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Biomedical subjects

S C Holt

Publications and source records attributed to S C Holt.

At least 19 recordsLinked to original sources
PubMed2000

Crystal structure of cystalysin from Treponema denticola: a pyridoxal 5'-phosphate-dependent protein acting as a haemolytic enzyme.

Cystalysin is a C(beta)-S(gamma) lyase from the oral pathogen Treponema denticola catabolyzing L-cysteine to produce pyruvate, ammonia and H(2)S. With its ability to induce cell lysis, cystalysin represents a new class of pyridoxal 5'-phosphate (PLP)-dependent virulence factors. The crystal structure of cystalysin was solved at 1.9 A resolution and revealed a folding and quaternary arrangement similar to aminotransferases. Based on the active site architecture, a detailed catalytic mechanism is proposed for the catabolism of S-containing amino acid substrates yielding H(2)S and cysteine persulfide. Since no homologies were observed with known haemolysins the cytotoxicity of cystalysin is attributed to this chemical reaction. Analysis of the cystalysin-L-aminoethoxyvinylglycine (AVG) complex revealed a 'dead end' ketimine PLP derivative, resulting in a total loss of enzyme activity. Cystalysin represents an essential factor of adult periodontitis, therefore the structure of the cystalysin-AVG complex may provide the chemical basis for rational drug design.

Binding Sites↗
PubMed2000

Gingival crevicular fluid inflammatory mediators and bacteriology of gingivitis in nonhuman primates related to susceptibility to periodontitis.

The hypothesis to be tested was that the microbiota and resulting local host inflammatory response characteristics in oral conditions of high levels of chronic gingival inflammation increases susceptibility to progressing periodontitis. This study used cynomolgus monkeys, Macaca fascicularis (nonhuman primates), with high and low levels of long-standing gingival inflammation to define the profiles of gingival crevicular fluid mediators, cytokines and immunoglobulins; describe the subgingival microbiota; and evaluate their susceptibility to ligature-induced periodontitis. Sixteen nonhuman primates were stratified into two groups (HI, LO) based upon Bleeding Index as a measure of the natural level of inflammation (HI = 1.26 +/- 0.45; LO = 0.22 +/- 0.16). The host mediator levels, subgingival microbiota, and clinical characteristics of the LO and HI groups were compared after 30 days of oral hygiene, during a 30 day experimental gingivitis (7, 14, and 30 days), and during periodontitis (30, 60, and 90 days). The results demonstrated that nonhuman primates with high levels of long-standing gingival inflammation when compared to those nonhuman primates with low inflammation show: 1) different inflammatory mediator profiles in gingival crevicular fluid (particularly for immunoglobulin A (IgA) and IgG levels), 2) a different quantitative and qualitative subgingival microbiota; and 3) a similar progression of periodontitis. Thus, while variations in host inflammatory responses to local factors exist in the nonhuman primates, an extensive subgingival challenge (such as ligation) may negate these individual differences.

Animals↗
PubMed2000

Porphyromonas gingivalis induction of mediator and cytokine secretion by human gingival fibroblasts.

We hypothesized that bacterial viability and strain characteristics of Porphyromonas gingivalis could affect the induction of pro-inflammatory mediator secretion by human gingival fibroblast cultures. Both killed and viable P. gingivalis elicited production of prostaglandin E2, interleukin-1 beta (IL-1 beta), IL-6 and IL-8, although killed P. gingivalis induced generally higher levels, particularly IL-6 and IL-8, compared with the viable bacteria. P. gingivalis strains, which exhibited wild-type levels of trypsin-like protease activity, stimulated human gingival fibroblasts to secrete increased levels of prostaglandin E2 and IL-1 beta, although minimal levels of IL-6 and IL-8 were noted in supernatants from the gingival fibroblast cells. P. gingivalis strains BEI and NG4B19, which have either decreased or undetectable levels of trypsin-like protease, respectively, induced significantly greater IL-6 and IL-8 levels in gingival fibroblast cultures compared with the other strains. The ability of antibody to P. gingivalis to alter human gingival fibroblast production of pro-inflammatory mediators was tested using nonhuman primate antisera. Both immune and nonimmune sera altered the P. gingivalis-generated pattern of mediators from the gingival fibroblasts. We conclude that: (i) viable and killed P. gingivalis were capable of inducing various pro-inflammatory cytokines from human gingival fibroblasts; (ii) strain differences in cytokine induction were noted, and the expression of a trypsin-like protease activity was related to decreased extracellular levels of IL-6 and IL-8; and (iii) the presence of serum, particularly with specific antibody to P. gingivalis, significantly altered human gingival fibroblast cytokine production compared with P. gingivalis alone.

