bluuebunny/crossref_metadata_2025 huggingface dataset:
Characterization of monoclonal antibodies to a crystal protein of Bacillus thuringiensis subsp. kurstaki
Authors: Huber-Lukac M , Jaquet F , Luethy P , Huetter R , Braun D G
Published: January 2020

Ten monoclonal antibodies were produced against a k-1-type crystal protein of Bacillus thuringiensis subsp. kurstaki. Eight of the antibodies belong to the immunoglobulin G1 (IgG1) subclass, with pI values ranging from 5.5 to 8.6, one could be assigned to the IgG2b subclass, and one could be assigned to the IgM class. Competitive antibody-binding assays and analysis of antibody specificity indicated that the 10 antibodies recognized at least nine distinct antigenic determinants. Eight antibodies bound to both protoxin and toxin, whereas the other two interacted with protoxin only. One antibody completely inhibited the biological activity of the delta-endotoxin, five antibodies reduced it by 15 to 82%, and four antibodies did not affect it at all. Based on cross-reaction studies, homologies and differences in the crystal protein structures of different B. thuringiensis subspecies were revealed. All of the monoclonal antibodies strongly cross-reacted with crystal proteins from strains of B. thuringiensis subsp. tolworthi, B. thuringiensis subsp. galleriae, B. thuringiensis subsp. dendrolimus, B. thuringiensis subsp. sotto, and B. thuringiensis subsp. subtoxicus. Some antibodies interacted only weakly with crystal proteins from strains of B. thuringiensis subsp. morrisoni and B. thuringiensis subsp. entomocidus, and some of these did not interact with B. thuringiensis subsp. kenyae and B. thuringiensis subsp. darmstadiensis. No cross-reaction was found with the parasporal inclusion protein of B. thuringiensis subsp. israelensis. Studies with the monoclonal antibodies also disclosed that crystal proteins from strains of the same subspecies can exhibit substantial differences in antigenic structure. In particular, striking strain-specific differences in the protoxins of B. thuringiensis subsp. kurstaki and B. thuringiensis subsp. thuringiensis were observed.
Role of capsule and O antigen in resistance of Klebsiella pneumoniae to serum bactericidal activity
Authors: Tomás J M , Benedí V J , Ciurana B , Jofre J
Published: January 2020

The ability of Klebsiella pneumoniae strains to resist the bactericidal activity of serum was quantitated. The K. pneumoniae strains tested included mutants lacking the capsular polysaccharide and mutants having a modified lipopolysaccharide structure. The last mutants were obtained as phage-resistant mutants, and their lipopolysaccharide was characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and chemical analysis. Serum-resistant mutants derived from phage-resistant mutants (lipopolysaccharide mutants) were also characterized. Resistance to the bactericidal activity of complement was mediated by the lipopolysaccharide, especially by the O-antigen polysaccharide chains. The capsular polysaccharide seemed not to play any important role in resistance to serum bactericidal activity in this bacterium.
Binding of Escherichia coli S fimbriae to human kidney epithelium
Authors: Korhonen T K , Parkkinen J , Hacker J , Finne J , Pere A , Rhen M , Holthöfer H
Published: January 2020

Purified S fimbriae and an Escherichia coli strain carrying the recombinant plasmid pANN801-4 that encodes S fimbriae were tested for adhesion to frozen sections of human kidney. The fimbriae and the bacteria bound to the same tissue domains, and in both cases the binding was specifically inhibited by the receptor analog of S fimbria, sialyl(alpha 2-3)lactose. S fimbriae bound specifically to the epithelial elements in the kidneys; to the epithelial cells of proximal and distal tubules as well as of the collecting ducts and to the visceral and parietal glomerular epithelium. In addition, they bound to the vascular endothelium of glomeruli and of the renal interstitium. No binding to connective tissue elements was observed. The results suggest that the biological function of S fimbriae is to mediate the adhesion of E. coli to human epithelial and vascular endothelial cells.
DNA sequence and product analysis of the virF locus responsible for congo red binding and cell invasion in Shigella flexneri 2a
Authors: Sakai T , Sasakawa C , Makino S , Yoshikawa M
Published: January 2020

The DNA sequence of virF, a locus associated with virulence and the ability to bind Congo red in Shigella flexneri 2a that is located on a 140-megadalton (230-kilobase) plasmid, was determined and analyzed. It was rich in A and T. The direction of transcription of virF was determined by using a chloramphenicol resistance cartridge. An open reading frame readable in this direction was found. Three proteins, 30, 27, and 21 kilodaltons, all corresponding to those predicted from the above sequence, were produced in minicells containing the virF locus. The three proteins were expressed only weakly in minicells with the 230-kilobase plasmid.
Vaccine potential of Haemophilus pleuropneumoniae oligosaccharide-tetanus toxoid conjugates
Authors: Fenwick B W , Osburn B I
Published: January 2020

Oligosaccharides of smooth-type lipopolysaccharide (LPS) and oligosaccharides of rough-type LPS were isolated from Haemophilus pleuropneumoniae and conjugated to tetanus toxoid by reductive amination. The antigenic and immunogenic characteristics of the oligosaccharides, the oligosaccharide-tetanus toxoid conjugates, and the LPS of H. pleuropneumoniae were determined by passive hemagglutination, enzyme-linked immunosorbent assay, and inhibition enzyme-linked immunosorbent assay with antisera produced by immunization of rabbits and pigs. The findings were compared with the immunologic response induced by immunization of pigs with an H. pleuropneumoniae whole-cell vaccine and by infection of pigs with viable H. pleuropneumoniae. The results show that conjugation of isolated oligosaccharides of H. pleuropneumoniae to tetanus toxoid improves the immunogenicity of the oligosaccharides without significantly altering their antigenic character. These findings extend the understanding of the immunobiology of H. pleuropneumoniae infection in pigs and suggest the potential of purified oligosaccharides as vaccines to prevent porcine pleuropneumonia.
Chromosomal map location of the alpha-hemolysin structural gene in Staphylococcus aureus NCTC 8325
Authors: Pattee P A
Published: January 2020

