bluuebunny/crossref_metadata_2025 huggingface dataset:
Cytoskeletal composition of attaching and effacing lesions associated with enteropathogenic Escherichia coli adherence to HeLa cells
Authors: Finlay B B , Rosenshine I , Donnenberg M S , Kaper J B
Published: January 2020

The cytoskeletal lesions associated with enteropathogenic Escherichia coli adhering to cultured HeLa epithelial cells were examined by immunofluorescence microscopy. The microfilament-associated proteins actin, alpha-actinin, talin, and ezrin were localized with adherent enteropathogenic E. coli, whereas tropomyosin, keratin and vimentin (intermediate filaments), tubulin (microtubules), and vinculin were not localized. These cytoskeletal structures differed significantly from those associated with Salmonella typhimurium internalization (invasion).
Opportunist mycobacteria express ligands that stimulate production of human V gamma 9V delta 2 T lymphocytes
Authors: Häcker G , Kromer S , Heeg K , Ivanyi J , Wagner H , Pfeffer K
Published: January 2020

Human gamma delta T cells are known to respond at high frequencies to pathogenic mycobacteria. Here we show that opportunistic strains of mycobacteria share with pathogenic mycobacteria the ability to trigger at high frequencies human V gamma 9V delta 2 T-cell-receptor-positive T lymphocytes. Stimulating ligands were present in part in a low-molecular-weight fraction of lysates from opportunistic mycobacteria, as has been found for pathogenic strains. These results support the view that postnatal exposure to ever-present opportunistic mycobacteria may be a driving force for the numerical expansion of the V gamma 9V delta 2 T-cell subset in adolescence.
Legionella pneumophila lipopolysaccharide activates the classical complement pathway
Authors: Mintz C S , Schultz D R , Arnold P I , Johnson W
Published: January 2020

Legionella pneumophila is a gram-negative bacterium capable of entering and growing in alveolar macrophages and monocytes. Complement and complement receptors are important in the uptake of L. pneumophila by human mononuclear phagocytes. The surface molecules of L. pneumophila that activate the complement system are unknown. To identify these factors, we investigated the effects of L. pneumophila lipopolysaccharide (LPS) on the classical and alternative complement pathways of normal human serum by functional hemolytic assays. Although incubation of LPS in normal human serum at 37 degrees C resulted in the activation of both pathways, complement activation proceeded primarily through the classical pathway. Activation of the classical pathway by LPS was dependent on natural antibodies of the immunoglobulin M class that were present in various quantities in sera from different normal individuals but were absent in an immunoglobulin-deficient serum obtained from an agammaglobulinemic patient. Additional studies using sheep erythrocytes coated with LPS suggested that the antibodies recognized antigenic sites in the carbohydrate portion of LPS. The ability of LPS to interact with the complement system suggests a role for LPS in the uptake of L. pneumophila by mononuclear phagocytes.
Role of aerobactin in systemic spread of an opportunistic strain of Escherichia coli from the intestinal tract of gnotobiotic lambs
Authors: Der Vartanian M , Jaffeux B , Contrepois M , Chavarot M , Girardeau J P , Bertin Y , Martin C
Published: January 2020

To assess the role of the aerobactin-related system in the virulence of bovine opportunistic Escherichia coli, and to determine the stage(s) of the overall infectious process at which it is acting, germfree lambs were mixedly infected orally with two derivative strains of this bacterium differing in their ability (Iut+) or inability (Iut-) to express a functional aerobactin-mediated iron transport system. The Iut- strain was compared with the Iut+ strain for colonization of the gut, translocation to the mesenteric lymph nodes (MLN), and spread to other organs and to the body fluids of diassociated lambs. The Iut- mutant was found in smaller numbers in the duodenum, suggesting that aerobactin conferred a significant selective advantage for colonization of this intestinal segment. Although the two challenge strains translocated to MLN, the population level in the MLN was always higher for the Iut+ strain. Moreover, experimental infections resulted in recovery of only the Iut+ strain in the organs other than the MLN and in the body fluids. These results indicate a role for aerobactin in promoting systemic spread of the bacteria from the intestine. Direct evidence was obtained that aerobactin secretion occurred in vivo at both intestinal and extraintestinal sites of infection. In contrast to enterobactin, aerobactin was detected in the duodenum, jejunum, ileum, cecum, liver, spleen, kidney, urine, cerebrospinal fluid, and bile. The highest concentration of aerobactin was found in the urine, even when the samples were devoid of infecting bacteria. All of these findings suggest that aerobactin is released in vivo in a diffusible form and that it may be an important step in the production of disease by intestinal opportunistic E. coli.
Comparison of adherence of Pseudomonas aeruginosa to respiratory epithelial cells from cystic fibrosis patients and healthy subjects
Authors: Saiman L , Cacalano G , Gruenert D , Prince A
Published: January 2020

