bluuebunny/crossref_metadata_2025 huggingface dataset:
Production of tumor necrosis factors alpha and beta by human mononuclear leukocytes stimulated with mitogens, bacteria, and malarial parasites
Authors: Ferrante A , Staugas R E , Rowan-Kelly B , Bresatz S , Kumaratilake L M , Rzepczyk C M , Adolf G R
Published: January 2020

Tumor necrosis factors alpha and beta (TNF-alpha and TNF-beta) are multifaceted polypeptide cytokines which may mediate some of the significant changes in cellular homeostasis which accompany the invasion of the mammalian host by viruses, bacteria, and parasites. Although it is well established that bacterial lipopolysaccharide is a potent inducer of TNF-alpha, there is still very little known of the types of agents which can trigger the production of TNFs in mononuclear leukocytes. Using an enzyme-linked immunosorbent assay for measuring TNF-alpha and TNF-beta, we examined the capacity of various T-lymphocyte and beta-lymphocyte mitogens as well as microbial components to stimulate production of these cytokines in culture. The mitogens phytohemagglutinin, concanavalin A, and pokeweed mitogen induced production of both TNF-alpha and TNF-beta, while whole-killed Staphylococcus aureus and Bordetella pertussis, like lipopolysaccharide, were potent inducers of TNF-alpha but failed to stimulate TNF-beta production. TNF-alpha production was detectable within 1 h after stimulation, while TNF-beta production was not detected until after 8 h of culture. The bacterial products tetanus toxoid, purified protein derivative, pertussis filamentous hemagglutinin, and pertussis toxin were all able to induce TNF-alpha and TNF-beta production. Disrupted (frozen-thawed) Plasmodium falciparum-infected erythrocytes were also potent inducers of TNF-alpha and TNF-beta. The results demonstrated that a wide variety of microbial components are inducers of TNF-alpha. Some may not only be more effective than lipopolysaccharide but can also induce TNF-beta production. Furthermore, evidence is presented showing that TNF-beta but not TNF-alpha production correlates with lymphoproliferation.
Mechanism of pertussis toxin B oligomer-mediated protection against Bordetella pertussis respiratory infection
Authors: Shahin R D , Witvliet M H , Manclark C R
Published: January 2020

Immunization with the B oligomer of pertussis toxin protected neonatal mice from a lethal respiratory challenge with Bordetella pertussis. All mice immunized with 8 micrograms of B oligomer survived aerosol challenge and had peripheral leukocyte counts and weight gains similar to those of mice immunized with pertussis toxoid before challenge and to those of control mice that were not challenged. Unprotected mice challenged with an aerosol of B. pertussis had an increase in peripheral leukocyte count, failed to gain weight, and died within 21 days of challenge. Protection appeared to be dose dependent, since a dose of 1 microgram of B oligomer per mouse prevented death in 100% of the mice challenged with B. pertussis, whereas 0.4 micrograms of B oligomer protected 50% of the challenged mice. Mice immunized with the B oligomer had increases in immunoglobulin G (IgG) anti-B oligomer in sera and in IgG and IgA anti-B oligomer in bronchoalveolar lavage fluids 1 to 3 weeks after respiratory challenge. Specific anti-B oligomer antibodies could not be detected in unimmunized, infected mice at the same time after challenge. Intravenous administration of the monoclonal antibody 170C4, which binds to the S3 subunit of the B oligomer, protected neonatal mice from B. pertussis respiratory challenge, while administration of an IgG1 anti-tetanus toxin monoclonal antibody, 18.1.7, was not protective. We conclude that anti-B-oligomer-mediated neutralization of pertussis toxin is one mechanism of protection in the mouse model of B. pertussis aerosol challenge.
Expression of Mycobacterium tuberculosis and Mycobacterium leprae proteins by vaccinia virus
Authors: Lyons J , Sinos C , Destree A , Caiazzo T , Havican K , McKenzie S , Panicali D , Mahr A
Published: January 2020

Eight Mycobacterium tuberculosis and M. leprae genes were inserted into the vaccinia virus genome by in vivo recombination. The resulting virus recombinants were shown to express five different M. tuberculosis proteins (71, 65, 35, 19, and 12 kDa) and three M. leprae proteins (65 and 18 kDa and a biotin-binding protein) by Western immunoblot analysis, radioimmunoprecipitation, or black-plaque assay. When injected into BALB/c mice, the recombinants expressing the M. tuberculosis 71-, 65-, or 35-kDa protein and the M. leprae 65-kDa protein or the biotin-binding protein elicited antibodies against the appropriate M. tuberculosis or M. leprae protein. These vaccinia virus recombinants are being tested for the ability to elicit immune protection against M. tuberculosis or M. leprae challenge in animal model systems. The recombinants are also useful in generating target cells for assays aimed at elucidating the cellular immune responses to mycobacterial proteins in leprosy and tuberculosis. Furthermore, the M. tuberculosis 65-kDa protein and four of the other mycobacterial proteins share homology with known eucaryotic and procaryotic stress proteins, some of which may play a role in autoimmunity.
Characterization of the gene for a 30-kilodalton adhesion-related protein of Mycoplasma pneumoniae
Authors: Dallo S F , Chavoya A , Baseman J B
Published: January 2020

