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P Stashenko

Publications and source records attributed to P Stashenko.

At least 55 records · Page 3Linked to original sources
PubMed1993

Characterization of bone-resorbing activity in human periapical lesions.

Extracts of human periapical granulomas were tested for the presence of bone-resorbing activity. All granulomas (10 of 10) contained low but significant levels of bone-resorbing activity, ranging from 2.1 to 4.9% treatment-% control/mg specific 45Ca release, as determined by the fetal rat long bone assay. Healthy periodontal ligament and dental pulp had no significant resorbing activity. In characterization studies, the resorbing activity in an extract pool was unaffected by the presence of polymyxin B, indicating an active moiety distinct from lipopolysaccharide. Resorbing activity was also unaffected by heating to 56 degrees C for 30 min, but was completely abolished by proteinase K treatment or heating to 70 degrees C, indicating that activity was largely protein mediated. Fast performance liquid chromatography gel filtration studies demonstrated that activity could be resolved to two major peaks, of M(r) 30,000 to 60,000 (I), and 15,000 to 20,000 (II), with a minor peak present at < 1,000 (III). Peak III was identified as prostaglandin E2 by radioimmunoassay. In inhibition studies, virtually all of the resorbing activity present was inhibited by anti-interleukin 1 beta (69%) and anti-tumor necrosis factor beta (66%) antisera, whereas anti-interleukin 1 alpha and antitumor necrosis factor alpha had no effect. Treatment with the cyclooxygenase inhibitor indomethacin also reduced activity by 74%. Taken together, these data demonstrate that most bone-resorbing activity present in chronic human periapical lesions is attributable to the action of resorptive cytokines interleukin 1 beta and tumor necrosis factor beta, acting via both indomethacin-dependent and independent pathways.(ABSTRACT TRUNCATED AT 250 WORDS)

Alveolar Bone Loss↗
PubMed1993

The role of interleukin-1 alpha in the pathogenesis of periapical bone destruction in a rat model system.

To identify the mediators that stimulate periapical bone resorption following infection, a rat model system was used in which active (rapid) and chronic (slow) phases of bone destruction can be distinguished. Extracts of inflammatory tissues from active lesions contained high levels of bone-resorbing activity, which was destroyed by proteinase K and heat (70 degrees C), but was unaffected by polymyxin B, indicating the presence of protein mediator(s) rather than lipopolysaccharide. Fast-performance liquid chromatography gel filtration of extracts of active lesions demonstrated that most activity was associated with macromolecules of MW 30-60 kDa and 15-20 kDa, consistent with bone resorptive cytokines, including interleukin 1 (IL-1) and tumor necrosis factor (TNF). Inhibition with cytokine-specific antisera demonstrated that resorbing activity in active lesions was significantly neutralized by anti-IL-1 alpha, whereas anti-IL-1 beta, anti-TNF alpha and anti-TNF beta had only slight effect. A lower amount of resorbing activity was present in extracts of chronic lesions, which was also neutralized only by anti-IL-1 alpha. Inflammatory tissue explants produced more IL-1 alpha than IL-1 beta in vitro, confirming findings with extracts, and high levels of IL-1 alpha were present in active lesions by radioimmunoassay. These data indicate that bone resorption stimulated by bacterial infection is primarily mediated by IL-1 alpha in this model. The similarity of cytokines in active and chronic lesions suggests that quantitative rather than qualitative differences in these mediators may account for lesion progression.

Alveolar Bone Loss↗
PubMed1993

Characterization of a tumor necrosis factor-responsive element which down-regulates the human osteocalcin gene.

Tumor necrosis factor (TNF) down-regulates the production of bone matrix proteins by osteoblasts, thereby inhibiting bone formation. Osteocalcin, the major noncollagenous protein in bone, is inhibited by TNF at the transcriptional level. Mapping studies were undertaken to characterize the TNF-responsive element (TNFRE) in the osteocalcin promoter. Deletion analysis localized the TNFRE to the -522/-511 region, which contains a 9-bp palindromic motif (AGGCTGCCT). Promoter segments containing this sequence down-regulated a heterologous simian virus 40 promoter. Site-specific mutagenesis of the TNFRE eliminated TNF down-regulation. Mobility shift assays demonstrated that a constitutively expressed nuclear factor bound to the TNFRE; this factor was tentatively identified as the p50 homodimer of NF-kappa B. TNF stimulation induced a second TNFRE-binding protein which displaced the constitutive factor. The TNF-induced protein was not inhibitable by the NF-kappa B consensus sequence and was unreactive with anti-NF-kappa B antiserum. DNase footprinting demonstrated that both factors protected the -522/-501 portion of the promoter, consistent with the results of mapping studies and competitive mobility shift assays. It is hypothesized that the generalized catabolic activities of TNF in infectious and malignant diseases may be regulated via this novel element.

