is negatively influenced by Lyt-2+ T cells and positively influenced by Lyt-2- spleen cells (i.e., L3T4+ T cells). These findings indicate that T cells appear to be involved in regulating the magnitude of the antibody response to some types of bacterial LPS.
Isolation of a membrane-associated Bacteroides gingivalis glycylprolyl protease
Authors: Grenier D , McBride B C
Published: January 2020

A low-molecular-weight proteolytic enzyme was purified 47-fold from outer membranes of Bacteroides gingivalis ATCC 33277 by preparative polyacrylamide gel electrophoresis. The enzyme was present in all B. gingivalis strains tested but was not found in other species of black-pigmented Bacteroides. The molecular weight, determined by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, was 19,500 when the enzyme was heated to 100 degrees C in SDS before electrophoresis and 29,000 when it was mixed with SDS but not heated. The optimum pH, with azocasein as the substrate, was between 6.0 and 6.5. The activity was inhibited by phenylmethylsulfonyl fluoride, N-alpha-p-tosyl-L-lysine chloromethyl ketone, Hg2+, and various reducing agents. The enzyme was active against azocasein, azocoll, proline-rich protein from saliva, and the synthetic peptide glycyl-L-proline-p-nitroanilide. The enzyme did not degrade acid-soluble collagen nor did it hydrolyze various arginine- and lysine-containing synthetic substrates.
Comparative study of Vibrio cholerae non-O1 protease and soluble hemagglutinin with those of Vibrio cholerae O1
Authors: Honda T , Booth B A , Boesman-Finkelstein M , Finkelstein R A
Published: January 2020

Protease and soluble hemagglutinating activities produced by a non-O1 Vibrio cholerae strain isolated from a patient with diarrhea were compared with similar activities produced by V. cholerae O1. The soluble protease activities were indistinguishable in heat stability, immunodiffusion, inhibition by antiserum, and electrophoretic analysis. On the other hand, the soluble hemagglutinating activities of both strains were not completely identical. The hemagglutinating activity of the non-O1 V. cholerae strain was not inhibited by Zincov; it was more sensitive to inhibition by normal serum, and it had an unusual pattern of heat stability. Heating at 100 degrees C resulted in some recovery of activity of a sample previously inactivated by heating at 60 degrees C.
Determinants of the immunogenicity of live virulent and mutant Vibrio cholerae O1 in rabbit intestine
Authors: Pierce N F , Kaper J B , Mekalanos J J , Cray W C , Richardson K
Published: January 2020

Determinants of the capacity of live Vibrio cholerae O1 isolates to evoke specific immune responses in intestinal mucosa were studied in rabbits, using mucosal immunoglobulin A (IgA) antitoxin as the measured immune response. Antitoxin responses were evoked mostly by the primary inoculation and were dose dependent; secondary-type responses were modest and occurred only when the booster inoculum was large, i.e., 10(10) CFU. The efficiency of mucosal immunization correlated closely with the mucosal colonizing capacity of the infecting strain and was otherwise independent of toxin genotype (A+ B+ or A- B+) or whether the strain was motile or nonmotile. Live bacteria evoked mucosal antitoxin more efficiently than did purified cholera toxin. Prior immunization with a nontoxinogenic (A- B-) V. cholera strain interfered significantly with the induction of mucosal antitoxin by subsequent immunization with its fully toxinogenic (A+ B+) parent. These results demonstrate the marked efficiency with which live V. cholerae stimulate a specific enteric mucosal secretory IgA response. They support the view that mucosal colonization aids efficient delivery of bacterial antigens to responsive subepithelial lymphoid tissue. This might occur by transfer of colonizing bacteria through M cells into Peyer patches or by efficient delivery of secreted toxin to M cells by mucosa-associated organisms. Preexisting antibacterial immunity interferes with colonization, which may prevent efficient antigenic stimulation and which may explain the relatively weak response to booster immunization. The same process may also limit the efficacy of hybrid enteric bacterial vaccines when there is preexisting mucosal immunity to the carrier organism due to either natural exposure or prior immunization with another vaccine that uses the same carrier.
Antigenic and immunogenic properties of recombinants from Salmonella typhimurium and Salmonella minnesota rough mutants expressing in their lipopolysaccharide a genus-specific chlamydial epitope
Authors: Brade L , Nano F E , Schlecht S , Schramek S , Brade H
Published: January 2020

