Differentiating zones at periodontal ligament-bone and periodontal ligament-cementum entheses.

BACKGROUND AND OBJECTIVE: The structural and functional integrity of bone-periodontal ligament (PDL)-cementum complex stems from the load-bearing attachment sites (entheses) between soft (PDL) and hard (bone, cementum) tissues. These attachment sites are responsible for the maintenance of a bone-PDL-cementum complex biomechanical function. The objective was to investigate changes in spatiotemporal expression of key biomolecules in developing and functionally active entheses. MATERIAL AND METHODS: Multilabeling technique was performed on hemimandibles of 3 wk and 3 mo-old scleraxis-GFP transgenic mice for CD146, CD31, NG2, osterix and bone sialoprotein. Regions of dominant stretch within the PDL were evaluated by identifying directionality of collagen fibrils, PDL fibroblasts and PDL cell cytoskeleton. RESULTS: CD146+ cells adjacent to CD31+ vasculature were identified at PDL-bone enthesis. NG2+ cells were located at coronal bone-PDL and apical cementum-PDL entheses in the 3-wk-old group, but at 3 mo, NG2 was positive at the entheses of the apical region and alveolar crest. NG2 and osterix were colocalized at the osteoid and cementoid regions of the PDL-bone and PDL-cementum entheses. Bone sialoprotein was prominent at the apical region of 3-wk-old mice. The directionality of collagen fibers, fibroblasts and their cytoskeleton overlapped, except in the apical region of 3 wk. CONCLUSION: Colocalization of biomolecules at zones of the PDL adjacent to attachment sites may be essential for the formation of precementum and osteoid interfaces at a load-bearing bone-PDL-tooth fibrous joint. Biophysical cues resulting from development and function can regulate recruitment and differentiation of stem cells potentially from a vascular origin toward osteo- and cemento-blastic lineages at the PDL-bone and PDL-cementum entheses. Investigating the coupled effect of biophysical and biochemical stimuli leading to cell differentiation at the functional attachment sites is critical for developing regeneration strategies to enable functional reconstruction of the periodontal complex.

Elastic discontinuity due to ectopic calcification in a human fibrous joint.

Disease can alter natural ramp-like elastic gradients to steeper step-like profiles at soft-hard tissue interfaces. Prolonged function can further mediate mechanochemical events that alter biomechanical response within diseased organs. In this study, a human bone-tooth fibrous joint was chosen as a model system, in which the effects of bacterial-induced disease, i.e. periodontitis, on natural elastic gradients were investigated. Specifically, the effects of ectopic biomineral, i.e. calculus, on innate chemical and elastic gradients within the cementum-dentin complex, both of which are fundamental parameters to load-bearing tissues, are investigated through comparisons with a healthy complex. Complementary techniques for mapping changes in physicochemical properties as a result of disease included micro X-ray computed tomography, microprobe micro X-ray fluorescence imaging, transmission electron and atomic force microscopy (AFM) techniques, and AFM-based nanoindentation. Results demonstrated primary effects as derivatives of ectopic mineralization within the diseased fibrous joint. Ectopic mineralization with no cementum resorption, but altered cementum physicochemical properties with increasing X-ray attenuation, exhibited stratified concretion with increasing X-ray fluorescence counts of calcium and phosphorus elements in the extracellular matrix in correlation with decreased hygroscopicity, indenter displacement, and apparent strain-relieving characteristics. Disease progression, identified as concretion through the periodontal ligament (PDL)-cementum enthesis, and sometimes the originally hygroscopic cementum-dentin junction, resulted in a significantly increased indentation elastic modulus (3.16±1.19 GPa) and a shift towards a discontinuous interface compared with healthy conditions (1.54±0.83 GPa) (Student's t-test, P<0.05). The observed primary effects could result in secondary downstream effects, such as compromised mechanobiology at the mechanically active PDL-cementum enthesis that can catalyze progression of disease.

