Suppressor cell function is preserved in pemphigus and pemphigoid.

Human peripheral blood lymphocytes (PBL) are activated to become suppressor T cells (S-T-C) by incubation with Concanavalin-A (Con-A). This has become the standard method for evaluation of suppressor function in patients. S-T-C function has been found to be impaired in several autoimmune diseases, including systemic lupus erythematosus (SLE). Using this assay, we have investigated suppressor-cell function in 2 autoimmune disorders, bullous pemphigoid (BP) and pemphigus vulgaris (PV), studying 6 patients from each group. Three patients with active SLE (positive controls), and 11 normal donors (negative controls) were also included. None of these patients had received systemic therapy with the exception of 2 patients with PV who were treated with gold in the past. PBL from these patients were incubated with and without 40 micrograms/ml Con-A for 72 hr to generate suppressor cells. Both groups of PBL were then irradiated wih 1500 r cobalt. Co-cultures were set up in sextuplicate using normal PBL as responders. Responder PBL were stimulated with 0.5, 1.0, and 2.0 micrograms/ml of phytohemagglutin (PHA) and 5.0, 10.0, and 20.0 micrograms/ml of Con-A. Cultures were pulsed on day 3 with 3H-thymidine and harvested on day 4. Data were analyzed using Student's t-test. S-T-C function was found to be significantly impaired in SLE vs normal (p = 0.0316). No statistically significant difference was seen in BP (p = 0.5883) and PV (p = 0.0921) as compared with normals. A defect in suppressor cell function may still be present in patients with PV and BP for the defect may be antigen-specific and therefore remain undetected by the Con-A suppressor assay.

Measurement of immunoglobulin concentration in cell culture supernates by computer-assisted ELISA.

The enzyme-linked immunosorbent assay (ELISA) is used extensively in immunologic research for obtaining quantitative estimates of immunoglobulin concentration in cell culture supernates. Through incorporation of a microcomputer for data acquisition, storage and rapid calculation of results, a substantial reduction in total assay time may be realized. Described here are a set of menu-driven programs written in Basic for the IBM-PC which provide advantages over existing software in simplicity, versatility and accuracy. Hardware requirements are minimal. These programs should encourage greater flexibility in terms of the size and complexity of experimental designs.

Cicletanine reverses vasoconstriction induced by the endogenous sodium pump ligand, marinobufagenin, via a protein kinase C dependent mechanism.

RATIONALE: Cicletanine (CIC), an anti-hypertensive compound with direct vascular and natriuretic actions, is especially effective in salt-sensitive hypertension, in which dysregulation of the sodium pump plays an important pathogenic role, and digitalis-like cardiotonic steroids contribute to increased vascular tone. The purpose of the present study was to investigate whether, and by what mechanisms, cicletanine antagonizes the vasoconstrictor effects of cardiotonic steroids in isolated human arteries. METHODS: The effects of cicletanine on vascular tone were studied in isolated, endothelium-denuded rings of 2nd-3rd-order branches of human mesenteric arteries pre-contracted with bufodienolide marinobufagenin (MBG), an Na/K-ATPase inhibitor, or endothelin-1 (ET-1). Na/K-ATPase activity was measured in sarcolemmal membranes from the mesenteric artery. Activity of rat brain protein kinase C (PKC) was measured using the PepTag phosphorylation assay. RESULTS: MBG and ET-1 both induced sustained vasoconstriction in human mesenteric artery rings, and cicletanine relaxed rings pre-contracted with either MBG (EC50 = 11 +/- 2 micromol/l) or ET-1 (EC50 = 6.4 +/- 1.1 micromol/l). Although 8-Br-cGMP (100 micromol/l) caused complete vasorelaxation of arterial rings pre-contracted with ET-1, it did not affect the MBG-induced vasoconstriction. An activator of PKC, phorbol diacetate (PDA) (50 nmol/l), attenuated CIC-induced vasorelaxation of mesenteric artery rings pre-contracted with MBG (EC50 > 100 micromol/l), but not rings pre-contracted with ET-1 (EC50 = 6.5 +/- 1.2 micromol/l). In mesenteric artery sarcolemma, 100 nmol/l MBG inhibited the Na/K-ATPase by 68 +/- 5% and cicletanine (100 micromol/l) attenuated this Na/K-ATPase inhibition by 85 +/- 6%. In the PepTag PKC assay, cicletanine produced a concentration-dependent inhibition of rat brain PKC activity (IC50 45 +/- 11 micromol/l). In the presence of 50 nmol/l PDA, 100 micromol/l cicletanine did not antagonize the Na/K-ATPase inhibition by MBG, and did not inhibit the PKC from rat brain. CONCLUSIONS: Cicletanine antagonizes vasoconstriction induced by Na/K-ATPase inhibition via a PKC-dependent mechanism that does not involve inhibition of cyclic GMP phosphodiesterase (cGMP-PDE). This mechanism of action may be relevant to the greater potency of cicletanine in salt-sensitive hypertension in which plasma levels of endogenous digitalis-like cardiotonic steroids are elevated. Our findings also suggest that PKC is an important factor for cardiotonic steroid-Na/K-ATPase interactions on the vascular tone, and is therefore a potential target for therapeutic intervention in hypertension.

