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Notes: Abstract Periodontal plaque is a complex bacterial ecosystem that carries an innate history of colonization, selection and maturation. Detailed examination of this balanced environment can reveal developmental sequences and certain interrelationships, much like an archeological record, that can provide insight in the understanding of plaque formation and maturation. For the present investigation, methods are employed which enable the retrospective elucidation of the historical data of plaque development and the nature of bacterial interactions. Non-parametric statistical methods are used to analyze risk, agreement and interdependence, following analytical techniques which are well established in medical epidemiology, but not generally employed in dentistry. The fundamental concept is that many organisms which are present in plaque prefer or require a preexisting bacterial milieu for colonization and growth to steady-state level. Plaque samples and Ramfjord attachment level measurements were obtained from 60 adult periodontitis patients. Loosely adherent plaque was sampled and different morphotypes were enumerated by darkfield1 microscopy. The colonization of small spirochetes (S-SP) within the loosely adherent plaque was essential for the colonization of medium spirochetes (M-SP), odds ratio – 15.7 and filaments (FIL), odds ratio = 22.2. Thus, a temporal colonization sequence is inferred for FIL and M-SP, both requiring S-SP as a prerequisite morphotype. Medium spirochetes, in turn, are required for fusiform (FUS) colonization. M-SP also enhance the colonization of FIL and large motile rods (L-MO-R) within the loosely adherent plaque. These morphotypes were inferred to be sequentially interdependent, each preferring or requiring the presence of the preceding morphotype. This interdependence was statistically significant at P〈0.05 as determined by the Z value of the κ, coefficient (intraclass correlation coefficient). The log mean number and % of small and medium spirochetes, as well as L-MO-R were found to be positively associated with increasing attachment loss and pocket depth. A model is proposed, demonstrating morphotype interaction, interdependence and colonization sequence.

Notes: Localized juvenile periodontitis (LJP) is characterized by severe, early onset alveolar bone loss, localized to the first molars and incisors and a high prevalence of infection with Actinobacillus actinomycetemcomitans and associated neutrophil functional abnormalities. Due to the frequent occurrence of this condition in families, it was the purpose of this investigation to determine the association of neutrophil chemotaxis abnormalities and clinical periodontal disease in families with LJP. Twenty-two families were studied in which the proband was selected based upon presentation of LJP. All siblings were examined for the presence of LJP and neutrophil chemotaxis was measured on all subjects. The results indicate that there is a high association of LJP and neutrophil chemotaxis disorders and that this association is consistent along family lines. Specially, in families in which the proband exhibits a neutrophil chemotaxis disorder, all affected siblings (LJP) also exhibit depressed neutrophil chemotaxis, whereas, non-affected siblings beyond the age of puberty have normal neutrophil chemotaxis. On the other hand, in families where the proband (LJP) exhibits normal chemotaxis, both normal and affected siblings exhibit normal chemotaxis. The finding among families with LJP that some exhibit neutrophil chemotactic depression and others exhibit normal neutrophils suggests heterogeneity of LJP. Accordingly, it is proposed that there is a syndrome of familial localized juvenile periodontitis with depressed neutrophil chemotaxis and another form of familial localized juvenile periodontitis with normal neutrophil chemotaxis. The chemotaxis disorder and LJP occur in nearly one-half of the siblings in families with LJP. which is consistent with a dominant trait. however, multigenerational studies are necessary to determine the mode of inheritance. Hence, neutrophil chemotaxis is a disease marker which can be used in genetic studies of familial LJP with depressed neutrophil chemotaxis. Furthermore, prepubertal siblings often exhibit defective neutrophil chemotaxis in the families in which the proband exhibits such a defect. Since the prepubertal siblings are not affected by clinically detectable LJP, the finding of neutrophil chemotactic depression in these children further suggests that the neutrophil chemotactic defect is genetic in origin, precedes and may predispose to localized juvenile periodontitis.

