PubMed Health⌕ Search

Biomedical subjects

P Stashenko

Publications and source records attributed to P Stashenko.

At least 37 records · Page 2Linked to original sources
PubMed1999

Infection-stimulated infraosseus inflammation and bone destruction is increased in P-/E-selectin knockout mice.

Infections of the dental pulp commonly result in infraosseus inflammation and bone destruction. However, the role of phagocytic leucocytes in the pathogenesis of pulpal infections has been uncertain. In this work we used P/E-/- selectin-deficient mice, which lack rolling adhesion of leucocytes to endothelium and mimic the human syndrome, leucocyte adhesion deficiency II (LAD-II), to test the hypothesis that phagocytic leucocytes protect against pulpal infection and subsequent periapical infraosseus bone resorption. P/E-/- mice and P/E+/+ wild-type controls were subjected to surgical pulp exposure, and both groups were infected with a mixture of pulpal pathogens including Prevotella intermedia, Fusobacterium nucleatum, Peptostreptococcus micros and Streptococcus intermedius. Animals were killed after 20 days, and the extent of infraosseus bone destruction was quantified by histomorphometry. In two separate experiments, P/E-/- mice had significantly greater bone resorption than P/E+/+ controls. The increased bone destruction correlated with a twofold decrease in polymorphonuclear (PMN) infiltration into periapical inflammatory tissues of P/E-/- mice. P/E-/- mice had higher tissue levels of the bone resorptive cytokine, interleukin (IL)-1alpha. Tissue levels of IL-2, IL-4, IL-10, tumour necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) were all higher in P/E-/- mice, but the increases were not statistically significant. Only IL-12 was higher in P/E+/+ mice, possibly reflecting a greater number of infiltrating monocytes in wild-type mice. These findings demonstrate that phagocytic leucocytes are protective in this model, and suggest that elevated expression of inflammatory cytokines is responsible for the observed bone destruction.

Animals↗
PubMed1998

zALK-8, a novel type I serine/threonine kinase receptor, is expressed throughout early zebrafish development.

Here, we report the isolation and characterization of zebrafish activin receptor-like kinase-8 (zALK-8), a novel type I serine/threonine (ser/thr) kinase receptor of the transforming growth factor beta (TGF-beta) family. zALK-8 is novel, in that it contains an extracellular domain that is quite distinct from that of previously identified ALK receptors 1 through 7. Analysis of the predicted amino acid sequence of the 506 amino acid zALK-8 receptor reveals an ser/thr kinase domain characteristic of type I TGF-beta family member receptors. zALK-8, therefore, is a traditional type I ser/thr kinase receptor of the TGF-beta family, but it may exhibit novel ligand-binding activities. The developmental expression of zALK-8 mRNA was examined by wholemount in situ hybridization analysis using a probe from the 3'-untranslated sequence of zALK-8, which does not cross react with other members of the highly conserved TGF-beta receptor family. zALK-8 mRNA is present as a maternal message that is expressed ubiquitously before the start of zygotic transcription. By 16 hr postfertilization (hpf), zALK-8 mRNA is still expressed fairly evenly throughout the embryo. In 24-hpf embryos, zALK-8 mRNA is expressed predominantly in the developing eye and neural structures. By 48 hpf, zALK-8 mRNA is faintly detectable as a diffuse signal throughout the head. zALK-8 mRNA is not detectable by this method in 72-hpf or 96-hpf embryos. Northern analysis of zALK-8 mRNA in poly(A+) mRNA isolated from 6-9 hpf embryos detects a major transcript of 3.6 kb and a minor transcript of 4.3 kb. zALK-8 mRNA expression correlates well with known functions of TGF-beta family members as early axial patterning and mesoderm-inducing growth factors and as potent growth and differentiation factors in craniofacial development.

Activin Receptors↗
PubMed1998

Periapical inflammatory responses and their modulation.