Animals↗
PubMed2000

Transfusion-related sepsis due to Serratia liquefaciens in the United States.

BACKGROUND: Severe, often fatal, transfusion reactions due to bacterial contamination of blood components continue to occur. Serratia liquefaciens, an unusual human pathogen, is a recently recognized potential cause of transfusion-related sepsis. CASE REPORTS: Five episodes of transfusion-related sepsis and endotoxic shock due to S. liquefaciens were reported to the CDC from July 1992 through January 1999. One episode has been described. The remaining four, all fatal, are described here: three associated with RBC transfusion and one associated with transfusion of platelets. In each instance, the source of contamination could not be found. The implicated units tended to be older (mean RBC age 28 days), and visual discoloration was noted in each RBC unit, although usually in retrospect. CONCLUSION: S. liquefaciens is an increasingly recognized cause of transfusion-related sepsis and is associated with a high mortality rate. S. liquefaciens can contaminate both RBCs and platelets, but the mechanism(s) of contamination remain unknown. Increased attention to pretransfusion visual inspection may avert the transfusion of some S. liquefaciens-contaminated RBC units. However, more sensitive rapid diagnostic tests are needed to further reduce the risk of transfusion-related sepsis and endotoxic shock.

Aged↗
PubMed2000

Effects of 0.12% chlorhexidine gluconate on experimental gingivitis in non-human primates: clinical and microbiological alterations.

OBJECTIVE: This study examined the efficacy of 0.12% chlorhexidine gluconate (Peridex) to reduce gingival inflammation in the absence of mechanical hygiene and its effect on the oral microbial ecology in a non-human primate (NhP) model of gingivitis. DESIGN: Twelve NhP were stratified based on existing inflammation into two groups of six NhP per group. Oral hygiene was performed on both groups so as to reach a level of gingival health (BOP < or = 0.3) at the conclusion of the hygiene phase. One group received 30 ml of 0.12% chlorhexidine gluconate twice daily 7 days/week, and a second group received 30 ml of placebo (distilled water colored to match the active) using the same regimen for 10 weeks. MEASUREMENT OUTCOMES: Clinical parameters including plaque (PLI), pocket depth (PD), attachment level (AL), and bleeding on probing (BOP) were evaluated at 2-week intervals. Subgingival plaque samples were collected by paper point at 2-week intervals and cultured for predominant cultivable bacteria. RESULTS: By week 2, there was a difference in BOP between the groups, which reached statistical significance by week 4. This difference in BOP was maintained throughout the course of the study. Chlorhexidine gluconate (0.12%) had no significant effect on PLI, PD, or AL; although PD was greater in the placebo group after week 2 and throughout the study. Microbiologically, at week 4, the treatment group had a reduction in total bacterial counts, as well as Gram positive bacteria, and total black pigmented bacteria, compared to the placebo group. However, only the differences in Actinomyces spp. reached significance. Interestingly, when both groups received only one treatment/day on the weekends (i.e., day 6 and 7), an associated loss of statistically significant differences between the two groups was observed. Additional experiments dosing the non-human primates once daily, 5 days/week yielded no significant differences in clinical parameters, including bleeding, when compared with the placebo group. CONCLUSION: Non-human primates provided a model system of gingivitis for testing antimicrobial agent effects on the subgingival ecology and accompanying inflammatory responses. Chlorhexidine gluconate (0.12%), even in the absence of mechanical hygiene, was effective in inhibiting clinical signs of inflammation, associated with alterations in the subgingival microbial ecology, most notably Actinomyces spp.

Actinomyces↗
PubMed1999

Sulfhemoglobin formation in human erythrocytes by cystalysin, an L-cysteine desulfhydrase from Treponema denticola.

