bluuebunny/crossref_metadata_2025 huggingface dataset:
Role of H-2 and non-H-2 genes in control of bacterial clearance from the spleen in Salmonella typhimurium-infected mice
Authors: Nauciel C , Ronco E , Guenet J L , Pla M
Published: January 2020

The ability of mice to clear Salmonella typhimurium from their spleens in the late phase of infection was studied after inoculation with a temperature-sensitive mutant. Clearance of bacteria was delayed in C57BL/6 mice compared with BALB/c, C3H/HeJ, DBA/2, A/J, and CBA mice. The responses of F1 hybrids, backcrosses, and recombinant inbred strains derived from C57BL/6 and BALB/c (both Itys) and of H-2 congenic mice were analyzed. The results showed that the low rate of bacterial clearance was recessive, that the rate of clearance was under polygenic control, and that an H-2-linked gene(s) plays a major role. Among H-2 congenic mice with a C57BL/10 background, three phenotypes of bacterial clearance could be distinguished: high (H-2j, H-2q, and H-2u), intermediate (H-2d, H-2f, H-2k, H-2p, H-2r, H-2s, and H-2v), and low (H-2b) rates. The effect of the H-2 complex was apparent with different genetic backgrounds (Itys and Ityr). In recombinant inbred strains derived from C57BL/6 (Itys) and A/J (Ityr) mice, the effect of the H-2b haplotype on bacterial clearance appeared to be fully expressed only in strains carrying the Itys allele.
Characterization of an adhesion antigen of Streptococcus sanguis G9B
Authors: Lamont R J , Rosan B , Baker C T , Nelson G M
Published: January 2020

An antigen possessing the attributes of an adhesion has been identified in Streptococcus sanguis G9B. Cell surface components were extracted from G9B and a spontaneously occurring nonadherent mutant of G9B, strain Adh-, with a 2 mM barbital buffer, pH 8.6. The extract of G9B but not of Adh- absorbed more than 80% of the adhesion-inhibitory activity of anti-G9B immunoglobulin G (IgG). Immunoblots revealed 80- and 52-kilodalton (kDa) antigens present in the G9B extract but not in the Adh- extract. Absorption of anti-G9B IgG with Adh- and G9B barbital extracts showed a correlation between the loss of the 80- and 52-kDa antibodies and the loss of adhesion-inhibitory activity. An antibody prepared against the 80-kDa antigen excised from sodium dodecyl sulfate-polyacrylamide gels recognized the 80- and 52-kDa antigens and another antigen of 62 kDa but did not inhibit adhesion. However, an antibody from an electroblot containing the native protein from which the 80-kDa and related antigens were derived (the 80-kDa antigen complex) inhibited adhesion to the same extent as anti-G9B IgG. Periodate oxidation of the G9B barbital extract modified the 80-kDa antigen complex and resulted in the loss of 40% of its absorbing activity. The barbital extract also contained an endogenous enzyme responsible for producing the 62- and 52-kDa antigens from the 80-kDa protein and which, under optimal conditions, degraded the antigen completely, resulting in the loss of antibody-absorbing activity. The 80-kDa antigen complex has a molecular mass of more than 200 kDa in native polyacrylamide gels and a pI of 4.1 to 4.8. These observations suggest that the adhesion antigen in S. sanguis G9B is a large glycoprotein from which an 80-kDa antigen complex is derived.
Induced engulfment of Neisseria gonorrhoeae by tissue culture cells
Authors: Richardson W P , Sadoff J C
Published: January 2020

Engulfment of gonococci by mammalian tissue culture cells was examined as a model of the penetration of host cells in gonorrhea. Engulfment required viable organisms; killing the gonococci with heat or refrigeration abolished the process. Engulfment also required tissue culture cell microtubule- and microfilament-dependent movement; treating the cells with cytochalasin B (0.5 micrograms/ml) or demecolcine (Colcemid; Ciba-Geigy AG, Basel, Switzerland) (10 micrograms/ml) also prevented his process.
Incorporation of recombinant gamma interferon into liposomes enhances its ability to induce peritoneal macrophage antitoxoplasma activity
Authors: Mellors J W , Debs R J , Ryan J L
Published: January 2020

In this study we compared the ability of free- and liposome-incorporated murine recombinant gamma interferon (rIFN-gamma) to enhance peritoneal macrophage H2O2 release and antitoxoplasma activity in vitro. rIFN-gamma was efficiently (37 to 47%) incorporated into multilamellar vesicles composed of phosphatidylglycerol/cholesterol in a 2:1 molar ratio. The amount of rIFN-gamma incorporated into multilamellar vesicles and added to macrophages (0.1 to 1,000 U/ml) was quantitated with [3H]rIFN-gamma. The concentration of liposomal rIFN-gamma required to enhance macrophage H2O2 release (1 U/ml) and maximally inhibit Toxoplasma gondii growth (10 U/ml) was one-tenth the concentration required for free rIFN-gamma (10 and 100 U/ml, respectively). This increase in potency was observed in both thioglycolate-elicited and resident peritoneal macrophages. Control liposomes containing encapsulated buffer had no effect on the potency of free rIFN-gamma. The duration of macrophage activation induced by 24 h of liposomal rIFN-gamma treatment was also considerably longer than that induced by free rIFN-gamma (2 days versus less than 1 day). These data indicate that liposomal rIFN-gamma is more active than free rIFN-gamma as an inducer of macrophage microbicidal properties in vitro. This enhanced activity, combined with the potential for selective delivery of liposomal rIFN-gamma to phagocytic cells in vivo, may improve the therapeutic efficacy of rIFN-gamma in infections characterized by parasitization of phagocytes.
In vitro and in vivo activity of polyclonal and monoclonal human immunoglobulins G, M, and A against Pseudomonas aeruginosa lipopolysaccharide
Authors: Pier G B , Thomas D , Small G , Siadak A , Zweerink H
Published: January 2020