Periapical inflammatory responses occur as a consequence of bacterial infection of the dental pulp, as a result of caries, trauma, or iatrogenic insult. Periapical inflammation stimulates the formation of granulomas and cysts, with the destruction of bone. These inflammatory responses are complex and consist of diverse elements. Immediate-type responses--including vasodilatation, increased vascular permeability, and leukocyte extravasation--are mediated by endogenous mediators, including prostanoids, kinins, and neuropeptides. Non-specific immune responses--including polymorphonuclear leukocyte and monocyte migration and activation, and cytokine production--are elicited in response to bacteria and their products. Interleukin-1 and prostaglandins in particular have been implicated as central mediators of periapical bone resorption. Chronic periapical inflammation further involves specific T- and B-cell-mediated anti-bacterial responses, and activates a network of regulatory cytokines which are produced by Th1- and Th2-type T-lymphocytes. Various naturally occurring and genetically engineered models of immunodeficiency are beginning to help elucidate those components of the immune system which protect the pulpal/periapical complex. Both specific and non-specific responses interface with and are regulated by the neural system. The modulation of these responses by immune response modifies, cytokine antagonists, and other novel therapeutic agents is discussed. As an experimental model, periapical inflammation has many advantages which permit it to be used in studies of microbial ecology and pathogenesis, host response, neuroimmunology, and bone resorption and regeneration.

Cytokines↗
PubMed1997

Bone-resorptive cytokine gene expression in periapical lesions in the rat.

Periapical bone destruction is an important pathogenic sequela of pulpal infection. Recent findings from this laboratory have demonstrated that most bone-resorbing activity in extracts of rat periapical lesions can be neutralized by an anti-interleukin (IL)-1 alpha antiserum. To further clarify pathogenic mechanisms, bone-resorptive cytokine messenger RNA (mRNA) expression was analyzed in developing rat periapical lesions. The molar teeth of 20 Sprague-Dawley rats were surgically exposed and left open to permit infection from the oral environment. Total cell RNA was isolated from periapical granuloma tissue obtained on days 3, 7, 15 and 30 after exposure. mRNA for IL-1 alpha, IL-1 beta and tumor necrosis factor alpha (TNF-alpha) was amplified by reverse transcription polymerase chain reaction, and levels were approximated by comparison to the parallel amplification of the housekeeping gene glyceraldehyde phosphate dehydrogenase. IL-1 alpha and TNF-alpha mRNA were both highly expressed beginning on day 7, increased on day 15, and declined somewhat on day 30. In contrast, IL-1 beta mRNA was expressed at much lower levels, but with similar kinetics. The kinetics of steady state IL-1 alpha and TNF-alpha mRNA levels were confirmed using the quantitative RNase protection assay, whereas IL-1 beta mRNA could not be detected by this technique. IL-1 alpha mRNA-expressing cells were identified using in situ hybridization and included infiltrating macrophages, as well as resident fibroblasts, endothelial cells and osteoclasts. These results demonstrate that the IL-1 alpha and TNF-alpha genes are highly expressed in developing periapical lesions in the rat and confirm previous studies at the protein level in this model.

Alveolar Bone Loss↗
PubMed1997

Increased susceptibility of RAG-2 SCID mice to dissemination of endodontic infections.

Specific immunity has been implicated in the pathogenesis of periapical lesions, although the extent to which these mechanisms are actually involved in either protection or destruction of the pulp-periapex complex is yet to be established. To investigate this question we compared periapical-lesion pathogenesis in RAG-2 severe combined immunodeficient (SCID) mice with immunocompetent control mice following surgical pulp exposure. In order to equalize the bacterial challenge, an infection protocol using Prevotella intermedia, Fusobacterium nucleatum, Peptostreptococcus micros, and Streptococcus intermedius was devised. The results demonstrated that after infection, the proportion of the root canal flora represented by the four pathogens was almost identical in both groups (39.9 and 42.2% for RAG-2 and immunocompetent control mice, respectively). The effects of abrogation of T- and B-cell mechanisms on periapical pathogenesis were then assessed. Approximately one-third of the RAG-2 mice developed endodontic abscesses, while no immunocompetent controls had abscesses, results which indicated regional dissemination of the infection. A similar incidence of abscesses was found in two additional experiments. Abscessed RAG-2 teeth had significantly larger periapical lesions than did nonabscessed RAG-2 teeth (P < or = 0.05) and exposed immunocompetent controls (P < or = 0.01), whereas nonabscessed RAG-2 teeth were not significantly different from those of exposed immunocompetent controls in periapical-lesion size. We conclude that B- and T-cell-mediated immunity protects the host from the dissemination of endodontic infections and that RAG-2 mice are more susceptible to infection-induced pulp-periapex destruction.