Animals↗
PubMed1992

Proinflammatory cytokines tumor necrosis factor-alpha and IL-6, but not IL-1, down-regulate the osteocalcin gene promoter.

A proinflammatory cytokine cascade, including IL-1 alpha, IL-1 beta, TNF-alpha, IL-6, and IL-8, is activated in response to infection or immunologic insult. Besides their immunologic effects, several of these mediators stimulate bone resorption and inhibit bone formation. Osteocalcin, the most abundant noncollagenous protein present in bone, is an osteoblast-specific product whose production closely correlates with bone formation, and which has also been implicated in control of bone resorption. IL-1 and TNF have previously been shown to down-regulate osteocalcin production in vitro and in vivo, although the mechanism of this inhibition is unknown. In the present studies, IL-1 beta and TNF-alpha both inhibited 1,25-dihydroxyvitamin D3-stimulated production of osteocalcin protein and mRNA by ROS 17/2.8 osteosarcoma cells, whereas IL-6 had no effect on protein and only weakly inhibited mRNA. To determine if down-regulation was exerted at the transcriptional level, an osteocalcin promoter-chloramphenicol acetyltransferase (CAT) fusion gene was constructed (PHOC-CAT). After transient transfection of PHOC-CAT into ROS 17/2.8 osteosarcoma cells, reporter CAT activity was up-regulated by vitamin D at concentrations above 10(-12) M. In screening studies, TNF-alpha (-57%) and IL-6 (-37%) inhibited vitamin D-stimulated osteocalcin transcription, whereas IL-1 alpha, IL-1 beta, and IL-8 had no effect. Other immune cytokines and growth factors, including IL-2, IL-3, IL-7, and M-CSF, also failed to regulate osteocalcin transcription. Despite their lack of promoter regulation, IL-1 alpha and IL-1 beta also stimulated PGE2 production by ROS 17/2.8, further confirming the ability of the host cell to respond to these mediators. In dose-response experiments, down-regulation by TNF-alpha was significant at concentrations as low as 0.14 pM (0.1 U/ml), whereas approximately 10(4)-fold higher concentration of IL-6 was required to exert a similar effect. TNF-alpha-mediated down-regulation was unaffected by indomethacin. These data demonstrate that of these cytokines, TNF-alpha alone potently down-regulates osteocalcin promoter function, whereas IL-1 acts post-transcriptionally, possibly by reducing mRNA stability. Heterogeneity therefore exists among the proinflammatory cytokines with respect to the level at which control of osteocalcin expression is exerted.

Cell Line↗
PubMed1992

Kinetics of immune cell and bone resorptive responses to endodontic infections.

Infection of the dental pulp stimulates a host immune response in the periapical region (the periapical "lesion") with the concomitant resorption of bone. The cell composition of human or rat periapical lesions is mixed, consisting of T, B, and "null" lymphocytes, plasma cells, macrophages, polymorphonuclear leukocytes, and mast cells. Cells are thus present which mediate a broad spectrum of immunological phenomena. In order to determine which of these mechanisms contribute to periapical bone resorption, a rat model system has been used in which periapical lesions are induced by pulp exposure and infection from the oral environment. In this model, a rapid period of lesion expansion and bone destruction occurs between days 1 and 15 after exposure (active phase), with a chronic phase characterized by lesion stabilization commencing thereafter. Phenotypic analysis of the cellular infiltrate has shown that T helper cells predominate during the active phase, whereas increased numbers of T suppressor cells are associated with chronicity. Extracts of periapical lesions contain bone-resorbing activity, as determined by the release of 45Ca from prelabeled a rat long bones in organ culture. Higher levels of bone-resorbing activity are present during the active than in the chronic phase. Characterization of bone-resorbing activity indicates the presence of resorptive mediators which are heat labile and protease sensitive and which are distinct from lipopolysaccharide. Preliminary biochemical fractionation indicates that resorptive mediators fractionate in the 15,000- to 60,000-dalton molecular mass range, presumably cytokines such as interleukin 1 and tumor necrosis factor. These studies demonstrate the utility of the rat model for studying mechanisms of periapical lesion pathogenesis and implicate T helper cell-mediated activities, in particular those leading to macrophage activation and cytokine production, in periapical lesion expansion.