We evaluated the in vitro opsonophagocytic killing activity of monoclonal human immunoglobulin G (IgG), IgM, and IgA specific for Pseudomonas aeruginosa lipopolysaccharide and the in vivo protective capacity in neutropenic mice of both monoclonal and purified polyclonal IgG, IgM, and IgA. Monoclonal IgM was efficacious in mediating opsonophagocytic killing only in conjunction with complement, whereas monoclonal IgG opsonic killing was potentiated by complement, and monoclonal IgA opsonic killing was independent of complement. These findings are similar to those previously reported for purified polyclonal IgM, IgG, and IgA. The monoclonal and polyclonal immunoglobulins had comparable 50% protective doses in neutropenic mice (range, 0.28 to 0.46 microgram per mouse). The protective activity of IgM in neutropenic mice was abolished by cobra venom factor treatment, whereas IgG and IgA maintained efficacy in cobra venom factor-treated mice. These data indicate that all three major human serum immunoglobulin isotypes have opsonophagocytic and protective activities against P. aeruginosa, with a critical role for complement in the function of IgM.
Sequence analysis of the 47-kilodalton major integral membrane immunogen of Treponema pallidum
Authors: Hsu P L , Chamberlain N R , Orth K , Moomaw C R , Zhang L Q , Slaughter C A , Radolf J D , Sell S , Norgard M V
Published: January 2020

The complete primary amino acid sequence for the 47-kilodalton (kDa) major integral membrane immunogen of Treponema pallidum subsp. pallidum was obtained by using a combined strategy of DNA sequencing (of the cloned gene in Escherichia coli) and N-terminal amino acid sequencing of the native (T. pallidum subsp. pallidum-derived) antigen. An open reading frame believed to encode the 47-kDa antigen comprised 367 amino acid codons, which gave rise to a calculated molecular weight for the corresponding antigen of 40,701. Of the 367 amino acids, 113 (31%) were sequenced by N-terminal amino acid sequencing of trypsin and hydroxylamine cleavage fragments of the native molecule isolated from T. pallidum subsp. pallidum; amino acid sequence data had a 100% correlation with that of the amino acid sequence predicted from DNA sequencing of the cloned gene in E. coli. Although no consensus sequences for the initiation of transcription or translation were readily identifiable immediately 5' to the putative methionine start codon, a 63-base-pair PstI fragment located 159 nucleotides upstream was required for expression of the 47-kDa antigen in E. coli. The 47-kDa antigen sequence did not reveal a typical leader sequence. The overall G+C content for the DNA corresponding to the structural gene was 53%. Hydrophilicity analysis identified at least one major hydrophilic domain of the protein near the N terminus of the molecule which potentially represents an immunodominant epitope. No repetitive primary sequence epitopes were found. The combined data provide the molecular basis for further structural and functional studies regarding the role of the antigen in the immunopathogenesis of treponemal disease.
Homology of the 70-kilodalton antigens from Mycobacterium leprae and Mycobacterium bovis with the Mycobacterium tuberculosis 71-kilodalton antigen and with the conserved heat shock protein 70 of eucaryotes
Authors: Garsia R J , Hellqvist L , Booth R J , Radford A J , Britton W J , Astbury L , Trent R J , Basten A
Published: January 2020

Two lambda gt11 recombinant clones, JKL2 and JKL15, each containing an insert coding for part of the highly immunogenic 70-kilodalton (kDa) protein antigen, were isolated from a Mycobacterium leprae genomic library by immunoscreening with the monoclonal antibody L7. Clone JKL2 contained the largest insert, 2.3 kilobase pairs. Nonoverlapping fragments of this insert were used as probes and showed strong hybridization to a number of Mycobacterium tuberculosis-lambda gt11 recombinants producing proteins recognized by an anti-M. tuberculosis 71-kDa monoclonal antibody, IT11. One clone from a recombinant Mycobacterium bovis library was also characterized by using L7, and the insert from this clone, B5bt, hybridized strongly to the M. leprae probes as well. The nucleotide sequence of the 1,037-base-pair coding region of the JKL2 M. leprae clone which encodes the carboxy-terminal half of the 70-kDa protein had extensive homology with genes from a number of species. In all cases, these genes, including the recently described Ag63 and Ag361 of Plasmodium falciparum, were found to be members of the heat shock protein 70 (hsp 70) family of genes. At the amino acid level, homology was maximal between amino acids 83 through 107 and 159 through 184, which showed extreme conservation (92 and 85% identity) with Escherichia coli DnaK amino acids 386 through 409 and 460 through 485, respectively, and was 51% homologous over the entire coding region (amino acids 1 through 344 of JKL2). In contrast, amino acids 129 through 158 had maximal homology with the phylogenetically more distant Xenopus laevis hsp70. Homology declined substantially in the carboxy-terminal 34 amino acids. The predicted ATP-binding functional activity of the 70-kDa antigen from M. bovis was confirmed with affinity purification of the antigen by binding to ATP-agarose and elution with ATP. In view of the conservation of sequences between these mycobacterial antigens and mammalian endogenous cellular enzymes, further evaluation of these molecules in vivo may aid in understanding tolerance to self-antigens as well as provide potentially useful immunodiagnostic reagents.
Enhancement of antibacterial resistance of neutropenic, bone marrow-suppressed mice by interleukin-1 alpha
Authors: McIntyre K W , Unowsky J , DeLorenzo W , Benjamin W
Published: January 2020