Circulating bufodienolide and cardenolide sodium pump inhibitors in preeclampsia.

OBJECTIVE: To determine plasma levels of the endogenous bufodienolide Na+/K+ ATPase inhibitor, marinobufagenin-like factor (MBG), in normotensive pregnancy and in preeclampsia, to compare changes of MBG with that of ouabain-like compound (OLC), and to characterize the purified MBG immunoreactive factor from preeclamptic plasma. DESIGN AND METHODS: Consecutive sample study. The levels of MBG and OLC compounds were measured in extracted plasma by solid phase fluoroimmunoassays. MBG and ouabain immunoreactive materials were partially purified from preeclamptic plasma via reverse-phase high-performance liquid chromatography (HPLC) and studied for their ability to cross react with MBG and ouabain antibodies, and to inhibit the Na+/K+ ATPase from human mesenteric arteries. Vasoconstrictor effect of authentic MBG was studied in isolated rings of human umbilical arteries. RESULTS: In 11 nonpregnant control individuals, plasma concentrations of MBG and OLC were 0.190+/-0.04 nmol/l and 0.297+/-0.037 nmol/l, respectively. In the third trimester of noncomplicated pregnancy (n = 6), plasma MBG increased (0.625+/-0.067 nmol/l, P<0.05), and OLC did not (0.32+/-0.07 nmol/l). In 15 patients with preeclampsia, plasma levels of both MBG and OLC increased dramatically (2.63+/-0.10 nmol/l and 0.697+/-0.16 nmol/l, respectively, P<0.01 versus both control groups). When fractionated by reverse phase HPLC, OLC was eluted by 18% acetonitrile, and MBG by 48% acetonitrile. Serially diluted samples of MBG and OLC immunoreactive materials from HPLC fractions reacted with MBG and ouabain antibody in solid phase immunoassay in a concentration dependent fashion. Authentic MBG caused contractile responses of isolated rings of human mesenteric arteries in a concentration-dependent manner. Similarly to the authentic MBG, HPLC purified MBG immunoreactive material from preeclamptic plasma inhibited Na+/K+ ATPase purified from human mesenteric artery. CONCLUSIONS: Our observations demonstrate the coexistence of two endogenous cardiotonic steroids in preeclamptic plasma, a more polar OLC and a less polar MBG-like compound. Substantial increases in plasma OLC and MBG immunoreactivity in preeclampsia, along with the vasoconstrictor properties of authentic MBG and Na+,K+ ATPase inhibitory activity of human MBG immunoreactive factor, suggest, that in preeclampsia, plasma concentrations of MBG are enough to substantially inhibit the sodium pump in cardiovascular tissues, and are in accordance with the views attributing endogenous digitalis-like factors a pathogenic role in the preeclamptic hypertension.

Changes in aqueous immunoglobulin and albumin levels following penetrating keratoplasty.