The adherence of Pseudomonas aeruginosa PAO1 to primary cultures of cystic fibrosis nasal polyp (CFNP), normal human nasal polyp (NHNP), and immortalized CF and normal cell lines was studied. PAO1 bound significantly more to primary CFNP cells than to NHNP cells as the mean adherence +/- standard deviation of 5 x 10(7) CFU of 35S-labeled bacteria per ml per well was 15.09 x 10(6) +/- 4.25 x 10(6) CFU/ml per well and 7.62 x 10(6) +/- 2.11 x 10(6) CFU/ml per well, respectively (Mann-Whitney U test, P less than 0.0001). There was no significant difference in PAO1 adherence to the immortalized CF and normal cell lines. The primary CFNP cells had more receptors (115 per cell) than did NHNP cells (34 per cell). P. aeruginosa binding to CFNP was blocked by GlcNAc, NeuAc, L-Fuc, and D-Gal, while binding to NHNP was blocked only by GlcNAc, suggesting that receptors on the two cell types were qualitatively different. Pseudomonas supernatants containing protease, phospholipase C, and neuraminidase activity increased adherence to CFNP and NHNP cells. The Pseudomonas exoproducts modified epithelial cell glycoconjugates, as characterized by binding of fluorescein isothiocyanate-labeled lectins and the release of sialic acid. There was minimal release of fibronectin by the bacterial supernatants. The affinity of P. aeruginosa for CF epithelial cells appeared to be due to an increased number of receptors and modification of the epithelial cell surface by P. aeruginosa exoproducts that exposed asialoganglioside binding sites.
Construction of Cu-Zn superoxide dismutase deletion mutants of Brucella abortus: analysis of survival in vitro in epithelial and phagocytic cells and in vivo in mice
Authors: Tatum F M , Detilleux P G , Sacks J M , Halling S M
Published: January 2020

Cu-Zn superoxide dismutase (SOD) deletion mutants of Brucella abortus S2308, a virulent strain, and S19, a vaccine strain, were generated by gene replacement. A deletion plasmid, pBA delta sodknr, was constructed by excising the Cu-Zn SOD gene (Cu-Zn sod) from a 2.3-kb B. abortus DNA fragment of plasmid pBA20-1527 and inserting a 1.4-kb DNA fragment encoding kanamycin resistance into the Cu-Zn sod excision site. The deletion plasmid was introduced into B. abortus by electroporation, and Southern blot analysis confirmed that the antibiotic resistance fragment had replaced Cu-Zn sod in kanamycin-resistant colonies. The survival and growth of Cu-Zn SOD mutant strains were compared with that of the parental strains in HeLa cells and in the mouse macrophagelike cell line J774. The survival and growth of the Cu-Zn SOD mutant strains were similar to those of their respective parental strains in HeLa and J774 cell lines. The kinetics of infection with these strains were examined in BALB/c mice. The splenic levels of the S19 Cu-Zn SOD mutant recovered from intraperitoneally infected BALB/c mice were approximately 10-fold lower than those of the parental strain through 26 days postinfection. Thereafter, infection sharply declined in both groups, and by 105 days postinfection, no organisms were detected. The splenic levels of the S2308 Cu-Zn SOD mutant were lower than those of wild-type S2308-infected mice. The spleen weights of mice infected with the S2308 Cu-Zn SOD mutant were consistently lower than those of wild-type S2308-infected mice. These results suggest that the antioxidant enzyme Cu-Zn SOD plays a role in the survival and pathogenicity of B. abortus in vivo.
Immunization with Porphyromonas (Bacteroides) gingivalis fimbriae protects against periodontal destruction
Authors: Evans R T , Klausen B , Sojar H T , Bedi G S , Sfintescu C , Ramamurthy N S , Golub L M , Genco R J
Published: January 2020

Adhesive fimbriae from Porphyromonas gingivalis are cell surface structures which may be important in the virulence of this oral pathogen and thus may serve as a critical or target antigen. Immunization with highly purified 43-kDa fimbrial protein protected against periodontal tissue destruction when tested in the P. gingivalis-infected gnotobiotic rat model. A similarly highly purified 75-kDa cell surface component did not provide protection. Heat-killed whole-cell and sonicated cell surface extracts which contain the 43-kDa protein as well as the 75-kDa component were protective also. This study indicates that the fimbrial protein may serve as a model for the development of effective vaccines against periodontitis, a major human oral disease.
Infection-induced airway fibrosis in two rat strains with differential susceptibility
Authors: McIntosh J C , Simecka J W , Ross S E , Davis J K , Miller E J , Cassell G H
Published: January 2020

Chronic infections play a significant role in the morbidity and mortality of patients with chronic airflow limitation. By stimulating airway inflammation, persistent infection has the potential to cause airway fibrosis. However, in patient this condition is most typically found in lungs damaged by other factors, such as smoking, abnormal secretions, or barotrauma. We report the characterization of Mycoplasma pulmonis infection-induced lung fibrosis in two immunocompetent rat strains with no preexisting lung disease. The fibrosis was predominantly in the airways, as demonstrated by the findings for infected animals of increased airway inflammation, airway fibrosis, and airway wall thickness, which correlated with the collagen content of the lungs. Also, the physiological alterations were the opposite of those found in interstitial fibrosis, with a positive correlation between lung compliance and collagen content. The airway fibrosis was noted earlier and to a greater extent in Lewis rats than in Fisher rats, and this result apparently was related to regulation of the inflammatory response. Airway wall thickness, airway inflammation, and airway fibrosis are commonly reported in tissue specimens from patients with chronic airway diseases and have been shown to correlate with airflow limitation in patients with chronic obstructive pulmonary disease. Thus, this model may be useful in furthering our understanding of the role of chronic infection and airway inflammation in airflow obstruction.
Phenotypic phase variation in Haemophilus somnus lipooligosaccharide during bovine pneumonia and after in vitro passage
Authors: Inzana T J , Gogolewski R P , Corbeil L B
Published: January 2020