Reduced functional loads alter the physical characteristics of the bone-periodontal ligament-cementum complex.

BACKGROUND AND OBJECTIVE: Adaptive properties of the bone-periodontal ligament-tooth complex have been identified by changing the magnitude of functional loads using small-scale animal models, such as rodents. Reported adaptive responses as a result of lower loads due to softer diet include decreased muscle development, change in structure-function relationship of the cranium, narrowed periodontal ligament space, and changes in the mineral level of the cortical bone and alveolar jaw bone and in the glycosaminoglycans of the alveolar bone. However, the adaptive role of the dynamic bone-periodontal ligament-cementum complex to prolonged reduced loads has not been fully explained to date, especially with regard to concurrent adaptations of bone, periodontal ligament and cementum. Therefore, in the present study, using a rat model, the temporal effect of reduced functional loads on physical characteristics, such as morphology and mechanical properties and the mineral profiles of the bone-periodontal ligament-cementum complex was investigated. MATERIAL AND METHODS: Two groups of 6-wk-old male Sprague-Dawley rats were fed nutritionally identical food with a stiffness range of 127-158 N/mm for hard pellet or 0.3-0.5 N/mm for soft powder forms. Spatio-temporal adaptation of the bone-periodontal ligament-cementum complex was identified by mapping changes in the following: (i) periodontal ligament collagen orientation and birefringence using polarized light microscopy, bone and cementum adaptation using histochemistry, and bone and cementum morphology using micro-X-ray computed tomography; (ii) mineral profiles of the periodontal ligament-cementum and periodontal ligament-bone interfaces by X-ray attenuation; and (iii) microhardness of bone and cementum by microindentation of specimens at ages 6, 8, 12 and 15 wk. RESULTS: Reduced functional loads over prolonged time resulted in the following adaptations: (i) altered periodontal ligament orientation and decreased periodontal ligament collagen birefringence, indicating decreased periodontal ligament turnover rate and decreased apical cementum resorption; (ii) a gradual increase in X-ray attenuation, owing to mineral differences, at the periodontal ligament-bone and periodontal ligament-cementum interfaces, without significant differences in the gradients for either group; (iii) significantly (p < 0.05) lower microhardness of alveolar bone (0.93 ± 0.16 GPa) and secondary cementum (0.803 ± 0.13 GPa) compared with the higher load group insert bone = (1.10 ± 0.17 and cementum = 0.940 ± 0.15 GPa, respectively) at 15 wk, indicating a temporal effect of loads on the local mineralization of bone and cementum. CONCLUSION: Based on the results from this study, the effect of reduced functional loads for a prolonged time could differentially affect morphology, mechanical properties and mineral variations of the local load-bearing sites in the bone-periodontal ligament-cementum complex. These observed local changes in turn could help to explain the overall biomechanical function and adaptations of the tooth-bone joint. From a clinical translation perspective, our study provides an insight into modulation of load on the complex for improved tooth function during periodontal disease and/or orthodontic and prosthodontic treatments.

Alteration of gene expression profiles of peripheral mononuclear blood cells by tobacco smoke: implications for periodontal diseases.