Rough mutants from Salmonella typhimurium and Salmonella minnesota were transformed with a plasmid containing a 6.5-kilobase insert of DNA from Chlamydia trachomatis assumed to encode a glycosyltransferase. Transformation resulted in the expression of a genus-specific chlamydial epitope on the lipopolysaccharide (LPS) of the recombinant strains. Proteinase K-digested whole-cell lysates of the recombinants and of controls were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by silver staining or Western blot analysis. Two LPS populations were detected in the recombinants, the parent LPS and a faster-migrating component. The latter stained with monoclonal antibody against the genus-specific chlamydial epitope and was not seen in the controls. LPS was extracted and purified from recombinants of S. minnesota R595 and R4 and characterized by the passive hemolysis and passive hemolysis inhibition assays and by hydrolysis kinetics. Different antigenic determinants could be distinguished from each other by the passive hemolysis inhibition test with monospecific antigen-antibody reactions. Rabbits were immunized with heat-killed recombinant bacteria to study the immunogenic properties of the recombinants. In all animals, antibodies were raised against the parent core specificity and against the chlamydia-specific epitope. The data show that the recombinant bacteria are useful as immunogens to prepare polyclonal antisera against chlamydiae and that LPS isolated from them exhibits the same antigenic determinants as chlamydial LPS and may thus be used as a substitute for chlamydial LPS in serological assays.
Candidacidal factors in murine bronchoalveolar lavage fluid
Authors: Nugent K M , Fick R B
Published: January 2020

Respiratory secretions provide an efficient method for protecting the large surface area of the lower respiratory tract. To determine whether lung secretions contribute to antifungal defenses, we tested bronchoalveolar lavage fluid for fungicidal activity. Candida albicans (blastoconidia) was incubated in unconcentrated cell-free lavage fluid from Swiss Webster mice and then cultured quantitatively to measure residual viability. In control buffer the residual fractions of viable fungi were 1.03 +/- 0.12 at 60 min and 0.84 +/- 0.05 at 120 min, whereas the residual fractions in lavage fluid were 0.64 +/- 0.07 and 0.23 +/- 0.05, respectively (P less than 0.05 by t tests). This activity was trypsin sensitive and heat stable (56 degrees C) and did not require divalent cations. It did not sediment with the surfactant fraction of lung lavage fluid. Unconcentrated lavage fluid reduced the adherence of C. albicans to serum-coated glass tubes to 2.3 +/- 1.5% of that of control Candida suspensions (n = 5, P less than 0.05 by t test). It did not alter Candida ingestion or intracellular processing by alveolar macrophages. Lavage fluid also killed clinical isolates of Candida tropicalis and Torulopsis glabrata but did not kill Candida krusei or Candida parapsilosis. Lavage fluid was concentrated and passed through an acrylamide-agarose gel matrix. The chromatogram indicated that the candidacidal activity eluted in a peak with a molecular weight range of 29,000 to 40,000. After electrophoresis on 15% sodium dodecyl sulfate-polyacrylamide gels, these fractions resolved into three bands. These were transferred to nitrocellulose and then eluted with Triton X-100; this procedure permitted the isolation of a single band of candidacidal activity with a molecular weight of 29,000. In summary, murine lavage fluid contains a heat-stable protein with direct antifungal activity. This soluble factor may contribute to lung defense processes by reducing fungal viability and adherence to tissue surfaces.
Extracellular release of antimicrobial defensins by human polymorphonuclear leukocytes
Authors: Ganz T
Published: January 2020