A high rate of phenotypic variation in the lipooligosaccharide (LOS) electrophoretic profile of Haemophilus somnus occurred in most isolates obtained at approximately weekly intervals from three calves intrabronchially challenged with a cloned isolate of H. somnus 2336. Daily subculturing for 2 weeks resulted in at least one major alteration in the LOS electrophoretic profiles for strain 2336 and both additional disease isolates examined, but no change occurred in the LOS electrophoretic profiles for any of three commensal isolates examined. None of the LOSs from any of the postchallenge intrabronchial isolates reacted with rabbit antiserum to the challenge strain LOS in immunoblotting, but LOSs from two nasopharyngeal isolates did. Antigenic variation in the extracted LOSs of most of the isolates was supported by the results of an enzyme-linked immunosorbent assay. Preimmune serum from each of the calves did not react with any of the isolates or the challenge strain, whereas sera obtained 35 days after challenge reacted with the challenge strain and zero to five additional isolates and sera obtained 74 days after challenge reacted with two to six additional isolates. Recognition of LOSs from isolates obtained near the end of the 70-day experiment by day-74 sera was related to clearance of the bacteria from the lungs. Isolates demonstrating major electrophoretic changes showed variations in the composition of the oligosaccharide, but not lipid A, moiety of their LOSs. The oligosaccharide of the LOS of each isolate was composed predominantly of glucose but varied substantially in the contents of galactose, arabinose, xylose, mannose, and 3-deoxy-D-manno-octulosonic acid. Therefore, the LOS of H. somnus is capable of undergoing compositional and antigenic variations, which may act as an important virulence mechanism for evading host immune defense mechanisms.
Further antigenic similarities of Neisseria gonorrhoeae lipooligosaccharides and human glycosphingolipids
Authors: Mandrell R E
Published: January 2020

Anticarbohydrate monoclonal antibodies were tested for their ability to bind to various strains of Neisseria. A monoclonal antibody that binds to the ganglio-series glycosphingolipid, ganglio-N-triaosylceramide, also bound to strains of Neisseria gonorrhoeae but not to other species of Neisseria. An antibody specific for the globo-series glycosphingolipid, globotriaosylceramide, also bound to strains of N. gonorrhoeae, Neisseria meningitidis, Neisseria lactamica, and Branhamella catarrhalis but not to any other strains of nonpathogenic Neisseria.
Site-specific growth of Nocardia asteroides in the murine brain
Authors: Ogata S A , Beaman B L
Published: January 2020

The growth of Nocardia asteroides GUH-2 and two mutants (NG-49 and I-38-syn) in regions of the brains of BALB/c mice was determined by microdissection and viable counting. GUH-2 grew throughout the murine brain but at different growth rates that depended on the specific location. The rate of increase in total CFU per brain during GUH-2 infection was unaffected by the inoculum size; however, in five of eight brain regions, an alteration in the inoculum size resulted in altered nocardial growth rates. Mutant NG-49 showed a significantly slower rate of increase in total CFU per brain than did the parental strain, GUH-2, and significantly decreased growth rates in seven brain regions. Mutant I-38-syn showed a rate of increase in total CFU per brain similar to that of the parental strain; however, this mutant grew significantly faster in the cerebellum and pons-medulla. Growth appeared to be a necessary precursor to the cellular damage that resulted in the variety of neurological disorders observed in mice infected with N. asteroides GUH-2, because mutant NG-49 exhibited a decreased ability to grow in specific regions of the brain and did not induce signs of neurological damage. In contrast, mutant I-38-syn induced neurological signs in a larger percentage of the infected animals than did parental strain GUH-2 and grew better in certain regions of the brain than did the parental strain. Furthermore, there appeared to be a relationship between the growth of N. asteroides in the substantia nigra and the induction of an L-dopa-responsive head shake that was observed in some of the mice following a sublethal intravenous injection of N. asteroides GUH-2.
Biological activity of synthetic phosphonooxyethyl analogs of lipid A and lipid A partial structures
Authors: Ulmer A J , Heine H , Feist W , Kusumoto S , Kusama T , Brade H , Schade U , Rietschel E T , Flad H D
Published: January 2020

We investigated the biological activity of four new synthetic analogs of lipid A, termed PE-1, PE-2, PE-3, and PE-4. All compounds contain an alpha-oxyethyl-linked (-O-CH2-CH2-) phosphoryl group in position 1 of the reducing glucosaminyl residue (GlcN I) of lipid A. PE-1 is a hexaacylated analog of Escherichia coli lipid A (compound 506). PE-2 differs from PE-1 in carrying two myristic acid residues at GlcN I. PE-3 has the same acylation pattern as PE-2, but GlcN I is present in the beta anomeric form. Finally, PE-4 represents an analog of tetraacyl precursor Ia (compound 406). Structure-activity relationships of these compounds were determined by measuring their capacity to induce tumor necrosis factor alpha, interleukin 1, and interleukin 6 release by human mononuclear cells and to cause mitogenicity of murine spleen cells. The results show that replacement of the glycosidic phosphoryl residue by a phosphonooxyethyl group had no substantial effect on the biological activity of compounds. However, the anomeric configuration of GlcN I was found to be of great biological relevance, as, in general, the alpha anomer (PE-2) expressed high activity, and the beta anomer (PE-3) expressed low mediator-inducing and mitogenic activity. The absence of the 3-hydroxyl groups within the acyl residues at GlcN I in PE-2 was found to only slightly affect the induction of monokines in human mononuclear cells compared with that of PE-1 or lipid A (506). These stable 1-phosphonooxyethyl analogs of lipid A may be candidates in the development of immunomodulators for the treatment of systemic endotoxicosis.
Glucose and nonmaintained pH decrease expression of the accessory gene regulator (agr) in Staphylococcus aureus
Authors: Regassa L B , Novick R P , Betley M J
Published: January 2020