The feline model of induced rejection of corneal allografts was employed to define the changes in the concentrations of immunoglobulins and albumin in the anterior chamber prior to, and concomitant with the rejection of the transplanted cornea. Fourteen animals received unilateral exchange corneal allografts. Aqueous humor obtained by anterior chamber paracentesis at regular intervals prior to and following the performance of the penetrating keratoplasties was analyzed for IgG, IgM and albumin concentrations using the micro enzyme-linked immunosorbant assay (ELISA). Two patterns of anterior chamber protein modulation were observed. Eight of the animals demonstrated a biphasic pattern in which both immunoglobulin and albumin concentrations were elevated two- to five-fold above presurgical values 14 days postkeratoplasty, returning to preoperative values by day 42. Three to 5 weeks after corneal rejection was induced increases in protein concentrations were observed that correlated with the appearance of clinical signs of rejection. A second, monophasic pattern of anterior chamber protein modulation following keratoplasty was observed in four of the animals. It was distinguishable from the biphasic pattern in that levels did not return to baseline values after the initial rise following keratoplasty until the rejection process was completed. The monophasic response was found to be characteristic of more rapid and vigorous corneal rejection. Examination of albumin to immunoglobulin ratios suggested that all changes in protein levels following keratoplasty were a result of increased influx of serum proteins into the anterior chamber, rather than due to local immunoglobulin synthesis.

Aspiration pneumonia: dental and oral risk factors in an older veteran population.

OBJECTIVES: To investigate the importance of medical and dental factors in aspiration pneumonia in an older veteran population. DESIGN: Prospective enrollment of subjects with retrospective analysis of data. SETTING: Department of Veterans Affairs outpatient clinic, inpatient ward, and nursing home. PARTICIPANTS: 358 veterans age 55 and older; 50 subjects with aspiration pneumonia. MEASUREMENTS: Demographic and medical data; functional status; health-related behaviors; dental care utilization; personal oral hygiene; comprehensive dental examination; salivary assays including IgA antibodies; and cultures of saliva, throat, and dental plaques. RESULTS: Two logistic regression models produced estimates of significant risk factors. One model using dentate patients included: requiring help with feeding (odds ratio (OR) = 13.9), chronic obstructive pulmonary disease (COPD) (OR = 4.7), diabetes mellitus (OR = 3.5), number of decayed teeth (OR = 1.2), number of functional dental units (OR = 1.2), presence of important organisms for decay, Streptococcus sobrinus in saliva (OR = 6.2), and periodontal disease, Porphyromonous gingivalis in dental plaque (OR = 4.2), and Staphylococcus aureus presence in saliva (OR = 7.4). The second model, containing both dentate and edentulous patients included: requiring help with feeding (OR = 4.7), COPD (OR = 2.5), diabetes mellitus (OR = 1.7), and presence of S. aureus in saliva (OR = 8.3). CONCLUSION: This study supports the significance of oral and dental factors while controlling for established medical risk factors in aspiration pneumonia incidence.

Cellular localization of a Hsp90 homologue in Porphyromonas gingivalis.

We previously reported an association between elevated serum antibody titers to the 90-kDa human heat shock protein (Hsp90), periodontal health and colonization by Porphyromonas gingivalis. In this study, we examined the cellular localization of the Hsp90 homologue of P. gingivalis. Cultures of P. gingivalis were heat-stressed (45 degrees C) and examined for localization of the Hsp90 homologue. Heat stress induced a 4-5-fold increase in anti-Hsp90 antibody reactivity over that of the unstressed controls. Western blot analysis revealed two bands (44 and 68 kDa) that reacted with anti-Hsp90 antibodies. The 68-kDa band was heat-inducible, while the 44-kDa band was not. Immunogold staining revealed that the Hsp90 homologue localized principally to the membrane and extracellular vesicles. Subcellular fractionation confirmed that the Hsp90 homologue was primarily membrane-associated.

Prevalence of Treponema denticola and Porphyromonas gingivalis in plaque from periodontally-healthy and periodontally-diseased sites.

Intracrevicular plaque from periodontally-healthy individuals who had refrained from oral hygiene measures for 24 h prior to sampling, and subgingival plaque from diseased sites of patients with chronic periodontitis were screened by ELISA for the presence of Porphyromonas gingivalis and Treponema denticola. The samples were also subjected to the PerioScan test to detect the presence of enzymes capable of degrading N-benzoyl-DL-arginine-2-naphthylamide (BANA). Of the 141 samples from periodontally-healthy sites, 73% contained T. denticola antigens and 78% P. gingivalis antigens, compared to 43% and 59%, respectively, in plaque samples from the 159 diseased sites. A positive reaction in the PerioScan test was obtained in 89% of plaque samples from diseased sites and in 60% of those from healthy sites. The correlation between the results of the two assays was poor in the case of intracrevicular plaque from healthy sites. However, with plaque samples from diseased sites, the results of the PerioScan test showed very strong correlation with those obtained with the ELISA, suggesting that the former may be a useful, rapid means of indicating the presence of T. denticola and P. gingivalis in such plaque samples.