The alpha-hemolysin structural gene, hly+, previously cloned, insertionally inactivated, and introduced into the chromosome by allele replacement (M.O. O'Reilly, J.C.S. De Azavedo, S. Kennedy, and T.J. Foster, Microb. Pathog. 1:125-138, 1986), was shown by protoplast fusion and transformation to be in the gene order purC-hly-uraB-omega[chr::Tn916]1101-thrB on the chromosome of Staphylococcus aureus NCTC 8325. This location is clearly distinct from that of the agr determinant, a regulatory gene affecting several extracellular proteins, including alpha-hemolysin, located between tmn and ilv.
Immunobiological activities of synthetic lipid A analogs with low endotoxicity
Authors: Kotani S , Takada H , Takahashi I , Ogawa T , Tsujimoto M , Shimauchi H , Ikeda T , Okamura H , Tamura T , Harada K
Published: January 2020

Synthetic lipid A analogs, beta(1-6)glucosamine disaccharide 1,4'-bisphosphates, which possesses four tetradecanoyl groups at the 2- and 2'-amino, and 3- and 3'-hydroxyl groups (LA-17-PP), and each two of the (R)-3-hydroxytetradecanoyl and tetradecanoyl groups at the 2- and 2'-amino and 3- and 3'-hydroxyl groups, respectively (LA-18-PP), were far less endotoxic than synthetic (506, LA-15-PP) and bacterial Escherichia coli type lipid A's; neither compound showed any detectable lethal toxicity in chicken embryos or preparatory activity for the local Shwartzman reaction in rabbits. Also both compounds were only weakly pyrogenic and comparably less lethally toxic in galactosamine-loaded mice than the reference synthetic and bacterial lipid A's and a synthetic counterpart to biosynthetic lipid A precursor Ia (406, LA-14-PP). Nevertheless, LA-17-PP and LA-18-PP exhibited definite in vivo immunoadjuvant activity in mice, and the ability to induce a possible tumor necrosis factor and alpha/beta interferon in Mycobacterium bovis BCG and Propionibacterium acnes-primed mice, respectively, although these activities were weaker than those of the reference lipid A's. 4'-Monophosphate analogs of the above two test compounds exhibited neither endotoxic nor beneficial activities, but they showed remarkable in vitro bioactivities comparable to those of the corresponding bisphosphate compounds; the ability to activate the human complement system and the clotting enzyme cascade of horseshoe crab amoebocyte lysate, stimulatory effects on guinea pig and murine peritoneal macrophages, and murine splenocytes.
Human immune response to iron-repressible outer membrane proteins of Neisseria meningitidis
Authors: Black J R , Dyer D W , Thompson M K , Sparling P F
Published: January 2020

Neisseria meningitidis grown under iron-limiting conditions in vitro expresses additional iron-repressible outer membrane proteins (FeRPs). To see which FeRPs were expressed and immunogenic in human infection, we examined purified membranes from four meningococcal disease isolates with Western blotting of patient sera. Convalescent serum from each patient contained immunoglobulin G (IgG) and IgM antibodies against the homologous 70-kilodalton (kDa) FeRP and IgG antibody to the homologous 94-kDa FeRPs. Three other immunoreactive FeRPs were identified in two or more strains. Neither acute-phase sera nor pooled normal human sera contained appreciable levels of these antibodies. Antigenic cross-reactivity among FeRPs was suggested by the observation that the convalescent sera of two patients contained IgG antibodies reactive with the 70- and 94-kDa FeRPs and IgM antibodies reactive with the 70-kDa FeRPs from all four strains. Additionally, rabbit antiserum against the 70-kDa FeRP from one of these disease isolates contained IgG and IgM antibodies that reacted in Western blots with the 70-kDa FeRPs in all four strains. These results demonstrate that meningococcal FeRPs are expressed and immunogenic in vivo and that certain of these proteins are immunologically cross-reactive.
Mycobacterium leprae-specific Lyt-2+ T lymphocytes with cytolytic activity
Authors: Chiplunkar S , De Libero G , Kaufmann S H
Published: January 2020

Mice were immunized intradermally with 10(7) irradiated Mycobacterium leprae organisms, and draining lymph nodes were collected after 4 weeks. Lymph node cells were restimulated in vitro with soluble M. leprae antigen and accessory cells. The resulting T-cell line was propagated in vitro in the presence of M. leprae antigen, accessory cells, and interleukin-2-containing supernatants from concanavalin A-stimulated rat spleen cells. Long-term cultured T cells were Thy-1+ L3T4- Lyt-2+ as revealed by analysis with the fluorescence-activated cell sorter. From this line, T-cell clones with the same phenotype were established. The T-cell clone A4 failed to secret interleukin-2 after stimulation with antigen and accessory cells, and its growth depended on exogeneous interleukin-2. A4 T cells produced gamma interferon in an antigen-specific, H-2-restricted, and interleukin-2-dependent way. Importantly, this T-cell clone was capable of lysing bone marrow macrophages presenting M. leprae antigen. Other T-cell clones as well as native Lyt-2+ T cells from M. leprae-immunized mice were also capable of lysing bone marrow macrophages expressing M. leprae antigens. These findings suggest that specific Lyt-2+ T cells participate in the immune response to M. leprae. It is postulated that cytolysis of M. leprae-infected macrophages or Schwann cells contributes to protection against and pathogenesis of leprosy.
The use of monoclonal antibodies to detect Bacteroides gingivalis in biological samples
Authors: Chen P , Bochacki V , Reynolds H S , Beanan J , Tatakis D N , Zambon J J , Genco R J
Published: January 2020