Notes: The Papillon-Lefevre and Haim Munk syndromes are characterized by the presence of both palmoplantar hyperkeratosis (PPK) and severe early onset periodontitis. It is the early onset periodontal disease component that distinguishes these from other more common forms of PPK. It has been proposed that the periodontal disease component may be a casual association in individuals with PPK. Genetic syndromes with palmoplantar keratosis and severe early onset periodontitis may be due to specific bacterial infections in individuals with PPK. Recently, keratin gene mutations have been identified in several conditions typified by palmoplantar keratosis. The present study sought to test the hypothesis that a keratin gene defect similar to those previously identified in other PPK conditions is responsible for the Haim Munk and the Papillon-Lefevre syndromes. We have performed genetic linkage studies to test for linkage between polymorphic DNA loci within 2 cytokeratin gene families and the disease phenotype in Haim Munk syndrome and Papillon-Lefevre syndrome. Families with individuals segregating for the Haim Munk syndrome and the Papillon-Lefevre syndrome were examined to determine disease status, and genotyped for microsatellite DNA markers closely linked to the acidic (type I) and the basic (type II) cytokeratin genes on chromosomes 12 and 17. Genotype data were evaluated for microsatellite allele homozygosity in affected individuals. Results of these preliminary genetic studies suggest that the gene defect in Haim Munk syndrome is not due to a gene defect in either the type I or the type II keratin gene clusters. These findings suggest that Haim Munk syndrome may be genetically distinct from other more common forms of PPK that have been linked to the cytokeratin gene families, and suggest that mutations in genes other than keratin genes are responsible. Additional family studies are needed to confirm these preliminary findings.

Notes: Longitudinal data were collected over a period of at least 18 months and up to 3 years on 41 adult periodontitis patients (AAP Type III and IV). Ramfjord attachment level measurements and sampling of crevicular fluid (CF) at each tooth were repeated every 3 months. A mean full mouth CF prostaglandin E2 (MCF-PGE) value was determined for each patient at each visit. The three-month monitoring was continued until a single site demonstrated a statistically and clinically significant attachment loss (ALOSS) episode. Results indicated that in ALOSS patients the MCF-PGE was significantly elevated at the ALOSS visit as compared to previous levels. Furthermore, the sites which had the ALOSS had elevated levels as compared to the contralateral no ALOSS control sites (305.6±56.5 vs. 65.7±6.89 ng/ml, mean ± SEM). One month following treatment the CF-PGE level dropped to 16.9±3.4 ng/ml at the ALOSS sites. Since the MCF-PGE level increases preceding the attachment loss episode, reaches a maximum at the sites which actually undergo ALOSS, and subsides following treatment, the possibility of using the MCF-PGE level to predict an oncoming future ALOSS episode was examined. The ALOSS patients had a MCF-PGE level of 113.4±9.0 ng/ml 6 months prior to the ALOSS episode, which was significantly higher than the no ALOSS patients’MCF-PGE level of 50.1±7.1. Analysis of MCF-PGE levels as a screening test indicate that this measurement has a high degree of sensitivity, specificity, and a predictive value of 0.92–0.95. Thus, this method has significant merit as a diagnostic tool to determine if a patient is in a state of remission or about to undergo an attachment loss episode.