Cystalysin, isolated from the oral pathogen Treponema denticola, is an L-cysteine desulfhydrase (producing pyruvate, ammonia and hydrogen sulfide from cysteine) that can modify hemoglobin and has hemolytic activity. Here, we show that enzymatic activity of recombinant cystalysin depends upon stochiometric pyridoxal phosphate. The enzyme was not functional as an L-alanine transaminase, and had a strong preference for L-cysteine over D-cysteine. Cystalysin preferred small alpha-L-amino acids as substrates or inhibitors and was far more active towards L-cysteine than towards the other standard amino acids that undergo pyridoxal phosphate-dependent beta-elimination reactions (serine, threonine, tryptophan and tyrosine). Cystalysin tolerated small modifications to the carboxylate of L-cysteine (i.e., the methyl and ethyl esters of L-cysteine were good substrates), but the smallest possible peptide with an N-terminal cysteine, L-cysteinylglycine, was a very poor substrate. These results, combined with the implicit requirement for a free amine for pyridoxal phosphate-dependent reactions, imply that cystalysin cannot catabolize cysteine residues located within peptides. Cystalysin has Michaelis-Menten kinetics towards L-cysteine, and there was little or no inhibition by ammonia, H2S, pyruvate and acetate. Human erythrocytes incubated with H2S or with cystalysin and cysteine primarily accumulated sulfhemoglobin and methemoglobin, along with minor amounts of choleglobin and protein aggregates. Erythrocytes retained the ability to reduce methemoglobin in the presence of H2S. Cystalysin could not modify hemoglobin when beta-chloroalanine was the substrate, indicating an absolute requirement for H2S production. Cystalysin appears to be an unregulated L-cysteine catabolizing enzyme, with the resulting H2S production being essential to the atypical hemolytic activity.

Cystathionine gamma-Lyase↗
PubMed1999

Studies on the binding of Treponema pectinovorum to HEp-2 epithelial cells.

We developed a radioassay to assess the adherence of the oral treponemes Treponema denticola and Treponema pectinovorum to live HEp-2 epithelial cells. T. pectinovorum bound firmly to the epithelial cell monolayer in a concentration-dependent manner. The results indicated that a subpopulation of T. pectinovorum appeared to bind and that the binding could be influenced by environmental factors. Increasing concentrations of fetal bovine serum inhibited binding, whereas T. pectinovorum membrane vesicles and co-incubation with T. denticola ATCC 35404 increased the number of cells bound to the monolayer. Treatment of T. pectinovorum with periodic acid, but not trypsin or proteinase K, decreased the binding suggesting that a cell surface carbohydrate, such as the O-antigenic component of the lipopolysaccharide, mediates attachment of the bacteria to the epithelial cells. Co-infection of the HEp-2 cells with both T. denticola and T. pectinovorum did not interfere with each other in attachment to the epithelial cell suggesting that they do not compete for the same cellular receptor on the host cell surface. This study demonstrates that T. pectinovorum is capable, in vitro, of forming a tight association with host cells and that this binding could represent an initial step in the pathogenesis of T. pectinovorum.

Adhesins, Bacterial↗
PubMed1999

Hemoxidation and binding of the 46-kDa cystalysin of Treponema denticola leads to a cysteine-dependent hemolysis of human erythrocytes.

Cystalysin, a 46-kDa protein isolated from the cytosol of Treponema denticola, was capable of both cysteine dependent hemoxidation and hemolysis of human and sheep red blood cells. The activities were characteristic of a cysteine desulfhydrase. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western immunoblotting analysis of the interaction of cystalysin with the red blood cells revealed an interaction of the protein with the red blood cell membrane. Substrates for the enzyme (including L-cysteine and beta-chloroalanine) enhanced the interaction, which occurred with both whole red blood cells as well as with isolated and purified red blood cell ghosts. SDS-PAGE and western immunoblotting employing anti-hemoglobin serum revealed that, during the hemoxidative events, the hemoglobin molecule associated with the red blood cell membrane, forming putative Heinz bodies. Spectrophotometric analysis of the hemoxidative events (cystalysin + cysteine + red blood cells) revealed a chemical modification of the native hemoglobin to sulfhemoglobin and methemoglobin. Hemoxidation also resulted in the degradation of both the red blood cell alpha- and beta-spectrin. The results presented suggest that the interaction of cystalysin with the red blood cell membrane results in the chemical oxidation of the hemoglobin molecule as well as an alteration in the red blood cell membrane itself.