Hybridomas were established which produce monoclonal antibodies specific for Bacteroides gingivalis, a pathogen associated with human periodontal disease. Spleen cells from BALB/c mice immunized with formalinized B. gingivalis were fused with Sp2/0-Ag14 myeloma cells. Of 1,050 wells with positive growth, 60 contained antibody reactive with the immunizing strain of B. gingivalis by enzyme-linked immunosorbent assay. Expansion of these cultures and cloning by limited dilution resulted in 28 clones which reacted with B. gingivalis but not with other orals and nonoral black-pigmented Bacteroides species or any of 29 representative strains of other oral bacteria. Of these 28 clones, 14 were also specific for B. gingivalis by indirect immunofluorescence microscopy. One clone, BBG-12 producing immunoglobulin G2b(kappa), was chosen to identify B. gingivalis in subgingival plaque because of its high reactivity in indirect immunofluorescence assays. This antibody reacted strongly with all 17 representative B. gingivalis strains obtained from diverse sources. Furthermore, when this reagent was applied to subgingival plaque samples, B. gingivalis was stained with high specificity and low background fluorescence, indicating that it may be useful for clinical identification of this organism.
Phagocytosis of Staphylococcus aureus by cultured bovine aortic endothelial cells: model for postadherence events in endovascular infections
Authors: Hamill R J , Vann J M , Proctor R A
Published: January 2020

We examined the interaction of Staphylococcus aureus with cultured bovine aortic endothelial cells as a model for the initial events in the pathogenesis of endovascular infections. Confluent monolayers of cultured endothelial cells were incubated with S. aureus. Cell-associated bacteria were measured by washing away nonadherent organisms, disrupting the monolayers, and performing quantitative cultures. Phagocytosis was differentiated from adherence by treating the cells with lysostaphin; approximately 60% of cell-associated bacteria was found to be intracellular. Phagocytosis could be blocked by using cytochalasin B, which interferes with microfilament function. Addition of fibronectin resulted in a 63% increase in adherence of S. aureus to the endothelial cells but did not increase ingestion. Transmission electron microscopy demonstrated a sequence of events similar to that which occurs during ingestion by professional phagocytes, including: adherence of bacteria to the endothelial cell; formation and elongation of surface extensions of the endothelial cell to surround the adherent bacteria; and complete enclosure within apparent phagosomes. Phagocytosis of bacteria by endothelial cells, followed by intracellular persistence, may be an important postadherence event in the pathogenesis and pathophysiology of endovascular infections.
Active immunization of hamsters against experimental infection with Borrelia burgdorferi
Authors: Johnson R C , Kodner C , Russell M
Published: January 2020

The immunogenicity of a single dose of a whole-cell vaccine of inactivated Borrelia burgdorferi HSF (human spinal fluid isolate) was evaluated. Syrian hamsters were vaccinated subcutaneously and challenged by the intraperitoneal injection of 1,000 50% infectious doses of B. burgdorferi HSF 30 or 90 days postvaccination. Animals were sacrificed 14 days after challenge, and the kidneys and spleens were examined for spirochetes by cultural procedures. At 30 days postvaccination, 86 to 100% protection against infection was achieved in hamsters receiving 50 and 100 micrograms (dry weight) of vaccine. Protection was decreased to 60% with 25 micrograms of vaccine and was absent with 10 micrograms of vaccine. Resistance to infection decreased to 25, 40, and 5% for the 100-, 50-, and 25-micrograms vaccine doses, respectively, at 90 days postvaccination.
Biosynthesis and disulfide cross-linking of outer membrane components during the growth cycle of Chlamydia trachomatis
Authors: Newhall W J
Published: January 2020

The synthesis and accumulation of Chlamydia trachomatis outer membrane proteins within infected HeLa 229 host cells were monitored by assessing the uptake of [35S]cysteine into chlamydial proteins during the 48-h growth cycle of a lymphogranuloma venereum strain, L2/434/Bu. Synthesis of the major outer membrane protein, a protein that accounts for about 60% of the outer membrane protein mass of elementary bodies (EB), was first detected between 12 and 18 h after infection. The uptake of [35S]cysteine into the 60,000-molecular-weight doublet (60K doublet) and 12.5K cysteine-rich proteins was not observed until 30 h after infection, when the intracellularly dividing reticulate bodies were beginning to transform into infectious EBs. By using a more sensitive immunoblotting method in conjunction with monoclonal antibodies specific for the 60K doublet proteins, synthesis of these proteins was detected even earlier, by 18 h after infection. These data suggest that the time and extent of synthesis of these outer membrane proteins are regulated by processes that coincide in time with the transformation of reticulate bodies into EBs. Additional studies were performed to determine the extent of disulfide cross-linking of outer membrane proteins during the growth cycle. Both the major outer membrane protein and the 12.5K protein became progressively cross-linked to about 60% during the last 24 h of the growth cycle, whereas the 60K doublet proteins were extensively cross-linked during most of the cycle. These data may indicate an intracellular cross-linking mechanism, possibly enzymatic, that exists in addition to an auto-oxidation mechanism that occurs upon host cell lysis and exposure to the extracellular environment.
Virulence factors of Bordetella bronchiseptica associated with the production of infectious atrophic rhinitis and pneumonia in experimentally infected neonatal swine
Authors: Roop R M , Veit H P , Sinsky R J , Veit S P , Hewlett E L , Kornegay E T
Published: January 2020