Animals↗
PubMed1996

Molecular cloning and characterization of a putative novel human osteoclast-specific 116-kDa vacuolar proton pump subunit.

A cDNA encoding a possible novel human 116-kDa polypeptide subunit of the osteoclastic proton pump (OC-116KDa) has been identified by differential screening of a human osteoclastoma cDNA library. The predicted sequence of OC-116kDa consists of 822 amino acids and is 46.9% and 47.2% identical at the amino acid level to the 116-KDa polypeptide of the vacuolar proton pump of rat and bovine brain respectively. OC-116KDa mRNA was found at high levels in osteoclastomas by Northern analysis but was not detected in tumor stromal cells or in other tissues including kidney, liver, skeletal muscle and brain. OC-116KDa mRNA was localized to multinucleated giant cells within the osteoclastoma tumor by in situ hybridization.

Amino Acid Sequence↗
PubMed1996

Extracts of periodontopathic microorganisms lack functional superantigenic activity for murine T cells.

Products of periodontopathic bacteria exert immunomodulatory effects on various lymphoid cell populations, some of which have been implicated in the pathogenesis of periodontitis. It has recently been suggested that some of these bacterial products may possess superantigenic (SAg) activity. SAg bind simultaneously to the V beta chain of T cell receptors and to class II major histocompatibility complex molecules, thereby activating as many as 35% of T cells to proliferate and produce cytokines. In order to examine this question, the proliferation of splenic and thymic T cells from immunologically naive, 3-6-wk-old Balb/c (H-2d), C57BL/6 (H-2b) and C3H/HeJ (H-2k) mice was assessed in response to sonic extracts of periodontopathogens. Laboratory and/or reference strains of a.o. Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, Prevotella intermedia and Prevotella nigrescens were used as stimulants. Staphylococcal enterotoxin B (SEB), a known superantigen, was utilized as a positive control. Unfractionated spleen cells responded to several of the tested preparations of the different bacteria, as well as to SEB, Con A and Escherichia coli LPS. Thymocytes responded to Con A and SEB, but not to LPS or to any sonic extract. Spleen cells depleted of B cells by panning responded to SEB and Con A, but not to LPS and showed a reduced response to sonicates. The residual response of B cell-depleted spleen cells was reduced essentially to background by treatment with anti-Thy 1.2 + C'. Similar results were obtained in the presence of 5% added mitomycin-treated antigen presenting cells, indicating that these cells were not limiting. These results demonstrate that extracts of periodontopathic bacteria do not stimulate murine T cells in a manner consistent with superantigenic activation.

Adjuvants, Immunologic↗
PubMed1995

Characterization of a silencer element in the first exon of the human osteocalcin gene.