Bacterial Proteins↗
PubMed1999

Isolation and chemical analysis of a lipopolysaccharide from the outer membrane of the oral anaerobic spirochete Treponema pectinovorum.

Isolation of a putative lipopolysaccharide from the surface of the oral treponeme, Treponema pectinovorum, revealed it to contain larger amounts of 3-deoxy-D-manno-octulosonic acid compared with other oral Treponema species. This molecule was isolated from the outer membrane of T. pectinovorum and had chemical characteristics of a putative lipopolysaccharide. The yield of lipopolysaccharide was between 0.6% and to 1.1% of the bacterial dry weight. The purified molecule was resistant to the action of proteinases and consisted of both sugars and lipids. 3-Deoxy-D-manno-octulosonic acid and hexoses accounted for 6.1-8.7% and 17.6-20.2%, respectively of the dry weight. Carbohydrate compositional analysis revealed the presence of glucose, galactose, 2-acetamido-2-deoxy-glucose, rhamnose and 6-deoxy-talose in the molar ratio of 1.00:0.96:0.19:0.88:0.98, respectively. No heptose was detected. The fatty acid analysis determined the presence of straight chain, C13:00, C14:00, C15:00 and C17:00 acids, as well as branched chain, C13:00, C14:00 and two species of C15:00, acids. Electrophoretic analysis indicated that the lipopolysaccharide was present as two major species.

Cell Membrane↗
PubMed1999

A multistate nosocomial outbreak of Ralstonia pickettii colonization associated with an intrinsically contaminated respiratory care solution.

From 1 February through 30 April 1998, 4 hospitals reported a total of 34 patients colonized with Ralstonia pickettii. All but 1 had been exposed to 0.9% saline solution manufactured by 1 company (Modudose; Kendall, Mainsfield, MA), which was used during endotracheal suctioning. Culture of saline solution from previously unopened vials yielded R. pickettii. All available product and patient isolates were genotypically related by pulsed-field gel electrophoresis (PFGE) analysis. The contaminated saline solution was manufactured at the same plant that had been associated with a similar outbreak in 1983. The 1983 and 1998 R. pickettii isolates were unrelated, as determined by PFGE. In both 1983 and 1998, a 0. 2-microm cartridge filter was used for terminal sterilization. The detection of R. pickettii should alert hospital personnel to the possibility of product contamination. In this outbreak, prompt notification of public health agencies resulted in rapid notification of other health care providers, which likely prevented additional outbreaks.

Adolescent↗
PubMed1999

Cystalysin, a 46-kDa L-cysteine desulfhydrase from Treponema denticola: biochemical and biophysical characterization.

A 46-kDa hemolytic protein referred to as cystalysin, from Treponema denticola ATCC 35404, was characterized and overexpressed in Escherichia coli LC-67. Cystalysin lysed erythrocytes, hemoxidized hemoglobin to sulfhemoglobin and methemoglobin, and removed the sulfhydryl and amino group from selected S-containing compounds (e.g., cysteine) producing H2S, NH3, and pyruvate. With L-cysteine as substrate, cystalysin obeys Michaelis-Menten kinetics. Cystathionine and s-aminoethyl-L-cysteine were also substrates. Several of the small alpha amino acids were found to be competitive inhibitors of cystalysin. The enzymatic activity was increased by beta-mercaptoethanol and was not inhibited by the proteinase inhibitor TLCK (N alpha-p-tosyl-L-lysine chloromethyl ketone), pronase, or proteinase K, suggesting the functional site was physically protected or located in a small fragment of the polypeptide. We hypothesize that cystalysin is a pyridoxal-5-phosphate-containing enzyme with the activity of an alphaC-N and betaC-S lyase (cystathionase). Since high amounts of H2S have been reported in deep periodontal pockets, this metabolic enzyme from T. denticola may also function in vivo as an important virulence molecule.

Cystathionine gamma-Lyase↗
PubMed1999

Systemic manifestations of periodontitis in the non-human primate.