Four strains of Bordetella bronchiseptica (CSU-P-1, 64-C-0406, 1120-A-83-013, and B205BT) with defined virulence for neonatal swine were examined, and an attempt was made to correlate the presence of certain in vitro phenotypic characteristics with the ability of a particular B. bronchiseptica strain to produce turbinate and lung lesions in piglets. All of the strains except CSU-P-1 colonized the nasal passages of the pigs heavily, and strains 1120-A-83-013 and B205BT produced moderate to severe nasal and lung lesions in experimentally infected piglets. All of the strains attached equally well to porcine tracheal ring explant cultures, and all of the strains except CSU-P-1 produced smooth, hemolytic colonies on Bordet-Gengou medium, agglutinated porcine erythrocytes, and possessed adenylate cyclase activity. Strains 1120-A-83-013 and B205BT produced considerably higher levels of dermonecrotic toxin activity than did strains CSU-P-1 and 64-C-0406. These results indicate that production of nasal and lung lesions in neonatal piglets by the B. bronchiseptica strains tested can be directly correlated with their level of dermonecrotic toxin production.
Isolation of Coccidioides immitis F antigen by immunoaffinity chromatography with monospecific antiserum
Authors: Cox R A , Britt L A , Michael R A
Published: January 2020

Detection of antibody to Coccidioides immitis F antigen is of proved value in the diagnosis of coccidioidomycosis. This antibody is demonstrable by use of an immunodiffusion assay with reference coccidioidin antigen and antiserum to C. immitis. Using a combination of lectin affinity and immunoaffinity chromatography, we isolated the F antigen from coccidioidin and prepared monospecific antibody to the purified antigen. The availability of these reagents will enable the development of a sensitive and specific assay for detecting serologic reactivity to this antigen.
Protection of gnotobiotic rats against dental caries by passive immunization with bovine milk antibodies to Streptococcus mutans
Authors: Michalek S M , Gregory R L , Harmon C C , Katz J , Richardson G J , Hilton T , Filler S J , McGhee J R
Published: January 2020

A multivalent vaccine consisting of whole cell antigens of seven strains, representing four serotypes (b, c, d and g), of mutans streptococci was used to hyperimmunize a group of cows. Serum samples from these animals contained immunoglobulin G1 (IgG1) antibody activity to seven serotypes (a to g) of mutans streptococci. Whey obtained from the animal with the highest serum antibody activity, which also contained high levels of IgG1 antibody, was used in passive caries immunity studies. Gnotobiotic rats monoinfected with Streptococcus mutans MT8148 serotype c or Streptococcus sobrinus OMZ176 (d) or 6715 (g) and provided a caries-promoting diet containing immune whey had lower plaque scores, numbers of streptococci in plaque, and degree of caries activity than similarly infected animals given a diet containing control whey obtained from nonimmunized cows. To establish the nature of the protective component(s) present in the immune whey, an ultrafiltrate fraction of the whey was prepared. This preparation contained higher levels of IgG1 anti-S. mutans antibody activity than the immune whey. Rats monoinfected with S. mutans MT8148 and provided with a diet supplemented with 0.1% of this fraction exhibited a degree of caries protection similar to that seen in animals provided a diet containing 100% immune whey. In fact, a diet containing as little as 0.01% of the ultrafiltrate fraction gave some degree of protection against oral S. mutans infection. The active component in the immune whey was the IgG1 anti-S. mutans antibody, since rats monoinfected with S. mutans MT8148 and provided a diet supplemented with purified immune whey IgG1 had significantly reduced plaque scores, numbers of S. mutans in plaque, and caries activity compared with control animals. Prior adsorption of the IgG fraction with killed S. mutans MT8148 whole cells removed antibody activity and abrogated caries protection.
Genes for immunodominant protein antigens are highly homologous in Mycobacterium tuberculosis, Mycobacterium africanum, and the vaccine strain Mycobacterium bovis BCG
Authors: Lu M C , Lien M H , Becker R E , Heine H C , Buggs A M , Lipovsek D , Gupta R , Robbins P W , Grosskinsky C M , Hubbard S C
Published: January 2020

The relatedness of immunodominant protein antigens in Mycobacterium tuberculosis, M. africanum, and M. bovis BCG was investigated by comparing the genes that encode major protein antigens in M. tuberculosis with their counterparts in the other two mycobacteria. Genes encoding homologs of M. tuberculosis major protein antigens were isolated from M. africanum and M. bovis BCG by constructing lambda gt11 recombinant DNA expression libraries and screening them with murine monoclonal antibodies and DNA probes. The antibodies were directed against four major protein antigens of M. tuberculosis with molecular masses of 71, 65, 19, and 14 kilodaltons. The isolated M. africanum and M. bovis BCG DNA clones were mapped with restriction endonucleases, and the maps of the mycobacterial genes were confirmed by Southern analysis of mycobacterial genomic DNA. The restriction maps of DNA containing the four genes in M. tuberculosis, M. africanum, and M. bovis BCG are identical, indicating that the immunodominant proteins that they encode are highly homologous in the three mycobacteria. Thus, the immunity against tuberculosis engendered by M. bovis BCG vaccination could be provided, at least in part, by the immune response to these homologous antigens.
Protective secretory immunoglobulin A antibodies in humans following oral immunization with Streptococcus mutans
Authors: Gregory R L , Filler S J
Published: January 2020

