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Background/objectives: Chronic periodontal infections have been suggested to contribute to the risk of adverse pregnancy outcomes., Material and Methods: This study describes the relationship of patterns of systemic inflammatory mediators and IgG antibody to 20 oral bacteria in pregnant female baboons (Papio anubis) coupled with clinical features of ligature-induced periodontitis, as risk indicators for adverse pregnancy outcomes. Animals showing a preterm delivery and/or low birth weight newborns, as well as those pregnancies resulting in spontaneous abortion, stillbirth, or fetal demise were tabulated as adverse pregnancy outcomes., Results: A significantly greater frequency of the periodontitis group neonates had a low birth weight (18.1%; p = 0.008) and decreased gestational age (9.8%). Spontaneous abortion/stillbirth/fetal demise were increased in the periodontitis (8.7%) versus the control group (3.8%) (p = 0.054). The baseline oral clinical presentation of the experimental animals did not relate to the adverse pregnancy outcomes. Animals with the greatest extent/severity of periodontitis progression during the initial ½ of gestation (ie. to mid-pregnancy) had the greatest risk for adverse pregnancy outcomes. Baseline biological parameters indicating historical responses of the animals to periodontal challenge demonstrated individual variation in selected mediators, some of which became more differential during ligature-induced periodontitis. The relationship of clinical parameters to systemic inflammatory responses was consistent with a temporal contribution to adverse pregnancy outcomes in a subset of the animals., Conclusions: These results support a link between periodontitis and adverse pregnancy outcomes in the baboons and provide a prospective experimental model for delineating the biologic parameters that contribute to a causal relationship between chronic oral infections and birth events., (© 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

The acquisition and development of the complex oral microbiome remain ill defined. While selected species of oral bacteria have been examined in relation to their initial colonization in neonates, a more detailed understanding of the dynamics of the microbiome has been developed only in adults. The current investigation used a nonhuman primate model to document the kinetics of colonization of the oral cavities of newborns and infants by a range of oral commensals and pathogens. Differences in colonization were evaluated in newborns from mothers who were maintained on an oral hygiene regimen pre- and postparturition with those displaying naturally acquired gingivitis/periodontitis. The results demonstrate distinct profiles of acquisition of selected oral bacteria, with the transmission of targeted pathogens, Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans, being passed on primarily from mothers with gingivitis/periodontitis. This colonization resulted in defined patterns of systemic antibody responses in the infants. The significant relative risk measures for infection with the pathogens, as well as the relationship of oral infection and blood serum antibody levels, were consistent with those of the newborns from mothers with gingivitis/periodontitis. These findings indicate that the early acquisition of potentially pathogenic oral bacterial species might impact the development of mucosal responses in the gingiva and may provide an enhanced risk for the development of periodontitis later in life.

This study tested the hypothesis that pregnant female baboons exhibit increased levels of various inflammatory mediators in serum resulting from ligature-induced periodontitis, and that these profiles would relate to periodontal disease severity/extent in the animals. The animals were sampled at baseline (B), mid-pregnancy (MP; two quadrants ligated) and at delivery (D; four quadrants ligated). All baboons developed increased plaque, gingival inflammation and bleeding, pocket depths and attachment loss following placement of the ligatures. By MP, both prostaglandin E(2) (PGE(2)) and bactericidal permeability inducing factor (BPI) were greater than baseline, while increased levels of interleukin (IL)-6 occurred in the experimental animals by the time of delivery. IL-8, MCP-1 and LBP all decreased from baseline through the ligation phase of the study. Stratification of the animals by baseline clinical presentation demonstrated that PGE(2), LBP, IL-8 and MCP-1 levels were altered throughout the ligation interval, irrespective of baseline clinical values. IL-6, IL-8 and LBP were significantly lower in the subset of animals that demonstrated the least clinical response to ligation, indicative of progressing periodontal disease. PGE(2), macrophage chemotactic protein (MCP)-1, regulated upon activation, normal T cell expressed and secreted (RANTES) and LBP were decreased in the most diseased subset of animals at delivery. Systemic antibody responses to Fusobacterium nucleatum, Porphyromonas gingivalis, Actinobacillus actinomycetemcomitans and Campylobacter rectus were associated most frequently with variations in inflammatory mediator levels. These results provide a profile of systemic inflammatory mediators during ligature-induced periodontitis in pregnant baboons. The relationship of the oral clinical parameters to systemic inflammatory responses is consistent with a contribution to adverse pregnancy outcomes in a subset of the animals., (© 2010 Authors. Clinical and Experimental Immunology © 2010 British Society for Immunology.)