The effect of glucose on accessory gene regulator (agr) expression in Staphylococcus aureus was examined. agr is a global regulator that affects the expression of numerous genes, including those for some factors implicated in virulence, such as toxic shock syndrome toxin 1, alpha-hemolysin, and protein A. The agr locus determines two divergent transcripts, designated RNAII and RNAIII. RNAII contains four open reading frames (agrABCD), and RNAIII encodes delta-hemolysin. The mechanisms responsible for agr-mediated regulation are not well understood, but it appears that the RNAIII transcript plays a central role in the regulation of a number of target genes, including those for alpha-hemolysin (hla), beta-hemolysin (hlb), protein A (spa), and staphylococcal enterotoxin B (seb+). In this study, S. aureus cultures were grown either in a shake flask system with a complex medium or in a fermentor system with a completely defined medium in which the pH and glucose concentration were maintained. Northern (RNA) blot analysis revealed that a dramatic reduction in agr expression was apparent only when the cultures contained glucose and when the pH was 5.5 or was not maintained. The effect of glucose on two agr target genes, sec+ and hla, was also studied. Glucose-containing cultures produced less sec+ and hla mRNAs at maintained pH (6.5). In addition, the glucose effect on sec+ and hla was enhanced under conditions that inhibited agr expression (i.e., pH 5.5 or a nonmaintained pH).
Synthetic peptides representing epitopes of outer membrane protein F of Pseudomonas aeruginosa that elicit antibodies reactive with whole cells of heterologous immunotype strains of P. aeruginosa
Authors: Hughes E E , Gilleland L B , Gilleland H E
Published: January 2020

By using the published amino acid sequence for mature outer membrane protein F of Pseudomonas aeruginosa, a computer-assisted analysis was performed to identify sites with potential as surface-exposed, antigenic regions located throughout the length of the protein molecule. Synthetic peptides 13 to 15 amino acid residues in length were synthesized for 10 such regions. Mice were immunized with each of the 10 synthetic peptides conjugated to keyhole limpet hemocyanin. An enzyme-linked immunosorbent assay (ELISA) of the antisera was performed by using each of the synthetic peptides as the ELISA antigen to verify that immunoglobulin G (IgG) antibodies capable of reacting with the peptide used as immunogen were elicited by each peptide. Each of the antipeptide antisera was screened for the presence of IgG antibodies that could bind to the surface of intact cells of strains representing the seven heterologous Fisher-Devlin immunotypes of P. aeruginosa by use of an ELISA with whole cells of the various strains as the ELISA antigen. Three peptides elicited antibodies capable of reacting with whole cells of all seven immunotype strains. Peptide 10, corresponding to amino acid residues 305 to 318, elicited whole-cell-reactive antibodies at high titers. Peptide 9, corresponding to amino acid residues 261 to 274, elicited whole-cell-reactive antibodies at more intermediate titers. Peptide 7, corresponding to amino acid residues 219 to 232, elicited such antibodies only at low titers. The carboxy-terminal portion of the mature protein appears to be the immunodominant portion. In particular, peptides 10 (NATAEGRAINRRVE) and 9 (TDAYNQKLSERRAN) appear to have potential for use as immunogens in a synthetic vaccine for immunoprophylaxis against infections caused by P. aeruginosa. Antisera from mice immunized with either peptide 9 or 10 mediated opsonophagocytic uptake by human polymorphonuclear leukocytes of wild-type cells of P. aeruginosa but exhibited no opsonic activity against a protein F-deficient mutant of P. aeruginosa.
Tumor necrosis factor and macrophage activation are important in clearance of Nocardia brasiliensis from the livers and spleens of mice
Authors: Silva C L , Faccioli L H
Published: January 2020

The roles of tumor necrosis factor (TNF) and macrophage activation in clearance of Nocardia brasiliensis from BALB/c mouse livers and spleens were evaluated. TNF activity was detectable in sera from animals at all stages of infection. Treatment of infected mice with an antiserum against TNF significantly enhanced the experimental infection as judged by enumeration of CFU in the spleens and livers of infected mice. In another set of experiments, a population of activated macrophages from the peritoneal cavities of N. brasiliensis-infected mice was studied by using a cytostatic assay. The observed cytotoxic activity of these activated macrophages against L929 cells was mediated by TNF, since this activity was inhibited by anti-TNF antiserum treatment. The level of TNF activity generated in vitro in the presence of lipopolysaccharide (LPS) by peritoneal macrophages from infected mice was higher than that of adherent peritoneal cells obtained from normal mice after challenge with LPS. When the nocardiacidal activity of peritoneal cells from N. brasiliensis-infected mice was estimated in vitro, a significant decrease in the number of CFU recovered was observed. Moreover, nocardiacidal activity of peritoneal cells obtained from N. brasiliensis-infected mice previously treated with anti-TNF antiserum was significantly reduced compared with the activity of cells obtained from infected mice previously treated with normal rabbit serum and that of cells from uninfected mice. These data suggest a role for TNF in resistance to N. brasiliensis infection.
Macrophage response to bacteria: induction of marked secretory and cellular activities by lipoteichoic acids
Authors: Keller R , Fischer W , Keist R , Bassetti S
Published: January 2020