Ingestion of a vaccine containing killed Streptococcus mutans, originally isolated from each volunteer, daily for 10 consecutive days induced increased levels of specific secretory immunoglobulin A (sIgA) antibodies to S. mutans cells and two cell surface proteins, glucosyltransferase and surface antigen I/II, in parotid saliva and tears of four healthy males and in parotid saliva, tears, colostrum, and milk of a pregnant woman. In addition, these antibodies inhibited glucosyltransferase activity. Both IgA1 and IgA2 antibodies were induced. The levels of IgA antibodies in all secretions remained significantly above preimmunization levels for more than 50 days after oral administration of antigen. A second series of immunizations for 7 consecutive days resulted in even higher levels of sIgA antibodies, which peaked earlier and persisted longer than those observed after the primary immunizations. No increase in levels of antibodies in serum were detected in any subject. Antibodies reactive with human heart and kidney antigens could not be detected in saliva, tears, colostrum, milk, or serum samples collected at any time during the immunization regimen. The numbers of viable S. mutans organisms in dental plaque and whole saliva decreased after each series of immunizations, which correlated with increased levels of IgA antibodies in saliva, suggesting that IgA antibodies in saliva were responsible for the reduced adherence of this bacterium. These results indicate that ingested S. mutans antigen induces secretion of specific IgA1 and IgA2 antibodies in saliva, tears, colostrum, and milk, providing further evidence for the existence of a common mucosal immune system.
Expression of M type 12 protein by a group A streptococcus exhibits phaselike variation: evidence for coregulation of colony opacity determinants and M protein
Authors: Simpson W J , Cleary P P
Published: January 2020

Three major categories of colony opacity were observed for natural variants of the M type 12 (M12) group A streptococcus strain CS24. Colony opacity variants that switched between two alternative categories at significantly high frequencies were identified and are referred to as switching between more opaque (Op+) and less opaque (Op-) phenotypes. Twenty lineages of such variants were derived for analysis and were assessed for resistance to phagocytosis, acid-extractable M12 antigen, and M12 mRNA, criteria which define the M protein-positive phenotype (M+). Transition from the M+ to the M protein-negative phenotype (M-) correlated with a change from Op+ to Op-. Reversion to the Op+ phenotype was accompanied by reversion to the M+ state in all variants except one and occurred at a higher frequency than the forward M+ to M- switch. These data demonstrate the existence of M12 protein phaselike switching in the group A streptococcus strain CS24. The discovery of an Op+ M- revertant confirmed that colony opacity and M protein can be expressed independently and are distinct gene products. We suggest that coregulation of colony opacity and M protein expression accounts for their association among descendents of strain CS24. Southern blot hybridization analyses of digested genomic DNA from 27 M- variants and 15 M+ revertants were performed with DNA probes containing M12 protein and adjacent upstream sequences. DNA deletions were identified only in two stable M- variants, approximately 1.3 and 1.4 kilobases upstream from the M12 gene, respectively, whereas all unstable M- variants lacked detectable rearrangements. This suggests that deletions within or adjacent to the structural gene are unlikely to be responsible for the reversible switch in M protein expression. However, the association with the stable M- phenotype and the location of these deletions, as well as two other deletions, approximately 0.5 kilobase upstream from the M12 promoter in two previously described variants of strain CS24 suggests that a second gene product is required for full expression of M12 protein synthesis in this strain.
Establishment and characterization of a T-cell line specific for Rickettsia tsutsugamushi
Authors: Kodama K , Kawamura S , Yasukawa M , Kobayashi Y
Published: January 2020

To study the immunological protective system against rickettsial infection, a T-cell line specific for Rickettsia tsutsugamushi antigen was established by long-term culture of splenocytes from mice immunized with live Gilliam strain R. tsutsugamushi and then propagated in the presence of homologous rickettsial antigen and syngeneic filler cells. The characteristics of the T-cell line and its capacity to induce antirickettsial protection in vivo were studied. Flow cytometric analysis demonstrated that the T-cell line showed the phenotype Thy-1.2+ L3T4+ Lyt-2-, suggestive of helper T cells. In a lymphocyte proliferation assay, this cell line showed a specific response to Gilliam antigen, partial cross-reactivity to Karp antigen, but no response to Kato antigen. The proliferative response of this T-cell line was filler cell dependent, and genetic restriction was observed between the T-cell line and filler cells. The T-cell line produced gamma interferon, one of the macrophage-activating factors, in cultures with specific antigen and was able to adoptively mediate antirickettsial protection in vivo. The data presented here suggest that antigen-specific helper T cells play an important role in protection against rickettsial infection.
The immunogenicity and antigenicity of lipid A are influenced by its physicochemical state and environment
Authors: Brade L , Brandenburg K , Kuhn H M , Kusumoto S , Macher I , Rietschel E T , Brade H
Published: January 2020