Alveolar Bone Loss↗
PubMed1992

Characterization of bone resorptive mediators in active periapical lesions.

The mechanism of bone destruction in periapical lesions was studied using a rat model system. Periapical lesions were induced by pulp exposure and infection from the oral environment. Lesions expanded most rapidly between induction on day 0 and day 15 ("active phase"), with enlargement occurring at a slower rate thereafter (days 20 and 30, "chronic phase"), as assessed by radiography and automated image analysis. Pooled extracts of day 15 periapical tissues contained significant levels of bone resorbing activity (BRA), as determined by 45Ca release from fetal rat long bones. Normal rat dental pulp and periodontal ligament contained no activity. In kinetic experiments, highest levels of BRA were detected in active phase tissues on days 10 and 15, with BRA declining thereafter in chronic phase tissues to near baseline levels by day 30. In characterization studies, BRA in pooled day 15 tissue extracts was unaffected by treatment with polymyxin B, but was completely abolished by proteinase K treatment or heating to 70 degrees C, indicating an active moiety distinct from bacterial LPS, probably a protein(s). FPLC gel filtration chromatography revealed that BRA could be resolved into four major peaks, of MW 30-60 kD (Peak I); 15-20 kD (II); 1-2 kD (III); less than 1kD (IV), consistent with the presence of the following bone resorptive mediators: (I) cytokines TNF alpha, TNF beta and/or unprocessed IL-1 alpha; (II) processed IL-1 alpha and/or IL-1 beta; (IV) PGE2. These findings demonstrate that bone resorbing activity is temporally related to bone destruction, and that the activity present during the rapid phase of periapical lesion expansion is primarily attributable to bone resorptive cytokines.

Animals↗
PubMed1991

Divergent effect of the anaerobic bacteria by-product butyric acid on the immune response: suppression of T-lymphocyte proliferation and stimulation of interleukin-1 beta production.

The effects of short-chain fatty acids (SCFA) produced by anaerobic bacteria, namely propionic, butyric and iso-butyric on T-cell proliferation was investigated. A dose-dependent inhibition of both phytohemagglutinin-induced blastogenesis and mixed lymphocyte culture was observed in the millimolar range of SCFA concentrations. The tested SCFA displayed different levels of suppression. The degree of activity was in the following order: butyrate greater than propionate greater than isobutyrate. T-cell inhibition was partially reversed, at least for propionic and isobutyric acids, by increasing the concentration of macrophages in the assay system. Furthermore, butyric acid displayed an interesting biphasic stimulation of monocyte interleukin-1 beta production, a cytokine with a powerful bone-resorbing activity. Since millimolar concentrations of SCFA are present in gingival fluid from periodontal pockets, the observed results support the role of these by-products of anaerobic metabolism in the pathogenesis of periodontal diseases.

Alveolar Bone Loss↗
PubMed1991

Levels of interleukin 1 beta in tissue from sites of active periodontal disease.