Lipoteichoic acids (LTAs) from various bacterial species, including Staphylococcus aureus, Streptococcus pyogenes, Streptococcus pneumoniae, Enterococcus faecalis, and Listeria monocytogenes, were examined for the ability to induce secretory and cellular responses in a pure population of bone marrow-derived mononuclear phagocytes. Some of the highly purified LTAs, in particular LTAs from Bacillus subtilis, S. pyogenes, E. faecalis, and Enterococcus hirae, were able to affect each of the macrophage parameters measured, i.e., reductive capacity, secretion of tumor necrosis factor and nitrite, and tumoricidal activity. As after stimulation with whole organisms or other bacterial products, secretion of tumor necrosis factor induced by these LTAs reached its maximum within the first few hours of the interaction, while secretion of nitrite and tumoricidal activity required 24 to 36 h for full expression. Other purified LTAs, i.e., LTAs from Streptococcus sanguis, S. pneumoniae, and L. monocytogenes, as well as lipomannan from Micrococcus luteus affected only some of these parameters, while native LTA from S. aureus was inactive. There was no obvious correlation between biological activity and chain length, kind of glycosyl substituents, glycolipid structures, or fatty acid composition of LTAs. Deacylation of LTAs resulted in a complete loss of activity, and deacylated LTAs did not impair the activity of their acylated counterparts, suggesting that acyl chains may be essential for binding of LTA to the cell surface. The results demonstrate that some LTA species are potent inducers of macrophage secretory and cellular activities.
Chelating agents inhibit activity and prevent expression of streptococcal glucan-binding lectins
Authors: Lü-Lü  , Singh J S , Galperin M Y , Drake D , Taylor K G , Doyle R J
Published: January 2020

Several of the cariogenic mutans streptococci produce cell wall-associated glucan-binding lectins (GBLs). The lectins bind alpha-1,6-linked glucans and have no affinity for other polysaccharides or anomeric linkages. When citrate or lactate was included in the growth medium, expression of the activities of the GBLs of Streptococcus cricetus and S. sobrinus was prevented. Furthermore, chelating agents, including citrate, lactate, EDTA, and acetylacetone, were able to reversibly inhibit glucan-induced aggregation of GBL+ streptococci. In addition, the chelating agents prevented sucrose-dependent streptococcal adhesion to glass surfaces and dispersed preformed adherent masses of the streptococci. Neither citrate nor other chelating agents modified the activities of glucosyltransferases. Expression of the lectin could only be achieved by the addition of manganous ion to the growth medium. Chloramphenicol and other metabolic inhibitors prevented synthesis of GBL in cells obtained from manganese-deficient medium and shifted to manganous ion-sufficient medium. The GBL may be a manganoprotein, the manganese of which may be perturbed, but not removed, by chelating agents. During synthesis of the GBL, manganous ion may be required in order for the protein to achieve an active conformation. Citrate or other chelating agents may have promise as anticaries agents.
Immune response to outer membrane antigens of Moraxella catarrhalis in children with otitis media
Authors: Faden H , Hong J , Murphy T
Published: January 2020

The systemic and local antibody responses to homologous strains of Moraxella catarrhalis were investigated in 14 children with otitis media. A total of 8 children (57%) demonstrated a rise in serum antibody of the immunoglobulin G (IgG) (5 of 14), IgM (5 of 14), or IgA (6 of 14) classes of immunoglobulin to outer membrane antigens. Local antibody consisted of IgG (100%), IgM (29%), and IgA (71%). The IgG and IgA specific antibody present in middle-ear effusions appeared to represent local production rather than passive diffusion from the systemic circulation. These data suggest that young children develop an antibody response to M. catarrhalis in the middle ear during otitis media but fail to develop systemic antibody in a uniform manner.
Structural and serological specificities of Pasteurella haemolytica lipopolysaccharides
Authors: Lacroix R P , Duncan J R , Jenkins R P , Leitch R A , Perry J A , Richards J C
Published: January 2020

Lipopolysaccharides (LPSs) from 16 serotypes of Pasteurella haemolytica were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and examined by silver staining and immunoblotting. Silver staining of proteinase K-digested cell lysates revealed two rough LPS serotypes (serotypes 2 and 8), which lacked demonstrable O-polysaccharide, while 14 others demonstrated a ladder pattern characteristic of smooth-type LPS. Purified LPSs from several serotypes yielded O-polysaccharide in addition to low-molecular-weight core oligosaccharide components when subjected to mild acid hydrolysis. Nuclear magnetic resonance spectroscopy revealed the O-chain polysaccharides of serotypes 1, 6, and 9 to be identical. Immunoblots using hyperimmune rabbit, mouse, bovine, and ovine sera from homologous and heterologous serotypes supported this finding and suggested that most of the A biotypes share common O-chain epitopes. Immunoblotting results also supported structural data which demonstrated that the O-polysaccharides of serotypes 3 and 15 and of serotypes 4 and 10 (T biotypes) are identical. Nuclear magnetic resonance analysis indicated that the core oligosaccharides of serotypes 1, 6, 8, 9, and 12 share similar structures, but that they are distinct from those of serotypes 3, 4, 10, and 15. Immunoblots with hyperimmune antisera and monoclonal antibody having specificity for the core region of serotype 1 LPS revealed shared epitopes in the core oligosaccharides of several A biotypes. Characterization of the molecular structure and antigenic specificities of LPS has been an important consideration in the development of purity and potency assays for veterinary vaccines which contain P. haemolytica.
Neuraminidase production by a Pasteurella haemolytica A1 strain associated with bovine pneumonia
Authors: Straus D C , Unbehagen P J , Purdy C W
Published: January 2020