Osteocalcin, the major non-collagenous protein in bone, is transcribed in osteoblasts at the onset of extracellular matrix mineralization. In this study it was demonstrated that sequences located in the first exon of the human osteocalcin gene possess a differentiation-related osteocalcin silencer element (OSE). Osteocalcin was rendered transcribable in UMR-106 cells and proliferating normal osteoblasts after deletion of the -3 to +51 region. Site-specific mutagenesis of this region revealed that a 7 bp sequence (TGGCCCT) (+29 to +35) is critical for silencing function. Mobility shift assays demonstrated that a nuclear factor bound to the OSE. The OSE binding protein was present in proliferating normal pre-osteoblasts and in UMR-106 and ROS 17/2.8 osteosarcoma cells, but was absent from post-proliferative normal osteoblasts. The binding protein was inhibited by fragments containing the +29/+35 sequence, but not by other promoter fragments or by the consensus oligomers of unrelated nuclear factors AP-1 and Sp1. DNase 1 footprinting demonstrated that the OSE binding-protein protected the +17 to +36 portion of the first exon, consistent with the results of mapping studies and competitive mobility shift assays. It is hypothesized that this silencer is activated by complexing of the OSE binding protein to the OSE during the osteoblast proliferation stage and that the OSE binding protein is down-regulated at the onset of extracellular matrix mineralization.

Animals↗
PubMed1995

Cloning and complete coding sequence of a novel human cathepsin expressed in giant cells of osteoclastomas.

A gene encoding a possible novel human cathepsin, a cysteine proteinase that is distinct from previously characterized enzymes, has been identified by differential screening of a human osteoclastoma cDNA library. This molecule, termed cathepsin X, appears to represent the human homolog of the osteoclast-expressed rabbit cathepsin OC-2. Cathepsin X (GenBank accession number U20280) is 93.9% identical to OC-2 at the amino acid level, and is 92% identical at the nucleotide level within the coding region. Cathepsin X is 52.2 and 46.9% identical to cathepsins S and L, respectively, and is therefore clearly distinct from these enzymes. Cathepsin X mRNA was localized to multinucleated giant cells within the osteoclastoma tumor by in situ hybridization. These data strongly support the hypothesis that cathepsin X represents a novel cysteine proteinase which is expressed at high levels in osteoclasts.

Amino Acid Sequence↗
PubMed1995

Immunolocalization of bone-resorptive cytokines in rat pulp and periapical lesions following surgical pulp exposure.

The bone-resorptive cytokines interleukin 1 (IL-1) and tumor necrosis factor (TNF) have been implicated in the pathogenesis of many chronic inflammatory diseases, including pulpitis and apical periodontitis.To further elucidate their role in these disorders, we have identified cells that express IL-1 alpha and TNF alpha in infected pulps and in developing rat periapical lesions after surgical pulp exposure. As detected by immunohistochemistry, IL-1 alpha- and TNF alpha-positive cells were present as early as 2 days after pulp exposure in both the pulp and periapical region. The numbers of cytokine-expressing cells increased up to day 4 in the pulp and up to day 30 in the periapex. In contrast, cells expressing IL-1 beta and TNF beta, the homologous forms of these mediators, were not found in pulp or periapical lesions during this period. Cells expressing IL-1 alpha and TNF alpha were identified primarily as macrophages and fibroblasts, with occasional staining of polymorphonuclear leukocytes. Osteoblasts and osteoclasts were also positive, whereas lymphocytes were negative. In general, cytokine-expressing cells were located proximal to abscesses and the root apex. These findings demonstrate that cells that express bone-resorptive cytokines IL-1 alpha and TNF alpha are present immediately after pulp exposure in this model, which supports the hypothesis that these mediators play a key role in pulpal and periapical pathogenesis, including the concomitant bone destruction. They also indicate that both resident connective tissue cells as well as infiltrating cells express bone-resorptive cytokines in response to infection in these lesions.

Alveolar Bone Loss↗
PubMed1995

Reduction of infection-stimulated periapical bone resorption by the biological response modifier PGG glucan.