The expression of the epithelial blood-group substances: normal and malignant tissues.

The blood-group isoantigens are macromolecules localized to the plasma membranes of certain epithelial tissues. These substances are not detectable on the epithelium once it has undergone malignant transformation. Results of this investigation have demonstrated that the loss of detectability of the blood-group isoantigens does not appear to be related to a "masking" effect by an increase in surface sialic acid. Using fluorescein-labeled lectins specific for sugar subunits which are components of the blood-group oligosaccharide chain, it was found that the malignant cells and cells of the parabasal layer of normal oral epithelium had high levels of N-acetyl-D-glucosamine (GlcNAC), the subterminal sugar residue of the blood-group chain. The basal cells of normal epithelium and a minority of the malignant cells demonstrated levels of D-galactose-N-acetyl-D-galactosamine, which are the most proximal blood-group sugar subunits, as well as subunits of other membrane antigens. Our results indicate that malignant cells seem to be capable of synthesizing the blood-group oligosaccharide chains to the same level as the normal cells of the parabasal layer of stratified squamous epithelium. This level is just subjacent to the terminal D-galactose residue of the blood-group precursor chain. Increased or decreased differentiation characteristics of squamous cell carcinomas did not alter the level of blood-group synthesis. However, there may be a correlation between the level of synthesis of these antigens and the ability of the cells to demonstrate motility and to proliferate.

Detection of two anaerobic periodontopathogens in children by means of the BANA and ELISA assays.

The mouths of young children become colonized by a variety of bacteria, but there have been only a few studies that have sought the presence of periodontopathic species in this population. Almost all of these studies used culturing techniques rather than the newer detection methodologies for various periodontopathogens. Studies in adults have shown that Treponema denticola and Porphyromonas (Bacteroides) gingivalis can be detected in dental plaque by use of the BANA and ELISA diagnostic tests. In the present study, plaque samples from four subgingival sites in each of 157 children (aged from two to 18 years) were tested for BANA hydrolysis with a BANA reagent card, and for T. denticola and P. gingivalis with an ELISA assay. Anaerobic periodontopathogens hydrolyzing the BANA substrate were found to be present in at least one of four plaque samples in 88 children (56%). T. denticola and/or P. gingivalis were detected by ELISA in at least one plaque sample in each of 135 children (86%). This study shows that children are widely colonized by these micro-organisms. A higher proportion of Black children than Caucasian children was colonized by these BANA-positive organisms. Also, children having a parent with a documented history of periodontal disease were more likely to be BANA-positive than were children of parents with unknown periodontal status.

The relationship between gingivitis and the serum antibodies to the microbiota associated with periodontal disease in children with Down's syndrome.

Gingival inflammation in Down's syndrome children (DS) develops earlier and is more rapid and extensive than in non-DS children. Abnormalities in host response to the oral flora have been proposed as etiological factors of this gingival inflammation. However, the relationship between gingivitis and the host response to oral microorganisms in DS by age has not been determined. The objective of this study was to clarify this relationship. Sera were obtained from 75 DS subjects (aged 2 to 18 years) and their gingival health assessed using a modified PMA Index (M-PMA). Antibody titers to Porphyromonas gingivalis (Pg), Prevotella intermedia (Pi), Treponema denticola (Td), Fusobacterium nucleatum (Fn), Selenomonas sputigena (Sel), Actinobacillus actinomycetemcomitans (Aa), and Streptococcus mitis (Mi) were determined using the micro-ELISA. DS subjects under 4 years old were found to have significantly more gingival inflammation than did normal children the same age. A significant positive correlation (r = 0.548, P < 0.0001) existed in the relationship between M-PMA score and plaque score for subjects in the G1 age group (deciduous dentition). At G1, the average antibody titers to Aa, Mi, and Fn exceeded those of the normal adult reference serum pool. In addition, IgG antibody titers to Pg, Aa, Fn, Sel, and Mi correlated significantly with the M-PMA scores in the G1 age group. There was a correlation between age (2 to 18 years) and these antibody titers. IgG antibody titers to Pg, Aa, Sel, and Mi increased significantly with increasing M-PMA score. Furthermore, the IgG antibody titers to Pg were higher (P < 0.05) in the most extensive disease group compared to the DS no-disease group. The IgG antibody titers to Pg at G3 (early puberty) were significantly higher when compared to G1 (preschool children). The IgM antibody titers to Aa at G3 were higher (P < 0.05) when compared to G1. This study suggests that colonization by Aa and Fn are closely associated with the onset of gingival inflammation in DS patients under 5 years old. Colonization by Pg, Aa, Sel, and Mi in DS appears to be associated with gingivitis at puberty.