Human polymorphonuclear leukocytes (PMN) contain three antimicrobial and cytotoxic peptides which belong to a family of mammalian granulocyte peptides named defensins. To determine their potential availability for extracellular microbicidal or cytotoxic events, we quantified the extracellular release of defensins after stimulation of human PMN with phorbol myristate acetate and opsonized zymosan. As determined by enzyme immunoassay and confirmed by polyacrylamide gel electrophoresis and densitometry, 10(6) human PMN contained 4 to 5 micrograms of defensins. After stimulation with a high concentration of phorbol myristate acetate (1 microgram/ml), about 8% of PMN defensins were found in the media. Release of defensins correlated best with the release of azurophil granule marker beta-glucuronidase or elastase and poorly with the release of either the specific granule marker lactoferrin or cytoplasmic lactate dehydrogenase. Phagocytosis of opsonized zymosan resulted in the extracellular release of less than 3% of PMN defensins. The factors responsible for less release of defensins into media relative to the release of other azurophil granule proteins may include heterogeneity of azurophil granules and the affinity of defensins for cellular surfaces and opsonized particles. In vivo, defensins are most likely to reach effective microbicidal or cytotoxic concentrations in PMN-rich exudates (pus), in confined environments of the phagolysosomes, or in intercellular clefts between PMN and their targets.
Release of outer membrane fragments by exponentially growing Brucella melitensis cells
Authors: Gamazo C , Moriyón I
Published: January 2020

Rough and smooth strains of Brucella melitensis released a membranous material that was devoid of detectable NADH oxidase and succinic dehydrogenase activity (cytoplasmic membrane markers) but that contained lipopolysaccharide, proteins, and phospholipids. This material was composed of two fractions that had similar chemical compositions but that were of different sizes which were separated by differential ultracentrifugation. Electron microscopy showed that both fractions are made of unit membrane structures. The membrane fragments were released during the exponential phase of growth, and no leakage of malic dehydrogenase activity (cytosol marker) was detected. Thus, the fragments were unlikely a result of cell lysis. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analysis showed that, although group 2 Brucella outer membrane proteins and lipoprotein were not detected, the proteins in the membranous material were outer membrane proteins. Gas-liquid chromatography analysis showed a similar fatty acid profile for the cell envelope and the outer membrane fragments of the smooth strain B. melitensis 16M. In contrast, the outer membrane fragments from the rough 115 strain were enriched in palmitic and stearic acids. With respect to the unfractionated cell envelope, outer membrane fragments were enriched in phosphatidylcholine, a phospholipid that is unusual in bacterial membranes.
Activation of murine macrophages and a bovine monocyte cell line by bovine lymphokines to kill the intracellular pathogens Eimeria bovis and Toxoplasma gondii
Authors: Hughes H P , Speer C A , Kyle J E , Dubey J P
Published: January 2020