We investigated the immunogenicity and antigenicity of synthetic lipid A and partial structures thereof. Included in the study were compounds which varied in the position of phosphate (1-mono-, 4'-mono-, and 1,4'-bisphosphates) and in the acylation (type, number, and distribution of fatty acids) and, in the case of monosaccharide compounds, the nature of the backbone sugar (D-glucosamine, D-glucose, 3-amino-3-deoxy-D-glucose, and 2,3-diamino-2,3-dideoxy-D-glucose). With the aid of the passive-hemolysis and passive-hemolysis-inhibition assays and by absorption experiments, five distinct antibody specificities were detected in polyclonal rabbit antisera raised against sheep erythrocyte-coated lipid A and lipid A incorporated into the membrane of liposomes (liposome-incorporated immunogens). Three antibody specificities reacted with disaccharide antigens specific for a 1-mono-, 4'-mono-, and 1,4'-bisphosphorylated beta-1,6-linked D-glucosamine disaccharide. Two antibodies reacted with either 1- or 4-phosphates of acylated D-gluco-configured monosaccharides and exhibited no cross-reaction with each other. However, they cross-reacted with disaccharide antigens with phosphate groups in the appropriate positions. We found that the physicochemical state and the environment of lipid A modulated its immunoreactivity. The immunogenicity was best expressed by erythrocyte-coated and liposome-incorporated immunogens. The antigenicity of lipid A was also greatly influenced by its physical surroundings. The reaction pattern of the above antibodies was highly specific in the hemolysis assay and in absorption experiments (the antibody reacted with antigen embedded in a cell membrane), whereas some cross-reactivities were observed in inhibition studies (the antibody reacts with antigen in aqueous solution). By using liposome-incorporated antigens as inhibitors, nonspecific reactions were avoided and specific ones were enhanced. Thus the antibodies described above against lipid A recognize epitopes in the hydrophilic backbone, the exposure of which depends on the intrinsic physicochemical properties of lipid A on the one hand and the physical environment on the other.
Entry of Shigella flexneri into HeLa cells: evidence for directed phagocytosis involving actin polymerization and myosin accumulation
Authors: Clerc P , Sansonetti P J
Published: January 2020

The enteroinvasive bacterium Shigella flexneri expresses a plasmid-mediated capacity to penetrate into nonphagocytic cells. By using 7-nitrobenz-2-oxa-1,3-diazole-phallacidin (NBD-phallacidin), a fluorescent dye which specifically stains microfilaments, we observed condensations of filamentous actin underneath the plasma membrane of HeLa cells which interacted with the invasive isolate M90T. As demonstrated by indirect immunofluorescence with the antimyosin monoclonal antibody CC-212, myosin accumulated at the same sites. The entry process could be synchronized by using strain SC301, a pIL22 transformant of M90T. pIL22, a recombinant plasmid encoding the Escherichia coli afimbrial adhesin AFA I, rendered shigellae highly adherent to HeLa cells. Using such a system, we demonstrated that the occurrence of bacterial penetration and the appearance of structures brightly stained by NBD-phallacidin were simultaneous events. Such microfilamentous structures resulted from de novo polymerization of the monomeric actin pool in a DNase I inhibition assay, as shown by measurement of the monomeric versus total actin content of infected HeLa cells. These data provide direct evidence that the penetration of S. flexneri into HeLa cells occurs through a mechanism similar to phagocytosis by professional phagocytes.
Expression of Campylobacter jejuni invasiveness in cell cultures coinfected with other bacteria
Authors: Bukholm G , Kapperud G
Published: January 2020

Enteroinvasive Salmonella, Shigella, and Escherichia coli strains were found to exert an effect which rendered Campylobacter jejuni capable of intracellular localization in epithelial cells in vitro. When monolayers of HEp-2 or A-549 cells were challenged with pure cultures of C. jejuni or Campylobacter coli, none of the eight strains tested invaded the cells. In contrast, four of these strains were able to localize intracellularly when the cells were challenged with a mixture of campylobacters and enteroinvasive Salmonella typhimurium, Shigella flexneri, Shigella boydii, Shigella sonnei, or E. coli strains. Invasiveness of campylobacters was also induced by one nonenteroinvasive strain of E. coli O124. Coinfection with other nonenteroinvasive E. coli strains did not induce invasiveness in C. jejuni. The degree of internalization induced by S. typhimurium was significantly higher than that induced by Shigella or E. coli strains. The invasive capacity of C. jejuni was found to differ considerably between strains. No evidence of an invasive potential was demonstrable for two C. coli strains or for two enterotoxigenic isolates of C. jejuni examined. C. jejuni was only able to localize intracellularly in cell cultures when the interaction occurred in a microaerobic atmosphere. None of the strains tested evoked keratoconjunctivitis in guinea pig eyes (Sereny test), regardless of the presence of coinfectants. The results indicate that a synergistic interaction that exists between C. jejuni and other enteropathogens facilitates invasion by C. jejuni.
Biologic activities of antibody to a peptidoglycan-associated lipoprotein of Haemophilus influenzae against multiple clinical isolates of H. influenzae type b
Authors: Green B A , Quinn-Dey T , Zlotnick G W
Published: January 2020

A peptidoglycan-associated lipoprotein of about 15 kilodaltons was purified from the outer membranes of Haemophilus influenzae by using nondenaturing detergents. To assess its vaccine potential, rabbit antiserum to the purified protein was obtained. The antiserum was specific for the peptidoglycan-associated lipoprotein in whole cell lysates of H. influenzae and was bactericidal for H. influenzae types a, b, d, e, and f and for 181 of 182 H. influenzae type b clinical strains isolated in widely dispersed geographic areas. The antibody protected infant rats from challenge with each of five clinical H. influenzae type b isolates and was additive to and did not interfere with bactericidal and protective activities of antibody against the type b capsule. These data indicate that the purified peptidoglycan-associated lipoprotein is a potentially valuable vaccine candidate for H. influenzae type b disease and may enhance the effectiveness of preexisting anticapsular antibody.
Plasmid-associated virulence of Salmonella typhimurium
Authors: Gulig P A , Curtiss R
Published: January 2020