Alterations of the host response by tobacco smoke adversely affect the periodontium. In this study, we examined the effects of in vitro acute smoke exposure on changes in m-RNA expression of primary peripheral mononuclear blood cells through microarray analysis. Mononuclear blood cells were isolated from four healthy non-smokers and plated in culture wells. Half of the cells were then exposed to 5 min of tobacco smoke. Fluorescent c-DNA probes were prepared from the linearly amplified m-RNAs for each sample and hybridized to cDNA microarrays representing approximately 30000 human genes. Significant increases or decreases in m-RNA gene expression between non-smoke-exposed and smoke-exposed samples were identified by permutation t-test, as implemented by the Significance Analysis of Microarrays software package. After smoke exposure, the expression of 90 genes with known function was significantly elevated and the expression of 19 genes with known function was significantly depressed. In addition, 18 upregulated and 26 downregulated transcripts were expressed sequence tags with little information available on function. Approximately 20 of the significantly elevated genes had previously been reported in the literature to be associated with periodontal pathogenesis (fold changes in parentheses). These included plasminogen activator (4.4), Heat Shock Protein (Hsp) 40 kD (2.2), thrombomodulin (4.2), cytochrome c (1.8), COX-2 (2.6), interleukin-1a (1.4), chemokine ligand 1 (3.8), cathepsin L (2.0), and calgranulin A (2.1). In addition, several significantly elevated genes not previously reported in the literature may also play a role in periodontal pathogenesis, and thus warrant further investigation. These include Diphtheria toxin receptor (heparin-binding epidermal growth factor-like growth factor) (7.8), Hsp 10 kDa (1.7), Hsp 105 kD (2.1), Hsp 70 kDa (1.6), and mitogen activated protein kinase 3 (1.5). Among the significantly depressed genes that may play a protective or destructive role in periodontal pathogenesis were interferon gamma receptor 2 (0.58) and chemokine receptor 2 (0.24). Our results may be of use in the search for the molecular mechanisms for the adverse effects of tobacco smoke on the host response.

Effects of tobacco smoke on the secretion of interleukin-1beta, tumor necrosis factor-alpha, and transforming growth factor-beta from peripheral blood mononuclear cells.

Alterations of the host response caused by short-term exposure to high levels of smoke during the act of smoking (acute smoke exposure) as well as long-term exposure to lower levels of tobacco substances in the bloodstream of smokers (chronic smoke exposure) may play a role in the pathogenesis of periodontal diseases in smokers. In this study, we examined the secretion of three cytokines [interleukin (IL)-1beta, tumor necrosis factor (TNF)-alpha, and transforming growth factor (TGF)-beta] from mononuclear blood cells from current smokers and non-smokers exposed to in vitro tobacco smoke (which may be comparable to in vivo acute smoke exposure) and mononuclear blood cells from current smokers not exposed to further in vitro smoke (which may be comparable to chronic smoke exposure). Peripheral blood mononuclear cells were isolated from eight healthy current smokers and eight healthy non-smokers, plated in culture wells, exposed in vitro for 1-5 min to cigarette smoke in a smoke box system or not exposed (baseline controls), and then incubated without further smoke exposure for another 24 h. Supernatants from each well were then collected and assayed for the concentrations of the three cytokines by enzyme-linked immunosorbent assay (ELISA). At baseline, mean IL-1beta levels were higher in smokers than in non-smokers (mean: 10.6 vs. 5.9 pg/ml, anova: P < 0.05). In both smokers and non-smokers, secreted levels of IL-1beta increased from 0 to 5 min of in vitro smoke exposure (mean: 5.9-9.9 pg/ml, t-test: P < 0.05 for non-smokers only) with levels in smokers higher than in non-smokers (P > 0.05). Mean TNF-alpha levels increased from 0 to 2 min of smoke exposure and decreased from 2 to 5 min in smokers and non-smokers, with higher levels in non-smokers than smokers at all time-points (P > 0.05). Mean TGF-beta levels were higher in smokers than in non-smokers at all time-points (mean: 180.5 vs. 132.0 pg/ml, P < 0.05 at 5 min only) with no significant alteration of the pattern of secretion with cigarette smoke exposure. These observed alterations in the secretion of cytokines from mononuclear blood cells in smokers, relative to non-smokers, and with in vitro smoke exposure may play a role in the pathogenesis of periodontal diseases in smokers.

An economic evaluation of a chlorhexidine chip for treating chronic periodontitis: the CHIP (chlorhexidine in periodontitis) study.