Pulpal and periodontal diseases are bacterial infections which result in local connective tissue and bone destruction. Effective host resistance to these infections is primarily mediated by neutrophils and other phagocytic cells. PGG glucan (poly-beta 1-6-glucotriosyl-beta 1-3-glucopyranose glucan) is a biological response modifier which stimulates the production of neutrophils and upregulates their phagocytic and bactericidal activity. In the present studies, the effect of PGG glucan on infection-stimulated alveolar bone resorption was tested in an in vivo model. Periapical bone resorption was induced in Sprague-Dawley rats by surgical pulp exposure and subsequent infection from the oral environment. Animals were administered PGG glucan (0.5 mg/kg) or saline (control) subcutaneously the day before and on days 2, 4, 6, 9, 11, 13, 16, and 18 following the pulp exposure procedure. PGG glucan enhanced the number of circulating neutrophils and monocytes and increased neutrophil phagocytic activity approximately two-fold. PGG glucan-treated animals had significantly less infection-stimulated periapical bone resorption than control animals, as determined radiographically (-48.0%; p < 0.001) and by histomorphometry (-40.8% and -42.4% for first and second molars, respectively; p < 0.001). PGG glucan-treated animals also had less soft tissue destruction, as indicated by decreased pulpal necrosis. Only 3.3% of the first molar pulps from PGG glucan-treated animals exhibited complete necrosis, as compared with 40.6% of pulps from controls. Finally, PGG glucan had no effect on either PTH- or IL-1-stimulated bone resorption in vitro.(ABSTRACT TRUNCATED AT 250 WORDS)

Alveolar Bone Loss↗
PubMed1995

Monocyte chemoattractant protein-1 expression and monocyte recruitment in osseous inflammation in the mouse.

In bone, early events in inflammation involve the production and release of primary proinflammatory cytokines, such as interleukin-1 beta. By activation of target cells, these cytokines are thought to induce a second wave of cytokines, including monocyte chemoattractant protein-1 (MCP-1). MCP-1 is a cytokine that stimulates chemotaxis of monocytes. Experiments were undertaken to examine the expression of MCP-1 in bone-associated cells in vivo. To observe in vivo expression of MCP-1, an inflammatory lesion was created in the murine mandible. Immunohistochemistry experiments using specific antibodies to MCP-1 were conducted to identify MCP-1-expressing cells in inflamed and noninflamed bone. We found that osteoblasts were the principal cells expressing MCP-1 in inflamed bone. There was little or no MCP-1 expression in noninflamed bone. Immunohistochemistry experiments were carried out to assess monocyte recruitment during osseous inflammation. The number of MCP-1-positive cells was significantly correlated to the number of monocytes/macrophages present (n = 15; r = 0.69; P < = 0.01). These in vivo results strongly suggest that MCP-1 is an important mediator involved in the recruitment of monocytes/macrophages in inflamed bone.

Animals↗
PubMed1994

Expression of 92 kD type IV collagenase/gelatinase B in human osteoclasts.

The digestion of type I collagen is an essential step in bone resorption. It is well established that osteoclasts solubilize the mineral phase of bone during the resorptive process, but the mechanism by which they degrade type I collagen, the major proteinaceous component of bone, is controversial. Differential screening of a human osteoclastoma cDNA library was performed to characterize genes specifically expressed in osteoclasts. A large number of cDNA clones obtained by this procedure were found to represent 92 kD type IV collagenase (gelatinase B; MMP-9, EC 3.4.24.35), as well as tartrate-resistant acid phosphatase. In situ hybridization localized mRNA for gelatinase B to multinucleated giant cells in human osteoclastomas. Gelatinase B immunoreactivity was demonstrated in giant cells from eight of eight osteoclastomas, osteoclasts in normal bone, and osteoclasts of Paget's disease by use of a polyclonal antiserum raised against a synthetic gelatinase B peptide. In contrast, no immunoreactivity for 72 kD type IV collagenase (gelatinase A; MMP-2, EC 3.4.24.24), which is the product of a separate gene, was detected in osteoclastomas or normal osteoclasts. We propose that the 92 kD type IV collagenase/gelatinase B plays an important role in the resorption of collagen during bone remodeling.

Base Sequence↗
PubMed1994

Pathogenesis of induced rat periapical lesions.