We investigated the role of the 100-kilobase (kb) plasmid of Salmonella typhimurium in the virulence of this organism for mice. Three strains, LT2-Z, SR-11, and SL1344, which possessed 100-kb plasmids with identical restriction enzyme digestion profiles, were cured of their respective 100-kb plasmids after Tnmini-tet was used to label plasmids. Curing wild-type virulent strains SR-11 and SL1344 raised peroral 50% lethal doses from 3 x 10(5) and 6 x 10(4) CFU, respectively, to greater than 10(8) CFU. Both wild-type strains had intraperitoneal 50% lethal doses of less than 50 CFU, whereas the intraperitoneal 50% lethal doses for cured SR-11 and SL1344 were less than 50 and 400 CFU, respectively. Reintroduction of the Tnmini-tet-labeled, 100-kb plasmid restored wild-type virulence. Invasion from Peyer's patches to mesenteric lymph nodes and spleens after peroral inoculation was the stage of pathogenesis most affected by curing S. typhimurium of the 100-kb plasmid. Wild-type S. typhimurium replicated in spleens of mice inoculated intravenously to a greater extent than did plasmid-cured derivatives. Wild-type and cured strains equally adhered to and invaded Henle-407, HEp-2, and CHO cells; furthermore, the presence of the 100-kb plasmid was not necessary for replication of S. typhimurium within CHO cells. The 100-kb plasmid had no effect on phagocytosis and killing of S. typhimurium by murine peritoneal macrophages in vitro and in vivo. Similarly, wild-type and plasmid-cured strains were resistant to killing by 90% normal human, rabbit, and guinea pig sera. All wild-type and plasmid-cured S. typhimurium strains possessed complete lipopolysaccharide, as determined by silver staining solubilized cells in sodium dodecyl sulfate-polyacrylamide gels. We have confirmed the role of the 100-kb plasmid of S. typhimurium in virulence, primarily in invasion to mesenteric lymph nodes and spleens after peroral inoculation of mice. Involvement of the 100-kb plasmid in infection of mesenteric lymph nodes and spleens suggests a role for the plasmid in the complex interaction of S. typhimurium with cells of the reticuloendothelial system.
Human immunoglobulin G antibody response to the major gonococcal iron-regulated protein
Authors: Fohn M J , Mietzner T A , Hubbard T W , Morse S A , Hook E W
Published: January 2020

In humans, gonococcal infection occurs in environments limited with respect to free iron. Neisseria gonorrhoeae produces increased quantities of iron-regulated membrane proteins when grown under in vitro conditions which restrict the availability of free iron. Using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot (immunoblot) techniques, we studied the reactivity of specific antibodies to the 37-kilodalton (kDa) major iron-regulated protein (MIRP) of gonococci grown under iron-limiting conditions. Antibodies reactive with the 37-kDa MIRP were distinguished from those reactive with protein I by using purified 37-kDa MIRP or gonococcal protein preparations. Acute-phase sera from patients with disseminated gonococcal infection (DGI) reacted strongly to both the 37-kDa MIRP and protein I. Acute sera from nine patients with uncomplicated gonorrhea did not exhibit strong reactivity with the 37-kDa MIRP and were indistinguishable from five control sera. When compared with acute-phase sera, convalescent-phase sera from patients with DGI failed to demonstrate increased reactivity, whereas convalescent-phase sera from one of nine patients with uncomplicated gonorrhea developed reactivity to the 37-kDa MIRP. These data indicate that (i) the 37-kDa MIRP is expressed and antigenic in vivo and (ii) humans with DGI consistently develop a systemic antibody response to the 37-kDa MIRP.
Molecular cloning of the Escherichia coli O75X adhesin
Authors: Nowicki B , Barrish J P , Korhonen T , Hull R A , Hull S I
Published: January 2020

The uropathogenic strain Escherichia coli IH11128 (O75:K5:H-) exhibits a mannose-resistant O75X adhesin. The genes encoding the O75X adhesin were cloned from a clinical strain and transferred to E. coli K-12 derivatives. The recombinant plasmids were found to express a 15-kilodalton fimbrial subunit protein, a fimbrialike extracellular structure, and mannose-resistant hemagglutination. An indirect immunofluorescence assay was used to study attachment of the clone and purified adhesin to frozen sections of human kidney. The clone bound selectively to the interstitial areas and notably to Bowman's capsule. The purified adhesin bound to the basement membrane of the tubules and to Bowman's capsule.
Opsonophagocytosis of Pseudomonas aeruginosa treated with antiflagellar serum
Authors: Anderson T R , Montie T C
Published: January 2020

Hyperimmune rabbit flagellar antisera were shown to enhance opsonophagocytosis of Pseudomonas aeruginosa by mouse peritoneal polymorphonuclear leukocytes. This response was specific for flagellar immunoglobulin G as indicated by cross-opsonization experiments.
Differentiation and interaction of secretory immunoglobulin A and a calcium-dependent parotid agglutinin for several bacterial strains
Authors: Rundegren J , Arnold R R
Published: January 2020