Objectives: Therapeutic hypothermia (TH) has been shown to improve survival and neurological outcome in patients resuscitated after out of hospital cardiac arrest (OHCA) from ventricular fibrillation/ventricular tachycardia (VF/VT). We evaluated the effects of using a TH protocol in a large community hospital emergency department (ED) for all patients with neurological impairment after resuscitated OHCA regardless of presenting rhythm. We hypothesized improved mortality and neurological outcomes without increased complication rates., Methods: Our TH protocol entails cooling to 33°C for 24 hours with an endovascular catheter. We studied patients treated with this protocol from November 2006 to November 2008. All non-pregnant, unresponsive adult patients resuscitated from any initial rhythm were included. Exclusion criteria were initial hypotension or temperature less than 30°C, trauma, primary intracranial event, and coagulopathy. Control patients treated during the 12 months before the institution of our TH protocol met the same inclusion and exclusion criteria. We recorded survival to hospital discharge, neurological status at discharge, and rates of bleeding, sepsis, pneumonia, renal failure, and dysrhythmias in the first 72 hours of treatment., Results: Mortality rates were 71.1% (95% CI, 56-86%) for 38 patients treated with TH and 72.3% (95% CI 59-86%) for 47 controls. In the TH group, 8% of patients (95% CI, 0-17%) had a good neurological outcome on discharge, compared to 0 (95% CI 0-8%) in the control group. In 17 patients with VF/VT treated with TH, mortality was 47% (95% CI 21-74%) and 18% (95% CI 0-38%) had good neurological outcome; in 9 control patients with VF/VT, mortality was 67% (95% CI 28-100%), and 0% (95% CI 0-30%) had good neurological outcome. The groups were well-matched with respect to sex and age. Complication rates were similar or favored the TH group., Conclusion: Instituting a TH protocol for OHCA patients with any presenting rhythm appears safe in a community hospital ED. A trend towards improved neurological outcome in TH patients was seen, but did not reach significance. Patients with VF appeared to derive more benefit from TH than patients with other rhythms.

Background: Chronic oral infections that elicit host responses leading to periodontal disease are linked with various sequelae of systemic diseases. This report provides seminal information on the clinical and adaptive immunologic characteristics of a baboon model of ligature-induced periodontitis during pregnancy., Methods: Female Papio anubis were evaluated for periodontal health at baseline. Ligatures were tied around selected teeth to initiate oral inflammation and periodontitis. Then the animals were bred. At midpregnancy ( approximately 90 days), a clinical evaluation was performed, and additional ligatures were tied on teeth in the contralateral quadrants to maintain progressing periodontitis throughout pregnancy. A final clinical evaluation was done for all experimental teeth after delivery, and ligatures were removed. Serum was collected at all sampling intervals for the determination of antibody levels to a group of 20 oral bacteria. Unligated animals served as controls., Results: At baseline, 16% of animals exhibited minimal plaque and gingival inflammation without periodontal disease. The remaining baboons demonstrated varying levels of inflammation/bleeding, and approximately 20% of the population had periodontal pocketing (>3 mm). Ligated animals expressed increased levels of inflammation and increased probing depths and clinical attachment loss (AL) and could be stratified into multiple subsets postligation based upon changes in clinical parameters at midpregnancy and at delivery. Baboons were categorized into disease susceptibility groups (periodontal disease susceptibility 1 through 4) that described the extent/severity of induced disease during pregnancy. Control animals showed minimal periodontal changes during gestation. Significant differences in serum antibody to multiple oral bacteria were found in animals presenting with periodontitis at baseline and during the 6 months of ligature-induced disease. A significant correlation to antibody to P. gingivalis, which was sustained throughout ligation and pregnancy, was observed with disease presentation., Conclusions: The clinical presentation at baseline, reflecting the natural history of oral disease in these animals, suggests individual variation that is reflected in the characteristics of the adaptive immune responses to oral bacteria. The variability in the response to ligation with resulting periodontal disease provides a model to document prospectively the relationship between oral and systemic health outcomes.