A previously identified trypsin-resistant surface protein of Mycoplasma pneumoniae clusters at the tip organelle of virulent mycoplasmas and appears to be essential for cytadherence and virulence. Monoclonal antibodies generated against this protein were used to identify positive recombinant clones from M. pneumoniae genomic DNA libraries. The structural gene was sequenced and contained an open reading frame of 825 nucleotides that encoded a protein of 275 amino acids with a calculated molecular mass of 29,743 Da. This protein (P30) contained three types of repeat sequences at the carboxy end, each consisting of six amino acids. In addition, the protein was proline rich (20.7%) and exhibited significant amino acid homology with the P1 cytadhesin of M. pneumoniae and with several matrix-associated eucaryotic proteins.
Immunization against anthrax with aromatic compound-dependent (Aro-) mutants of Bacillus anthracis and with recombinant strains of Bacillus subtilis that produce anthrax protective antigen
Authors: Ivins B E , Welkos S L , Knudson G B , Little S F
Published: January 2020

The safety and efficacy of five prototype, live anthrax vaccines were studied in Hartley guinea pigs and CBA/J and A/J mice. Two of the strains, Bacillus anthracis FD111 and FD112, are Aro- mutants derived by Tn916 mutagenesis of B. anthracis UM23-1. Bacillus subtilis PA1 and PA2 contain a recombinant plasmid, pPA101 or pPA102, respectively, that carries the gene from B. anthracis encoding synthesis of protective antigen (PA). The final strain, B. subtilis PA7, was isolated in this study from B. subtilis DB104 transformed with pPA101. All five strains were less virulent in guinea pigs and A/J and CBA/J mice than the toxinogenic, nonencapsulated B. anthracis veterinary vaccine Sterne strain. A/J and CBA/J inbred mice represent strains that are innately susceptible and resistant, respectively, to the Sterne strain. These differences in susceptibility are due to differences in ability to produce complement component 5. In guinea pigs, immunization with PA1 or PA2 vegetative cells or PA7 spores protected greater than or equal to 95% from an intramuscular spore challenge with the virulent, "vaccine-resistant" B. anthracis Ames strain. Strain PA2 vegetative cells and strain PA7 spores were as effective as the Sterne strain in Sterne-resistant CBA/J mice, protecting 70% of the mice from Ames strain spore challenge. Immunization with FD111 or FD112 vegetative cells fully protected guinea pigs from challenge. Immunization with FD111 cells protected up to 100% of CBA/J mice and up to 70% of A/J mice.
Comparative production and rapid purification of Candida acid proteinase from protein-supplemented cultures
Authors: Ray T L , Payne C D
Published: January 2020

Six Candida spp. that were previously characterized for cutaneous pathogenicity were assessed for Candida acid proteinase (CAP) production in albumin-supplemented, nitrogen-restricted media. C. albicans CAP production was compared in media supplemented with albumin, casein, collagen, hemoglobin, or keratin and in TC medium 199. C. albicans, C. stellatoidea, and C. tropicalis, which are cutaneous pathogens in murine infections, produced 3.3 to 4.7 times more CAP than did the nonpathogens C. parapsilosis and C. guilliermondii. C. krusei, a nonpathogen, produced negligible amounts of enzyme. C. albicans CAP production was similar in each protein-supplemented medium, and only a single acid proteinase was recovered from each one. Rapid CAP purification from culture supernatants was achieved by hollow fiber and stirred cell ultrafiltration followed by Affi-Gel blue and Sephacryl column chromatographies. The highest yield, purity, and specific activity of CAP were obtained from keratin-supplemented medium supernatants, producing 2.86 mg of purified CAP from a 7-liter culture. CAP was characterized as a 41,500-dalton glycoprotein, with a pI of 4.5; a pH range of 2.5 to 5.5; and broad substrate specificity, including that for keratin, denatured collagen, hemoglobin, casein, and albumin. Isolation of CAP also isolated the keratinolytic proteinase of Candida spp. CAP was inhibited by pepstatin A, but not by EDTA or phenylmethylsulfonyl fluoride. Monospecific antibody to CAP was produced in mice and reacted only to the 41,500-dalton protein, as determined by immunoblot analysis. High CAP production by cutaneous pathogenic Candida spp. supports the fact that CAP is a potential virulence factor that may facilitate Candida colonization and invasion of skin. CAP production from keratin-supplemented medium was superior to that from the other media that were tested and yielded sufficient and suitable enzyme for use in immunoassays of CAP antigen and antibody.
Chronic arthritis induced in rats by cell wall fragments of Eubacterium species from the human intestinal flora
Authors: Severijnen A J , van Kleef R , Hazenberg M P , van de Merwe J P
Published: January 2020

To investigate arthritis-inducing properties of Eubacterium species, which are major residents of the human intestinal flora, cell wall fragments (CWF) of several Eubacterium strains were prepared and tested in an animal model. After a single intraperitoneal injection in the rat, CWF of E. aerofaciens, E. contortum, and E. lentum induced a chronic polyarthritis. E. limosum and E. tortuosum CWF induced an acute self-limiting joint inflammation, whereas E. rectale CWF failed to do so. The rhamnose contents of the isolated CWF were not related to their arthritis-inducing properties. Paradoxically, the sensitivity of CWF to lysozyme digestion, which is regarded as a parameter for the clearance of CWF in tissues, appeared to be positively correlated with the ability of Eubacterium CWF to induce chronic joint inflammation. Our findings show the diversity in arthritis-inducing properties among different species of the anaerobic genus Eubacterium and underline the importance of the anaerobic intestinal flora in the induction of joint inflammation.
Influence of different regions of the H-2 complex on the rate of clearance of Salmonella typhimurium
Authors: Nauciel C , Ronco E , Pla M
Published: January 2020

The rate of clearance of Salmonella typhimurium from the mouse spleen is under H-2 linked genetic control. The results of the present study, with H-2 recombinant mice on a C57BL/10 background, suggest the involvement of at least two loci, one in the D region and the other in the K-A alpha chromosomal segment.
Identification of a T-cell epitope on the circumsporozoite protein of Plasmodium vivax
Authors: George F W , Law J L , Rich K A , Martin W J
Published: January 2020