The effect of recombinant human interleukin-1 alpha (IL-1) on the resistance of normal and bone marrow-suppressed mice against bacterial infection was evaluated. IL-1 induced neutrophilia and enhanced the resistance of normal mice against acute, systemic intraperitoneal infection with Klebsiella pneumoniae and methicillin-resistant Staphylococcus aureus. Mice with cyclophosphamide-induced bone marrow suppression were neutropenic and exhibited increased susceptibility to infection. Treatment of neutropenic C57BL/6 and C3H/HeJ mice with IL-1 before infection accelerated recovery of peripheral neutrophil counts and stimulated resistance against infection. Increases in neutrophils and enhancement of resistance induced by IL-1 were both dose and time dependent. Both neutrophilia and augmented resistance to infection were eliminated by a second dose of cyclophosphamide administered during the IL-1 treatments. Bone marrow-suppressed mice treated with IL-1 showed, at 4 h postinfection, greater increases in peripheral blood neutrophils and in numbers of peritoneal exudate neutrophils than suppressed mice treated with vehicle. The data suggest that the IL-1-stimulated recovery of myelopoiesis is an important factor in the enhancement of antibacterial resistance in bone marrow-suppressed, neutropenic mice. These findings indicate that IL-1 may be efficacious in limiting the duration of the neutropenia and of the increased risk for the development of bacterial infection associated with bone marrow suppression.
Pellicle receptors for Actinomyces viscosus type 1 fimbriae in vitro
Authors: Clark W B , Beem J E , Nesbitt W E , Cisar J O , Tseng C C , Levine M J
Published: January 2020

Actinomyces viscosus T14V-J1 and its fimbria-deficient mutant strain possessing type 1 fimbriae strongly aggregated with latex beads treated with acidic proline-rich protein 1, basic proline-rich proteins, and proline-rich glycoprotein and its deglycosylated derivative. These type 1+ strains did not aggregate with latex beads treated with other proteins, such as salivary amylase, salivary histidine-rich polypeptides, laminin, type 1 collagen, fibronectin, or C1q. The type 1+ strains also adsorbed well to experimental pellicles formed with acidic proline-rich protein 1, basic proline-rich proteins, and proline-rich glycoprotein and its deglycosylated derivative on hydroxyapatite (HA) surfaces. These interactions were inhibited with immunoglobulins and Fabs specific for type 1 fimbriae. Type 1- actinomyces exhibited feeble adsorption to latex beads or HA treated with any of the aforementioned proteins. Collectively, these data indicate that actinomyces type 1 fimbriae may specifically interact with several proline-rich salivary molecules, forming experimental pellicles on HA or polystyrene surfaces.
Activation of macrophages in an experimental rat model of arthritis induced by Erysipelothrix rhusiopathiae infection
Authors: Renz H , Gentz U , Schmidt A , Dapper T , Nain M , Gemsa D
Published: January 2020

Infection of Lewis rats with Erysipelothrix rhusiopathiae represents an experimental model system of acute and chronic arthritis. We studied here the acute inflammatory phase with respect to stimulation of macrophages and lymphocytes. Intragluteal injection of viable E. rhusiopathiae (10(2) to 10(4) bacteria) rapidly induced generalized inflammation, loss of body weight, hind leg arthritis, and systemic macrophage activation within 2 to 3 days. The same symptoms could also be evoked by injection of dead E. rhusiopathiae. Ex vivo, peritoneal macrophages released large amounts of tumor necrosis factor alpha on day 2 and interleukin-1 on day 3, whereas production of prostaglandin E2 was delayed to days 5 to 7 and appeared to counteract tumor necrosis factor alpha synthesis. The inflammatory response and development of arthritis were strongly dependent on T lymphocytes, as evidenced by the following findings: (i) lymphocytes released lymphokines that activated macrophages to enhanced mediator release; (ii) treatment of rats with cyclosporin A reduced infection-induced macrophage activation; (iii) mitogen-stimulated thymocyte proliferation was enhanced, indicating an infection-induced maturation-differentiation process in the thymus; and (iv) in T-cell-deficient nude rats, a higher dose of bacteria was required for infection, the inflammatory response was less severe, and only mild, but not chronic, arthritis developed. Thus, an E. rhusiopathiae-induced inflammation in rats provides a useful tool to characterize activated macrophages and T lymphocytes during the development of acute arthritis and its transition into the chronic form.
Effect of glutaraldehyde fixation on cell surface binding capacity of Candida albicans
Authors: Mleczko J , Litke L L , Larsen H S , Chaffin W L
Published: January 2020

The ability of viable and glutaraldehyde-fixed, stationary-phase yeast cells of Candida albicans to bind concanavalin A and monospecific antiserum for antigenic factor 1 was examined. Both fluorescence flow cytometric analysis and transmission electron microscopy indicated that glutaraldehyde-fixed cells bound less of the two reagents than did unfixed viable cells.
Molecular cloning and characterization of a hemolysin gene from Actinobacillus (Haemophilus) pleuropneumoniae
Authors: Lian C J , Rosendal S , MacInnes J I
Published: January 2020