Objective: Dietary caloric restriction (CR) has been found to reduce systemic markers of inflammation and may attenuate the effects of chronic inflammatory conditions. The purpose of this study was to examine the effects of long-term CR on naturally occurring chronic inflammatory periodontal disease in a nonhuman primate model., Methods: The effects of long-term CR on extent and severity of naturally occurring chronic periodontal disease, local inflammatory and immune responses, and periodontal microbiology, were evaluated in a cohort of 81 (35 female and 46 male; 13-40 y of age) rhesus monkeys (Macaca mulatta) with no previous exposure to routine oral hygiene. CR monkeys had been subjected to 30% CR for 13-17 y relative to control-fed (CON) animals starting at 3-5 y of age., Results: Same sex CR and CON monkeys exhibited similar levels of plaque, calculus, and bleeding on probing. Among CON animals, males showed significantly greater periodontal breakdown, as reflected by higher mean clinical attachment level and periodontal probing depth scores, than females. CR males exhibited significantly less periodontal pocketing, lower IgG antibody response, and lower IL-8 and ss-glucuronidase levels compared to CON males, whereas CR females showed a lower IgG antibody response but comparable clinical parameters and inflammatory marker levels relative to CON females. Long-term CR had no demonstrable effect on the periodontal microbiota., Conclusion: Males demonstrated greater risk for naturally occurring periodontal disease than females. Long-term CR may differentially reduce the production of local inflammatory mediators and risk for inflammatory periodontal disease among males but not females.

Background: The microbiology of periodontitis in type 1 diabetes has been reported, but less is known about type 2 diabetes. Moreover, these data have not linked microbial colonization, host response, and clinical presentation in type 1 or type 2 diabetes. The objectives of this study were to relate periodontal status, periodontal microorganisms, and host-response characteristics in Hispanic Americans with type 2 diabetes., Methods: Plaque and serum samples were obtained from 63 Hispanic American subjects with and without type 2 diabetes. The microbiology of subgingival plaque samples was evaluated using DNA checkerboard hybridization, and serum antibody to a battery of oral microorganisms was determined using an enzyme-linked immunosorbent assay., Results: In general, similar pathogens were present in periodontitis sites from subjects with and without type 2 diabetes, although the periodontitis sites in diabetes showed a higher frequency of Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans (previously Actinobacillus actinomycetemcomitans), and Campylobacter spp. Serum antibody to Campylobacter rectus was elevated in type 2 diabetes, whereas antibody to P. gingivalis and C. rectus were elevated in subjects with periodontitis, irrespective of diabetes status. Stratification of the population based upon antibody to P. gingivalis or C. rectus suggested a linkage between elevated antibody to P. gingivalis, increased frequency of diabetes, and significantly worse periodontitis., Conclusion: The increased severity of periodontal disease with type 2 diabetes may reflect an alteration of the pathogenic potential of periodontal bacteria and/or a modification of the characteristics of the host's inflammatory response that may contribute to a breakdown in the homeostasis of the periodontium.

Porphyromonas gingivalis appears to be a major contributor to periodontal disease, especially soft tissue destruction, which is reflected by the ability to cause invasive, spreading lesions, and tissue inflammation in a murine abscess model. This study investigated the role of hemin on the regulation of growth and virulence of P. gingivalis strains. P. gingivalis strains W50, A7A1-28, 3079, 381, W50/BEI, and NG4B19 were grown in broth and on blood agar plates. P. gingivalis cells grown under iron-depleted conditions for multiple passages showed significantly decreased lesion size in mice, in contrast to cells grown under iron-normal (5 microg/ml) and iron-elevated conditions. Statistically significant (P < 0.01) decreases in gingipain enzyme activity were found among the strains grown under iron-depleted conditions. P. gingivalis grown in the presence of blood induced significantly different lesion type, lesion size, lesion onset, and mortality. Elevated hemin resulted in increased cell-associated iron in P. gingivalis, which increased the capacity of the microorganism to survive at times of iron deprivation. These results indicate that hemin or iron availability regulates multiple aspects related to P. gingivalis virulence, including growth, survival, gingipain levels, and iron accumulation.