A series of overlapping peptides, representing sequences in the vicinity of region II on the Plasmodium vivax circumsporozoite protein, was synthesized. One of the peptides (PV-23), a 20-mer containing the 6 C-terminal amino acids of region II, was found to evoke an in vitro T-cell proliferative response in spleen cells from C3Hf (H-2km2) mice immunized with the peptide. These results demonstrate that PV-23 contains a T-cell epitope. To our knowledge, this is the first report of a T-cell epitope on the circumsporozoite protein of P. vivax.
A monoclonal antibody that defines a surface antigen on Candida albicans hyphae cross-reacts with yeast cell protoplasts
Authors: Ollert M W , Calderone R A
Published: January 2020

Female BALB/c mice were immunized with a whole-hyphal-cell extract obtained from Candida albicans wild-type strain 4918 grown in Lee medium. Monoclonal antibody (MAb)-producing hybridomas were prepared by fusing immune splenocytes with NS-1 myeloma cells. One of the hybrid cell clones (1.183) secreted an immunoglobulin G1 antibody that reacted with C. albicans hyphae in an indirect immunofluorescence assay but not with yeast cells and pseudohyphal segments directly originating from parent blastoconidia. In the same assay eight of nine recent clinical C. albicans isolates and Candida stellatoidea tested positive for hyphal cell-specific reactivity with MAb 1.183. The recognized antigen on hyphal cells was sensitive to heat treatment, beta-mercaptoethanol reduction, and proteolysis with pronase, trypsin, and subtilisin. Western blot (immunoblot) analysis of hyphal whole-cell and dithiothreitol extracts with MAb 1.183 revealed two major proteins with approximate molecular masses of 55 and 60 kilodaltons (kDa) under reducing conditions. Endo-alpha-N-acetylgalactosaminidase (O-glycanase) treatment reduced the molecular mass of the 60-kDa protein slightly but did not affect recognition by MAb 1.183, whereas peptide:N-glycosidase F (N-glycanase) had no effect on either protein. When exponentially growing yeast cells were treated sequentially with EDTA, beta-mercaptoethanol, and Zymolase to form protoplasts, a specific immunofluorescence signal was obtained with MAb 1.183. In a Western blot, MAb 1.183 showed reactivity with a 20-kDa protein in the sodium dodecyl sulfate extract from protoplasts, whereas no reactivity was found with cell wall material obtained from yeast cells. In summary, these experiments indicated that specific cell surface components from C. albicans hyphae are related to antigens which are present in yeast cells but are not detectable on the surface of the latter.
Nonpathogenic isolates of Yersinia enterocolitica do not contain functional inv-homologous sequences
Authors: Pierson D E , Falkow S
Published: January 2020

Previous studies have demonstrated a correlation between the ability of isolates of Yersinia enterocolitica to cause disease and to invade tissue culture cells in vitro. Two genes, inv and ail, isolated from a pathogenic strain of Y. enterocolitica have each been shown to confer this invasive phenotype upon Escherichia coli. Eighty pathogenic, invasive isolates studied by Miller et al. (Infect. Immun. 57:121-131, 1989) contained sequences homologous to both of these genes. Thirty-five nonpathogenic, noninvasive isolates similarly studied had no ail homology but carried inv-homologous sequences. We investigated inv-homologous sequences from four nonpathogenic isolates. Recombinant clones of these inv-homologous sequences did not confer the invasive phenotype upon E. coli. No RNA transcripts capable of encoding a full-length Inv protein were detected in the four noninvasive Yersinia strains. When the inv gene from a pathogenic isolate was introduced into two of these strains, the resulting transformants invaded tissue culture cells in vitro. The inv gene was transcribed in a pathogenic Yersinia isolate grown at 30 degrees C but not at all in these cells grown at 37 degrees C. The production of RNA transcripts homologous to inv in transformants was not regulated by temperature to the same degree as was seen for pathogenic isolates. We conclude that the inv gene in nonpathogenic strains of Y. enterocolitica is nonfunctional. Y. enterocolitica isolates epidemiologically linked to disease contain both a functional inv gene and a functional ail gene. Environmental isolates not associated with disease have a nonfunctional inv gene and no ail gene.
Construction of a urease-negative mutant of Proteus mirabilis: analysis of virulence in a mouse model of ascending urinary tract infection
Authors: Jones B D , Lockatell C V , Johnson D E , Warren J W , Mobley H L
Published: January 2020

Proteus mirabilis, a urease-producing uropathogen, causes serious urinary tract infections in humans. To specifically evaluate the contribution of urease to virulence, a mutation was introduced into P. mirabilis HI4320 by homologous recombination. Virulence was assessed in the CBA mouse model of ascending urinary tract infection. Twenty mice each were challenged transurethrally with P. mirabilis HI4320 and its urease-negative derivative (1 x 10(9) to 2 x 10(9) CFU). At 48 h animals were sacrificed and the mean log10 CFU per milliliter of urine (parent, 6.23; mutant, 4.19; P = 0.0014) or per gram of bladder (parent, 6.29; mutant, 4.28; P = 0.0002), left kidney (parent, 4.11; mutant, 1.02; P = 0.00009), and right kidney (parent, 4.11; mutant, 2.43; P = 0.036) were all shown to be significantly different. These data demonstrate a role for urease as a critical virulence determinant for uropathogenic P. mirabilis.
Adhesion of enterotoxigenic Escherichia coli to the human colon carcinoma cell line Caco-2 in culture
Authors: Darfeuille-Michaud A , Aubel D , Chauviere G , Rich C , Bourges M , Servin A , Joly B
Published: January 2020