BACKGROUND: The authors previously suggested that an adjunctive, controlled-release chlorhexidine, or CHX, chip may reduce periodontal surgical needs at little additional cost. This article presents an economic analysis of the CHX chip in general dental practice. METHODS: In a one-year prospective clinical trial, 484 chronic periodontitis patients in 52 general practices across the United States were treated with either scaling and root planing, or SRP, plus any therapy prescribed by treating, unblinded dentists; or SRP plus other therapy as above but including the CHX chip. Economic data were collected from bills, case report forms and 12-month treatment recommendations from blinded periodontist evaluators. RESULTS: Total dental charges were higher for SRP + CHX chip patients vs. SRP patients when CHX chip costs were included (P = .027) but lower when CHX chip costs were excluded (P = .012). About one-half of the CHX chip acquisition cost was offset by savings in other charges. SRP + CHX chip patients were about 50 percent less likely to undergo surgical procedures than were SRP patients (P = .021). At the end of the trial, periodontist evaluators recommended similar additional procedures for both groups: SRP, about 46 percent; maintenance, about 37 percent; surgery, 56 percent for SRP alone and 63 percent for SRP + CHX chip. CONCLUSIONS: Adjunctive CHX chip use for general-practice patients with periodontitis increased costs but reduced surgeries over one year. At study's end, periodontists recommended similar additional surgical treatment for both groups. CLINICAL IMPLICATIONS: In general practice, routine use of the CHX chip suggests that costs will be partially offset by reduced surgery over at least one year.

Periodontal management of HIV-infected patients.

Recognition and diagnosis of the general oral manifestations and specific periodontal manifestations of HIV infection will continue to be a major responsibility of the dental practitioner. The dental team needs to understand the underlying principles of care for the necrotizing and inflammatory periodontal changes associated with HIV. In addition, the practitioner must take into consideration how the presence of opportunistic infections in the periodontium such as Candida as well as the patient's level of immunosuppression may affect conventional approaches to the common forms of periodontal diseases. By combining local and systemic therapy aimed at both preventing and treating oral lesions and periodontal diseases, combined with new systemic antiviral and vaccine therapies, dental and medical practitioners may together help reduce both the dental morbidity and the overall patient morbidity in the HIV infected patient.

Effects of smoking on local delivery of controlled-release doxycycline as compared to scaling and root planing.

This paper examines the effects of smoking on the treatment outcomes of two nonsurgical therapies: (1) scaling and root planing alone (SRP) or (2) controlled-release of subgingivally delivered doxycycline hyclate in a polylactic acid based polymer gel. Subjects from 2 9-month multicenter studies were classified as nonsmokers (never smoked: 100 subjects), former smokers (137 subjects), and current smokers (> or = 10 cigarettes/day: 121 subjects). Clinical parameters were analyzed for treated sites with baseline probing depths > or = 5 mm and for a subset of treated sites with baseline probing depths of > or = 7 mm. Clinical parameters (plaque levels, clinical attachment levels, pocket depths, and bleeding on probing) were analyzed at baseline, 4, 6, and 9 months. In the doxycycline treated group in general, there were neither marked significant differences in clinical attachment gain nor differences in probing depth reduction among the 3 smoking groups. On the other hand, in the scaling and root planing treated group in general, there were significant differences in clinical attachment gain and pocket depth reduction, with non-smokers responding better than former smokers and current smokers at 6 and 9 months. These differences in clinical response between scaling and root planing alone versus controlled-release of locally-delivered doxycycline hyclate among these 3 smoking groups are discussed in relation to treatment implications for smokers.

Cysteine protease inhibitory activity and levels of salivary cystatins in whole saliva of periodontally diseased patients.