Macrophage (M phi)-activating lymphokines present in concanavalin A-stimulated bovine T-lymphocyte cultures (ConAS) were studied by assessing their effects on Eimeria bovis and Toxoplasma gondii growth in cultured bovine monocytes (BM) and mouse M phi. The in vitro development of both parasites was assessed by incorporation of [3H]uracil and by microscopic examination of parallel cultures. Incorporation of [3H]uracil into infected cultures was an accurate indicator of growth of both E. bovis and T. gondii in BM and mouse M phi. Sporozoites of E. bovis underwent merogony in untreated BM but not in mouse M phi, whereas T. gondii developed in both cell types. Inhibition of T. gondii growth was greatest in ConAS-treated BM, whereas preincubation of mouse M phi with ConAS resulted in about 80% growth inhibition. There was no significant difference between the inhibition of either T. gondii sporozoite- or tachyzoite-induced growth in ConAS-treated cells, showing that activation pathways are equally effective against both stages. Treatment of ConAS with glycine-hydrochloride buffer (pH 2) resulted in a total loss of antiviral activity mediated by gamma interferon (IFN-gamma). When pH 2 dialyzed ConAS was used to activate BM, inhibition of T. gondii growth was only partially affected. Because bovine IFN-gamma does not activate mouse M phi and due to the partial effects of pH 2 on ConAS-induced growth inhibition, the major component(s) of ConAS responsible for T. gondii growth inhibition is distinct from IFN-gamma. Furthermore, IFN-gamma may act synergistically rather than being part of a priming sequence for M phi responsiveness to other lymphokines. Murine recombinant granulocyte-macrophage colony-stimulating factor (rGM-CSF) was tested for any microbistatic activity against T. gondii sporozoites and tachyzoites. There was no significant difference in either colony formation or [3H]uracil incorporation between rGM-CSF-treated and control cultures, regardless of host cell type. Thus, rGM-CSF does not induce adequate M phi activation to kill T. gondii and is not a major microbistatic component of ConAS. rGM-CSF also had no effect on T. gondii infection in vivo.
Demonstration of a flagellar antigen shared by a diverse group of spiral-shaped bacteria that colonize intestinal mucus
Authors: Lee A , Logan S M , Trust T J
Published: January 2020

Western blot analysis showed that there is little immunological cross-reactivity of the human pathogens Campylobacter jejuni and Campylobacter coli with Campylobacter pyloridis, except for a very strong cross-reaction between the flagellins. This same antigenic cross-reaction was found with two isolates of gram-negative spiral microaerophilic bacteria that are known to colonize the intestinal mucosa of rodents, but not with the flagellins of a number of other motile bacteria. It is proposed that this shared flagellin antigen may be important structurally and functionally.
Comparison of Bacteroides vulgatus strains in the enhancement of experimental ulcerative colitis
Authors: Onderdonk A B , Bronson R , Cisneros R
Published: January 2020

Strains of Bacteroides vulgatus from a variety of sources were tested for their abilities to enhance the inflammatory response in an experimental model for ulcerative colitis. Although there were considerable differences noted in inflammatory responses when guinea pigs were immunized with the various strains, there did not appear to be any correlation between the source of the isolates and the severity of the carrageenan-induced lesions. Strains from patients with ulcerative colitis were no more active in the model system than were strains from patients with antibiotic-associated colitis or strains from a healthy human source. The antibody titer to the strain used for immunization did not correlate with the severity of the cecal ulcerations, as determined by histopathologic evaluation.
Inhibition of the intracellular growth of Histoplasma capsulatum by recombinant murine gamma interferon
Authors: Wu-Hsieh B A , Howard D H
Published: January 2020

Recombinant murine gamma interferon as well as lymphokines prepared from immune splenocytes and concanavalin A-stimulated T-cell hybridoma activated normal mouse peritoneal macrophages to inhibit the intracellular growth of Histoplasma capsulatum. The activities of the lymphokine from immune splenocytes and of recombinant murine gamma interferon were neutralized by rabbit anti-murine gamma interferon antibody. The intracellular yeasts were not killed by the interaction even though growth was completely inhibited.
Cloning and functional expression of the Coxiella burnetii citrate synthase gene in Escherichia coli
Authors: Heinzen R A , Mallavia L P
Published: January 2020

