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S C Holt

Publications and source records attributed to S C Holt.

At least 55 records · Page 3Linked to original sources
PubMed1996

Characteristics of systemic antibody responses of nonhuman primates following active immunization with Porphyromonas gingivalis, Prevotella intermedia and Bacteroides fragilis.

Periodontal disease is an infectious disease manifested by the progressive change of a healthy resident commensal microbiota to a pathogenic one characterized by a specific microbiota. Thus, the prospect for the use of selected bacteria or their antigens as a vaccine to interfere with the microbial changes and resulting progression of periodontal tissue destruction has been proposed. As a first step in examining the use of bacterial antigens as immunogens in periodontitis, this study characterized the humoral immune response in Macaca fascicularis after systemic immunization with intact Porphyromonas gingivalis, Prevotella intermedia and Bacteroides fragilis. Parental immunization of the nonhuman primate with the intact bacteria resulted in the production of specific and significantly elevated levels of antibodies to P. gingivalis and P. intermedia, with the predominant isotype being immunoglobulin G (IgG). In contrast, the principal response to the nonoral, intestinal bacterium, B. fragilis, was of the IgM isotype. Immunization increased IgG, IgM, and IgA antibody by 14-227 fold to P. gingivalis and 8-108 fold to P. intermedia. The level of serum IgA antibody increased (77-227 fold). The kinetics of the antibody response post-immunization and post-ligation differed with respect to each of the bacteria tested. IgG antibody to P. gingivalis increased through week 16 of the experiment and remained elevated above baseline through week 32. The IgG antibody level to P. intermedia peaked at 4 weeks following the third immunization and decreased post-ligation to near baseline levels by week 16. Characterization of the immune response after active immunization in the nonhuman primate has demonstrated a substantial and specific increase in antibody response which was sustained for several weeks. The insights obtained from these studies should help optimize the potential for immunologic interference with progressing periodontitis.

Analysis of Variance↗
PubMed1996

Mixed infection with Porphyromonas gingivalis and Fusobacterium nucleatum in a murine lesion model: potential synergistic effects on virulence.

These studies determined the characteristics of tissue destruction in a murine abscess model elicited by mixed infection with the periodontopathogens Fusobacterium nucleatum and Porphyromonas gingivalis. The interbacterial effects of this synergism, the kinetics of the relationship of the bacterial interaction, and the characteristics of the bacteria required for the tissue destruction were studied. Infection of mice with P. gingivalis and F. nucleatum strains elicited lesions of various sizes as a function of infective dose. Primary infection with F. nucleatum plus P. gingivalis at various ratios (i.e., <1:1) resulted in a significantly greater lesion size (P < 0.001) compared with that resulting from primary infection with P. gingivalis alone. At F. nucleatum/P. gingivalis ratios of > or = 1:1, spreading lesion formation and progression were significantly (P < 0.001) decreased, suggesting that bacterial interaction (i.e., coaggregation) may have inhibited the spread of the P. gingivalis infection to a site distant from the initial injection. Infection with F. nucleatum and P. gingivalis simultaneously (at different sites) or F. nucleatum administered within 4 h prior to or 1 h following P. gingivalis infection significantly enhanced the ability of P. gingivalis to form large phlegmonous lesions. Chemical inhibition of the P. gingivalis trypsin-like protease activity or the use of a trypsin-negative P. gingivalis strain abrogated tissue destruction either alone or in combination with F. nucleatum. Therefore, it was possible to examine aspects of virulence of these pathogens in a murine lesion model by either altering bacterial ratios, manipulating the time of infection, or targeting vital bacterial virulence factors.

Animals↗
PubMed1996

Yolk sac number, size and morphologic features in monochorionic monoamniotic twin pregnancy.