Interleukin 1 beta is a potent bone resorptive cytokine which also mediates soft tissue destruction through the stimulation of prostaglandin production, and the induction of collagenase and other proteases. This constellation of activities suggests a role for IL-1 beta in the pathogenesis of human periodontitis. Levels of IL-1 beta were therefore determined in tissue obtained from (1) diseased, active (2) diseased, inactive and (3) healthy sites from 12 patients with destructive adult periodontitis. Disease activity was defined as attachment loss of greater than or equal to 2.5 mm, as determined by sequential probing and the tolerance method. IL-1 beta was extracted from homogenates of tissue biopsies taken at surgery, and levels were quantified by ELISA. IL-1 beta was found to be present in most patient tissue samples, with levels ranging from 0-82 ng/ml. Disease active sites had higher IL-1 beta levels (p less than 0.05) than inactive and healthy sites. Diseased inactive sites were divided into 2 groups, those losing small amounts of attachment (0.5-2.0 mm, worsening sites) and those which showed no change or attachment gain (stable sites). Stable diseased sites had IL-1 beta levels which were comparable to those found in healthy sites, and which were significantly different from active sites (p less than 0.02). Worsening sites had IL-1 beta levels intermediate between the levels in stable and active sites. Detection of disease activity occurred more frequently at sites with IL-1 beta levels greater than 25 ng/ml (p less than 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1991

Kinetics of bone-resorbing activity in developing periapical lesions.

The presence and kinetics of bone-resorbing activity in periapical lesions were studied with a rat model system. Lesions were found to expand most rapidly between induction on day 0 and day 15 ("active phase"), with enlargement occurring at a slower rate thereafter (days 20 and 30, "chronic phase"), as assessed by measurement of magnified radiographs and automated image analysis. Pooled extracts of periapical tissues obtained on day 15 contained significant levels of bone-resorbing activity, as determined by 45Ca release from pre-labeled fetal rat long bones. Normal rat dental pulp and periodontal ligament contained no activity. In two kinetic experiments, highest levels of bone-resorbing activity were detected in tissues on days 10 (10.4% and 11.6% specific 45Ca release/mg/mL) and 15 (8.9%). Activity declined thereafter on day 20 (4.4% and 6.1%) to near baseline levels by day 30 (1.4% and 3.3%). Identical levels of resorbing activity were found in the presence or absence of polymyxin-B, an inhibitor of bacterial lipopolysaccharide. These findings demonstrate that bone-resorbing activity is temporarily related to periapical bone destruction and suggest that this model may be useful for studies of mediators that are involved in the pathogenesis of inflammatory bone resorption.

Animals↗
PubMed1991

Localization of interleukin-1 beta in human periodontal tissue.

Interleukin-1 beta (IL-1 beta) is the predominant form of IL-1 produced by macrophages. IL-1 beta possesses numerous and diverse biological activities. Several of these activities, including fibroblast proliferation, potentiation of the immune response, and stimulation of bone resorption may be of relevance to the pathogenesis of periodontal disease. This study was designed to examine the presence of IL-1 beta in human periodontal tissue. An antiserum directed against the N-terminal segment (117-131) of human IL-1 beta was used to detect IL-1 beta using immunofluorescent staining techniques. IL-1 beta positive staining cells were observed in both normal and diseased tissue and were limited to the lamina propria. Brightly staining cells were increased by almost 3-fold in periodontally diseased tissue when compared to normal tissue. Low intensity staining cells were equally distributed in the normal and diseased specimens. We propose that IL-1 beta and IL-1 beta produced by cells in periodontal tissues may be related to the pathological processes associated with periodontal disease.

Adult↗
PubMed1991

Tissue levels of bone resorptive cytokines in periodontal disease.

The levels of 3 bone resorptive cytokines, interleukin 1 alpha (IL-1 alpha), IL-1 beta, and tumor necrosis factor alpha (TNF alpha), were assessed in tissues from sites of periodontal disease. As determined by ELISA of tissue extracts, IL-1 beta and TNF alpha were detected in all diseased sites, whereas IL-1 alpha was present in 8/22 sites, IL-1 beta was present in highest concentration (mean +/- SEM: 11,695 +/- 2,888 pg/ml; 672 pM), followed by TNF alpha (434 +/- 135 pg/ml; 26 pM), and IL-1 alpha (342 +/- 160 pg/ml; 20 pM). The levels of all 3 mediators were significantly lower in clinically healthy tissues. There was a highly significant correlation between levels of IL-1 beta and TNF alpha (rs = 0.61, P less than 0.001), suggesting coordinated expression of these 2 mediators. The numbers of cells containing each mediator was also determined by indirect immunofluorescence on frozen tissue sections. Consistent with findings from tissue extracts, IL-1 beta-containing cells were present in approximately 5-fold higher numbers than TNF alpha-containing cells, and 40-fold higher numbers than IL-1-alpha-containing cells. Taken together with previous findings, these results indicate that IL-1 beta is likely to be an important mediator in the pathogenesis of periodontal disease.