The citrate synthase gene from the obligate intracellular rickettsial parasite Coxiella burnetii was cloned and expressed in Escherichia coli. Transduction into E. coli with a C. burnetii gene library constructed in the cosmid vector pHK17 resulted in the functional complementation of the gltA mutation of E. coli MOB154. A GltA+ clone carrying 16.4 kilobase pairs of C. burnetii DNA and designated pJCC959 was isolated and characterized. Southern hybridization analysis confirmed that the pJCC959 cloned insert consists of C. burnetii DNA and that homology exists with the Rickettsia prowazekii citrate synthase gene. Subcloning analysis with the multicopy expression vector pUC8 revealed that citrate synthase expression was under control of a C. burnetti promoter. In vitro transcription-translation of subclones pLPM20 and pLPM30 established a molecular weight of ca. 46,000 for the monomer form of the cloned enzyme. Transposon Tn5 mutagenesis of pLPM30 defined the coding region to approximately 1.2 kilobase pairs of C. burnetii DNA. Maxicell analysis of selected pLPM30::Tn5 insertion derivatives identified the direction of transcription and the relative translational start and stop sites and substantiated the molecular weight value calculated from the in vitro analysis. Inhibition studies showed that citrate synthase activity in crude cell extracts obtained from strain MOB154 transformed with the cloned C. burnetii gene was markedly inhibited by 4 mM ATP, while 4 mM alpha-ketoglutarate had virtually no effect. These data indicate that the C. burnetii enzyme displays regulatory behavior characteristic of the small gram-positive bacterial and eucaryotic enzyme.
Effect of monoclonal antibody to pertussis toxin on toxin activity
Authors: Sato H , Sato Y , Ito A , Ohishi I
Published: January 2020

Two distinct monoclonal antibodies, one to pertussis toxin subunit S2, called 9G8, and another to subunits S2 and S3, called 11E6, were generated from the hybridomas of myeloma SP2/0 and spleen cells of BALB/c mice immunized mainly with the subunit S234 complex. Binding ability of 9G8 and 11E6 to the subunits was confirmed by the enzyme-linked immunosorbent assay and immunoblotting analysis. Generation of 11E6 bound to both S2 and S3 might mean that there is common antigenicity between S2 and S3. Neutralizing activities of 9G8 and 11E6 on various biological activities of pertussis toxin, including ADP-ribosyltransferase and leukocytosis-promoting, islet-activating, permeability-increasing. Chinese hamster ovary (CHO) cell-clustering, and hemagglutinating activities, were compared with those of anti-S1 monoclonal antibodies 1B7 and 3F10, which were isolated and characterized in a previous study (H. Sato, A. Ito, J. Chiba, and Y. Sato, Infect. Immun. 46:422-428, 1984). 1B7 and 3F10 neutralized ADP-ribosyltransferase activity of pertussis toxin or S1, but 9G8 and 11E6 did not. 1B7 showed very potent neutralization against leukocytosis-promoting, islet-activating, permeability-increasing, and CHO cell-clustering activities of pertussis toxin, but 3F10 did not, although anti-ADP-ribosyltransferase activities of both antibodies were identical. 11E6 neutralized leukocytosis-promoting, islet-activating, CHO cell-clustering, and hemagglutinating activities but not permeability-increasing activity. 9G8 showed slight neutralization of leukocytosis-promoting and CHO cell-clustering activities. Specific activities of 1B7 and 11E6 in each neutralization test were higher than or almost comparable to those of polyclonal antibodies to pertussis toxin. The neutralizing mechanism of 1B7 and 11E6 in leukocytosis-promoting activity was compared. 11E6 seemed to interfere with the binding of pertussis toxin to receptors on mouse spleen cells.
Spontaneous production of thymocyte-activating factor by human gingival fibroblasts and its autoregulatory effect on their proliferation
Authors: Ohmori Y , Hanazawa S , Amano S , Miyoshi T , Hirose K , Kitano S
Published: January 2020