The relationship between gingivitis and colonization by Porphyromonas gingivalis and Actinobacillus actinomycetemcomitans in children.

BACKGROUND: Porphyromonas gingivalis and Actinobacillus actinomycetemcomitans are closely associated with the onset and severity of adult periodontal disease. However, little is known regarding the colonization by, and host antibody response to, these microorganisms in children. METHODS: Plaque and sera were obtained from 40 healthy children, 2 to 18 years old. Gingival health was assessed by the periodontal disease index (PDI), papillary bleeding score (BS) and the modified total papillary margin attachment index (M-PMA). P. gingivalis and A. actinomycetemcomitans in plaque samples were detected by slot immunoblotting (SIB). Serum antibody levels against these microorganisms were evaluated using ELISA. RESULTS: More than 60% of the children had detectable levels of P. gingivalis in their plaque. Those having detectable levels had more gingival inflammation than those having none; however, these differences were significant only in children over the age of 12 years (PDI, BS). In contrast, while 75% of the children had detectable A. actinomycetemcomitans, there were significant differences in gingival inflammation associated with colonization in children from 3 to 7 years of age (PDI) and over 12 years of age (M-PMA). Serum antibody levels to P. gingivalis were inversely correlated with gingival inflammation in all age groups, while A. actinomycetemcomitans titers were positively correlated with gingival inflammation only in the children over 12 years. No significant relationship between the presence of either A. actinomycetemcomitans or P. gingivalis and antibodies to them was found. CONCLUSIONS: Our findings show that P. gingivalis and A. actinomycetemcomitans are readily detected as early as 3 years of age and that their presence is associated with the onset and severity of gingivitis.

Humoral immune response to selected subgingival plaque microorganisms in insulin-dependent diabetic children.

Juvenile diabetics have been shown to have an increased susceptibility to gingivitis and periodontitis following puberty. However, little data are available on changes in the microbial flora that occur at the onset of puberty. This study was performed to determine if antibacterial antibody titers to selected periodontal disease-associated microorganisms might be helpful in revealing changes in plaque flora at the onset and conclusion of puberty. Sera was obtained from 35 subjects (ages 7 to 18 years) selected from a population of insulin-dependent diabetics. The subjects were given a thorough medical examination which included an assessment of sexual maturation and a dental examination which included the recording of onset and magnitude of bleeding according to the papillary bleeding score. Antibody titers to A. naeslundii (AN), B. intermedius (BI), B. gingivalis (BG), F. nucleatum (FN), A. actinomycetemcomitans (AA), C. ochracea (CO) and T. denticola (TD) were determined using the microELISA. Stratification of antibody titers by age groups (less than or equal to 12 years, 12 to 15 years, greater than 15 years) revealed that titers to AN increased significantly (P less than 0.025, ANOVA) and progressively (P less than 0.05, regression analysis) with increasing age. In contrast, the titers to FN were maximal in the under 12 year group and decreased with age (ANOVA, P less than 0.05; regression analysis, P less than 0.05). There were no significant variations in titers observed for the other microorganisms. Stratification by sexual maturity revealed a similar progressive decrease of the titer to FN (ANOVA, P less than 0.05; regression analysis, P less than 0.005).(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of lyophilized autologous plasma on periodontal healing of replanted teeth.