This article describes the molecular cloning and expression of a hemolysin gene from a serotype 1 strain of Actinobacillus pleuropneumoniae. The hemolysin was a thermolabile protein with an apparent molecular weight of 29,500 (29.5K hemolysin). Unlike expression of the recently described 105K hemolysin of A. pleuropneumoniae (J. Frey and J. Nicolet, FEMS Microbiol. Lett. 55:41-46, 1988), expression of this hemolysin was not regulated by Ca2+. Antiserum prepared against the 105K hemolysin did not neutralize the activity of the 29.5K hemolysin; conversely, antiserum prepared against the 29.5K hemolysin did not neutralize the activity of the 105K hemolysin. The hemolytic activity was not neutralized with antisera against hemolytic Escherichia coli, Streptococcus agalactiae, or purified streptolysin O, but antisera prepared against recombinants containing the 29.5K gene and convalescent pig sera abrogated hemolytic activity. Although hemolytic activity could be detected in several strains of E. coli K-12 and in minicells expressing several different constructs encoding the 29.5K hemolysin, we could not rigorously exclude the possibility that the gene which we have isolated encodes a regulator of hemolytic activity rather than a hemolysin per se.
Antibody response in white-footed mice (Peromyscus leucopus) experimentally infected with the Lyme disease spirochete (Borrelia burgdorferi)
Authors: Schwan T G , Kime K K , Schrumpf M E , Coe J E , Simpson W J
Published: January 2020

White-footed mice (Peromyscus leucopus), the primary reservoir for Borrelia burgdorferi in the northern midwest and northeastern United States, were experimentally inoculated with an infectious strain or a noninfectious strain of the Lyme disease spirochete and examined for their specific antibody response with the enzyme-linked immunosorbent assay and Western blot (immunoblot) analysis. Immunoglobulin M (IgM) anti-B. burgdorferi antibodies were detected in mice 1 to 2 days after inoculation with either the infectious or noninfectious strain of spirochetes and peaked on days 4 and 5. Mice inoculated with the infectious strain of spirochete had a secondary increase in IgM 21 days after inoculation. Mice also produced both IgG1 and IgG2 antibodies beginning 5 to 7 days after inoculation and they increased in titer until 84 days after inoculation when the experiment was terminated. Western blot analysis of sequential plasma samples from mice inoculated with the infectious strain of spirochete demonstrated the development of IgM, IgG1, and IgG2 antibodies to numerous spirochetal antigens, whereas mice inoculated with the noninfectious strain had reduced blot patterns with antibodies reactive primarily to the 31,000-kilodalton outer surface protein A. Persistent spirochetal infection in some mice, in spite of a strong and diverse antibody response, deserves further investigation.
In vivo formation of hybrid toxins comprising Shiga toxin and the Shiga-like toxins and role of the B subunit in localization and cytotoxic activity
Authors: Weinstein D L , Jackson M P , Perera L P , Holmes R K , O'Brien A D
Published: January 2020

Shiga toxin, Shiga-like toxin I (SLT-I) and Shiga-like toxin II (SLT-II) are cell-associated cytotoxins that kill both Vero cells and HeLa cells, whereas Shiga-like toxin II variant (SLT-IIv) is an extracellular cytotoxin that is more cytotoxic for Vero cells than for HeLa cells. The basis for these differences in cytotoxin localization and host cell specificity were examined in this study. The A and B subunit genes of Shiga toxin and the SLTs were recombined by two methods so that hybrid toxins would be formed in vivo. Complementation of heterologous subunits was accomplished by cloning the individual A and B subunit genes of SLT-I, SLT-II, and SLT-IIv on plasmid vectors of different incompatibility groups so that they could be maintained in double transformants of Escherichia coli. In addition, six operon fusions were constructed so that the A and B subunit genes of Shiga toxin, SLT-II, and SLT-IIv could be expressed as a single operon. The activities of the hybrid cytotoxins were assessed in three ways: (i) level of cytotoxicity, (ii) ratio of HeLa to Vero cell cytotoxicity, and (iii) ratio of extracellular to cell-associated cytotoxicity. Neither the A subunit of Shiga toxin nor SLT-I associated with a heterologous B subunit to form an active cytotoxin. However, in all other cases the hybrid molecules formed by subunit complementation or operon fusion were cytotoxic. Furthermore, the cytotoxic specificity and localization of the hybrid cytotoxins always corresponded to the activities of the native toxin possessing the same B subunit.
Reduction of capsular polysaccharide production in Klebsiella pneumoniae by sodium salicylate
Authors: Domenico P , Schwartz S , Cunha B A
Published: January 2020

Heavily encapsulated Klebsiella pneumoniae (serotypes 1 and 2) was cultured in the presence of sodium salicylate. The addition of salicylate (2 to 30 micrograms/ml) progressively decreased the amount of capsular polysaccharide produced by all strains without significantly inhibiting cell growth. Further addition of salicylate (50 to 200 micrograms/ml) was progressively inhibitory to cell growth and decreased the production of polysaccharide only slightly. The optimal concentration of salicylate that could reduce the polysaccharide production of heavily encapsulated, virulent strains by 50% or more was 30 micrograms/ml. Mutants of these bacteria that produced less capsule were affected by salicylate to a lesser degree. All concentrations of salicylate tested were physiologically achievable in humans and within the therapeutic range of aspirin. The addition of calcium and magnesium partially reversed the effects of salicylate on polysaccharide production. Chelating agents, particularly EGTA [ethylene-bis(oxyethylenenitrile)tetraacetic acid], reduce capsule production as salicylate did. Thus, the chelation of calcium and magnesium by salicylate could account, at least in part, for the reduction of capsule. Optical density measurements allowed for rapid monitoring of capsule production in various culture media because a large part of culture turbidity was apparently due to the capsule. Decreased production of the primary K. pneumoniae virulence factor with salicylate may have therapeutic potential.
Relative susceptibilities of inbred mouse strains C57BL/6 and A/J to infection with Histoplasma capsulatum
Authors: Wu-Hsieh B
Published: January 2020