The 3 human salivary cystatins S, SA and SN are multifunctional proteins that possess a cysteine protease inhibitory property, but their ability to act as such is very different (SN > SA >> S). One form, S, also appears to possess antibacterial properties towards the bacterium Porphyromonas gingivalis, often associated with periodontal diseases. In this study we measured the total cystatin inhibitory activity and the levels of each salivary cystatin in the whole saliva of 8 periodontally diseased patients and 2 groups of control subjects (n = 6 and n = 10). The total cystatin inhibitory activity and the total salivary cystatin concentration in the periodontally diseased patients were found to be lower than the controls (p < or = 0.005). The concentration of S was depleted to levels that would not allow it to be an effective antibacterial agent, and the concentration of SA, although depleted in some cases, was still present at sufficient levels to allow it to act as an effective physiological inhibitor of cathepsin L. The concentration of cystatin SN was also depleted in the periodontally diseased patients, but was still present in sufficient quantities to act as an effective physiological cysteine protease inhibitor of cathepsins H and L. In comparison, the concentration of all 3 salivary cystatins in the control subjects were sufficient to enable these proteins to be both effective physiological cysteine protease inhibitors and antibacterial agents.

Alterations of neutrophil L-selectin and CD18 expression by tobacco smoke: implications for periodontal diseases.

Alterations in neutrophil functions by both chronic low levels of tobacco and by acute short-term higher levels of tobacco smoke, as encountered during the act of smoking, may play a role in the pathogenesis of periodontal diseases in smokers. Among the early migration events of neutrophil function is the alteration in surface expression of L-selectin and the CD11/18 integrins. In the present study we examined the effect of in vitro smoke exposure and nicotine alone on the expression of these 2 adhesion molecules in neutrophils from smokers and non-smokers. We also determined the physiological relevance of this in vitro system by assessing the levels of nicotine exposure in this in vitro system and comparing these levels to acute and chronic levels of nicotine in saliva and gingival crevicular fluid. Peripheral neutrophils were isolated from the blood of smokers (> 1 pack/d) and non-smokers and incubated in vitro with either cigarette smoke (0-5 min), 10(-7) M F-met-leu-phe, or nicotine alone at 1.62 mg/ml to 162 ng/ml (10(-2) M-10(-6) M). The neutrophils were then incubated with fluoresceine conjugated anti-Leu8 (L-selectin), anti-CD18 (CD18 integrin), or gamma-4 (non-specific control), fixed and analyzed by flow cytometry. With cigarette smoke exposure, there was an approximate 75% shedding of L-selectin in both smokers and non-smokers with no marked difference between groups at 1-5 min of smoke exposure. Cigarette smoke exposure resulted in a 15-20% increase in CD18 expression in both smokers and non-smokers. At all time points, there was slightly greater but statistically insignificant expression of CD18 integrin in non-smokers when compared to smokers. These patterns of CD18 increases and L-selectin shedding were similar in magnitude to incubations with 10(-7) M F-met-leu-phe. Acute smoke exposure resulted in elevation of nicotine in the smoke box to 529 ng/ml at 5 min, in saliva from 109.2 ng/ml before smoking to 1821.4 ng/ml after smoking, and in gingival crevicular fluid to 5961 ng/ml after smoking. No significant alterations in L-selectin or CD18 expression were noted with in vitro nicotine from 1.62 mg/ml to 162 ng/ml.

Alterations of neutrophil oxidative burst by in vitro smoke exposure: implications for oral and systemic diseases.