The properties of an extracellular neuroaminidase produced by a Pasteurella haemolytica A1 strain (isolated from a case of bovine pneumonia) during growth in a defined medium were examined in this investigation. This enzyme, isolated from concentrated culture supernatants of P. haemolytica A1, was active against N-acetylneuramin lactose, human alpha 1-acid glycoprotein, fetuin, and bovine submaxillary mucin. Neuraminidase production paralleled bacterial growth in a defined medium and was maximal in the stationary phase of growth. The enzyme was purified to homogeneity by a combination of salt fractionation, ion-exchange chromatography on DEAE-Sephacel, and gel filtration on Sephadex G-200. These procedures yielded an enzyme preparation that possessed a specific activity of 100.62 mumol of sialic acid released per min per mg of protein against human alpha 1-acid glycoprotein. The Km value for this enzyme with human alpha 1-acid glycoprotein as the substrate was 1.1 mg/ml, and the enzyme possessed a pH optimum of 6.5. The P. haemolytica A1 neuraminidase had a molecular weight of approximately 150,000 as estimated by gel filtration and approximately 170,000 when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme was stable at 4 degrees C for 3 h. At 37 degrees C for 3 h, 25% of enzymatic activity was lost. Approximately 55% of the enzyme activity was lost within 30 min at 50 degrees C, with greater than 70% of the enzyme activity being destroyed within 10 min at temperatures of > or = 65 degrees C.
Apathogenic, intestinal, segmented, filamentous bacteria stimulate the mucosal immune system of mice
Authors: Klaasen H L , Van der Heijden P J , Stok W , Poelma F G , Koopman J P , Van den Brink M E , Bakker M H , Eling W M , Beynen A C
Published: January 2020

Segmented filamentous bacteria (SFBs) are apathogenic autochthonous bacteria in the murine small intestine that preferentially attach to Peyer's patch epithelium. SFBs have never been cultured in vitro. We have studied the effects of SFBs on the immune system of the host. Mice monoassociated with SFBs were compared with germ-free mice and with mice without SFBs but with a specific-pathogen-free (SPF) gut flora. SFBs versus no microbial flora raised the number of lymphoid cells in the lamina propria of the ileal and cecal mucosa, raised the number of immunoglobulin A (IgA)-secreting cells in the intestinal mucosa, produced elevated IgA titers in serum and intestinal secretions, and enhanced the concanavalin A-induced proliferative responses of mesenteric lymph node cells. The SPF flora had effects similar to but less pronounced than those mediated by SFBs. The results indicate that SFBs stimulate the mucosal immune system to a greater extent than do other autochthonous gut bacteria.
Inhibition of growth of Mycobacterium avium in murine and human mononuclear phagocytes by migration inhibitory factor
Authors: Orme I M , Furney S K , Skinner P S , Roberts A D , Brennan P J , Russell D G , Shiratsuchi H , Ellner J J , Weiser W Y
Published: January 2020

Infections caused by Mycobacterium avium, the most common form of diseminated bacterial disease in AIDS patients, are difficult to treat because of their resistance to many antimycobacterial drugs. The results of the present study show that recombinant migration inhibitory factor, a 12-kDa molecule recently isolated by COS-1 cell expression screening of cDNA from a human T-cell hybridoma, has potent inhibitory activity on the growth of a panel of clinical isolates of M. avium within both bone-marrow-derived murine macrophages and cultured human blood monocytes. These cells cultured in recombinant migration inhibitory factor exhibit various signs of activation, including cell division, morphological changes such as evidence of substantial phagolysosomal fusion, and enhanced secretion of tumor necrosis factor.
Purification and partial characterization of the major outer membrane protein of Haemophilus somnus
Authors: Tagawa Y , Ishikawa H , Yuasa N
Published: January 2020

We purified the major outer membrane protein (MOMP), which is the most abundant OMP (with an apparent molecular mass of 40 kDa), from Haemophilus somnus strain 8025. The method involves solubilization of the MOMP with Zwittergent 3-14 and further purification accomplished by ion-exchange and molecular-sieve chromatographies. The amino-terminal sequence of the MOMP showed considerable similarity to those of porin proteins from other gram-negative bacteria. The MOMP of H. somnus is immunogenic to rabbits and calves. Hyperimmune sera from rabbits and calves reacted with both the MOMP and lipopolysaccharides in enzyme-linked immunosorbent assay (ELISA) and immunoblot analysis. The rabbit antiserum to the MOMP was cross-reactive with whole-cell preparations from strains 8025, D1238, NT2301, and 540 at a band with a molecular mass of 40 kDa in immunoblot analysis, although the reactivity of the rabbit antiserum with strain 540 was lower than those with the other strains tested. Two murine monoclonal antibodies (MAbs) to the MOMP were developed. ELISA with the OMP fractions as the antigens showed that one MAb was cross-reactive with the four strains but that the other MAb was reactive with the three strains other than strain 540. These results indicate that the MOMP of H. somnus possesses at least two antigenic determinants and that the MOMP of strain 540 is antigenically different from those of the other strains. The antigenic heterogeneity of the H. somnus MOMP has implications regarding the development of a serotyping system with MAbs that is based on the MOMP epitopes.
Antibody-mediated enhancement of Legionella pneumophila-induced interleukin 1 activity
Authors: Widen R H , Newton C A , Klein T W , Friedman H
Published: January 2020