Enterotoxigenic Escherichia coli (ETEC) strains possessing colonization factor antigen I (CFA/I), CFA/II, CFA/III, and antigen 2230 were tested for their ability to adhere to the following cell lines: HeLa, HEp-2, HRT 18, Hutu 80, MDBK, MDCK, Vero, and Caco-2. ETEC strains adhered only to the Caco-2 cell line. Irrespective of the known adhesive factors, the ETEC strains that adhered to the brush border of human enterocytes also adhered to the Caco-2 cell line. The negative variants, which were cured of the plasmid encoding the adhesive factor, did not adhere. Adhesion of ETEC strains no longer occurred when the Caco-2 cells were pretreated with the homologous colonization factor antigen or when the bacterial cells were pretreated with homologous antibodies raised against the adhesive factors. This indicates that this adhesion is specific and that a different receptor exists for each type of adhesion factor. Electron micrographs of cross sections of the monolayer showed that the adhesion of ETEC strains to the brush border microvilli does not induce any lesion. Therefore, the Caco-2 cell line behaves in the same way as human enterocytes do.
Effects of neutrophils and in vitro oxidants on survival and phenotypic switching of Candida albicans WO-1
Authors: Kolotila M P , Diamond R D
Published: January 2020

The relationship to pathogenesis of the spontaneous phenotypic switching of Candida albicans is uncertain. Since neutrophils are critical in containment of disseminated candidiasis, we used these cells and some of their potentially microbicidal oxidative products to define effects on a C. albicans strain (WO-1) that exhibits characteristic, easily recognized switching between the white and opaque phenotypes. Blastoconidia of the opaque phenotypes were more susceptible than those of the white to killing by either intact neutrophils or cell-free oxidants, including reagent hydrogen peroxide or the myeloperoxidase-H2O2-Cl- system. Paralleling these findings, opaque blastoconidia were 2.8- to 3.6-fold more potent stimuli of neutrophil superoxide generation than were the white cells. In addition, both neutrophils and oxidants (reagent H2O2 or hypochlorous acid as well as the myeloperoxidase-H2O2-Cl- system) induced unidirectional increases in spontaneous rates of switching from white to opaque phenotypes. Differences in expression of C. albicans phenotypes therefore may determine relative susceptibility to neutrophil fungicidal mechanisms, and neutrophils themselves appear to be capable of selectively augmenting the switching process.
Influence of the 60-megadalton plasmid on adherence of Escherichia coli O157:H7 and genetic derivatives
Authors: Toth I , Cohen M L , Rumschlag H S , Riley L W , White E H , Carr J H , Bond W W , Wachsmuth I K
Published: January 2020

The adherence of enterohemorrhagic Escherichia coli serotype O157:H7 and various genetic derivatives to Henle 407 intestinal and HEp-2 epithelial cell lines was examined by light and electron microscopy. The parent outbreak strain, 7785, harbors a 60-megadalton serotype-specific plasmid designated pO157 and adhered to both cell lines, as determined by light microscopy. A plasmidless derivative, 2-45, showed reduced adherence to both cell lines. After being labeled with Tn801, pO157 was transformed into E. coli C600, E. coli HB101, and E. coli GH42, and back into 2-45. Both E. coli C600 and HB101 transformants adhered weakly; full adherence was restored to the 2-45(pO157::Tn801) transformant. Transmission electron microscopy (EM) demonstrated the intimate attachment of HB101(pO157::Tn801) to Henle 407 cells which formed cuplike structures and areas of possible actin polymerization adjacent to adhering bacterial cells; scanning EM further extended these observations. EM studies of E. coli O157:H7 strains were hampered by extensive intestinal cell damage, presumably due to the action of Shiga-like toxins. EM also demonstrated that 7785 and its plasmidless derivative 2-45 were piliated and that no pili were apparent on HB101(pO157::Tn801) or GH42//(pO157::Tn801). The plasmid pO157 appears to modify the eucaryotic cell adherence of E. coli O157:H7 and to confer that adherence on E. coli HB101 through surface structures other than pili. These findings, when compared with other published reports, also suggest similarities between enterohemorrhagic and enteropathogenic E. coli adherence properties.
Net fluid secretion and impaired villous function induced by colonization of the small intestine by nontoxigenic colonizing Escherichia coli
Authors: Schlager T A , Wanke C A , Guerrant R L
Published: January 2020

The role of colonizing bacteria in the small bowel in causing diarrhea remains unclear. We examined whether colonizing, nontoxigenic Escherichia coli could alter small bowel function by determining net water and electrolyte fluxes and sucrase activity in colonized and noncolonized ileal segments by using the reversible-ileal-tie adult rabbit model. Colonization of the ileum with nontoxigenic E. coli for greater than or equal to 72 h at greater than or equal to 10(4)/cm2 was associated with significant functional derangements, as follows: (i) overt liquid diarrhea in 50% of animals colonized at greater than 10(4)/cm2; (ii) reversal of normal net ileal absorption to net secretion of water, sodium, and chloride; and (iii) significant decrease in mucosal sucrase activity. We conclude that small bowel colonization by colonizing, nontoxigenic E. coli impairs water and electrolyte absorption and sucrase activity in the absence of recognized enterotoxin, cytotoxin, invasion, or effacement traits.
Molecular cloning and sequence analysis of the Sta58 major antigen gene of Rickettsia tsutsugamushi: sequence homology and antigenic comparison of Sta58 to the 60-kilodalton family of stress proteins
Authors: Stover C K , Marana D P , Dasch G A , Oaks E V
Published: January 2020