OBJECTIVE: To determine the relation between three characteristics of the yolk sac (number, size and morphologic features) and outcome in monochorionic monoamniotic twin pregnancy. METHODS: The authors reviewed data for the four sets of monochorionic monoamniotic twins detected by first-trimester ultrasonography between January 1990 and June 1994 at their institution. Data analysed included yolk sac number, size and morphologic features, as well as the outcome of the pregnancy. RESULTS: In all four sets of twins, only one yolk sac was identified. In one case the yolk sac was irregular in contour, and in two it was abnormally large. Two of the four mothers delivered healthy twins at 34 weeks gestational age, one had conjoined twins ( and underwent elective termination of the pregnancy), and one had a twin ectopic pregnancy (and underwent salpingectomy). CONCLUSION: A single yolk sac in cases of monochorionic monoamniotic twins may be a normal finding.

Adult↗
PubMed1995

Cell envelope and cell wall immunization of Macaca fascicularis: effect on the progression of ligature-induced periodontitis.

The nonhuman primate, Macaca fascicularis, was used to study the role of immunization with selected members of the periodontopathic microbiota in the longitudinal progression of ligature-induced periodontitis. Animals were immunized with cell envelope antigens prepared from Porphyromonas gingivalis and Prevotella intermedia, and a mixture prepared from Fusobacterium nucleatum, Campylobacter rectus, and Actinomyces viscosus. Serum immunoglobulin G (IgG), IgM and IgA isotype antibodies increased significantly in all immunization groups and were specific for each of the immunogens. P. gingivalis and P. intermedia immunization resulted in a stabilization of the proportions of these species throughout most of the experiment. The high P. gingivalis antibody titer resulted in low P. gingivalis numbers being recovered. P. gingivalis immunization, while lowering recoverable viable P. gingivalis, resulted in significantly increased levels of Prevotella loescheii, Prevotella buccae, Bacteroides macacae and Prevotella melaninogenica compared with preligation and preimmunization levels. Actinobacillus actinomycetemcomitans, Capnocytophaga spp. and Eikenella spp. remained at preligation levels postimmunization. Campylobacter spp. increased significantly during the course of the experiment in all groups, whereas the levels of Fusobacterium spp. decreased. Plaque indices and bleeding on probing showed significant increases in all groups following ligation, with the placebo group showing the greatest increase. Pocket depth measurements revealed that , whereas the placebo animals showed an approximate 5% increase, the P. gingivalis- and P. intermedia-immunized groups showed nearly a 20% increase in pocket depth. Attachment level measurements showed significantly greater attachment loss in the P. gingivalis- and P. intermedia-immunized groups, and the F. nucleatum + C. rectus + A. viscosus immunization appeared to prevent significant changes in pocket depth/attachment level loss. Radiographic measurement of bone loss by computer-assisted densitometric image analysis revealed that the placebo group lost bone throughout the experiment. P. gingivalis- and P. intermedia-immunized groups showed an exacerbated loss of bone density and the group immunized with F. nucleatum + C. rectus + A. viscosus exhibited significantly lower amounts of bone loss when analyzed by computer-assisted densitometric image analysis, compared with the other immunized groups. Although immunization with P. gingivalis and P. intermedia cell envelope antigens had an effect on their emergence in the complex microbiota of the developing periodontal pocket, this immunization also resulted in greater bone loss than immunization with F. nucleatum + C. rectus + A. viscosus, suggesting that, whereas selective members of the putative periodontopathic microbiota may play a direct role in periodontal tissue destruction, the complexity of the subgingival microbiota dictates that considerable scrutiny is required to select useful immunogens that can elicit functional protection from periodontal tissue destruction induced by oral microorganisms that already colonize or infect the host.

Actinomyces viscosus↗
PubMed1995

Comparative virulence of periodontopathogens in a mouse abscess model.