This study investigated the endotoxic and biological properties of purified lipopolysaccharide (LPS) isolated from an oral spirochete, Treponema pectinovorum. Endotoxicity, measured by Limulus amoebocyte lysate kinetic assay, showed that the LPS contained 1.28 endotoxin units per microg of purified LPS, which was approximately 4,000 times less than Escherichia coli O55:B5 LPS. To determine in vivo endotoxicity, LPS responder mice were administered LPS following galactosamine (GalN) sensitization. The LPS induced neither endotoxic symptoms nor lethality for 96 h, suggesting negligible or very low endotoxicity. In contrast, infection with live T. pectinovorum induced 100% lethality within 12 h in GalN-sensitized LPS responder mice, indicating an endotoxin-like property of this treponeme. Heat-killed microorganisms exhibited no lethality in GalN-sensitized mice, suggesting that the endotoxicity was associated with heat-labile components. To determine cytokine and chemokine induction by LPS, human gingival fibroblasts were stimulated and secretion of interleukin 1beta (IL-1beta), granulocyte-macrophage colony-stimulating factor, gamma interferon, IL-6, IL-8, and monocyte chemoattractant protein 1 (MCP-1) was assessed. The purified LPS induced significant amounts of only IL-6, IL-8, and MCP-1, although they were substantially lower than levels after challenge with live T. pectinovorum. After injection of LPS or live or heat-killed T. pectinovorum, serum was collected from mice and analyzed for proinflammatory cytokines IL-1beta, tumor necrosis factor alpha (TNF-alpha), and IL-6. LPS induced only IL-6 consistently. Both live and heat-killed T. pectinovorum induced serum IL-6, which was higher than the level detected following LPS administration. Importantly, live bacteria elicited systemic TNF-alpha and IL-1beta levels similar to those induced by a lethal dose of live E. coli O111. The results indicated that T. pectinovorum LPS has very low or no endotoxicity, although it can elicit low levels of cytokines from host cells. In contrast to the LPS, live T. pectinovorum demonstrated in vivo toxicity, which was associated with serum IL-1beta, TNF-alpha, and IL-6, suggesting an endotoxin-like property of a heat-labile molecule(s) of the spirochete.

Background: Bacterial contamination of blood components can result in transfusion-transmitted infection, but the risk is not established., Study Design and Methods: Suspected cases of transfusion-transmitted bacteremia were reported to the CDC by participating blood collection facilities and transfusion services affiliated with the American Red Cross, AABB, or Department of Defense blood programs from 1998 through 2000. A case was defined as any transfusion reaction meeting clinical criteria in which the same organism species was cultured from a blood component and from recipient blood, with the organism pair confirmed as identical by molecular typing., Results: There were 34 cases and 9 deaths. The rate of transfusion-transmitted bacteremia (in events/million units) was 9.98 for single-donor platelets, 10.64 for pooled platelets, and 0.21 for RBC units; for fatal reactions, the rates were 1.94, 2.22, and 0.13, respectively. Patients at greatest risk for death received components containing gram-negative organisms (OR, 7.5; 95% CI, 1.3-64.2; p = 0.009)., Conclusion: Bacterial contamination of blood is an important cause of transfusion-transmitted infection; infection risk from platelet transfusion is higher compared with that from RBCs, and, overall, the risk of infection from bacterial contamination now may exceed that from viral agents. Recipients of components containing gram-negative organisms are at highest risk for transfusion-related death. The results of this study may help direct efforts to improve transfusion-related patient safety.

Background: The transfusion of blood components contaminated with bacteria may have serious clinical consequences, but few data are available on the incidence of these events. A national effort to assess the frequency of blood component bacterial contamination associated with transfusion reaction (the BaCon Study) was initiated to better estimate their occurrence., Study Design and Methods: Standard reporting criteria, data collection forms, and a standardized reporting protocol were developed in collaboration with the American Red Cross, AABB, and the Department of Defense. Episodes reported to the BaCon Study were compared with those reported to the FDA's national reporting systems to estimate the extent to which all serious reactions associated with bacterial contamination were captured., Results: During the first 2 years, 38 episodes meeting study criteria were reported; 21 were laboratory-confirmed. The estimated proportion of episodes reported to the BaCon Study (i.e., completeness of coverage) was lower than that reported to the FDA during the same period (0.33 vs. 0.68), but the positive predictive value was higher (0.66 vs.0.28)., Conclusion: Despite the complexity of obtaining reports from a large number of United States hospitals and transfusion centers, the feasibility and usefulness of the BaCon Study were shown. This study was the only national study in the United States to monitor adverse clinical events associated with bacterial contamination of blood components. By building on hospital-based reporting of transfusion-related adverse events, the BaCon Study serves as a model for the study of other complications associated with blood and blood components.