Adult↗
PubMed1991

Interleukin-1 beta stimulates bone resorption and inhibits bone formation in vivo.

Interleukin-1 beta (IL-1 beta) and several other cytokines, including IL-1 alpha, tumor necrosis factor (TNF), and lymphotoxin, stimulate bone resorption and also inhibit bone formation in vitro. These effects are consistent with an uncoupling function for these mediators, although the effect of in vivo regulatory mechanism(s) that couple resorption and formation cannot be adequately evaluated in vitro. In the present studies, the effect of IL-1 beta on bone resorption and formation was determined in adult rats in vivo. Resorption was assessed by serum and urinary calcium levels, osteoclast number, and active resorption surface. Bone formation was determined by measurement of serum osteocalcin levels, and by quantitation of bone apposition rate using tetracycline labeling. A modest dose of IL-1 beta (1 microgram/kg) was found to stimulate transient increases in serum calcium, and a persistent elevation of urinary calcium excretion. IL-1 beta treatment also resulted in decreases in serum iron levels and in the albumin/globulin ratio, well-established in vivo effects of IL-1. SGOT, SGPT, BUN, creatinine, and total protein were unaffected, indicating that IL-1 beta treatment did not compromise kidney or liver function, and that animals were systemically healthy. This was further evidenced by normal weight gain in IL-1 beta-treated animals. Low doses (50 micrograms/kg) of synthetic human parathyroid hormone (PTH 1-34) also stimulated resorption, as shown by a sustained increase in serum calcium without increased urinary excretion. Both IL-1 beta and parathyroid hormone (PTH) stimulated similar increases in osteoclast number (N.Oc) and active resorption surface [Oc.S(%)].(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1990

Interleukin 1 interacts synergistically with forskolin and isobutylmethylxanthine in stimulating bone resorption in organ culture.

This study examined the interaction of interleukin 1 (IL-1) with forskolin and isobutyl-methylxanthine (3-isobutyl-1-methyl-xanthine) (IBMX) in stimulating bone resorption in 5-day fetal rat long bone organ culture. Forskolin and IBMX are pharmacologic agents that elevate cyclic adenosine monophosphate (AMP) levels in many cell types, including osteoblasts and osteoclasts. The interaction of IL-1 with forskolin and IBMX are synergistic when submaximal resorptive concentrations of agonists were examined. Stimulated resorption was 2 to 5 times that expected for an additive response. When maximally resorptive concentrations of agonists were examined, the interaction between IL-1 and the other agents was, at most, additive. We have previously reported that parathyroid hormone and prostaglandin E, agents that also activate the cyclic AMP pathway in bone cells, interact synergistically with IL-1 in stimulating bone resorption. The results of this study, together with our previous studies, suggest that activation of the cyclic AMP pathway is a sufficient signal for an agent to interact synergistically with IL-1 in stimulating bone resorption.

1-Methyl-3-isobutylxanthine↗
PubMed1990

Limit of resolution of flow cytometry for the detection of selected bacterial species.

The enumeration of bacteria in dental plaque samples is a vital but time-consuming procedure that uses standard cultural methods. Flow cytometry has proven to be a useful tool for the analysis of eukaryotic cells. In the present investigation, the utility of this technology for the enumeration of bacteria in mixtures was explored. Rabbit antisera were produced against the putative periodontal pathogens A. actinomycetemcomitans, B. intermedius, B. gingivalis, E. corrodens, W. recta, B. forsythus, as well as the frequently isolated supragingival species S. sanguis. Cross-reactive antibodies were removed by absorption, and the specificity of each antiserum was confirmed by being tested against a panel of 235 oral microbial strains (79 genera; 94 species) by means of ELISA. Conditions were established for the indirect immunofluorescent labeling of cells without agglutination with use of a goat anti-rabbit Ig-FITC second antibody. When an internal bead standard was used, it was found that unstained bacteria were enumerated by light-scattering parameters with poor efficiency (less than 3%). However, cells exposed to FITC either in the presence of specific or non-specific first antibody were enumerated with high efficiency (102.6 +/- 29.3%), indicating that a small amount of non-specific binding of fluorochrome facilitates bacterial detection. Clear discrimination between specifically- and non-specifically-stained bacteria was achieved with all six rabbit antisera. Mixtures of known composition were made (1) with pure cultures or (2) with a known species and supragingival plaque devoid of that species by culture. The results from both approaches with various species combinations revealed that the limit of resolution for accurate quantitation of a selected species was approximately 5%, although specific organisms could be detected qualitatively when present at approximately 1%.