Differences in the 30-day survival of Histoplasma capsulatum after intravenous injection indicated that the A/J strain of inbred mouse was more resistant to experimental infection than was the C57BL/6 strain. CFU from the spleens of infected animals increased during the first week after injection but gradually declined over the next 3 weeks. The CFU per gram of tissue in the C57BL/6 animals were 10- to 100-fold higher than were those in the A/J mice during the time between 7 and 28 days after infection. The units of gamma interferon (IFN-gamma) in supernatants of spleen cells stimulated with heat-killed yeast cells of H. capsulatum reached a peak at the time of the largest number of CFU per gram of tissue. The titers of IFN-gamma at days 3 to 5 were higher in the A/J mice than they were in the C57BL/6 mice, but from days 7 to 28, the titers of IFN-gamma were not correlated with the more efficient clearance of the fungus from the spleens of A/J mice. The L3T4+ spleen cells were shown to be active IFN-gamma producers. Treatment of Histoplasma-infected mice with anti-IFN-gamma antibody resulted in much larger tissue burdens of the fungus in the lungs and spleens of treated animals than in untreated animals. There was no marked difference in the result of treatment with anti-IFN-gamma antibody between A/J and C57BL/6 mice. Treatment of Histoplasma-infected mice with recombinant murine IFN-gamma did not alter the course of infection in either inbred strain of mouse.
Isolation of a corncob (coaggregation) receptor polypeptide from Fusobacterium nucleatum
Authors: Kaufman J , DiRienzo J M
Published: January 2020

Corncobs, which are distinct morphological units formed by the ordered coaggregation of a filamentous microorganism and streptococci, can be made in vitro by using oral strains of Fusobacterium nucleatum and Streptococcus sanguis. Previous studies have shown that strains of F. nucleatum contain one of at least two different types of corncob receptor. The objective of this study was to isolate the receptor from F. nucleatum ATCC 10953 as the first step in the elucidation of the molecular basis of corncob formation. The cell envelope fraction from this bacterium was treated with trypsin, delipidated with chloroform-methanol, and subjected to ion-exchange chromatography. A single polypeptide (apparent Mr, 39,500), which was eluted from the column with 0.5 M sodium chloride and extracted with dodecyltrimethylammonium bromide to remove contaminating lipopolysaccharide, inhibited corncob formation between strain ATCC 10953 and S. sanguis CC5A. Similarly derived cell fractions from either F. nucleatum FDC 364 or Fusobacterium necrophorum failed to effect coaggregation in the inhibition assay. Amino acid analysis of the polypeptide showed a moderately hydrophobic character (polarity index, 41) and 11% basic residues. Antiserum made against the purified polypeptide agglutinated F. nucleatum ATCC 10953, neutralized the ability of this bacterium to form corncobs, and agglutinated whole cells of S. sanguis CC5A that were precoated with the receptor polypeptide. The identification and isolation of this receptor should greatly enhance our ability to define some of the complex intergeneric coaggregation mechanisms that are thought to occur in the human oral cavity.
Antibody response to Staphylococcus aureus surface proteins in rabbits with persistent osteomyelitis after treatment with demineralized bone implants
Authors: Thomas V L , Sanford B A , Keogh B S , Triplett R G
Published: January 2020

A rabbit model was used to study the effect of allogeneic demineralized bone powder (DBP) implants on the persistence of Staphylococcus aureus osteomyelitis. Thirty-one rabbits with chronic osteomyelitis of the tibia established by day 21, were started on systemic antibiotics followed by either no additional treatment or debridement plus either DBP (with or without supplemental antibiotics) or supplemental antibiotics only. On day 21, cultures showed a mean of 2 x 10(4) CFU/mg of debrided osseous material. By day 70, the treatment most effective in clearing infection was found in animals treated with supplemental antibiotics only (mean of 142 +/- 116 CFU/mg). In contrast, infection persisted at a 7- to 10-fold-higher level in animals receiving DBP with and without supplemental antibiotics; these results suggest that DBP contributed to persistence of infection. Longitudinal sera were tested again staphylococcal sonic extracts by immunoblot. Detection of numerous probe-positive bands indicated complex but remarkably similar antibody responses among infected animals. Antibodies attached directly to the cell surfaces of staphylococci as shown by immunogold and blocked the binding of organisms to HEp-2 and human fetal lung cells in a radioadherence assay. Antibodies could be absorbed out by intact organisms and were unreactive by immunoblot against antigens derived from cells pretreated with pronase, proteinase K, or lysostaphin. These results indicate that the major response was directed against staphylococcal cell surface proteins. Surprisingly, only one major band (molecular weight, approximately 12,000) was detected when a homologous in vivo antigen preparation was studied by immunoblot. Antibody reactive against this peptide did not appear to react with staphylococci grown in vitro.
Immunological studies of the disulfide bridge region of Pseudomonas aeruginosa PAK and PAO pilins, using anti-PAK pilus and antipeptide antibodies
Authors: Lee K K , Sastry P A , Paranchych W , Hodges R S
Published: January 2020