Background: Vancomycin-resistant enterococci (VRE) are increasing in prevalence at many institutions, and are often reported in dialysis patients. We studied the prevalence of and risk factors for VRE at seven outpatient hemodialysis centers (three in Baltimore, MD, USA, and four in Richmond, VA, USA)., Methods: Rectal or stool cultures were performed on consenting hemodialysis patients during December 1997 to April 1998. Consenting patients were recultured during May to July 1998 (median 120 days later). Clinical and laboratory data and functional status (1 to 10 scale: 1, normal function; 9, home attendant, not totally disabled; 10, disabled, living at home) were recorded., Results: Of 478 cultures performed, 20 (4.2%) were positive for VRE. Among the seven centers, the prevalence of VRE-positive cultures varied from 1.0 to 7.9%. Independently significant risk factors for a VRE-positive culture were a functional score of 9 to 10 (odds ratio 6.9, P < 0.001), antimicrobial receipt within 90 days before culture (odds ratio 6.1, P < 0.001), and a history of injection drug use (odds ratio 5.4, P = 0.004)., Conclusions: VRE-colonized patients were present at all seven participating centers, suggesting that careful infection-control precautions should be used at all centers to limit transmission. In agreement with previous studies, VRE colonization was more frequent in patients who had received antimicrobial agents recently, underscoring the importance of judicious antimicrobial use in limiting selection for this potential pathogen.

Treponema denticola does not appear to produce siderophores, so it must acquire iron by other pathways. Indeed, T. denticola has been shown to have an iron-regulated 44-kDa outer membrane protein (HbpA) with hemin binding ability. To characterize the HbpA protein, its gene was cloned from genomic DNA libraries of T. denticola. Sequence analysis of the hbpA open reading frame indicated that it encoded a 42.8-kDa protein with a 23-amino-acid signal peptide. HbpA has no significant homology to any proteins in the databases. Southern blot analysis demonstrated that hbpA is present in several T. denticola ATCC strains and clinical isolates, but not in Treponema pectinovorum, Treponema socranskii, or Escherichia coli. HbpA, expressed as a recombinant protein in E. coli and purified by antibody affinity chromatography, has hemin binding activity as determined by lithium dodecyl sulfate-polyacrylamide gel electrophoresis with tetramethylbenzidine staining. Northern blot analysis showed that there were two hbpA-containing transcripts, of approximately 1.3 and 2.6 kb, and that the RNA levels were low-iron induced. Interestingly, the 2.6-kb mRNA also encoded a second protein with significant homology to hbpA. This downstream gene, called hbpB, was cloned and sequenced and its product was expressed as a fusion protein in E. coli. The hbpB gene product is 49% identical to HbpA and binds hemin. Thus, T. denticola has two novel hemin binding proteins which may be part of a previously unrecognized iron acquisition pathway.

For decades, investigations have identified local and systemic humoral immune responses to microorganisms comprising the supra- and subgingival biofilms in the oral cavity. Inflammation and tissue destruction in the periodontium are accompanied by alterations in the quantity, quality, and specificity of antibody. The conundrum in this scenario is the existence of a substantial plasma cell infiltrate at sites of periodontal lesions and a seemingly robust antibody response in the oral cavity and the serum, apparently coincident with progressing disease. Consequently, much effort has been expended to elucidate the critical characteristics of protective humoral responses and to develop strategies for enhancing these unique features. We and others have conducted studies attempting to distinguish disease susceptibility associated with: i) variations in response levels significantly increased to some species with disease, minimal response to others; ii) functional comparisons of antibody subclass differences, genetic regulation, and maturation of responses; iii) microbial and antigenic specificity of the antibody focus on specific pathogens and identification of selected antigens as targets for immunoprotection; and, iv) kinetics of responses during disease and therapeutic interventions linking immune changes with infection and as a measure of treatment success. This report summarizes varied research designs and results, to provide a profile of antibody in health, gingivitis, and periodontitis. These profiles may be used to provide a framework focusing on the humoral response to commensal microorganisms and likely pathogens, as they emerge in the biofilm--etiologic for or in response to disease processes. Models for antibody as a diagnostic adjunct and for predicting protective antibody responses are suggested. These concepts are likely relevant for considering vaccine approaches to periodontitis.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