The ability of antibody specific for Legionella pneumophila to enhance the induction of interleukin 1 (IL-1) production by murine peritoneal, splenic, and pulmonary macrophages in response to the bacterium was examined. Two preparations of L. pneumophila were utilized, a formalin-killed whole-cell preparation and viable bacteria. We measured both secreted (sIL-1) and cell membrane-associated (mIL-1) activities after incubation of the macrophages with the bacterial preparations in the presence or absence of the antibody. Both bacterial preparations induced sIL-1 and mIL-1 activities in each of the macrophage populations tested. These activities were generally enhanced by pretreating the bacteria with antibody, with the greatest enhancing activity observed for the formalin-killed preparations at lower doses of bacteria.
Adhesion of Helicobacter pylori to gastric epithelial cells in primary cultures obtained from stomachs of various animals
Authors: Kobayashi Y , Okazaki K , Murakami K
Published: January 2020

Of 35 strains of Helicobacter pylori tested, 5 were found to adhere well to HEp-2 cells. We selected three of these adhesive strains and four from the remaining strains to examine their ability to adhere to gastric epithelial cells in primary cultures obtained by collagenase digestion of stomachs from mice, rats, Mongolian gerbils, guinea pigs, pigs, and cynomolgus monkeys. The three adhesive strains adhered well to epithelial cells from monkey and pig gastric antra. The adhesion was inhibited by incubating the bacterial cells with fetuin, and this inhibition was further confirmed by the binding of gold-labeled fetuin to the surface of the adhesive strains. However, these adhesive strains only weakly adhered to fundic epithelial cells from monkeys and pigs and to gastric epithelial cells from the other animals. As for the four strains poorly adhesive to HEp-2 cells, they adhered weakly to gastric epithelial cells from all of the animals tested. They had higher hemagglutination titers than the adhesive strains, showing that there was no correlation between hemagglutination titers and the ability to adhere to gastric cells. Taking the similarities of human and monkey or pig stomachs into consideration, these results suggest that the primary target cell of H. pylori in colonization in human stomachs is the antral epithelial cell and that the putative adhesin involved in adhesion has affinity for fetuin.
Modulation of adjuvant arthritis in Lewis rats by recombinant vaccinia virus expressing the human 60-kilodalton heat shock protein
Authors: López-Guerrero J A , López-Bote J P , Ortiz M A , Gupta R S , Páez E , Bernabeu C
Published: January 2020

The immune response to the mycobacterial 65-kDa heat shock protein (hsp65) is considered an important event in the induction of adjuvant arthritis (AA) in rats; this induction probably occurs through a molecular mimicry mechanism involving cross-reactivity against the rat homolog hsp60. To analyze the role of mammalian molecule hsp60 in arthritis, we generated a recombinant vaccinia virus (hsp60-VV) carrying the human hsp60 gene inserted into the thymidine kinase locus under the control of the 7.5k vaccinia virus promoter. Human hsp60 is almost identical to its rat homolog (97.4% linear amino acid homology) and shares about 50% of amino acid positions with Mycobacterium tuberculosis hsp65. The latter supposedly carries a critical epitope for AA induction that is not present in human hsp60. Infections with hsp60-VV of monkey cell cultures led to the expression of the human hsp60 molecule, as evidenced by immunoblotting analysis with specific monoclonal antibodies. Also, Lewis rats infected with hsp60-VV produced specific antibodies, demonstrating the in vivo expression of human hsp60 in the infected animals. Therefore, we used hsp60-VV to analyze whether the delivery of hsp60 could affect the induction of AA in Lewis rats. hsp60-VV clearly reduced and retarded arthritic symptoms when administered to rats at day 7 after AA induction. In contrast, inoculation of rats with a control recombinant vaccinia virus did not affect the course of the disease. The improvement in AA with hsp60-VV administration was associated with a specific immune response, as determined by the presence of antibodies to hsp60 in the sera and the proliferation induced by hsp60 of T cells from popliteal lymph nodes. These results support a critical role for immunity to heat shock proteins in AA. Since the protective construct is virtually identical to rat homolog hsp60, we conclude that immunity directed to conserved areas of this family of proteins is directly involved in the pathogenesis of AA.
Optimizing oral vaccines: induction of systemic and mucosal B-cell and antibody responses to tetanus toxoid by use of cholera toxin as an adjuvant
Authors: Jackson R J , Fujihashi K , Xu-Amano J , Kiyono H , Elson C O , McGhee J R
Published: January 2020