The purpose of this histologic and autoradiographic study of replanted teeth was to evaluate the beneficial effect, if any, of lyophilized autologous plasma (LAP) application on periodontal healing and to re-examine rates of repair in different areas of the associated periodontium following replantation. Maxillary and mandibular incisors and premolars of three rhesus monkeys were used. Teeth were extracted with forceps and placed in sterile physiologic saline. After 5 minutes each tooth was returned to its socket and immobilized by interproximal acid-etch splints. Splints were removed after 1 week. Of the 48 replants performed, 24 (controls) were replanted as described. Of the 24 experimental teeth, during the 5 minute interval between tooth extraction and replantation, the root surface and the inner socket walls were bathed with 1 ml of the reconstituted LAP-saline solution (800 mg/ml). Replants and animal sacrifice were scheduled to provide observations at 1, 3, 7, 14, 28 and 45 days following replantation. One hour prior to sacrifice, each monkey received an intravenous injection of tritiated thymidine, 1 microCi/gm body weight. Tissue specimens were processed for evaluation following standard procedures. Eight replanted teeth were available for evaluation for each of the six time-points. Four teeth were treated with LAP and four without it. Histologically, tissue sections were examined for epithelial proliferation and attachment, periodontal fibers organization and maturation, inflammatory cell types, presence or absence of cementum resorption and dentoalveolar ankylosis and degree of vascularity of the tissues. For autoradiographic evaluation, the periodontium associated with the replanted tooth was divided into nine spatial cell compartments. In each compartment, labeled tissue cells, epithelial or connective, were counted and recorded. Differences between the control (untreated) replanted teeth and the LAP-treated teeth, at each time-point and within each compartment, were analyzed for significance using the paired t-test. The findings of this study indicate that LAP use enhanced healing by early replacement of the fibrin clot, increased connective tissue cell proliferation, reduction of the inflammatory response and inhibition of root cementum resorption. Periodontal healing and repair occurred more rapidly in the supracrestal or transseptal connective tissue region than within the periodontal membrane space.

Cell proliferation after flap surgery, root conditioning and fibronectin application.

This study evaluated the effects of citric acid demineralization and autologous fibronectin application on cell proliferation after mucoperiosteal flap surgery. Three adult rhesus monkeys were used. After flaps were raised, the roots were surgically exposed and planed. Surfaces on the experimental sides were decalcified with citric acid, and after thorough rinsing, the inner aspect of the flaps and the roots were bathed with 1 ml of autologous plasma fibronectin in normal saline (400 micrograms m/ml) and the flaps sutured. Contralateral teeth, acting as controls, were treated only with the surgical procedure. One hour prior to sacrifice, the animals were injected with an intravenous injection of tritiated thymidine (1 microCi/gm body weight). Surgeries were staggered to produce the following time periods: 3, 7, 15, 21 and 28 days. After processing, autoradiographs were obtained for evaluation, and labeled cells were counted in five compartments at 400 x: (1) oral epithelium, (2) crevicular area, (3) supracrestal connective tissue, (4) coronal periodontal membrane and (5) coronal bone marrow. Forty tissue sections per procedure (20 slides per tooth) were counted and means obtained for the three monkeys. Differences between experimental and control values were statistically evaluated for each component, at each time interval, using pairwise t tests. Fibronectin-treated areas showed significantly increased cellular proliferation (P less than 0.01) during the first 2 weeks, affecting mainly all the supracrestal tissues. Histologically, the establishment of a well-organized fibrinous clot at 3 days was noted in these areas. Results show a faster healing after surgery with the use of citric acid and fibronectin. It was concluded that citric acid followed by fibronectin enhanced cellular proliferation.

Occurrence of Porphyromonas gingivalis, Bacteroides forsythus, and Treponema denticola in periodontally healthy and diseased subjects as determined by an ELISA technique.