Pseudomonas aeruginosa is an opportunistic pathogen that attaches to host cells via their pili. The pilus of P. aeruginosa PAK consists of a polymer of a single subunit, pilin, which is a 144-residue polypeptide. The C-terminal end of this protein is semiconserved in a number of strains and contains a disulfide bridge. We have synthesized the C-terminal peptide PAK (128-144)-OH in both its reduced and oxidized forms and the analog PAK(A-129) (128-144)-OH, in which cysteine-129 was substituted by alanine. These three peptides were used to immunize rabbits and prepare antipeptide antisera. It was found that antipeptide antisera to reduced peptide (17-R) and to oxidized peptide (17-O) bound to native PAK pili and cross-reacted with strain PAO pili in direct enzyme-linked immunosorbent assay (ELISA) and immunoblot experiments. However, the antiserum to the peptide immunogen PAK(A-129)(128-144)-OH, which does not have the ability to form the disulfide bridge, did not bind to either PAK or PAO pili. Competitive ELISA experiments with reduced and oxidized peptides of Ac-PAK(128-144)-OH showed that there was no difference in binding between the two peptides for 17-R or 17-O immunoglobulin G. When immunoglobulin G from native PAK antipilus antiserum was used in competitive or direct ELISA experiments, there was also no preference in binding to reduced or oxidized Ac-PAK(128-144)-OH or to PAK(A-129)(128-144)-OH. This result showed that the disulfide bridge in Pseudomonas pili is not critical to the immunogenicity of this region. However, the disulfide bridge is important in the immunogenicity of the C-terminal peptide when preparing antipeptide antisera that are cross-reactive with pili from different strains, since only the disulfide bridge peptide antisera cross-reacted well with the PAO pili as shown by competitive ELISA, suggesting that this region could be an important candidate for development of a synthetic vaccine.
C3bi-binding protein on Candida albicans: temperature-dependent expression and relationship to human complement receptor type 3
Authors: Eigentler A , Schulz T F , Larcher C , Breitwieser E M , Myones B L , Petzer A L , Dierich M P
Published: January 2020

We investigated in detail the previously described capacity of pseudohyphae of Candida albicans to bind C3-coated particles. We show that the expression of the C3bi receptor of C. albicans was dependent upon the growth temperature of the fungi. C. albicans grown at 30 degrees C bound strongly to EAC1423bi, whereas those cells grown at 38.5 degrees C were completely devoid of this capacity. The molecule responsible for the attachment of EAC1423bi was heat labile and trypsin sensitive. Several, but not all, monoclonal antibodies to the alpha-chain of human complement receptor type 3 (CR3) stained C. albicans, and this reactivity was expressed in parallel with the capacity of C. albicans to bind EAC1423bi, i.e., both were dependent on the growth temperature of the fungi and were trypsin sensitive. In contrast to CR3, the binding of EAC1423bi to C. albicans did not require the presence of divalent cations. Rabbit immunoglobulin G antibodies directed against C. albicans inhibited the binding of EAC1423bi to C. albicans but not to human CR3. These inhibiting IgG antibodies recognized antigens expressed on the surface of pseudohyphae but not those of yeast cells. OKM-1, a monoclonal antibody to human CR3 inhibited the attachment of EAC1423bi to CR3 and also to C. albicans. OKM-1 precipitated a 130-kilodalton band from solubilized 125I-labeled C. albicans. We conclude that the complement receptors on C. albicans and human CR3 were antigenically related but not identical and that they differed in their functional characteristics.
Vaccination of mice against Mycobacterium leprae infection
Authors: Singh N B , Lowe A C , Rees R J , Colston M J
Published: January 2020

Intradermal immunization with killed Mycobacterium leprae renders mice immune to infection with viable M. leprae. This protection is long lasting and systemic in that immunization in the left flank results in protection in both the left and right footpads. Immunization with Mycobacterium vaccae was ineffective in protecting mice against M. leprae infection, while Mycobacterium bovis BCG provided partial protection. Mycobacterium habana TMC 5135 (now known as Mycobacterium simiae) was found to be as effective as M. leprae in protecting mice against footpad infection.
Pasteurella haemolytica antigens associated with resistance to pneumonic pasteurellosis
Authors: Mosier D A , Simons K R , Confer A W , Panciera R J , Clinkenbeard K D
Published: January 2020

Antigens associated with whole Pasteurella haemolytica biotype A serotype 1, a capsular carbohydrate-protein extract of the organism, and P. haemolytica leukotoxin were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Antigens of the electrophoresed preparations were detected by Western blotting (immunoblotting) with sera from cattle which were either nonvaccinated or vaccinated with live or killed P. haemolytica vaccines and had variable degrees of resistance to experimental pneumonic pasteurellosis. Distinct, easily recognizable antigens of these preparations were identified, and the antibody responses to these antigens were quantified by densitometry. To determine their importance to disease resistance, we then compared antibody responses with experimental lesion scores. Antibody reactivity to surface antigens which were significantly correlated with resistance and present in two or more of the preparations were detected at 86, 66, 51, 49, 34, 31, and 16 kilodaltons (kDa). Of these, antibody responses to antigens at 86, 49, and 31 kDa appeared most important based on their concentration and significance levels. Antibody reactivity to leukotoxin antigens which were significantly correlated with resistance and common with important surface antigens were detected at 86, 66, and 49 kDa. Antibody responses to unique leukotoxin antigens which were significantly correlated with resistance were present at 92 and 58 kDa.
Inhibition of Leishmania donovani promastigote internalization into murine macrophages by chemically defined parasite glycoconjugate ligands
Authors: Palatnik C B , Borojevic R , Previato J O , Mendonça-Previato L
Published: January 2020

Leishmania donovani, the agent of human visceral leishmaniasis, is an intracellular parasite that must be recognized and internalized by host macrophages to complete its biological cycle. In a search for possible ligands for macrophage surface receptors, glycoconjugates were obtained from Leishmania promastigotes by aqueous, phenol-aqueous, and alkaline extraction. A fucose-mannose glycoproteic ligand, a lipopeptidephosphoglycan, and a phosphate mannogalactan ligand were purified from promastigotes and analyzed for their chemical contents, with special attention to their glycidic moieties. Sugars that were identified as components of these glycoconjugates were tested for their capacity to inhibit promastigote internalization by BALB/c peritoneal macrophages in vitro. Neutral hexoses showed little inhibitory activity; fucose, charged monosaccharides, and a mannose polymer showed the highest activity. Two of the glycoconjugates (fucose-mannose glycoproteic ligand and phosphate mannogalactan ligand) purified from promastigotes were potent inhibitors of internalization, 75% inhibition being obtained at concentrations of 6 to 10 micrograms/ml. The simultaneous presence of both ligands in low concentrations yielded an increase in inhibitory activity above that found for each ligand alone, indicating that promastigotes may use at least two receptor sites for penetration into macrophages. These ligands are specific inhibitors of L. donovani promastigote phagocytosis, since 10 micrograms of each ligand per ml interfered neither with internalization of yeast cells nor with phagocytosis of Leishmania adleri promastigotes.
Role of tryptophan degradation in respiratory burst-independent antimicrobial activity of gamma interferon-stimulated human macrophages
Authors: Murray H W , Szuro-Sudol A , Wellner D , Oca M J , Granger A M , Libby D M , Rothermel C D , Rubin B Y
Published: January 2020