OBJECTIVE(S): This report compares the virulence of selected strains of P. gingivalis, A. actinomycetemcomitans, C. rectus, F. nucleatum and T. denticola in a murine model as a measure of pathogenic potential of these oral microorganisms. The characteristics of the tissue destruction associated with these monoinfections were then related to a potential model for bacterial synergism in progressing periodontitis. DESIGN AND METHODS: All bacterial strains were grown to mid-logarithmic to early stationary growth phase, harvested and used at various doses to challenge BALB/c normal and BALB/c dexamethasone (DEX) treated mice to mimic a neutrophil dysfunction. The characteristics of tissue destruction, and overt tissue destructive capacity of these species were examined as a function of challenge dose and time. OUTCOME MEASURES: The mice were examined for an interval of approximately 15 days post-challenge and the presence/absence of lesions, localized or generalized nature of the lesion (including size in mm2), and lethality of the infection were assessed. RESULTS: Comparison of the virulence of the various P. gingivalis strains related to lethality and lesion size associated with destruction of the connective tissue, indicated a virulence capacity of P. gingivalis strains 53977>W50 = T22>3079>33277>381. C. rectus elicited localized necrotic lesions which were limited to the epithelial layers of the skin. The size of the lesions also indicated a graded difference in virulence, such that C. rectus strains 234>576>>33238. A. actinomycetemcomitans caused the formation of classic localized abscesses with a PMN infiltrate and inflammatory exudates. Although each of the A. actinomycetemcomitans strains exhibited a similar virulence pattern in this murine model, A. actinomycetemcomitans serotype b representative strains were potentially more pathogenic with a virulence capacity of 3113D-N = 3975A>JP2 > or = Y4>29523>33384. Both C. rectus and A. actinomycetemcomitans strains showed clear evidence that recent clinical isolates were more virulent than laboratory strains. Challenge with F. nucleatum resulted in tissue destructive responses which were different from those observed with the other strains used in this study. A rapid onset of dose-dependent lesion development, related to the formation of either closed abscesses or open lesions, was observed with F. nucleatum. Tissue involvement was also greater at lower F. nucleatum doses when compared to the other bacteria. F. nucleatum challenge of DEX-treated mice resulted in a shift to open lesions. T. denticola appeared to be more tissue invasive than the other species examined in this study. Challenge of mice with T. denticola resulted in involvement of multiple tissues, including epithelial and connective tissues, as well as appearing to invade muscle layers and deeper tissues. In addition to invading deeper tissues, the resulting lesions took considerably longer to resolve. In the DEX-treated mice (neutrophil depleted), P. gingivalis, C. rectus, and A. actinomycetemcomitans were significantly more virulent. In contrast, while DEX treatment altered the characteristics of lesions caused by F. nucleatum, the extent of lesions produced by F. nucleatum and T. denticola was not substantially enhanced. CONCLUSIONS: The results obtained from this study suggest that different microorganisms have the ability to provide individual pathologies which may act in an additive/synergistic fashion contributing to the tissue destruction noted in periodontitis.

Abscess↗
PubMed1995

The 46-kilodalton-hemolysin gene from Treponema denticola encodes a novel hemolysin homologous to aminotransferases.

The 46-kDa hemolysin produced by Treponema denticola may be involved in the etiology of periodontitis. In order to initiate a genetic analysis of the role of this protein in disease, its gene has been cloned. Synthetic oligonucleotides, designed on the basis of the previously reported amino-terminal amino acid sequence of the 45-kDa hemolysin, were used as primers in a PCR to amplify part of the hemolysin (hly) gene. This PCR product was then used to clone the entire hly gene from libraries of T. denticola genomic DNA. Constructs containing the entire cloned region on plasmids in Escherichia coli produced both hemolysis and hemoxidation activities either on sheep blood agar plates or in liquid assays. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot (immunoblot) analysis revealed that the constructs synthesized a protein with molecular size of about 46 kDa which was reactive with anti-T. denticola hemolysin. Nucleotide sequence analysis indicated that the largest open reading frame could encode a protein with a calculated molecular size of 46.2 kDa. The first 31 amino acids encoded by this open reading frame were identical to the experimentally determined amino-terminal sequence of the 45-kDa hemolysin. These results indicate that the entire hly gene has been cloned. The deduced amino acid sequence of the T. denticola hly gene is homologous (23 to 37% identity) to those of proteins that are members of a family of pyridoxal-phosphate-dependent aminotransferases. This suggests that the 46-kDa hemolysin may be related to an aminotransferase and have a novel mechanism of hemolysis. However, the functional aspects of this relationship remain to be investigated.

