Periodontitis in pregnant baboons: systemic inflammation and adaptive immune responses and pregnancy outcomes in a baboon model.

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.

Systemic inflammatory responses in progressing periodontitis during pregnancy in a baboon model.

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.

Implantation of Bacteroides gingivalis in nonhuman primates initiates progression of periodontitis.

Although periodontitis is a bacterial disease, its multidimensional nature and its bacterial complexity have made it difficult to definitively prove that specific microorganisms initiate the disease process. The successful implantation of a rifampin-resistant strain of the putative periodontal pathogen Bacteroides gingivalis into the periodontal microbiota of monkeys (Macaca fascicularis) resulted in an increase in the systemic levels of antibody to the microorganism and rapid and significant bone loss.

Periodontitis in pregnancy: clinical and serum antibody observations from a baboon model of ligature-induced disease.

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.

A flagella-less mutant of Borrelia burgdorferi. Structural, molecular, and in vitro functional characterization.

A nonmotile mutant of Borrelia burgdorferi, the etiologic agent of Lyme disease, was isolated and characterized. The mutant was compared with the wild-type predecessor as well as with a motile back-revertant of the same genetic background. The mutant lacked, by morphologic, biochemical, and immunologic criteria, the major structural protein of flagella, flagellin. This mutation was not associated with major DNA rearrangements or with failure of transcription. An apparent consequence of a loss of flagella was reduced ability to penetrate human endothelial cell layers in vitro. In another assessment of functional significance, the flagella-less mutant was equal if not superior to flagella-bearing, isogenic isolates when examined in an enzyme-linked immunosorbent assay for anti-B. burgdorferi antibodies in the sera of Lyme disease patients. These studies of a mutant, the first among pathogenic Borrelia spp. to be characterized, indicate that the flagellum and motility it confers play a role in B. burgdorferi's invasion of human tissues. A flagella-less B. burgdorferi may be useful as the basis of a more specific immunoassay and a vaccine for protection against Lyme disease.

Coaggregation between bacterial species.

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

Capillary hemangioblastoma. An immunohistochemical study.

Previous studies using immunohistochemical methods to determine the presence of glial fibrillary acidic protein (GFAP) and Factor VIII-related antigen (FVIIIR:Ag) in stromal cells of capillary hemangioblastomas have yielded conflicting results with respect to the possible astrocytic and endothelial origins of these cells. This study included Ulex europaeus I lectin (UEAI), a more sensitive marker of endothelial cells. Antibodies were also used as markers of pericytes and a variety of markers were employed to identify different populations of histiocytes. Results of this investigation indicate that FVIIIR:Ag and UEAI are limited only to endothelial cells, and that GFAP is present in entrapped astrocytes only. Positivity of stromal cells was found with some of the histiocytic markers, but the authors were unable to conclude that these cells have a histiocytic origin. It was concluded that currently there is no evidence that stromal cells are derived from endothelial, pericytic, or astrocytic cells-their origin remains uncertain.

ULTRASTRUCTURE OF THE THERMOPHILIC BLUE-GREEN ALGA, SYNECHOCOCCUS LIVIDUS COPELAND(1).

The ultrastructure of Synechococcus lividus Copeland, a thermophilic blue-green alga, was studied in thin sections. The cell envelope reveals striking similarities with that of some gram-negative bacteria. In contrast to bacteria and to many other species of blue-green algae, ribosomes are predominantly found in the central nuclear region and appear to be associated with the DNA fibrils. Thylakoids (photo-synthetic lamellae) are arranged as concentric shells, around the nuclear equivalent, lying nearly parallel to one another and to the plasma membrane. Both plasma and thylakoidal membranes, as described by other authors for different Cyanophyceae, are of the unit membrane dimension and morphology. Various types of intracellular inclusions are found: (1) Lipid inclusions, located in the cytoplasm are similar to the osmiophilic globules of higher plant chloroplasts. (2) Polyphosphate inclusions (or volutin) resembling those of other species are generally found at the cell poles but within the nuclear region. (3) Polyhedral inclusions also located in the nuclear region are clearly recognized to be different from the polyphosphate bodies, but their function remains unknown.

Lipopolysaccharide-stimulated PGE2 release from human monocytes. Comparison of lipopolysaccharides prepared from suspected periodontal pathogens.

Lipopolysaccharides (LPS) prepared from the suspected periodontal pathogens Actinobacillus actinomycetemcomitans (A. a.), Bacteroides gingivalis, B. intermedius and Wolinella recta were compared to Salmonella typhimurium LPS for their capacity to stimulate prostaglandin E2 (PGE2) release from human monocytes. Counterflow isolated monocytes were cultured with control medium or media containing 10 micrograms/ml LPS. Media were then exchanged every 24 hours for a total of 72 hours. Salmonella and Wolinella LPS preparations demonstrated seven-fold greater PGE2 release than B. gingivalis and two-fold greater than A. a. and B. intermedius. PGE2 release was found to decrease over time with all LPS preparations except Wolinella. The potency of the LPS preparations is tentatively ranked as follows: Wolinella greater than or equal to Salmonella greater than A. a. greater than B. intermedius greater than or equal to B. gingivalis. These findings demonstrate that LPS preparations from suspected periodontal pathogens are capable of stimulating PGE2 release from human monocytes. The high potency and prolonged stimulation of PGE2 release with Wolinella LPS suggests unusual toxic properties that may exert a greater influence in the pathogenesis of destructive periodontal diseases.

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.

Host responses in patients with generalized refractory periodontitis.

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

Oral bone loss is increased in ovariectomized rats.

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.

Normal pelvic anatomy using transvaginal scanning.

The fine spatial and contrast resolution accorded by TVS provides us with the opportunity to see the uterus, ovary, and larger pelvic vessels with detail and accuracy. Other structures may also be visualized but not with the same finesse, regularity, or attention. Transvaginal color Doppler imaging and pulsed waveform analysis capabilities now provide us with the opportunity of distinguishing vascular and nonvascular structures, and gives us the ability to determine the normal and abnormal flow characteristics of pelvic viscera. Improvements in TVS technology will undoubtedly expand our future capabilities and applications.

Acquisition of oral microbes and associated systemic responses of newborn nonhuman primates.

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.