Bacteria↗
PubMed1989

T helper and T suppressor cell reversal during the development of induced rat periapical lesions.

T helper (TH) and T suppressor (TS) cells were enumerated in developing rat periapical lesions in order to identify the cell type associated with the active phase of lesion formation. Experimental pulp exposures were made in all molar teeth of 29 Sprague-Dawley rats, and teeth were left open to the oral environment. On days 15, 20, 30, and 90 after induction, inflammatory cells were isolated from periapical lesions, and T helper (W3/25+), T suppressor (OX8+), and total T cells (W3/13+) were enumerated by reactivity with monoclonal antibodies. During the early, most active phase of lesion development (day 15), TH cells outnumbered TS cells (TH/TS = 1.7). However, by day 20, when lesion expansion had slowed, the TH/TS ratio was reversed (0.9) and remained so for up to 90 days after induction (TH/TS = 0.7). Total T cell numbers did not change over the period of observation. TH cells thus predominated during the active phase of lesion development, whereas TS cells were associated with the chronic phase. This dynamic TH/TS reversal indicates that immune responses which occur within periapical lesions are highly regulated. In addition, the predominance of TH during the active phase of lesion development suggests that TH-mediated activities may be involved in the bone destruction and immune cell activation within periapical lesions.

Animals↗
PubMed1987

Synergistic interactions between interleukin 1, tumor necrosis factor, and lymphotoxin in bone resorption.

Cytokines with bone-resorbing activity include IL 1 beta (pI 7), IL 1 alpha (pI 5), tumor necrosis factor (TNF), and lymphotoxin (LT). Possible interaction between IL 1 beta, the major mediator with osteoclast-activating factor (OAF) activity, and other cytokines was studied. By itself, IL 1 beta was 13-fold more potent than IL 1 alpha and 1000-fold more potent than either TNF or LT in stimulating bone resorption. Suboptimal concentrations of IL 1 beta or IL 1 alpha in combination with suboptimal concentrations of TNF or LT resulted in synergistic bone-resorptive responses (1.5 to 10 times the expected responses if their effects were additive). Synergy between either form of IL 1 and TNF or LT resulted in a twofold increase in activity of IL 1, and a 100-fold increase in activity of TNF or LT. However, even with optimal synergy, IL 1 beta remained 20-fold more potent in inducing bone resorption than TNF or LT. Because IL 1 beta is considerably more potent than TNF and LT in stimulating bone resorption either alone or under synergistic conditions, it is unlikely that TNF and LT are responsible for more than a minor proportion of the total bone-resorbing activity formerly referred to as OAF.

Animals↗
PubMed1987

Synergism between parathyroid hormone and interleukin 1 in stimulating bone resorption in organ culture.

The interaction of interleukin 1 (IL-1), a locally produced factor, and parathyroid hormone (PTH), a systemic factor, in stimulating bone resorption was examined using fetal rat long bone organ culture. Concentrations of IL-1 and PTH, which stimulated little bone resorption when present singly, produced marked resorption when present simultaneously. This synergistic interaction of IL-1 and PTH was not affected by the presence of the prostaglandin synthetase inhibitor indomethacin. Both interleukin 1 alpha and interleukin 1 beta were capable of producing synergy. Synergy was not produced by sequential exposure of bone to IL-1 and PTH, but required the simultaneous presence of both mediators. The leftward shift in the dose response curve of PTH produced by IL-1 may be an important mechanism controlling localized bone resorption. A role for IL-1 in stimulating bone resorption in pathologic conditions, such as arthritis and periodontal disease, is strengthened by the finding that even low concentrations of IL-1 can produce resorptive effects by synergistic interaction with PTH.

Animals↗