Amino Acid Sequence↗
PubMed1994

Characterization of hemolysis and hemoxidation activities by Treponema denticola.

This study determined the presence of two hemolytic activities in the oral treponeme, Treponema denticola, strains ATCC 35404 (TD-4), ATCC 33520, GM-1, and MS25. These activities, referred to as hemolytic and hemoxidative (HeA, HeO, respectively), were found to be both secreted into the extracellular environment, and cell associated. The extracellular activity was associated with small molecules with relative molecular weights of < 1000 Da, and its activity was cysteine independent; the cell-associated HeA and HeO activities were associated with a molecular weight fraction > 10 kDa, and were cysteine dependent. The HeO activity of the fractionated material observed was due to the oxidation of hemoglobin to methemoglobin, and preceded the HeA lysis of the RBCs by approximately 2 h. Heating at 80 degrees C and treatment with proteinase K resulted in the complete destruction of these activities in the fraction > 10 kDa, while lipase at high concentration (800 micrograms/ml) reduced the HeA and HeO activities in the extracellular fraction by approximately 50%. Proteinase inhibitors had a variable effect on HeA and HeO activities in both extracellular and cell-associated fractions. Scanning and transmission electron microscopy revealed a progressive destruction of the RBC membrane, with membrane protrusions formed early in the interaction, which progressed to irregular holes in the membrane, and the complete loss of membrane integrity.

Erythrocytes↗
PubMed1994

Purification and characterization of a 45 kDa hemolysin from Treponema denticola ATCC 35404.

A 45 kDa polypeptide capable of erythrocyte (RBC) lysis and hemoglobin oxidation was isolated from Treponema denticola, ATCC 35404 (TD-4) after sequential ammonium sulfate (2.8-3.6 M) precipitation and preparative electrophoresis. The purified polypeptide produced a single protein band on PAGE at a relative molecular weight of 45 kDa in the presence and absence of SDS. The polypeptide was sensitive to proteinase K and pronase, and heating at 80 degrees C. The protease inhibitors, PMSF, TLCK and benzamidine had no inhibitory affect on activity. It was non heat-modifiable, and lost all hemolytic and hemoxidative function in SDS. Cysteine and other sulfhydryl-containing compounds were required for hemolytic and hemoxidative activities. The isoelectric point of the polypeptide was 5.3 and N'-terminal sequence analysis indicated it to belong to a new, so far undescribed group of peptides possessing hemoxidation and hemolytic activities. Functionally, it was capable of rapid hemoxidation of sheep and human erythrocytes (hemoglobin to methemoglobin) coupled to erythrocyte lysis, or hemolysis.

Amino Acid Sequence↗
PubMed1994

Effect of iron regulation on expression and hemin-binding function of outer-sheath proteins from Treponema denticola.

The effect of the iron-chelating compounds EDDA and BPD on polypeptide regulation in the putative oral pathogen Treponema denticola was studied. SDS-PAGE analysis of the T. denticola strains grown in the presence of EDDA or BPD, i.e. iron-limiting environmental conditions, revealed the expression of 44 and 43 kDa polypeptides in the outer sheath, a 73 kDa polypeptide in the cell membrane, and a 16 kDa polypeptide in the soluble cell fraction. The hemin-binding activity of purified outer sheaths from T. denticola TD-4 grown in the presence of 6.4 mM EDDA was significantly greater than that observed in control (absence of EDDA) outer sheaths. Both activities were inhibited by proteinase K. SDS-PAGE, LDS-PAGE and TMBZ staining revealed the 44 and 43 kDa outer-sheath polypeptides to be expressed by T. denticola strains GM-1. MS-25, ATCC 33520 and ATCC 33404 (TD-4), strains which possessed strong hemin-binding activity. The 44 kDa hemin-binding polypeptide was purified by 1% CHAPS solubilization, HPLC, and SDS-preparative electrophoresis. N'-terminal sequence analysis indicated the purified 44 kDa polypeptide to belong to a new, undescribed group of polypeptides possessing hemin-binding activity.

2,2'-Dipyridyl↗
PubMed1994

Cloning and expression of hemolysin genes from Treponema denticola strains ATCC 35404 (TD-4) and human clinical isolate GM-1 in Escherichia coli.