The aim of this study was to assess by means of an ELISA technique, the occurrence of 3 putative periodontopathogens, Porphyromonas gingivalis, Bacteroides forsythus, and Treponema denticola, in 3 clinically-defined adult periodontal conditions. Thirty systemically-healthy subjects were selected and grouped into 3 categories according to their periodontal health: 1) 10 periodontitis subjects (PS), having moderate adult chronic periodontitis; 2) 10 untreated gingivitis subjects (UGS), exhibiting no signs of periodontal destruction but presenting with clinical signs of mild gingivitis; and, 3) 10 treated gingivitis subjects (TGS), having the same clinical status as UGS, but who received a thorough prophylaxis treatment within the past 7 to 14 days prior to the baseline examination. A total of 60 samples were collected subgingivally from the six Ramfjord teeth per subject in each group and ELISA analysis was carried out to give a semiquantitative estimate of P. gingivalis. B. forsythus, and T. denticola. The immunologic detection method suggested the presence of antigens of P. gingivalis, B. forsythus, and T. denticola in subjects from each of the 3 groups. When a global analysis for the 3 disease groups was performed at one time, statistically significant differences were found among the ELISA scores of the 3 bacterial species. For example, comparisons of the ELISA scores showed that the concentrations of P. gingivalis differed significantly when comparing TGS to UGS and PS, but not when examining UGS/PS. The ELISA scores for B. forsythus were significantly different between TGS and PS. Mean concentrations of T. denticola were significantly different when comparing PS to TGS or UGS, whereas no difference was found between the latter categories. Within the limited scope of this study, the concentration of antigens detectable from putative periodontopathogens like P. gingivalis, B. forsythus, and T. denticola differed among the 3 diseased groups, with periodontitis subjects often showing the greatest level of antigens. Thus, it is reasonable to expect that, when using sensitive immunological detection methods, antigens of suspected periodontal pathogens can be found irrespective of the individual's clinical status. However, while detectable in the periodontal sites, the concentrations of these microorganisms are most likely to be above the threshold necessary to induce clinically-significant disease. Studies with larger sample size and standardized antigens are necessary to determine if the groups we found not to differ, were, in fact, different.

Bacterial adherence to guided tissue regeneration barrier membranes exposed to the oral environment.

Microbial colonization of barrier materials used in guided tissue regeneration (GTR) is known to adversely affect treatment outcomes. The purpose of this study was to compare the rate at which 11 commonly-occurring oral bacteria species colonize three different barrier materials (collagen, expanded polytetrafluoroethylene, and polylactic acid). The study group consisted of 10 systemically healthy individuals with no history of periodontal disease and absence of antimicrobial therapy within the previous 3 months. In each patient, 4 teeth per quadrant (P1, P2, M1, M2) were selected and 3 teeth were randomly assigned as test teeth while the remaining tooth acted as a control site (i.e., natural colonization of the tooth surface). These teeth were then randomly assigned to receive one of the three barrier types (i.e., each patient received 4 barriers of each type, 1 per quadrant). A 2 x 5 mm piece of barrier material was positioned over the oral surface of the buccal marginal gingiva and secured with an external sling suture. With oral hygiene procedures suspended, one barrier of each type was collected at 1, 3, 7, and 14 days. Slot immunoblot assay demonstrated that all species types (A. actinomycetemcomitans, A. viscosus, B. melaninogenicus, F. nucleatum, P. gingivalis, P. intermedia, S. mutans, S. sanguis, Selenomonas sputigena, T. denticola, and T. vincentii) were present. Semi-quantitative scoring (scale 0 to 3) of slot blot results and analysis by chi-square ratio and Pearson correlation test indicated that while total bacteria adherence increased over time (P < 0.05), the 3 barrier types and the control sites did not differ in numbers or species of colonizing bacteria detected per time point. These results suggest that under these experimental conditions the barrier materials tested do not differ in bacteria adherence or antimicrobial properties.

Humoral immunity to stress proteins and periodontal disease.

BACKGROUND: There is evidence that microbial heat shock (stress) proteins (Hsp) are immunodominant antigens of many microorganisms. Immunity to these proteins has been shown in non-oral infections to contribute to protection. This study was undertaken to assess the relationship(s) between immunity to human and microbial heat shock proteins, periodontal disease status, and colonization by periodontal disease-associated microorganisms. METHODS: Subgingival plaque and blood samples obtained from 198 patients during an earlier clinical study were examined for the presence of specific periodontal disease-associated microorganisms and antibodies to selected human and microbial heat shock proteins (Hsp70, Hsp90, DnaK, and GroEL). Particle concentration immunofluorescence assay (PCFIA) was used to detect anti-Hsp antibodies and slot immunoblot assay (SIB) was used to detect subgingival plaque species. Regression models were used to examine the contribution of age, gender, gingival index, probing depth, attachment loss, calculus index, plaque index, and microbial colonization to the anti-Hsp antibody concentrations. RESULTS: Our studies demonstrated that, when evaluated by ANOVA, patients with higher anti-Hsp (Hsp90, DnaK, and GroEL) antibody concentrations tended to have significantly (P< or =0.05) healthier periodontal tissues. This was most obvious when the relationship between mean probing depths and antibody concentrations were studied. For Hsp90 antibodies, 2 variables (probing depth and P. gingivalis concentration) were found to have significant contributions (R = 0.293, P<0.0002). The equation derived from the regression model was y = 12558-2070*PD +1842*PG. This confirmed the inverse relationship with probing depth and the positive relationship with colonization by P. gingivalis. Attempts to model the other stress protein antibodies were not successful. CONCLUSIONS: We believe that the present observations reflect the presence of protective anti-Hsp antibodies, rather than simply the presence of the microorganism in the gingival sulcus. The clinical significance of these observations lies in the potential of identifying patients who are at risk for developing periodontal disease based on their inability to mount an immune response to specific Hsp or Hsp epitopes, as well as the development of vaccines based on Hsp epitopes.