This study was designed to investigate the virulence characteristics of Treponema denticola, T. socranskii, T. pectinovorum, and T. vincentii following challenge infection of mice. These microorganisms induced well-demarcated, dose-dependent, raised subcutaneous (s.c.) abscesses which were similar in time of onset, lesion progression, and duration of healing. Only viable cells were capable of inducing these characteristic s.c. abscesses. Histological examination of the skin lesion 3 and 5 days postinfection revealed abscess formation in the s.c. tissues, and abundant spiral organisms were demonstrated to be present in the abscess. Host resistance modulation by dexamethasone (neutrophil alteration) and cyclophosphamide (neutrophil depletion) pretreatment had a minimal effect on the virulence expression by any of these treponemes. The T. denticola isolates demonstrated significant trypsin-like protease (TLPase) activity, while both T. socranskii and T. vincentii were devoid of this activity. Interestingly, T. pectinovorum strains were heterogeneous with respect to TLPase as high producers, low producers, and nonproducers. However, no differences in lesion formation were noted regardless of whether the species expressed this proteolytic activity or whether treatment with N alpha-p-tosyl-L-lysine chloromethyl ketone (TLCK) and dithiothreitol was performed. These results showed that (i) a murine model may be used to evaluate virulence expression by oral treponemes; (ii) while TLPase activity varies among the oral treponemes, this protease does not appear to participate in abscess induction in the mouse model; and (iii) T. pectinovorum strains show variation in TLPase activity.

This study utilized various mouse strains with documented alterations in immune system components to assess their contribution to modify the virulence of Porphyromonas gingivalis. P. gingivalis W50 was cultivated on blood agar plates, harvested and used to challenge mice by subcutaneous injection on the dorsolateral surface of the back. Soft tissue lesion development was estimated by measuring the area of the spreading lesion formed by this microorganism over a period of 15 days. Challenge of various normal inbred and outbred mouse strains including: BALB/cN, BALB/cJ, BALB/c nu/+, ICR, B10.A(4R), B10.MBR, A/J, C57BL/6J, CBA/CaH, C.B-17/Icv Tacf DF and C3H/HeN with 2 x 10(10) bacteria showed similar lesion size among these strains (approximately 400 mm2). Genetically deficient mouse strains [C.B-17/Icr Tac (SCID); DBA/2 (C5 deficient); BALB/c nu/nu (T cell deficient); CBA/CaHN-XID/J (B cell deficient) and C3H/HeJ (LPS hyporesponsive)] demonstrated a lesion size which was similar to normal animals. C57BL/6J-BgJ (NK cell deficient) mice exhibited a significantly more severe lesion than the other strains tested. Following healing of the lesions, we initiated a secondary infection of the surviving animals to estimate the acquisition of protective immunity following recovery from the primary infection. Normal mice demonstrated a delayed onset and decrease in lesion size of 15 to 30% compared with the primary infection. In contrast, each of the immunodeficient strains appeared unable to develop immune protection to the secondary challenge. The findings suggest that protection against primary infections with P. gingivalis are mediated by innate immune mechanisms (PMN. NK cells). Additionally, it appears that T-cell-dependent humoral responses are critical to developing immunity to subsequent P. gingivalis infection., (Copyright 1997 Academic Press Limited.)

The major protein present in the isolated outer membrane of Treponema pectinovorum ATCC 33768, MompA, was identified, purified, and characterized. Immuno-gold electron microscopy, using anti-MompA serum, and cell fractionation experiments confirmed the localization of MompA to the outer membrane. MompA was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to have a molecular mass of 42 kDa when heat denatured, whereas native MompA formed a number of detergent-stable forms with molecular masses of 71, 76, and 83 kDa. A temperature of 60 degrees C was required to convert the native protein to the 42-kDa form. A number of detergents and chemical agents that are capable of breaking ionic and hydrogen bonds of proteins did not convert native MompA to the 42-kDa species. The native forms of the protein were resistant to the combined action of proteinase K, trypsin, and chymotrypsin, whereas the 42-kDa form of MompA was not. The N-terminal amino acid sequence of MompA was determined to be DVTVNINSRVRPVLYTT, and database searches did not identify any homology with known protein sequences. Amino acid compositional analysis showed the protein to be rich in proline and glycine, with these amino acids accounting for 28 and 13%, respectively, of the total amino acids. Antiserum raised against the major outer membrane protein of T. denticola GM-1 and ATCC 35405 did not cross-react with MompA, and antiserum raised against MompA did not react with any cellular components of Treponema denticola, Treponema vincentii, or Treponema socranskii. A major outer membrane protein similar in molecular mass to MompA was identified in eight clinical isolates of T. pectinovorum. The major outer membrane protein produced by four of the clinical isolates reacted strongly, by Western blotting, with anti-MompA serum, whereas proteins of the other strains did not.