The scrub typhus 58-kilodalton (kDa) antigen (Sta58) of Rickettsia tsutsugamushi is a major protein antigen often recognized by humans infected with scrub typhus rickettsiae. A 2.9-kilobase HindIII fragment containing a complete sta58 gene was cloned in Escherichia coli and found to express the entire Sta58 antigen and a smaller protein with an apparent molecular mass of 11 kDa (Stp11). DNA sequence analysis of the 2.9-kilobase HindIII fragment revealed two adjacent open reading frames encoding proteins of 11 (Stp11) and 60 (Sta58) kDa. Comparisons of deduced amino acid sequences disclosed a high degree of homology between the R. tsutsugamushi proteins Stp11 and Sta58 and the E. coli proteins GroES and GroEL, respectively, and the family of primordial heat shock proteins designated Hsp10 Hsp60. Although the sequence homology between the Sta58 antigen and the Hsp60 protein family is striking, the Sta58 protein appeared to be antigenically distinct among a sample of other bacterial Hsp60 homologs, including the typhus group of rickettsiae. The antigenic uniqueness of the Sta58 antigen indicates that this protein may be a potentially protective antigen and a useful diagnostic reagent for scrub typhus fever.
Coaggregation of Streptococcus sanguis and other streptococci with Candida albicans
Authors: Jenkinson H F , Lala H C , Shepherd M G
Published: January 2020

Thirteen strains of viridans group streptococci and two strains of other streptococci were tested for coaggregation with Candida albicans. Streptococcus sanguis strains generally exhibited low levels of adherence to 28 degrees C-grown exponential-phase yeast cells, but starvation of yeast cells for glucose at 37 degrees C (or at 28 degrees C) increased their coaggregating activity with these streptococci by at least tenfold. This was a property common to four C. albicans strains tested, two of which were able to form mycelia (6406 and MEN) and two of which were not (MM2002 and CA2). The expression of the coaggregation adhesin during yeast cell starvation was inhibited by addition of trichodermin or amphotericin B. The strains of S. sanguis, Streptococcus gordonii, and Streptococcus oralis tested for coaggregating activity encompassed a diverse range of physiological and morphological types, yet all exhibited saturable coaggregation with starved C. albicans cells. There was no correlation of cell surface hydrophobicity, of either yeast or streptococcal cells, with their abilities to coaggregate. Strains of Streptococcus anginosus also coaggregated with starved yeast cells; Streptococcus salivarius and Streptococcus pyogenes coaggregated to a lesser degree with C. albicans, and the coaggregation with S. pyogenes was not promoted by yeast cell starvation; Streptococcus mutans and Enterococcus faecalis did not coaggregate with yeast. The coaggregation reactions of S. sanguis and S. gordonii with C. albicans were inhibited by EDTA and by heat or protease treatment of the yeast cells and were not reversible by the addition of lactose or other simple sugars. These observations extend the range of intergeneric coaggregations that are known to occur between oral microbes and suggest that coaggregations of C. albicans with viridans group streptococci may be important for colonization of oral surfaces by the yeast.
Cross-reactivity and sequence homology between the 65-kilodalton mycobacterial heat shock protein and human lactoferrin, transferrin, and DR beta subsets of major histocompatibility complex class II molecules
Authors: Aguas A , Esaguy N , Sunkel C E , Silva M T
Published: January 2020

Immunogold ultracytochemistry and Western immunoblotting showed that polyclonal antibodies against human lactoferrin bind to the highly immunogenic 65-kilodalton (kDa) heat shock protein of mycobacteria. The fast-growing mycobacterial species Mycobacterium smegmatis showed a higher density of these receptors for antilactoferrin sera than the slow-growing M. avium. Polyclonal antibodies against mycobacteria (M. bovis BCG) recognized human lactoferrin. Comparison of the amino acid sequence of lactoferrin with that of the 65-kDa protein of M. tuberculosis revealed seven instances of four amino acid sequence homology between the microbial and the human iron-binding protein. Four of these tetrapeptide sequences were also shared with the human transferrin molecule. The shared amino acid sequence KDLL was also present in the DR1, DR3, and DR4 subsets of the DR beta subregion of major histocompatibility complex (MHC) class II molecules. The molecular mimicry between the 65-kDa mycobacterial protein and the human proteins (lactoferrin, transferrin, and MHC class II molecules) offers a molecular setting for mycobacteria-associated, T-cell-dependent autoimmune disease, namely, for rheumatoid arthritis.
Pili of Vibrio cholerae non-O1
Authors: Nakasone N , Iwanaga M
Published: January 2020

Pili of Vibrio cholerae non-O1 strain S7 were purified and characterized. The pili of S7 were morphologically, electrophoretically, and immunologically (as far as polyclonal antibody was used) indistinguishable from the 16-kilodalton pili of V. cholerae O1 strain 82P7. The purified pili and organisms had D-mannose- and L-fucose-resistant hemagglutinin. The hemagglutinating activity of the purified pili was inhibited by the Fab fraction of antipilus antibody, but the hemagglutinating activity of live organisms was not inhibited completely. The purified pili or Fab fraction of antipilus antibody did not inhibit the adhesion of V. cholerae non-O1 to rabbit intestines. Therefore, the pili were not regarded as a colonization factor of V. cholerae non-O1. A total of 148 V. cholerae non-O1 and O1 clinical isolates were screened for the presence of S7 pili by using an agglutination test with anti-S7 pilus serum; 12 of 49 V. cholerae non-O1 strains and 25 of 99 V. cholerae O1 strains were positive for agglutination. These agglutination reactions were not correlated with adhesion of the organisms to intestines.
Adherence of mutans streptococci to other oral bacteria
Authors: Lamont R J , Rosan B
Published: January 2020