To determine whether extracellular tryptophan degradation represents an oxygen-independent antimicrobial mechanism, we examined the effect of exogenous tryptophan on the intracellular antimicrobial activity of gamma interferon (IFN-gamma)-stimulated human macrophages. IFN-gamma readily induced normal monocyte-derived macrophages (MDM) to express indoleamine 2,3-dioxygenase (IDO) activity and stimulated MDM, alveolar macrophages, and oxidatively deficient chronic granulomatous disease MDM to degrade tryptophan. All IFN-gamma-activated, tryptophan-degrading macrophages killed or inhibited Toxoplasma gondii, Chlamydia psittaci, and Leishmania donovani. Although exogenous tryptophan partially reversed this activity, the increases in intracellular replication were variable for normal MDM (T. gondii [5-fold], C. psittaci [3-fold], L. donovani [2-fold]), chronic granulomatous disease MDM (T. gondii [2.5-fold], C. psittaci [5-fold]), and alveolar macrophages (T. gondii [1.5-fold], C. psittaci [1.5-fold]). In addition, IFN-alpha and IFN-beta also stimulated normal MDM to express IDO and degrade tryptophan but failed to induce antimicrobial activity, and IFN-gamma-treated mouse macrophages showed neither IDO activity nor tryptophan degradation but killed T. gondii and L. donovani. These results suggest that while tryptophan depletion contributes to the oxygen-independent antimicrobial effects of the activated human macrophage, in certain cytokine-stimulated cells, tryptophan degradation may be neither sufficient nor required for antimicrobial activity.
Characterization of gamma interferon-mediated cytotoxicity to chlamydia-infected fibroblasts
Authors: Byrne G I , Schobert C S , Williams D M , Krueger D A
Published: January 2020

Addition of murine recombinant gamma interferon (IFN-gamma) to mouse fibroblast cultures infected with Chlamydia psittaci was found to induce a cytotoxic response that was dependent on the concentration of IFN-gamma added and the multiplicity of infection given. No cytotoxicity was observed for uninfected cells treated with IFN-gamma, nor did infection alone elicit cytotoxicity. Cytotoxicity was detected only if IFN-gamma was present for at least the first 18 h of a 30-h incubation period. Cytotoxic activity was not observed when infected cells were treated with 50 micrograms of chloramphenicol per ml, a drug which inhibits differentiation of infectious elementary bodies to noninfectious reticulate bodies. Cytotoxic activity was restored if addition of chloramphenicol was delayed until 18 h postinfection. Addition of 100 U of penicillin per ml to infected host cells reduced but did not abolish cytotoxic activity. Treatment of host cells with as little as 0.2 microgram of cycloheximide per ml inhibited cytotoxicity without interfering with chlamydial growth. When addition of cycloheximide was delayed until 12 h after infection and IFN-gamma treatment, cytotoxicity was restored. These data indicate that IFN-gamma functions as a cytotoxic cytokine against chlamydia-infected fibroblasts. Cytotoxicity was found to be dependent on chlamydial multiplicity of infection, differentiation of chlamydiae to the metabolically active form, and host cell protein synthesis.
Pseudomonas aeruginosa cytotoxin: periplasmic localization and inhibition of macrophages
Authors: Kluftinger J L , Lutz F , Hancock R E
Published: January 2020

Pseudomonas aeruginosa cytotoxin has been isolated previously from cell autolysates. Both purified cytotoxin and periplasmic contents (osmotic shock fluid) cross-reacted on Western immunoblots with antibodies specific for cytotoxin. In addition, both preparations caused a significant reduction in antibody-mediated phagocytosis of P. aeruginosa M2 by mouse macrophage cell line P388D1. Phagocytosis was restored in each case on preincubation of cytotoxin or periplasmic contents with anti-cytotoxin serum. Both cytotoxin and periplasmic contents caused depolarization of the P388D1 cell membrane, as demonstrated with a polarization-sensitive fluorescent probe. Similar correlations were not observed for other P. aeruginosa cell fractions or for osmotic shock fluid from Escherichia coli C600. These data indicate that P. aeruginosa cytotoxin is localized in the periplasm and has the potential to inhibit macrophage-mediated phagocytosis, possibly by perturbing ion gradients across the macrophage plasma membrane.
Ureaplasma urealyticum pneumonia: experimental production and demonstration of age-related susceptibility
Authors: Rudd P T , Cassell G H , Waites K B , Davis J K , Duffy L B
Published: January 2020

Two different strains of Ureaplasma urealyticum isolated in pure culture from the lungs of newborn human infants were shown to produce an acute, self-limiting, interstitial pneumonia in newborn C3H/HeN and C57BL/6 mice that were free of other respiratory pathogens. Lesion severity peaked 3 to 6 days following intranasal inoculation of ureaplasmas and was resolved by 12 days. Rhinitis and otitis also occurred but did so less frequently than pneumonia. Organisms were localized within the alveoli in areas of inflammation. In comparison with newborn mice, 14-day-old mice were less susceptible to either colonization or disease.
Hemagglutination activity of Campylobacter pylori
Authors: Nakazawa T , Ishibashi M , Konishi H , Takemoto T , Shigeeda M , Kochiyama T
Published: January 2020