A 46-kDa hemolytic protein, referred to as cystalysin, from Treponema denticola ATCC 35404 was overexpressed in Escherichia coli LC-67. Both the native and recombinant 46-kDa proteins were purified to homogeneity. Both proteins expressed identical biological and functional characteristics. In addition to its biological function of lysing erythrocytes and hemoxidizing the hemoglobin to methemoglobin, cystalysin was also capable of removing the sulfhydryl and amino groups from selected S-containing compounds (e.g., cysteine) producing H2S, NH3, and pyruvate. This cysteine desulfhydrase resulted in the following Michaelis-Menten kinetics: Km = 3.6 mM and k(cat) = 12 s(-1). Cystathionine and S-aminoethyl-L-cysteine were also substrates for the protein. Gas chromatography-mass spectrometry and high-performance liquid chromatography analysis of the end products revealed NH3, pyruvate, homocysteine (from cystathionine), and cysteamine (from S-aminoethyl-L-cysteine). The enzyme was active over a broad pH range, with highest activity at pH 7.8 to 8.0. The enzymatic activity was increased by beta-mercaptoethanol. It was not inhibited by the proteinase inhibitor TLCK (N alpha-p-tosyl-L-lysine chloromethyl ketone), pronase, or proteinase K, suggesting that 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 activity of an alphaC-N and betaC-S lyase (cystathionase) type. Since large amounts of H2S have been reported in deep periodontal pockets, cystalysin may also function in vivo as an important virulence molecule.

The immune response of the primate, Macaca fascicularis, to cell envelope (CEA) or cell wall (CWA) antigens of several periodontal pathogens was examined to develop a strategy to interfere with ligature-induced periodontitis. Animals were parenterally immunized with CEA of either Porphyromonas gingivalis, Prevotella intermedia or a combination of CEA/CWA of Campylobacter rectus, Fusobacterium nucleatum and Actinomyces viscosus. Serum samples were taken every 2-4 weeks over a 4-month period, which included a 13-week interval with molar teeth ligated. All of the nonhuman primates in the study exhibited baseline levels of IgG, IgM and IgA antibody to formalinized whole cells of the bacteria. These levels increased significantly following immunization and were elevated above baseline throughout the remainder of the experiment. The largest change in antibody responses was seen in IgA antibody levels of P. gingivalis and C. rectus (42-fold above baseline), IgM antibody to P. intermedia, (41-fold increase) and IgG antibody to F. nucleatum and A. viscosus (32 and 63-fold increases). Moreover, the nonhuman primates exhibited differences in isotype response levels to whole microorganisms compared with the cell envelope antigens. These findings demonstrate the capacity of these nonhuman primates to produce an active immune response to microorganisms chronically colonizing the subgingival microbiota. Additionally, it appears that the bacteria may exhibit some unique differences in their immunogenicity as detected by the nonhuman primate and may contribute to the ability of the immune responses to effectively interact with these pathogens.

These studies determined the ability of selected periodontopathogens to synergistically initiate soft tissue destruction in a murine abscess model. The development of immunity following recovery from infection or by active immunization was also examined. Mice were infected with P. gingivalis W50, F. nucleatum T18, or a combination of the two microorganisms. F. nucleatum caused only a localized lesion in contrast to P. gingivalis which caused a spreading suppurative inflammatory lesion of the skin and subcutaneous tissues, which, depending upon the dose, could result in death. Infection of mice with a combination of P. gingivalis and F. nucleatum elicited a significantly greater lesion size (P<0.001) and lethality compared with P. gingivalis alone. Mice infected with a subclinical dose (no visible lesion) of P. gingivalis failed to develop protective immunity to a secondary P. gingivalis challenge. Mice that had recovered from P. gingivalis lesions demonstrated partial protection against subsequent P. gingivalis challenge; however, the immunity was less protective against the mixed F. nucleatum + P. gingivalis infection. Active immunization with P. gingivalis protected against both the P. gingivalis and F. nucleatum + P. gingivalis challenges and this protection was correlated with the levels of specific serum immunoglobulin G (IgG) antibody. The results indicated that the murine model is ideally suited to examine bacterially-mediated mixed infections that result in soft tissue destruction. This destruction can be minimized, but not abrogated, with development of immunity. Challenge with sufficient numbers of the pathogens can overwhelm the acquired immunity.