Alterations of neutrophil functions by tobacco products may play a central role in the pathogenesis of periodontal diseases and several smoking-related systemic diseases. In the present study, we examined the in vitro effects of cigarette smoke on neutrophils at times and concentrations that may be encountered during smoke exposure. We measured the level of smoke exposure in the in vitro system by measuring the levels of nicotine and comparing these to levels in the oral cavity in smokers before and after smoking. We examined both the unstimulated and stimulated release of 2 oxidative burst products: superoxide (O-2) and hydrogen peroxide (H2O2). Salivary washings were collected from 7 smokers (> 1 pack/day) before smoking a cigarette. Immediately after they smoked a cigarette, a second set of washings was collected. In vitro exposure to smoke involved incubating aliquots of neutrophils in phosphate-buffered saline for 1 to 5 minutes. Nicotine and cotinine levels were quantitated using gas chromatography, with detection by electron impact mass spectrometry. Peripheral neutrophils were isolated from medically healthy non-smoking volunteers via a double-density gradient technique and incubated in vitro with whole cigarette smoke for 0 to 5 minutes. Phorbol myristate acetate (PMA; 10(-7) M) was used to stimulate half of the cell aliquots. Superoxide generation was assessed through the superoxide dismutase (SOD) inhibitable reduction of ferricytochrome c. H2O2 production was assessed through the H2O2-dependent breakdown of dichlorofluorescin diacetate to its fluorophore and measured by flow cytometry. There was a marked elevation in salivary nicotine concentration from before smoking (mean: 80.8 ng) to after smoking (mean 1,685 ng/mL). In the in vitro smoke box system, there was a time-related elevation in nicotine from 1 to 5 minutes (50-->136 ng/mL). In PMA-stimulated cells exposed to smoke, there was a time-related inhibition of both superoxide and H2O2 production. However, in unstimulated cells exposed to smoke, there was a time-related increase in the release of superoxide and H2O2. A novel finding in unstimulated cells exposed to smoke was that there appeared to be 2 distinct populations of cells--one of "high" H2O2 producers and one of "low" H2O2 producers. The proportion of high H2O2 producers increased relative to smoke exposure. The relative production of H2O2 in the unstimulated high producers was comparable to PMA-stimulated cells at 5 minutes. This release of superoxide and H2O2 in unstimulated cells exposed to smoke may alter the pathogenic processes both in periodontal diseases and other systemic diseases.

Nicotine effects on neutrophil F-actin formation and calcium release: implications for tobacco use and pulmonary diseases.

Alterations in neutrophil functions by tobacco components may play a pivotal role in pulmonary emphysema. This study examined the role of nicotine in altering F-actin formation and calcium (Ca2+) release (two early events in neutrophil motility). The effects of these alterations on the motile function of phagocytosis were also examined. Human peripheral neutrophils from medically healthy nonsmoking subjects were incubated with nicotine at concentrations normally encountered during acute exposure to cigarette smoke (10(-2) to 10(-5) M) and/or the chemotactic peptide FLPEP (10(-7) M). Relative F-actin stain was determined by NBD phallacidin staining followed by flow cytometry. Intracellular Ca2+ was determined by INDO-1 AM loading followed by emission ratio quantitation by fluorometry. Phagocytosis was determined by the % phagocytic cells with carboxylated microspheres. Incubation of neutrophils with varying concentrations of nicotine resulted in a significant elevation of the relative F-actin stain at 30 s at 10(-2) and 10(-3) M (p < .05, ANOVA) and at 30 min at 10(-2) to 10(-4) M (p < 0.05). In time course studies with 10(-7) M FLPEP stimulation, there was a approximately 325% rise in relative F-actin stain at 30-60 s, followed by a gradual decrease to near baseline levels. There was an immediate rise in Ca2+ to approximately 150% over baseline values, followed by a gradual decrease to baseline. By contrast, stimulation with nicotine demonstrated a approximately 105% increase in relative F-actin staining at 10(-2) M (p < .001, ANOVA) and a smaller increase at 10(-3) M, which remained elevated up to 600 s. Intracellular Ca2+ levels also rose in a dose-dependent manner with an increased of 700% over baseline with 10(-2) M nicotine, and remained elevated up to 600 s. Coincubation with both FLPEP and nicotine demonstrated additive effects in relative F-actin staining at both maximal and submaximal concentrations. Preincubation with 10(-2) or 10(-3) M nicotine suppressed the % phagocytic cells by 32% and 16%, respectively (p < .001, ANOVA) with only a 1-4% reduction in cell viability (trypan blue exclusion). The results demonstrate that the concentration of nicotine during acute cigarette exposure can directly stimulate neutrophil F-actin formation and intracellular Ca2+ release by a mechanism different from peptide stimulation. The alteration of these two pivotal neutrophil signaling events by nicotine may in turn alter other neutrophil functions in tobacco-related pulmonary emphysema.