Forty-five strains of Campylobacter pylori isolated from gastric biopsy specimens showed distinct hemagglutination activity. The activity was partially decreased by treatment with heat, trypsin, or an alkylating agent and was inhibited by porcine gastric mucin but not by various compounds, including D-mannose.
Impairment of melibiose utilization in Streptococcus mutans serotype c gtfA mutants
Authors: Barletta R G , Curtiss R
Published: January 2020

The Streptococcus mutans serotype c gtfA gene encodes a 55-kilodalton sucrose-hydrolyzing enzyme. Analysis of S. mutans gtfA mutants revealed that the mutant strains were specifically impaired in the ability to use melibiose as a sole carbon source. S. mutans gtfA mutant strains synthesized less alpha-galactosidase activity inducible by raffinose than wild-type strains. Melibiose (an inducer in wild-type strains) failed to induce significant levels of alpha-galactosidase in the mutant strains. We hypothesize that melibiose use by S. mutans requires the interaction of the GtfA enzyme, or another gene product under the control of the gtfA promoter, with other gene product(s) involved in melibiose transport or hydrolysis.
Development of a rat model for respiratory infection with Bordetella pertussis
Authors: Woods D E , Franklin R , Cryz S J , Ganss M , Peppler M , Ewanowich C
Published: January 2020

Considerable effort directed toward designing a safe and effective vaccine for Bordetella pertussis in which the cellular and/or acellular antigens necessary to confer immunity are known has been hampered by lack of information on the pathogenesis of the natural infection. The study presented here describes an animal model of lung infection by B. pertussis encased in agar beads in adult (200- to 220-g) male Sprague-Dawley rats. At 3 and 7 days after inoculation with phase I B. pertussis, organisms could be recovered from the lungs of rats; however, organisms were not recoverable at days 10 and 14 but reappeared in lung homogenates at day 21. Histopathological examination revealed findings similar to those seen in human disease. At day 3, a mild lymphocytic infiltrate was present in the bronchi, with progressive lymphoid hyperplasia peribronchially. By day 7, a necrotizing inflammation of the tracheobronchial mucous membranes, characterized by both mononuclear and polymorphonuclear cells, was noted. Phase III B. pertussis organisms were not recoverable from the lungs of inoculated rats at day 3 after inoculation, nor were histological changes noted in these animals. Clinical findings in phase I B. pertussis-infected rats included hypoglycemia, circulating lymphocytosis, and paroxysms in which air was forcibly expelled from the mouth or nose.
Modulation of invasiveness and catalytic activity of Bordetella pertussis adenylate cyclase by polycations
Authors: Raptis A , Knipling L G , Gentile F , Wolff J
Published: January 2020

Penetration of Bordetella pertussis adenylate cyclase into CHO cells was monotonically inhibited by polylysines, with a minimum degree of polymerization of greater than 6 and less than or equal to 9 to 10. Above this level, inhibitory potency per lysyl residue was independent of polymer length; 50% inhibition was obtained with 60 microM lysine monomer. Other polycations were also potent inhibitors. The adenylate cyclase itself showed a biphasic (stimulation-inhibition) response, with a similar independence of polymer length above a certain minimum. Half-maximal inhibitory concentrations for cyclic AMP accumulation corresponded to half-maximal stimulatory concentrations of poly-L-lysine for the cyclase. The inhibitory effect of polylysines on cyclic AMP accumulation was not reversed by washing or enzymatic removal of neuraminic acid. We conclude that charge-charge interactions play an important role in the penetration of B. pertussis adenylate cyclase into host cells.
A Legionella pneumophila gene encoding a species-specific surface protein potentiates initiation of intracellular infection
Authors: Cianciotto N P , Eisenstein B I , Mody C H , Toews G B , Engleberg N C
Published: January 2020

To investigate the pathogenesis of Legionnaires disease at a molecular level, we mutated by directed allelic exchange a gene encoding a Legionella pneumophila-specific 24,000-dalton (Da) surface protein. Southern hybridization and immunoblot analyses demonstrated that the predicted DNA rearrangement occurred in L. pneumophila with a specific loss of 24-kDa antigen expression. Compared with its isogenic parent, the mutant was significantly impaired in its ability to infect transformed U937 cells, a human macrophagelike cell line; i.e., the bacterial inoculum of the mutant strain that was required to initiate infection of the macrophage monolayer was ca. 80-fold greater than that of the isogenic parent strain. The mutant strain regained full infectivity on reintroduction of a cloned 24-kDa protein gene, indicating that the reduced infectivity was due specifically to the mutation in that gene. Compared with the parent strain, the mutant strain was recovered at titers that were ca. 10-fold lower shortly after infection, but it exhibited a similar intracellular growth rate over the next 40 h, indicating that the mutant was defective in its ability to initiate macrophage infection rather than in its ability to replicate intracellularly. When opsonized, the mutant strain was still significantly less infectious than the parent strain, despite equivalent macrophage association, suggesting that the mutant was not merely missing a ligand for macrophage attachment. The mutant also exhibited reduced infectivity in explanted human alveolar macrophages, demonstrating the relevance of the U937 cell model for analyzing this mutant phenotype. These results represent the first identification of a cloned L. pneumophila gene that is necessary for optimal intracellular infection; we designate this gene mip, for macrophage infectivity potentiator.