Adherence of mutans streptococci to strains of Actinomyces viscosus, Streptococcus sanguis, and Streptococcus mitis immobilized on a nitrocellulose membrane was measured. Strains of Streptococcus mutans, S. sobrinus, and S. rattus bound in a lactose-independent manner to a variety of the actinomyces and streptococci. Most of these reactions could proceed in the presence of whole saliva although adherence of S. rattus BHT to the streptococci was inhibited by salivary molecules. In contrast, adherence of S. mutans 10449 and KPSK2 to A. viscosus, S. sanguis, and S. mitis was enhanced by salivary molecules. S. mutans KPSK2, S. sobrinus OMZ 176, and S. rattus FA-1 binding to A. viscosus NC3 and S. sanguis G9B exhibited saturation kinetics. Adherence to A. viscosus NC3 was of a higher avidity than adherence to S. sanguis G9B. Attachment of S. mutans KPSK2 to S. sanguis G9B and of S. mutans OMZ 176 to A. viscosus NC3 and S. sanguis G9B was inhibited by heat treatment of the mutans streptococci. Attachment of S. mutans KPSK2 to A. viscosus NC3 and of S. rattus FA-1 to A. viscosus NC3 and S. sanguis G9B was unaffected by heat. These observations suggest that the mutans streptococci can adhere to a variety of early plaque bacteria by several distinct mechanisms. Such interactions may be important in the colonization of tooth surfaces by the mutans streptococci.
Effects of cations and osmotic protectants on cytolytic activity of Actinobacillus actinomycetemcomitans leukotoxin
Authors: Iwase M , Lally E T , Berthold P , Korchak H M , Taichman N S
Published: January 2020

Actinobacillus actinomycetemcomitans leukotoxin permeabilized the plasma membrane of HL-60 promyelocytic leukemia cells, resulting in colloid osmotic lysis. These events were associated with efflux of 51chromium (from prelabeled cells), influx of propidium iodide, and ultrastructural evidence of cellular damage. Target cell lysis was inhibited by procedures which may interfere with the initial interaction of the toxin with the plasma membrane. For example, washing cultures (to dilute and remove toxin) or the addition of monoclonal antibodies (to neutralize toxin) or trypsin (to inactivate toxin) limited lysis when undertaken within the first 5 min of the reaction. The extent of injury was also diminished when radiolabeled HL-60 cells were exposed to toxin in the presence of unlabeled, toxin-sensitive cells (e.g., HL-60 cells or human neutrophils) or certain toxin-resistant target cells (e.g., human K562 erythroleukemia cells). This suggests that the association of the toxin with the cell membrane may not be sufficient to cause lysis without activation of additional effector mechanisms. The addition of specific trivalent (e.g., La3+) or divalent (e.g., Ca2+ and Zn2+) cations to toxin-treated cells appeared to enhance their capacity to repair or minimize the extent of toxin-mediated membrane damage. Depending on size, certain saccharides served as osmotic protectants: maltose almost completely inhibited radiolabel release, while smaller molecules provided correspondingly less protection. The results imply that the leukotoxin has membranolytic activity, producing pores in target cells with a functional diameter approximately the size of maltose (0.96 nm).
A domestic ferret model of immunity to Campylobacter jejuni-induced enteric disease
Authors: Bell J A , Manning D D
Published: January 2020

Oral or intravenous inoculation of previously unexposed juvenile and adult ferrets with Campylobacter jejuni uniformly resulted in intestinal colonization lasting 2 to 12 days. Disease varied from mild to moderate diarrhea, which resolved in 2 to 3 days. Orally infected animals developed agglutinin titers of 8 to 256 within 3 weeks, while those infected intravenously developed titers of 256 to 2,048. Ferrets which had recovered from campylobacteriosis all developed high titers of agglutinating and bacterial antibodies but were readily colonized by subsequent oral inoculation with the same strain of C. jejuni. Orally infected ferret kits 3 to 6 weeks of age exhibited the same general pattern of infection and disease as adults, but diarrhea was somewhat more severe. Kits resolved their diarrhea in 1 to 6 days and developed agglutinin titers in serum of 16 to 32 within 3 weeks. A series of five oral or rectal inoculations of kits during the 5- to 9-week age interval resulted in progressively shorter clearance times and eventual strain-specific resistance against infection, as well as disease. Gnotobiotic adults showed the same pattern of strain-specific accelerated clearance and resistance to disease. Kits born to immune dams with high levels of whey antibodies had passively acquired serum agglutinin titers of 256 to 2,048. These kits showed no resistance to colonization with the homologous strain of C. jejuni but were completely refractory to diarrhea. These observations suggest that (i) some form(s) of specific immunity, rather than factors relating solely to age or normal flora, is responsible for resistance to C. jejuni colonization and disease production and (ii) humoral immunity at a level that does not prevent colonization can protect against enteric disease caused by this organism.
A proteolytic enzyme secreted by Proteus mirabilis degrades immunoglobulins of the immunoglobulin A1 (IgA1), IgA2, and IgG isotypes
Authors: Loomes L M , Senior B W , Kerr M A
Published: January 2020