Epithelial cell kinetics with atelocollagen membranes: a study in rats.

A recent development in guided tissue regeneration procedures is the use of resorbable membranes, which eliminate the need for subsequent surgical removal. In this study we performed flap surgery in rats with (experimental) or without (control) implantation of one of the newer materials, atelocollagen. We observed the gingival epithelial cell kinetics using 3H-thymidine and examined the extent of gingival epithelium migration. Histological observations at day 1 on the experimental side demonstrated regenerated epithelium apposed to the collagen membrane with an intervening layer of necrotic tissues and/or fibrinous exudate. There was no observable proliferation of regenerated epithelium toward the root apex. On day 14, the regenerated epithelium migrated apically along the treated root surface in the control group. By contrast, on day 14 in the experimental group, the regenerated epithelium contacted the root surface at the cemento-enamel junction (CEJ). Apical to the CEJ, there was new cementum formation with inserting connective tissue fibers. Autoradiographs from day 1 experimental sides demonstrated labeled cells in the basal cell layers from oral, crevicular, and junctional epithelium. From day 1 to day 5, labeling indices of oral epithelium and regenerating crevicular epithelium on experimental sides were lower than on control sides. These histological and autoradiographic findings suggest that atelocollagen membrane inhibits apical migration of regenerating epithelium and accelerates connective tissue reattachment in part by inhibiting the mitotic function of basal epithelial cells in early stages of wound healing.

Periodontal considerations in the patient with HIV.

The HIV-associated periodontal diseases present unique challenges to the dental practitioner. The presence and severity of HIV-associated gingivitis, HIV-associated periodontitis, and possibly necrotizing ulcerative gingivostomatitis, and other oral lesions may indicate the presence and staging of HIV infection. In general, a similar relationship does not appear to exist between adult periodontitis and HIV staging. There is a wide variation in recent reports on the epidemiology of HIV-associated diseases. These variations point to the need for a standard definition for HIV-associated periodontal diseases using both conventional periodontal evaluation criteria and criteria designed specifically for the characteristics of HIV-associated gingivitis and HIV-associated periodontitis. HIV staging, geographic location of the study, antiviral and antimicrobial therapies, and oral habits may also account for many of these differences. Both the HIV-associated gingivitis and periodontitis lesions have similar microbiologic profiles to adult periodontitis lesions for previously identified periodontopathic bacteria. In addition, these lesions may have a unique opportunistic microflora. The pathogenesis of HIV-associated periodontal diseases may be due to the microflora, the effects of HIV and other viral agents, or alterations in the host response. These factors should be taken into consideration in the treatment and prevention of these HIV-associated periodontal lesions.

Cytokeratin patterns in corneal, limbal, and conjunctival epithelium. An immunofluorescence study with PKK-1, 8.12, 8.60, and 4.62 anticytokeratin antibodies.

The authors examined immunofluorescently the specific cytokeratin staining patterns of corneal, limbal, and conjunctival epithelium with PKK-1, 8.12, 4.62, and 8.60 monoclonal anticytokeratin antibodies. Observations were made on unfixed frozen postmortem human tissue. The PKK-1 antibody stain was observed in all layers of corneal epithelium but only in suprabasal layers of limbal and conjunctival epithelium. By contrast, the 8.12 antibody stain was observed only in the superficial layer of corneal epithelium but through all layers of limbal and conjunctival epithelium. The 4.60 antibody stain was seen in focal areas of corneal and limbal epithelium and through all layers of conjunctival epithelium. The 8.60 antibody stain was not present in the three epithelia. These immunofluorescence studies showed unique cytokeratin patterns among layers in corneal, limbal, and conjunctival epithelium.

In situ localization of cytoskeletal elements in the human trabecular meshwork and cornea.