Cholera toxin (CT) is an effective mucosal antigen and acts as an adjuvant when given orally with various antigens; however, few studies have compared the levels of antibody responses to CT and coadministered protein in systemic and mucosal tissues. In this study, we used tetanus toxoid (TT) for assessment of immune responses. Time course and dose-response studies established that 250 micrograms of TT given orally with 10 micrograms of CT three times at weekly intervals induced high serum and gastrointestinal tract anti-TT and anti-CT antibody responses. Oral immunization with TT alone induced no detectable mucosal immunoglobulin A (IgA) antibodies in fecal extracts and only weak serum IgG anti-TT responses. The coadministration of CT and TT induced peak serum IgG anti-TT responses following two oral doses that remained constant after the third oral immunization, while optimal mucosal IgA responses were seen after the third oral immunization. The serum anti-TT response obtained with CT and TT proved protective against TT challenge (100 minimum lethal doses), whereas mice orally given CT or TT alone died. Antigen-specific B-cell responses were assessed with an isotype-specific Elispot assay of isolated lymphoid cells from the spleen, Peyer's patches, and the small intestinal lamina propria. Interestingly, approximately fourfold-higher numbers of IgA anti-CT than of anti-TT antibody-producing (spot-forming) cells occurred in lymphocytes from the lamina propria of mice orally immunized with both TT and CT. The adjuvant CT did not induce polyclonal B-cell responses in mice given CT by the oral route, since no significant differences in total numbers of B cells producing IgA, IgG, or IgM were found compared with the numbers in mice given TT alone. The results clearly indicate that serum and mucosal antibody responses develop with different kinetics and that protective TT-specific antibody responses are generated in the systemic compartment when TT is administered with CT via the oral route.
Reproduction of porcine proliferative enteropathy with pure cultures of ileal symbiont intracellularis
Authors: McOrist S , Jasni S , Mackie R A , MacIntyre N , Neef N , Lawson G H
Published: January 2020

Porcine proliferative enteropathy is consistently associated with the presence of intracellular curved bacteria in epithelial cells in affected portions of intestine. Two strains of these intracellular bacteria were cultured in a cell culture system with rat enterocytes (IEC-18) and passaged several times and used as oral inocula for 14 gnotobiotic and 8 conventional pigs. DNA and immunological studies had identified these bacteria as belonging to a new taxon, Ileal symbiont (IS) intracellularis. Conventional pigs dosed with approximately 3.7 x 10(6) of these organisms passaged six times in cell culture developed severe lesions of proliferative enteropathy in the ileum. Other conventional pigs dosed with a lower titer or with organisms passaged 13 times developed moderate and minor lesions, respectively. All gnotobiotic pigs dosed with organisms failed to develop lesions. Control pigs, eight conventional and two gnotobiotic, dosed with diluent, uninfected cell material or left undosed failed to develop lesions also. Reisolation of IS intracellularis and demonstration of the organism in mucosal and fecal samples only occurred in conventional pigs dosed with organisms. Gnotobiotic pigs lacking a normal intestinal flora have not been shown to be colonized by the organism. Seroconversion to IS intracellularis or mucosal infiltration by inflammatory cells was not observed in experimentally affected pigs, confirming the weak immune response characteristic of the natural disease. These results support the identification of IS intracellularis as an etiological agent of proliferative enteropathy in pigs.
Activation of the complement system in baboons challenged with live Escherichia coli: correlation with mortality and evidence for a biphasic activation pattern
Authors: de Boer J P , Creasey A A , Chang A , Roem D , Eerenberg A J , Hack C E , Taylor F B
Published: January 2020

Activation of the complement system was studied in baboons that were challenged with live Escherichia coli. In the group challenged with a lethal dose (n = 4), the complement activation parameters C3b/c, C4b/c, and C5b-9 increased 13, 5, and 12 times the baseline value, respectively, during the first 6 h after the E. coli infusion, whereas in the group challenged with a sublethal dose (n = 10), they increased only moderately, by 2 to 3 times the baseline value. However, in this latter group, a more pronounced activation occurred at 24 h. Subsequent experiments showed that this second phase in complement activation started at 6 h after the challenge, at which time infused microorganisms had been cleared from the circulation. The simultaneous increase in C-reactive protein with this second phase suggested an endogenous activation mechanism involving this acute-phase protein. Levels of inactivated (modified) C1 inhibitor also increased in both groups, with peak levels of 2.5 times the baseline value at 24 h in the sublethal group and of 4 times at 6 h after the challenge in the lethal group. Thus, activation of complement in this animal model for sepsis occurs in a biphasic pattern, the initial phase mediated by the bacteria and the later phase mediated by an endogenous mechanism possibly involving C-reactive protein. The differences in complement activation between animals with lethal or sublethal sepsis support the hypothesis that complement activation contributes to the lethal complications of sepsis.
Expression and secretion of an Arthrobacter dextranase in the oral bacterium Streptococcus gordonii
Authors: Kubo S , Kubota H , Ohnishi Y , Morita T , Matsuya T , Matsushiro A
Published: January 2020

We have constructed a plasmid to express and secrete dextranase in the oral bacterium Streptococcus gordonii. The dextranase gene from Arthrobacter sp. strain CB-8 was linked to a promoter and a DNA sequence encoding the signal peptide of Streptococcus downei glucosyltransferase I (gtfI) followed by the Escherichia coli rrnBt1t2 terminator and inserted in the shuttle vector pVA838. S. gordonii transformed with this plasmid (pMNK-4) expressed and secreted mature Arthrobacter dextranase. The transformant was found to repress the firm adherence of water-insoluble glucan in a coculture experiment with cariogenic bacteria, Streptococcus sobrinus, in the presence of sucrose. Such genetically engineered oral bacteria could provide a therapy to prevent dental caries.