This report describes our findings regarding the potential contribution of periodontitis to atherosclerotic processes using a nonhuman primate model. The goal of the investigations was to target general mechanisms which could describe the association of these disease processes, including: (i) systemic translocation of bacteria/products during periodontitis; (ii) alterations in systemic inflammatory biomarkers during periodontitis; and (iii) the relationship of periodontitis to serum lipids/lipoproteins. Increases in serum endotoxin (e.g. LPS) during ligature-induced periodontitis were observed in these animals. We determined serum levels of various acute phase reactants and chemokines (e.g. CRP, alpha 1-antitrypsin, haptoglobin, fibrinogen, IL-8). A number of these host factors were significantly increased during gingivitis and/or periodontitis. Finally, we observed specific changes in serum lipid levels (cholesterol, triglycerides, HDL, LDL) and lipoproteins (apoA-I) during periodontitis, which were exacerbated by exposure of the animals to a diet with elevated fat content. Thus, we have described systemic manifestations of periodontitis that include detection of bacterial products, inflammatory biomarkers, and dyslipoproteinemia consistent with an increased atherogenic risk.

Animals↗
PubMed1999

Lack of humoral immune protection against Treponema denticola virulence in a murine model.

This study investigated the characteristics of humoral immune responses to Treponema denticola following primary infection, reinfection, and active immunization, as well as immune protection in mice. Primary infection with T. denticola induced a significant (400-fold) serum immunoglobulin G (IgG) response compared to that in control uninfected mice. The IgG response to reinfection was 20, 000-fold higher than that for control mice and 10-fold higher than that for primary infection. Mice actively immunized with formalin-killed treponemes developed serum antibody levels seven- to eightfold greater than those in animals after primary infection. Nevertheless, mice with this acquired antibody following primary infection or active immunization demonstrated no significant alterations of lesion induction or decreased size of the abscesses following a challenge infection. Mice with primary infection developed increased levels of IgG3, IgG2b, and IgG2a antibodies, with IgG1 being lower than the other subclasses. Reinfected mice developed enhanced IgG2b, IgG2a, and IgG3 and less IgG1. In contrast, immunized mice developed higher IgG1 and lower IgG3 antibody responses to infection. These IgG subclass distributions indicate a stimulation of both Th1 and Th2 activities in development of the humoral immune response to infection and immunization. Our findings also demonstrated a broad antigen reactivity of the serum antibody, which was significantly increased with reinfection and active immunization. Furthermore, serum antibody was effective in vitro in immobilizing and clumping the bacteria but did not inhibit growth or passively prevent the treponemal infection. These observations suggest that humoral immune responses, as manifested by antibody levels, isotype, and antigenic specificity, were not capable of resolving a T. denticola infection.

Animals↗
PubMed1999

Environmental modulation of oral treponeme virulence in a murine model.

This investigation examined the effects of environmental alteration on the virulence of the oral treponemes Treponema denticola and Treponema pectinovorum. The environmental effects were assessed by using a model of localized inflammatory abscesses in mice. In vitro growth of T. denticola and T. pectinovorum as a function of modification of the cysteine concentration significantly enhanced abscess formation and size. In contrast, growth of T. denticola or T. pectinovorum under iron-limiting conditions (e.g., dipyridyl chelation) had no effect on abscess induction in comparison to that when the strains were grown under normal iron conditions. In vivo modulation of the microenvironment at the focus of infection with Cytodex beads demonstrated that increasing the local inflammation had no effect on lesion induction or size. In vivo studies involved the determination of the effects of increased systemic iron availability (e.g., iron dextran or phenylhydrazine) on the induction, kinetics, and size of lesions. T. denticola induced significantly larger lesions in mice with iron pretreatment and demonstrated systemic manifestations of the infectious challenge and an accompanying spreading lesion with phenylhydrazine pretreatment (e.g., increases in circulating free hemoglobin). In contrast, T. pectinovorum virulence was minimally affected by this in vivo treatment to increase iron availability. T. denticola virulence, as evaluated by lesion size, was increased additively by in vivo iron availability, and cysteine modified growth of the microorganism. Additionally, galactosamine sensitized mice to a lethal outcome following infection with both T. denticola and T. pectinovorum, suggesting an endotoxin-like activity in these treponemes. These findings demonstrated the ability to modify the virulence capacity of T. denticola and T. pectinovorum by environmental conditions which can be evaluated by using in vivo murine models.

Abscess↗
PubMed1998

Virulence of a polymicrobic complex, Treponema denticola and Porphyromonas gingivalis, in a murine model.