Notes: An accumulation of elevated numbers of macrophages (Mø) and Ig producing cells is associated with localized and chronically inflamed gingiva of patients with adult periodontitis. When gingival lymphocytes were isolated from inflamed tissues and examined by flow cytometry, approximately 20–30% of lymphocytes were CD4+ T cells. For the analysis of Thl and Th2 cytokine expression by these CD4+ T cells, RNA was extracted and reverse transcriptase polymerase chain reaction (RT-PCR) was performed by using specific 5′ and 3′primers for IFN-γ and IL-2 (Thl), IL-4, IL-5, IL-6, IL-10 and IL-13 (Th2) and β-actin (housekeeping gene). Two distinct cytokine profiles were noted based on the expression of selected Thl and Th2 cytokines. Thus, one pattern was represented by the expression of mRNA for IFN-γ, IL-6. IL-10 and IL-13. while the other case consisted of mRNA for IFN-γ, IL-6 and IL-13. Except for a few cases, messages for IL-2, IL-4 and IL-5 were not detected by cytokinespecific RT-PCR. The predominant expression of Th2 cytokines (e.g. IL-6. IL-10 and IL-13) may contribute to the induction of high B cell responses in local disease sites. On the other hand, lack of IL-4 may be responsible for the accumulation of Mφ in diseased periodontium. We also investigated whether a relationship exists between IL-4 receptor (IL-4R) expression and Mø persistence in the absence of exogenous IL-4. Gingival Mγ, when compared with monocytes (MN)/Mø from peripheral blood mononuclear cells (PBMC), expressed high levels of IL-4R mRNA. When gingival Mø were incubated with recombinant IL-4 (rIL-4). the cell viability was dramatically reduced by apoptosis. These findings clearly show that the lack of IL-4 may contribute to the persistant occurrence of Mø at the disease site and addition of exogenous rIL-4 to gingival Mø cultures leads to cell death by apoptosis.

Notes: Guided tissue regeneration (GTR) is a concept that evolved from the development of membrane-barrier techniques, which allow the repopulation of periodontal wounds by specific cells, resulting in a new attachment apparatus. To help understand the biological mechanisms involved in membrane barrierled periodontal healing, the present study investigated the macromolecules phenotypic of bone and cementum formation in tissues grown under the GTR barrier by immunolocalization. Periodontal regeneration was initiated by placing barriers on experimentally induced periodontal defects in a Rhesus monkey model. Samples were harvested 6 wk after healing and sections of soft tissues grown under GTR barriers (membrane tissue) were stained with antibodies to bone morphogenetic proteins-2 and 4 (BMP-2, BMP-4), bone morphogenetic protein-7 (OP-1), cementum attachment protein (CAP), osteonectin (OTN) and bone sialoprotein (BSP). Tissues grown in the absence of any barrier device served as a control (control tissue). Membrane periodontal tissues from beneath the ePTFE membrane were comprised of spindle-shaped fibroblast-like cells encased in a dense fibrillar extracellular matrix (ECM). Round-shaped cells aggregated to form nodules. Newly formed hard tissue was conspicuous. A similar, but very disorganized, fiber network was observed in control tissues, but neither nodule formation nor hard tissue was observed. Osteonectin staining was observed in the ECM of membrane tissues and particularly in the area of the connective tissue adjacent to newly formed hard tissue. The dense network of connective tissue fibers was also stained. In control tissues, cells and fiber network had a significantly weaker signal for osteonectin. An intense reaction was observed in membrane tissues stained for BSP, particularly the connective tissue adjacent to the newly formed hard tissue, while the control tissues did not stain for BSP. Cementum attachment protein (CAP) was observed in the connective tissue adjacent to the newly formed hard tissue of the membrane tissues whereas control tissues exhibited no CAP staining. In membrane tissues, BMP-2 and 4 distribution was found to concentrate in nodule areas, in the newly formed hard tissue and in the fiber network, while very faint staining was observed in control sections. The distribution of OP-1 in membrane and control tissues was found to mimic the BMP-2 pattern, but staining was more distributed in hard tissue matrix. When the profile of BMP-2, BMP-4, OP-1, OTN. CAP and BSP staining was analyzed on membrane tissue sections, striking similarities were noted in the connective tissue adjacent to the newly formed hard tissue and in nodular areas. In addition, the localization of BMP-2 and BMP-4 mRNA was investigated in both tissues by in situ hybridization. An intense expression of BMP-2 and 4 transcripts was observed in membrane tissues while control tissues never yielded any positive hybridization signal. The correlation between these histochemical findings strongly suggests that the forming soft tissues under ePTFE membranes contain cells and ECM macromolecules normally associated with bone and cementum.