The purpose of this study was to examine whether human gingival fibroblasts produce a cytokine which modulates in immune and inflammatory responses including alterations in connective tissue metabolism in periodontal tissue. We found that a cultured human gingival fibroblast cell line (Gin-1) and freshly isolated human gingival fibroblasts produced thymocyte-activating factor(s), so we called the factor(s) fibroblast-derived thymocyte-activating factor (FTAF). Growth of the producing cell was itself modulated by the factor(s). Gin-1 cells spontaneously produced a significant amount of FTAF in a cell growth-dependent manner. Maximum activity was observed in conditioned medium from stationary-phase cells. The activity in conditioned medium of cultures lacking serum was significantly higher than that in those containing serum. Treatment of Gin-1 cell cultures with cycloheximide, an inhibitor of protein synthesis, markedly inhibited FTAF production. When Gin-1 cells were stimulated by triggering with muramyl dipeptide or sonicated extracts of Bacteroides gingivalis, FTAF production was significantly stimulated. Freshly isolated human gingival fibroblasts from gingival biopsies of healthy donors also produced FTAF which enhanced thymocyte proliferation. Peaks of thymocyte proliferation activity in conditioned medium from Gin-1 cells were observed in fractions having molecular weights of 25,000, 35,000, and 45,000, as determined by Sephadex G-75 column chromatography. The peak fractions (partially purified FTAF) significantly suppressed the proliferation of Gin-1 cells themselves as evaluated by [3H]thymidine uptake. The suppressive effect of partially purified FTAF was, at least partially, mediated by endogenous prostaglandin for the following reasons: addition of indomethacin, and inhibitor of prostaglandin synthesis, abrogated the suppressive effect; partially purified FTAF stimulated the production of prostaglandin E2 by the cells; and the suppression of cell proliferation was reinforced by addition of exogenous prostaglandins. These observations suggest that gingival fibroblasts play a significant role in regulation of cell growth of lymphocytes and in their own growth under physiological conditions and in pathological states in periodontal connective tissue.
Dephosphorylation of the lipid A moiety of Escherichia coli lipopolysaccharide by mouse macrophages
Authors: Peterson A A , Munford R S
Published: January 2020

An Escherichia coli deep rough lipopolysaccharide (LPS), biosynthetically labeled with 32PO4 and [3H]glucosamine, was used to study dephosphorylation of the lipid A moiety by murine macrophages. Over a 48-h incubation period, the macrophages removed approximately two-thirds of the 32P from [3H32P]LPS that was added to the culture medium. The LPS-derived phosphate was incorporated into cell components (e.g., phospholipids), as well as released from the cells. Cell lysates were also able to remove phosphate from [3H32P]LPS. The phosphatase activity was optimal at acidic pH and was greatly reduced by 10 mM sodium fluoride or heating at 80 degrees C. There was no evident difference in the LPS-dephosphorylating ability of macrophages from LPS-responsive and -hyporesponsive mice. The results indicate that murine macrophages dephosphorylate the lipid A moiety of deep rough E. coli LPS and raise the possibility that enzymatic dephosphorylation may modify LPS bioactivity.
Purification, morphology, and genetics of a new fimbrial putative colonization factor of enterotoxigenic Escherichia coli O159:H4
Authors: Tacket C O , Maneval D R , Levine M M
Published: January 2020

The ability to colonize the small intestine is essential for the pathogenesis of diarrhea caused by enterotoxigenic Escherichia coli (ETEC). Colonization is mediated by fimbriae (pili), of which there are several antigenically distinct types, including colonization factor antigen I, colonization factor antigen II (CS1, CS2, and CS3), and PCF8775 (CS4, CS5, and CS6). These fimbriae are associated with certain ETEC O serogroups. Serogroup O159 has had no known colonization factor. We found a distinct plasmid-encoded fimbria composed of 19-kilodalton protein subunits associated with ETEC serotype O159:H4. Rabbit antibody against this purified fimbria reacted with a single 19-kilodalton protein band as seen by Western immunoblot of sheared-cell preparations. The rabbit antibody, treated with colloidal-gold-labeled goat anti-rabbit immunoglobulin G, bound specifically to fimbriae when cells were examined with an electron microscope. Of 10 available ETEC O159:H4 strains from Europe, Bangladesh, and Kenya, 6 expressed this type of fimbria; its true prevalence among ETEC strains is unknown. This putative colonization factor of O159:H4 joins other ETEC fimbriae as potentially useful immunogens against human diarrhea.