Clinical evaluation of the use of citric acid and autologous fibronectin in periodontal surgery.

This study evaluated the effects of citric acid demineralization and autologous fibronectin application in association with a modified Widman flap in the treatment of periodontitis. The study population comprised 29 patients under treatment for moderate to advanced periodontitis who reached the one-year posttherapy evaluation. After thorough scaling and root planing, a split mouth design was used in which two quadrants were treated by modified Widman flap alone, and the other two randomly assigned quadrants were treated by modified Widman flap combined with citric acid demineralization and autologous fibronectin application. Fibronectin, which had previously been isolated from the patient's own plasma, was applied with a tuberculin syringe on the citric acid demineralized root surfaces and the inner aspect of the flap. After suturing provided good flap adaptation, additional fibronectin was again applied under the flap and external pressure was applied. Patients were clinically evaluated at baseline and at one year. Statistical evaluation of the data using paired t test and Chi-square analysis indicated that both approaches, modified Widman flap alone or in combination with citric acid and fibronectin, significantly reduced probing pocket depth and increased clinical attachment. However, the changes achieved with citric acid and fibronectin were statistically greater than those obtained with the flap alone. Furthermore, the number of sites gaining 2 mm or more of clinical attachment were significantly increased. The results suggest that the use of citric acid and fibronectin holds promise in promoting reattachment after periodontal therapy.

Effect of citric acid and lyophilized autologous plasma on healing following periodontal flap surgery in monkeys.

The purpose of this histologic, histometric, and autoradiographic study was to examine the effect of citric acid conditioning and lyophilized autologous plasma (LAP) application on healing following periodontal flap surgery. Mucoperiosteal flaps were elevated in six rhesus monkeys using the modified Widman flap procedure. A total of 24 quadrants were treated, each included the first and second premolar and first and second molar teeth. Cementum was removed from the exposed root surfaces, and reference notches were inscribed into the roots at the alveolar bone margin. Two treatment modalities were employed: (1) surgery plus citric acid conditioning, to serve as control and (2) surgery plus citric acid followed by LAP application (400 mg/ml saline). Flaps were returned to their preoperative positions and sutured. Animal sacrifices were scheduled to provide observations 3, 7, 14, 21, 28, and 45 days after surgery. Each monkey received an intravenous injection of tritiated thymidine, 1 microCi/gm of body weight, 1 hour before it was killed. Tissue specimens were processed for evaluation following standard procedures. Histologically, tissue sections were examined for: (1) proliferation and attachment of epithelium, (2) organization and maturation of periodontal fibers, (3) inflammatory cell types, (4) presence or absence of new cementum deposition, and (5) degree of vascularity of the tissues. For histometric evaluation, the radicular notches were used as reference points. The distances examined histometrically were: (1) from the root surface notch to the alveolar bone crest, (2) from the root surface notch to the apical extent of the junctional epithelium, and (3) from the free gingival margin to the apical extent of the junctional epithelium. For autoradiographic evaluation labeled cells were counted in five spatial compartments at 400 X magnification: (1) oral epithelium, (2) crevicular area, (3) supracrestal connective tissue, (4) coronal periodontal membrane, and (5) coronal bone marrow. For each the histometric and autoradiographic evaluation involved a total of 36 tissue sections per quadrant (9 sections per tooth). Tooth and quadrant means were obtained for each monkey. The plasma-treated and control quadrants were compared at each time point by the paired t test. N = 2 monkeys were used for each comparison. Histologic results showed that in teeth that were acid-conditioned after root planing, the epithelium often migrated apically reaching the radicular notch. Those teeth that were conditioned and subsequently treated with LAP demonstrated fiber attachment to the planed root surface and little or no epithelial downgrowth.(ABSTRACT TRUNCATED AT 400 WORDS)