The effect of a polymicrobic infection employing Treponema denticola and Porphyromonas gingivalis in the murine lesion model was used to determine the synergistic virulence of these two periodontopathic bacteria. At high doses of P. gingivalis W50, addition of T. denticola in the infection mixture had no effect on the formation and size of the spreading lesion caused by this microorganism. However, at low P. gingivalis challenge doses, T. denticola significantly enhanced the virulence of P. gingivalis compared with monoinfection of this microorganism. A potential role of the trypsin-like protease enzyme activity of P. gingivalis in this synergistic virulence was tested using P. gingivalis mutants deficient (i.e., BEI) or devoid (i.e., NG4B19) of this protease activity. These findings demonstrated that T. denticola-P. gingivalis complexes exhibit enhanced virulence in this model and that even using a polymicrobic challenge infection, the trypsin-like protease activity was important to P. gingivalis virulence expression.

Abscess↗
PubMed1998

Bone resorption caused by three periodontal pathogens in vivo in mice is mediated in part by prostaglandin.

Gingival inflammation, bacterial infection, alveolar bone destruction, and subsequent tooth loss are characteristic features of periodontal disease, but the precise mechanisms of bone loss are poorly understood. Most animal models of the disease require injury to gingival tissues or teeth, and the effects of microorganisms are thus complicated by host responses to tissue destruction. To determine whether three putative periodontal pathogens, Porphyromonas gingivalis, Campylobacter rectus, and Fusobacterium nucleatum, could cause localized bone resorption in vivo in the absence of tissue injury, we injected live or heat-killed preparations of these microorganisms into the subcutaneous tissues overlying the calvaria of normal mice once daily for 6 days and then examined the bones histologically. We found that all three microorganisms (both live and heat killed) stimulated bone resorption and that the strain of F. nucleatum used appeared to be the strongest inducer of osteoclast activity. Treatment of the mice concomitantly with indomethacin reduced but did not completely inhibit bone resorption by these microorganisms, suggesting that their effects were mediated, in part, by arachidonic acid metabolites (e.g., prostaglandins). Our findings indicate that these potential pathogens can stimulate bone resorption locally when placed beside a bone surface in vivo in the absence of prior tissue injury and support a role for them in the pathogenesis of bone loss around teeth in periodontitis.

Alveolar Bone Loss↗
PubMed1998

Immunization with Porphyromonas gingivalis cysteine protease: effects on experimental gingivitis and ligature-induced periodontitis in Macaca fascicularis.

Targeting bacterial virulence factors such as proteases for immunization may hold the key to limiting or preventing loss of attachment and alveolar bone in periodontal disease. This study examined the clinical, microbiological, and immununological responses following active immunization with a purified Porphyromonas gingivalis cysteine protease (porphypain-2) in the nonhuman primate (Nhp) Macaca fascicularis. One group of Nhp was immunized with porphypain-2 antigen while control Nhp received placebo injections. All Nhp were subjected to experimental gingivitis followed by ligature-induced periodontitis in a split-mouth design. An enzyme-linked immunosorbent assay demonstrated that immunization elicited a significantly elevated and specific IgG antibody response to both whole cell P. gingivalis (36-fold) and to porphypain-2 (194-fold). Checkerboard hybridization DNA analysis of subgingival plaque from ligated sextants demonstrated that 25% more Gram-negative anaerobic species became significantly elevated from baseline and at earlier timepoints in the control group than in the immununized group. Immunization with this protease did not suppress the emergence of P. gingivalis. Clinical indices showed few changes related to immunization. Alveolar bone density changes demonstrated a highly significant loss in ligated sextants compared to non-ligated sextants within the control group (P < 0.001), and a smaller but significant difference within the immunized group (P = 0.043). Comparison of ligated sextants only demonstrated more bone loss in the control group versus the immunized group (-13.07+/-9.51 versus -9.41+/-6.18; computer-assisted densitometric image analysis units +/- SD); the difference approached, but did not reach, significance. The results suggest that porphypain-2 may contribute to the pathogenic potential of the subgingival plaque microbiota in the Nhp model of ligature-induced periodontitis, and that active immunization with porphypain-2 appeared capable of altering this pathogenic response.

Absorptiometry, Photon↗