The oral spirochete, Treponema denticola is a putative etiologic agent in adult periodontitis, and acute necrotizing ulcerative gingivitis. In vitro, the oral treponeme produces several factors including proteases, hemolysins, hemin-binding proteins, which could potentially be involved in the virulence of this spirochete. Our laboratory has been investigating the pathobiology of T. denticola, and has demonstrated the production of several hemolysins by T. denticola. In this report two hemolysin genes from T. denticola strains ATCC 35404 (TD-4) and GM-1 were isolated by screening genomic DNA libraries of T. denticola on sheep blood agar plates. Physical maps of the insert fragments were not identical. Southern blot analyses suggested some degree of homology in the nucleotide sequence. Maxicell analyses of [35S]-methionine-labeled polypeptides from the recombinant plasmids have suggested the synthesis of an approximately 62.5 kDa polypeptide. Biochemical characterization of the T. denticola hemolysin genes indicated the activity to be inhibited by Mg2+, Ca2+ and Zn2+ but not by EDTA. Dithiothreitol and glutathione moderately enhanced the hemolytic activity of the recombinant plasmids. Iron partially inhibited the hemolytic activities. Addition of 2-2' bipyridyl moderately enhanced the activities, possibly by iron limitation. These results suggest the isolation of an identical hemolysin gene from T. denticola strains TD-4 and GM-1.

Animals↗
PubMed1994

Coaggregation between bacterial species.

Bacterial coaggregation, or interbacterial adherence, is one mechanism involved in the development of bacterial biofilms that are found on surfaces in nature. Assays used to measure coaggregation rely on the interaction of bacterial cells in suspension or attachment of one species to a second species that has been fixed to a solid substrate. Both semiquantitative and quantitative assays are described. These methods have also been used to determine the nature of the adherence and molecules involved in mediating the interaction, to characterize potential inhibitors, to isolate the bacterial adhesins and receptors, and to isolate adherence-deficient mutant strains. Each of the assay systems offers different advantages, with significant variations in sensitivity. Selection of a particular assay system should depend on the goals of the study to be performed.

Actinomyces↗
PubMed1994

Identification of lactoferrin-binding proteins from Treponema pallidum subspecies pallidum and Treponema denticola.

Lactoferrin-binding or -associated proteins were identified in Treponema pallidum subspecies pallidum and Treponema denticola by affinity column chromatography using human lactoferrin and detergent-solubilized, radiolabelled spirochaetes. Two discrete polypeptides of T. pallidum with masses of 45 and 40 kDa and a broad band from 29-34 kDa exhibited association with human apo- and partially ferrated lactoferrin. T. denticola produced two proteins that associated with a lactoferrin affinity matrix (50 and 35 kDa). T. pallidum and T. denticola did not associate with soluble, human transferrin in parallel experiments. Soluble human lactoferrin competed with all lactoferrin-associated proteins from T. pallidum and T. denticola in competitive-binding assays. However, the T. denticola proteins dissociated from a lactoferrin-affinity matrix in the presence of differing concentrations of unlabelled, soluble lactoferrin competitor. Treatment with phospholipase D altered migration of the diffuse 29-34 kDa band of T. pallidum suggesting that the polypeptide was lipid-modified. Each of the lactoferrin-binding proteins from T. pallidum and T. denticola reacted with pooled rabbit syphilitic antisera. The lactoferrin-binding proteins of T. pallidum reacted with human sera from patients at all stages of syphilis. In addition, a monoclonal antibody generated against the 45 kDa polypeptide of T. pallidum crossreacted with the 29-34 kDa protein.

Antibodies, Bacterial↗
PubMed1994

Host responses in patients with generalized refractory periodontitis.