Proteus mirabilis strains associated with human urinary tract infections have previously been shown to secrete an extracellular metalloproteinase which cleaves serum immunoglobulin A (IgA). The enzyme has now been purified to apparent homogeneity from culture supernatants of P. mirabilis 64676. The protease activity is associated with a 50-kilodalton (kDa) protein. Unlike that of the classic IgA1 proteases, the substrate specificity of the P. mirabilis protease has been found to extend to both sublcasses of IgA, IgG, and the nonimmunoglobulin substrates, secretory component and casein. Cleavage of IgA1 and IgA2 by the P. mirabilis protease yielded fragments on sodium dodecyl sulfate-polyacrylamide gel electrophoresis whose sizes were consistent with a cleavage site outside the hinge region. Both secretory IgA1 and IgA2 were also cleaved by P. mirabilis protease, although the secretory IgA2 molecule was less readily cleaved than secretory IgA1. Free and IgA-bound secretory components were degraded to some extent by P. mirabilis protease. Cleavage of IgG, however, occurred at the hinge region as a two-stage process. The first stage was pepsinlike and generated an F(ab')2 fragment of 120 kDa and a small pFc fragment detected on nonreduced polyacrylamide gels. In the second stage, the F(ab')2 product was cleaved to yield papainlike Fab and Fc fragments, visualized as a diffuse band of 40 to 50 kDa.
Specific immunoglobulin A-secreting cells in peripheral blood of humans following oral immunization with a bivalent Salmonella typhi-Shigella sonnei vaccine or infection by pathogenic S. sonnei
Authors: Van de Verg L , Herrington D A , Murphy J R , Wasserman S S , Formal S B , Levine M M
Published: January 2020

The ability of bivalent Salmonella typhi-Shigella sonnei vaccine strain 5076-1C to stimulate an intestinal immunoglobulin A response in humans was evaluated by detecting gut-derived, trafficking antibody-secreting cells (ASC) in peripheral blood. Following vaccination, an immunoglobulin A-ASC response to O antigens of S. typhi and S. sonnei was observed in 10 of 13 and 13 of 13 vaccine recipients, respectively. Experimental challenge with pathogenic S. sonnei stimulated an ASC response to the S. sonnei O antigen in all subjects who developed clinical illness. The magnitude of the ASC response to challenge was significantly greater than that resulting from vaccination. Furthermore, compared with the response of the unimmunized controls, individuals previously immunized with 5076-1C demonstrated a significantly greater ASC response following challenge with S. sonnei.
Identification of isolate-specific sporozoite proteins of Cryptosporidium parvum by two-dimensional gel electrophoresis
Authors: Mead J R , Humphreys R C , Sammons D W , Sterling C R
Published: January 2020

Five isolates of Cryptosporidium parvum collected from human, horse, and calf sources were compared for differences in sporozoite protein patterns by using two-dimensional gel electrophoresis. Silver-stained two-dimensional gels contained over 300 protein spots from detergent-solubilized sporozoites. A distinguishing 106-kilodalton peptide that shifted in isoelectric point was detected in four of the five isolates. Computerized two-dimensional gel analysis was performed to obtain objective quantitation of the pI shift. Three of these four isolates could be differentiated from one other by the pI shift in this peptide. The fifth isolate was distinguished by the absence of the 106-kilodalton peptide and the presence of a 40-kilodalton peptide that was not observed in any other isolate.
Production and characterization of monoclonal antibodies to cell wall antigens of Aspergillus fumigatus
Authors: Ste-Marie L , Sénéchal S , Boushira M , Garzon S , Strykowski H , Pedneault L , de Repentigny L
Published: January 2020

Two murine monoclonal antibodies (MAbs) against Aspergillus fumigatus were produced and characterized. Splenocytes from cell wall-immunized BALB/c mice were fused with SP2/0 myeloma cells. The hybridomas were screened with a cold alkali (CA) extract of mycelium containing protein, mannose, and galactose, and two MAbs of the immunoglobulin M class were purified from ascites fluid. MAbs 1 and 40 were characterized by double immunodiffusion against CA antigen, indirect enzyme immunoassay with mannans of Candida albicans serotypes A or B or Candida tropicalis, indirect immunofluorescence with C. albicans- or A. fumigatus-infected tissues, indirect immunofluorescence with smears of other pathogenic fungi, Western blotting (immunoblotting) with the lectin concanavalin A or BS-1 from the seeds of Bandeirea simplicifolia, and immunoelectron microscopy. MAb 1 did not cross-react with Candida mannan and recognized a periodate-sensitive, pronase- and heat-resistant epitope in CA antigen and three mannose- and galactose-containing components (80, 62, and 49 kilodaltons) of a mycelial homogenate. Immunoelectron microscopy demonstrated binding of MAb 1 to the inner cell wall and intracellular membranes of hyphae and conidia of A. fumigatus. Circulating antigen was detected in experimental invasive aspergillosis by inhibition enzyme immunoassay with MAb 1 and CA antigen. MAb 40 was a nonprecipitating antibody cross-reactive with Candida species, and competition for an epitope located diffusely in the cell wall of A. fumigatus hyphae was demonstrated by incubating MAb 40 with mannan of C. albicans serotype A. These results suggest that MAb 1 recognizes immunodominant oligogalactoside side chains of A. fumigatus galactomannan, while MAb 40 binds to mannopyranosyl side chains common to A. fumigatus galactomannan and C. albicans mannan.
Study of virulence and vector transmission of Babesia bovis by use of cloned parasite lines
Authors: Timms P , Stewart N P , De Vos A J
Published: January 2020

Cloned lines of Babesia bovis were prepared from the avirulent vaccine strain, Ka, by an in vivo limiting dilution procedure. The virulence of these clones for adult Bos taurus cattle varied from completely avirulent to highly virulent. This suggests that the parent strain, Ka, is composed of a mixture of subpopulations of varied virulence. Passage of the avirulent clone K-19-47 in intact (nonsplenectomized) cattle resulted in its full reversion to virulence. This suggests that two mechanisms are operating to enable virulence to be a readily modified characteristic in this parasite: differential gene expression and phenotypic selection of subpopulations. A series of experiments demonstrated that all clones were non-tick transmissible. This lack of vector transmission was a stable characteristic and could not be altered by the passage of K-19-47 in intact cattle, despite the fact that passage in intact animals caused this clone to revert to the fully virulent phenotype. A mechanism is suggested for the virulence and vector transmission variations observed in B. bovis.
Molecular analysis of a sphingomyelinase C gene from Leptospira interrogans serovar hardjo
Authors: Segers R P , van der Drift A , de Nijs A , Corcione P , van der Zeijst B A , Gaastra W
Published: January 2020