Previous studies have suggested that both secretory immunoglobulin A (sIgA) and various nonimmunoglobulin salivary glycoproteins are capable of agglutinating a variety of bacteria. The present study was designed to compare the nature of the agglutinins for Streptococcus mutans and Salmonella typhimurium in parotid saliva and colostrum. S. mutans was aggregated by saliva and colostrum, whereas S. typhimurium was aggregated only by saliva as detected by a spectrophotometric method. The principal salivary agglutinin for both S. mutans and S. typhimurium was calcium dependent and could be desorbed in phosphate-buffered saline (pH 6.8). In contrast, the colostral agglutinin was calcium independent and not readily desorbed. The agglutinin activities of saliva and colostrum for S. mutans were additive, suggesting independent target sites on the bacterial surface. The agglutinin activity of colostrum was totally associated with sIgA as was suggested by blocking of the agglutinating activity with anti-alpha-chain serum and the absence of blocking with an antibody specific for salivary agglutinin. Interestingly, anti-alpha-chain serum removed all agglutinating activity from saliva, but not from the phosphate-buffered saline-desorbed agglutinin. Dialysis of parotid saliva against 0.1 M disodium EDTA eliminated the agglutinin blocking activity of anti-alpha-chain serum but not that of the antiagglutinin antibody. The ability of anti-alpha-chain serum to block agglutination of the EDTA-dialyzed saliva could be restored by the addition of calcium chloride, suggesting that sIgA and salivary agglutinin are associated through a calcium-mediated interaction. These results indicate that bacterial agglutinating activity of colostrum, as detected spectrophotometrically, is mediated by sIgA, and that of saliva is mainly dependent upon a calcium-dependent nonimmunoglobulin agglutinin. The agglutinating activities of sIgA and parotid agglutinin seem to be additive, and their calcium-dependent association may favor the enhancement of their respective activities.
Contribution of Salmonella gallinarum large plasmid toward virulence in fowl typhoid
Authors: Barrow P A , Simpson J M , Lovell M A , Binns M M
Published: January 2020

Four strains of Salmonella gallinarum isolated from independent cases of fowl typhoid all possessed both an 85-kilobase and a 2.5-kilobase plasmid. Each plasmid was eliminated in turn from one of the strains by transposon labeling and curing at 42 degrees C. Elimination of the small plasmid had no effect on the high virulence of the strain for newly hatched and 2-week-old chickens. Whereas oral inoculation of 2-week-old chickens with the parent strain produced 90% mortality with characteristic signs of fowl typhoid, inoculation of the large-plasmid-minus strain produced 0% mortality. A corresponding increase in the 50% lethal dose from log10 1.1 to greater than log10 7.3 was seen with the large-plasmid-minus strain after intramuscular inoculation. Reintroduction of the large plasmid completely restored virulence. A role for the plasmid-linked virulence genes in both invasion and growth in the reticuloendothelial system is suggested by the failure of the large-plasmid-minus strain to penetrate to the liver and spleen after oral inoculation and by its increased clearance from the reticuloendothelial system after intravenous inoculation. These results clearly demonstrate that the large plasmid of S. gallinarum contributes toward virulence in fowl typhoid of chickens.
Susceptibilities of macrophage populations to infection in vitro by Leishmania donovani
Authors: Olivier M , Tanner C E
Published: January 2020

Many studies have demonstrated differences in the resistance of strains of mice to infection by Leishmania donovani, Salmonella typhimurium, and Mycobacterium bovis BCG; this resistance/susceptibility phenotype seems to be controlled by a single gene. The present study investigated the susceptibility of liver, lung, peritoneal, and spleen macrophages to infection by L. donovani promastigotes in vitro; the objective was to determine if the susceptibility of animals was expressed by their macrophages when infected in vitro. This study indicated that the Lsh phenotype was only expressed by liver macrophages. The liver macrophages of the susceptible C57BL/6J strain were significantly more phagocytic than those of the resistant C57L/J strain; infection affected the phagocytic activity of the macrophage population. These results indicated that only liver macrophages can express the Lsh gene. Recognition of expression is in part due to its effect on the phagocytic activity of the macrophages.
Purification of Pseudomonas aeruginosa exoenzyme S
Authors: Woods D E , Que J U
Published: January 2020

Pseudomonas aeruginosa produces two distinct ADP-ribosyl transferases, exotoxin A and exoenzyme S, which differ in a number of properties including substrate specificity. Exoenzyme S was purified from culture supernatants of P. aeruginosa DG1. The procedure for purification consists of four major steps: ammonium sulfate precipitation, anion-exchange chromatography on DEAE-Sephacel, acetone precipitation in the presence of 1 M NaCl, and G-100 Superfine gel filtration chromatography. Exoenzyme S was monitored during purification by an assay for ADP-ribosyl transferase activity, mouse toxicity, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified material exhibited ADP-ribosyl transferase activity, reacted with monoclonal antibodies to exoenzyme S, and was toxic to mice and a variety of tissue culture cell lines.
Pseudomonas aeruginosa adhesins for tracheobronchial mucin
Authors: Ramphal R , Guay C , Pier G B
Published: January 2020

Mucins of the tracheobronchial tree are preferential sites for adherence and colonization by Pseudomonas aeruginosa. They possess specific receptors for this organism that have amino sugars as their principal constituents. Since mucins probably reflect the receptors on the cellular surfaces, we hypothesized that the bacterial adhesins previously shown to mediate the binding of P. aeruginosa to cells would also mediate bacterial binding to mucins. We therefore tested the roles of the exopolysaccharide from mucoid strains of P. aeruginosa and pili from nonmucoid strains to see whether they are indeed the adhesins for mucins. Using a microtiter plate assay of adherence to mucins, we demonstrated that the mucoid exopolysaccharide bound to mucins and enhanced the adherence of mucoid strains to this substance. Antibodies raised against the exopolysaccharide from a single mucoid strain inhibited the adherence of all mucoid strains tested. Purified pili from nonmucoid strains inhibited the binding of nonmucoid strains but not of mucoid strains. Inhibition of adherence by antibody to pili was quite specific, antibody being able to inhibit only the binding of the homologous nonmucoid strain. These data support our previous observations with tracheal cells, confirming the similarity of the adhesins for respiratory tract cells and the mucins which cover them.