Although patients with refractory periodontitis have been widely reported, no clear biologic profile of these patients has been noted. The purpose of the present study was to evaluate host responsiveness of a well-defined group of refractory periodontitis patients by determining the effect of a lipopolysaccharide (LPS) challenge on monocyte surface receptor density and on the release of inflammatory mediators. Venous blood was obtained from 7 refractory periodontitis, 8 stable periodontal maintenance, and 8 gingivitis patients with no evidence of periodontitis. Mononuclear cells were cultured in either control media or media treated with Actinobacillus actinomycetemcomitans (Aa), Porphyromonas gingivalis (Pg), or Salmonella typhimurium (S. typh) LPS. At 0 and 24 hours supernatants were assayed for prostaglandin-E2 (PGE2) and interleukin-1 beta (Il-1 beta) release by ELISA. Using flow cytometry the density of specific monocyte surface receptors were assayed with Mo3e and LeuM3 monoclonal antibodies (mAb); T-cell CD4/CD8 ratios were assayed with OKT-3, OKT-4, and OKT-8 mAb. After 24 hours incubation with Pg or S. typh LPS, the upregulation of the Mo3e receptor was significantly decreased for refractory periodontitis patients (P < 0.05) when compared to gingivitis and to stable maintenance patients. In refractory periodontitis patients the T-cell CD4/CD8 ratio was decreased. Upon stimulation with Pg or S. typh LPS, monocytes from stable maintenance and refractory periodontitis patients released more Il-1 beta (P < 0.05) and PGE2 (P = 0.13 and 0.15) than monocytes from gingivitis subjects.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1993

Cloning of two distinct hemolysin genes from Porphyromonas (Bacteroides) gingivalis in Escherichia coli.

Hemolysin is considered a potent virulence factor in a large number of Gram-positive and Gram-negative bacterial pathogens. The hemolysin produced by the oral pathogen Porphyromonas gingivalis functions to provide the cell with its required heme-containing molecules for growth in the periodontal pocket. Two distinct P. gingivalis genes, each of which confers a hemolytic phenotype in Escherichia coli, were isolated by screening genomic DNA libraries of P. gingivalis on sheep blood agar plates. The results obtained from physical maps and Southern blots indicated a considerable degree of divergence in the nucleotide sequences of these two genes. Maxicell analyses of the recombinant plasmids in E. coli suggested that plasmid pPGH5 encoded a polypeptide of molecular weight 48 kDa, while an 18-kDa polypeptide was obtained with pPGH1 and pPGH7. When E. coli harboring these hemolysin genes were subjected to iron starvation, the levels of hemolysin activity increased. Biochemical characterization of hemolytic activities indicated that the activity of both hemolysins was inhibited by Mg2+ and Ca2+; but not by EDTA. Elevated levels of hemolytic activity were obtained from the E. coli recombinant strains in the presence of glutathione, DTT and 2-mercaptoethanol. Cholesterol inhibited the activity.

Bacterial Proteins↗
PubMed1993

Properties of adhering and nonadhering populations of Mycoplasma genitalium.

The interaction between Mycoplasma genitalium and human lung fibroblasts (HLFs) was studied with the use of wild-type and hemadsorption-negative (HA-) mycoplasmas. [35S]-methionine-labeled M. genitalium adhered to HLFs by first-order kinetics, with maximal interaction occurring at approximately 2 hours. Electron microscopy of chemically fixed cells revealed an almost immediate association of mycoplasmas with the HLF plasma membrane that was mediated by the mycoplasma tip and a nap-like layer, which appeared to extend from the tip around much of the mycoplasmal unit membrane. Following cytadherence, M. genitalium appeared capable of invading the intracellular spaces of targeted HLF cells, possibly by receptor-mediated endocytosis. Spontaneously arising HA- variants of M. genitalium strain G37 failed to adhere to HLF cells and were distinguished on the basis of their protein profiles. SDS-PAGE analysis of the class I (lacking the 140-kd protein but containing a polypeptide doublet at approximately 140-kd) and class II (lacking the 140-kd protein and doublet) variants of M. genitalium revealed that class I variants contain a doublet protein in the 140-kd region, which reacted with a monoclonal antibody generated to the adhesin-implicated 140-kd protein (P140) of wild-type M. genitalium. Class II variants completely lacked the 140-kd protein or immunologically related peptides.

Bacterial Adhesion↗