A thermolabile hemolysin from Leptospira interrogans serovar hardjo, strain Sponselee, was shown to specifically degrade sphingomyelin. Nucleotide sequence determination revealed that sphingomyelinase activity was encoded by an open reading frame of 1,668 nucleotides. Although a putative signal sequence could be identified, no evidence for protein export in either L. interrogans or Escherichia coli was obtained. The apparent molecular mass of the expression product in E. coli minicells was 41.2 kilodaltons, whereas open reading frame 1 encoded a protein of 63,268 daltons. The observed difference may be explained by processing at the carboxy-terminal part of the hemolysin in E. coli. A high degree of similarity on the DNA and protein levels with Staphylococcus aureus beta-hemolysin and sphingomyelinase C from three Bacillus cereus strains was observed. The presence of various sphingomyelinase genes within the L. interrogans species is demonstrated.
Binding competition of toxic shock syndrome toxin 1 and other staphylococcal exoproteins for receptors on human peripheral blood mononuclear cells
Authors: See R H , Krystal G , Chow A W
Published: January 2020

Binding of toxic shock toxin 1 (TSST-1) and staphylococcal enterotoxin A (SEA) to human peripheral blood mononuclear cells (PBMC) was investigated by using 125I-labeled ligands. Scatchard analyses revealed similar numbers of receptors (approximately 5,000 to 8,000) and similar dissociation constants (Kd, approximately 20 to 25 nM) per PBMC. SEA but not enterotoxin B, C1, C2, C3, D, or E significantly inhibited binding of 125I-TSST-1 to PBMC. Cross-competition of TSST-1 and SEA in binding assays suggests that they may bind to overlapping or separate epitopes on the same receptor.
The cytolysin gene of Vibrio vulnificus: sequence and relationship to the Vibrio cholerae E1 Tor hemolysin gene
Authors: Yamamoto K , Wright A C , Kaper J B , Morris J G
Published: January 2020

A cytolysin of ca. 56 kilodaltons has been suggested as a possible virulence factor in Vibrio vulnificus infections. We sequenced the DNA encoding cytolytic activity and found that the sequence contained two open reading frames, vvhA and vvhB. vvhA encoded the structural gene for the cytolysin and contained the N-terminal amino acid sequence previously reported for the protein. Regions of the vvhA gene showed homology to the structural gene for the Vibrio cholerae E1 Tor hemolysin.
Electron microscopic study of coexpression of adhesive protein capsules and polysaccharide capsules in Escherichia coli
Authors: Kröncke K D , Orskov I , Orskov F , Jann B , Jann K
Published: January 2020

Escherichia coli 21535 (O21:K4:H4 with nonfimbrial adhesin NFA-6) and 21511 (O7:K98:H6 with nonfimbrial adhesin NFA-4) were analyzed by immunoelectron microscopy with a K98-specific antiserum and K4- and NFA-4-specific and NFA-6-reactive monoclonal antibodies. The bacteria were analyzed in ultrathin sections after stabilization of the capsules with specific antibodies by embedding in Epon 812 as well as in Lowicryl K4M. With the Lowicryl-embedded samples, the polysaccharide K antigens were labeled by the immunogold technique. It was found that with both strains all bacteria expressed the polysaccharide capsule, while in each case about 20% expressed the protein capsule in addition. Thus, in both invasive E. coli strains, bacteria are present which express composite capsules with the adhesin (recognition protein) at the cell-distal outer region and the K antigen (acidic polysaccharide) at the cell-proximal inner region. These findings are discussed with respect to the participation of the capsular compartments in unspecific host defense.
Stimulatory effect of staphylococcal leukocidin on phosphoinositide metabolism in rabbit polymorphonuclear leukocytes
Authors: Wang X , Noda M , Kato I
Published: January 2020

When rabbit polymorphonuclear leukocytes (PMNs) were incubated with staphylococcal leukocidin (F and S components) in the presence of 32Pi at 37 degrees C, incorporation of 32Pi into phosphatidylinositol 4-phosphate (PIP) and phosphatidylinositol 4,5-bisphosphate (PIP2) occurred after a lag phase of 10 s and reached a maximal level at 60 s of 50- and 30-fold increase, respectively, compared with that of the control in the absence of the toxin. Whereas the amount of 32P radioactivity incorporated in PIP and PIP2 decreased to control levels in a few minutes, 32P incorporation into phosphatidic acid (PA) continuously increased over 3 min. These findings suggested an early activation of phosphoinositide-specific phospholipase C in rabbit PMNs by leukocidin as shown by the rapid breakdown of PIP and PIP2 accompanied by the appearance of PA. The stimulatory effect of leukocidin on some enzymatic activities of the phosphatidylinositol pathway was further investigated by using PMN cell membrane preparations. In the presence of both the F and S components, enhanced 32P incorporation was observed not only in PIP2 and PA but also in PIP. While the F component mainly enhanced 32P incorporation into PIP2 and PA, the S component alone had no effect on 32P incorporation into PIP, PIP2, and PA. The F component alone enhanced conversion of PIP to [32P]PIP2 in the presence of unlabeled PIP and [gamma-32P]ATP, through the activation of PIP kinase. PIP kinase activity was potentiated by the addition of NAD and GTP. Subsequent formation of [32P]PA was also enhanced by the F component, resulting from activation of the phosphoinositide-specific phospholipase C. These results suggested that the F component of staphylococcal leukocidin is responsible for the enhancement of phosphoinositide metabolism in rabbit PMN cell membranes.