Studies of the mechanisms of pathogenesis of periapical lesions were undertaken using a rat model of surgical pulp exposure. In this model, periapical lesions develop rapidly between days 0 and 15 (active phase) and more slowly thereafter (chronic phase). A Gram-negative anaerobic flora, similar to that seen in human beings, are quickly established. Lesions contain a mixed inflammatory cell infiltrate consisting of T cells, neutrophils, B cells, macrophages, and plasma cells. Helper T cells predominate during the active phase, whereas suppressor T cells are more frequent in the chronic phase. Extracts of periapical lesions contain bone-resorbing activity, the highest levels of which are present when lesions are actively expanding. Most bone-resorbing activity is mediated by the cytokine interleukin-1 alpha, as determined by biochemical criteria and antibody neutralization studies. Prostaglandin2 accounts for 10% to 15% of resorptive activity. Cells that express interleukin-1 alpha were identified in pulp beginning on day 2 after exposure and in periapical tissue beginning on day 7, as determined by in situ hybridization and immunostaining. Macrophages, fibroblasts, neutrophils, and osteoclasts were positive for interleukin-1 alpha mRNA and protein. Cells that express tumor necrosis factor alpha were also detected, whereas cells expressing interleukin-1 beta or tumor necrosis factor beta were absent. Finally, periapical bone destruction was inhibited by 60% by treatment with interleukin-1 receptor antagonist. These studies establish a key role for interleukin-1 alpha in the pathogenesis of periapical lesions in the rat model.

Alveolar Bone Loss↗
PubMed1994

Changes in root canal microbiota during the development of rat periapical lesions.

Periapical lesions are reproducibly induced in rats by pulp exposure and infection from the oral cavity. Lesions expand rapidly between day 7 and day 15-20 (active phase), with slowed expansion thereafter. In the present study we characterized the root canal microbiota present during the active phase of lesion development in this system. The mandibular first molars of Sprague-Dawley rats were exposed on day 0. The teeth were extracted after 7 days (n = 10 animals) and 15 days (n = 10), and the microbiota present in roots was isolated and characterized. The number of colonies isolated per tooth was similar on day 7 (1.53 +/- 0.64 colony-forming units x 10(3)) and day 15 (1.49 +/- 0.37 colony-forming units x 10(3)). No colonies were isolated from unexposed control teeth. Anaerobic bacteria increased significantly between day 7 (24.3 +/- 5.7%) and day 15 (47.3 +/- 7.5%), and the proportion of gram-negative organisms increased from day 7 (24.3 +/- 6.1) to day 15 (46.9 +/- 6.8). The predominant bacteria included, on day 7: Streptococcus and Bacteroides species; on day 15: species of Streptococcus, Bacteroides, Prevotella, Neisseria and Peptostreptococcus. Streptococcus oralis, Streptococcus mitis, Streptococcus rattus, Bacteroides pneumosintes and Bacteroides ureolyticus were frequently isolated at both points. Although approximately the same mean number of different species (approximately 3.5) was isolated per tooth on both day 7 and 15, the overall diversity of the isolates increased on day 15.(ABSTRACT TRUNCATED AT 250 WORDS)

Alveolar Bone Loss↗
PubMed1993

Platelet activating factor increases intracellular calcium in isolated osteoclasts but does not modify bone resorption.

Platelet activating factor (PAF) is an inflammatory mediator secreted by activated macrophages and granulocytes. Since PAF has been previously demonstrated to increase intracellular calcium in isolated osteoclasts, the effect of PAF on bone resorption was examined. This was compared to effects seen after addition of calcium ionophores and calcitonin, an inhibitor of bone resorption, in order to determine if an increase in [Ca++]i inhibits osteoclast-mediated resorption. The results demonstrate that PAF has no effect on either unstimulated or PTH-induced bone resorption. Calcium ionophores, ionomycin, and A23187, at concentrations that increased [Ca++]i, failed to modulate resorption in the fetal long bone assay. In addition, salmon calcitonin did not change [Ca++]i in spite of its strong inhibition of bone resorption. Taken together, these results demonstrate that the second messenger pathways involved in osteoclast downregulation can be independent of [Ca++]i, and may involve other messenger systems.

Animals↗