Alveolar bone loss associated with periodontal disease occurs frequently in postmenopausal females, the same group that is predisposed to osteoporosis. To determine if the estrogen-deficient state enhances oral bone loss, we studied ovariectomized rats administered the potent bone-resorbing cytokine interleukin-1 or the periodontal pathogen Campylobacter rectus lipopolysaccharide (LPS). Distal root canals of first mandibular molars were instrumented with endodontic files, and bone resorbing factors were deposited and sealed into the root canal. Radiographs of periapical bone loss were evaluated using computer assisted image analysis to determine lesion size. Both interleukin-1 and C. rectus LPS caused a significant increase in lesion area in both ovariectomized and normal rats when compared with controls and a significant increase in ovariectomized animals compared to nonovariectomized animals receiving LPS. Using this endodontic model, we have demonstrated that estrogen deficiency results in increased oral bone loss in rats.

Campylobacter rectus is a putative periodontopathogen which expresses a proteinaceous surface layer (S-layer) external to the outer membrane. S-layers are considered to play a protective role for the microorganism in hostile environments. The S-layer proteins from six different C. rectus strains (five human isolates and a nonhuman primate [NHP] isolate) were isolated, purified, and characterized. The S-layer proteins of these strains varied in molecular mass (ca. 150 to 166 kDa) as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. They all reacted with monospecific rabbit antiserum to the purified S-layer of C. rectus 314, but a quantitative enzyme-linked immunosorbent assay demonstrated a strong antigenic relationship among the five human strains, while the NHP strain, 6250, showed weaker reactivity. Amino acid composition analysis showed that the S-layers of four C. rectus strains contained large proportions of acidic amino acids (13 to 27%) and that >34% of the amino acid residues were hydrophobic. Amino acid sequence analysis of six S-layer proteins revealed that the first 15 amino-terminal amino acids were identical and showed seven residues of identity with the amino-terminal sequence of the Campylobacter fetus S-layer protein SapA1. CNBr peptide profiles of the S-layer proteins from C. rectus 314, ATCC 33238, and 6250 confirmed that the S-layer proteins from the human strains were similar to each other and somewhat different from that of the NHP isolate (strain 6250). However, the S-layer proteins from the two human isolates do show some structural heterogeneity. For example, there was a 17-kDa fragment unique to the C. rectus 314 S-layer. The amino-terminal sequence of this peptide had homology with the C. rectus 51-kDa porin and was composed of nearly 50% hydrophobic residues. Thus, the S-layer protein from C. rectus has structural heterogeneity among different human strains and immunoheterogeneity with the NHP strain.

One way prokaryotes respond to environmental stresses is by modifying selected outer membrane components. Iron, in the form of hemin, has been shown to be a significant regulator of Porphyromonas gingivalis growth and virulence and of the expression of outer membrane proteins and lipopoly saccharide. Since lipopoly saccharide has profound effects on host immune cells, this study compared the effect of hemin-restricted and hemin-normal P. gingivalis growth conditions on lipopolysaccharide priming of N-formylmethionyl-leucyl-phenylalanine-induced superoxide generation by human neutrophils. P. gingivalis was grown in a chemostat under normal (5 micrograms hemin/ml) and hemin-restricted (0.08 microgram hemin/ml) conditions. Purified lipopolysaccharide from both P. gingivalis normal and hemin-limited environments increased N-formylmethionyl-leucyl-phenylalanine-induced superoxide release by neutrophils in a dose-dependent manner. Lipopolysaccharide isolated from the hemin-normal conditions was a significantly more potent neutrophil priming agent than the lipopolysaccharide isolated from hemin-restricted conditions. Addition of normal human serum enhanced the priming effect of both lipopolysaccharide preparations; this effect, however, was more evident with the hemin-normal lipopolysaccharide. Further, this enhancing effect of serum was partly reduced in the presence of antibodies raised against the serum lipopolysaccharide-binding protein. The differences in the biological activity of the two lipopolysaccharide preparations could be associated with structural differences detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. These results indicate that hemin availability affects regulation of an aspect of P. gingivalis virulence, lipopolysaccharide-human neutrophils priming. The reduced capacity for neutrophil priming by hemin-restricted lipopolysaccharide appears to be related to lipopolysaccharide-neutrophil interactions and not to serum factors Targeting bacterial cell-surface components involved in hemin transport might be effective therapy for P. gingivalis-associated periodontal diseases.

A 30 kDa (heated 24 kDa) hemin-binding protein whose expression is both hemin and iron regulated was identified and purified in Porphyromonas gingivalis 381. A strong hemin-binding function was found by LDS-PAGE and TMBZ staining when cells were grown under hemin (iron)-limited conditions. N-terminal amino acid sequence analysis of CNBr-digested 24 kDa hemin binding protein revealed that this protein belongs to a new, so far undescribed hemin-binding class of proteins.

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.

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.