The authors compared cytoskeletal elements of the in situ human trabecular-meshwork cell with in situ human corneal cells using indirect immunofluorescence staining for tubulin and intermediate filaments (vimentin, cytokeratin, and desmin) and NBD-phallacidin staining for f-actin using both fixed frozen and unfixed frozen sections from postmortem eyes. Both f-actin and tubulin were found throughout the cell body of trabecular-meshwork cells, keratocytes, corneal endothelium, and corneal epithelium. The f-actin staining pattern was concentrated at the cell periphery of these four cell types. Vimentin stain was intensely localized in focal areas of the trabecular-meshwork cell, keratocytes, and throughout the corneal endothelium. A general anticytokeratin antibody was intensely localized in corneal epithelium and endothelium. However, PKK-1 anticytokeratin antibody was seen only in superficial layers of corneal epithelium and not in corneal endothelium. The 4.62 anticytokeratin antibody was not observed in either corneal epithelium or endothelium. None of these three cytokeratin antibodies were seen in trabecular-meshwork cells or keratocytes. Desmin stain was not noted in any of these cell types. In general, cytoskeletal staining of unfixed frozen sections showed a similar staining pattern for f-actin and tubulin but a more uniform and intense staining pattern for vimentin and cytokeratin compared with fixed frozen material. The authors conclude that these cytoskeletal stains can differentiate human trabecular-meshwork cells from cells of the cornea in situ.

Ammonia as a potential mediator of adult human periodontal infection: inhibition of neutrophil function.

Neutrophils (polymorphonuclear leucocytes) are the principal cell of the host defence system. Consequently, if periodontal pathogen-derived substances in the gingival crevice significantly inhibit their function, they could shift the bacterial-host balance in favour of the bacteria. The hypothesis that ammonia can inhibit neutrophil function was tested. Ammonia was specifically selected because periodontal pathogens produce substantial amounts of ammonia. The findings indicated that ammonia can inhibit neutrophil phagocytosis, degranulation and oxygen metabolism. Ammonia decreased the total number of phagocytosing polymorphonuclear neutrophils (66% of control) and also decreased degranulation (61% of control). Ammonia decreased oxygen metabolism of both resting and stimulated neutrophils (33 and 42% of control, respectively). These observations support the hypothesis that ammonia can inhibit the function of polymorphonuclear leukocytes. They suggest that the presence of ammonia in the gingival crevice may increase the risk of development of periodontal disease.

Microtubule-granule relationships in motile human polymorphonuclear leukocytes.

We examined the relationship of microtubules to the granule organization in stimulated human polymorphonuclear leukocytes (PMNs). Electron microscopic (EM) observations of critical-point-dried PMNs revealed that only a portion of the granules appeared in close association to microtubules. These closely associated granules appeared to be attached to the microtubule via smaller-diameter filaments. The remaining granules appeared either attached to microtubules at a further distance, via smaller-diameter filaments such as actin, or unassociated with microtubules. EM observations of PMNs treated with either the microtubule promoter drug taxol or the microtubule depolymerization drugs nocodozole and colchicine revealed a redistribution of granules towards the nucleus. Granule clustering at the periphery of the cell was also noted with nocodozole and colchicine. With cytochalasin B, a uniform distribution of granules was noted. However, granule clustering was noted when PMNs were coincubated with cytochalasin B and colchicine. These results indicate that microtubules may have both a direct and indirect role (through other cytoskeletal elements) in the organization of PMN granules.

Quantitative assessment of cynomolgus monkey trabecular cell phagocytosis and adsorption.

A simple double fluorescence technique is described which allows quantification of cynomolgus monkey trabecular cell phagocytosis and adsorption. Within the first hour of exposure, significant adsorbance and minimal phagocytosis of polystyrene beads was observed. Peak internalization occurred at six hours after increasing linearly at a rate of 20% of maximum per hour; adsorbance was constant and minimal.