Philip Stashenko

Atp6i-deficient mice exhibit severe osteopetrosis due to loss of osteoclast-mediated extracellular acidification.

Solubilization of bone mineral by osteoclasts depends on the formation of an acidic extracellular compartment through the action of a V-proton pump that has not yet been characterized at the molecular level. We previously cloned a gene (Atp6i, for V-proton pump, H+ transporting (vacuolar proton pump) member I) encoding a putative osteoclast-specific proton pump subunit, termed OC-116kD (ref. 4). Here we show that targeted disruption of Atp6i in mice results in severe osteopetrosis. Atp6i–/– osteoclast-like cells (OCLs) lose the function of extracellular acidification, but retain intracellular lysosomal proton pump activity. The pH in Atp6i–/– liver lysosomes and proton transport in microsomes of Atp6i–/– kidney are identical to that in wild-type mice. Atp6i–/– mice exhibit a normal acid-base balance in blood and urine. Our results demonstrate that Atp6i is unique and necessary for osteoclast-mediated extracellular acidification.

A unique cell surface antigen identifying lymphoid malignancies of B cell origin.

A monoclonal antibody (anti-B1) specific for a unique B cell surface differentiation antigen was used to characterize the malignant cells from patients with leukemias or lymphomas. All tumor cells from patients with lymphomas or chronic lymphocytic leukemias, bearing either monoclonal kappa lambda light chain, expressed the B1 antigen. In contrast, tumor cells from T cell leukemias and lymphomas or acute myeloblastic leukemia were unreactive. Approximately 50% of acute lymphoblastic leukemias (ALL) of non-T origin and 50% of chronic myelocytic leukemia in blast crisis were also anti-B1 reactive. moreover, 21 of 28 patients with the common ALL antigen (CALLA) positive form of ALL were anti-B1 positive, whereas 0 of 13 patients with CALLA negative ALL were reactive. These observations demonstrate that an antigen present on normal B cells is expressed on the vast majority of B cell lymphomas and on approximately 75% of CALLA positive ALL, suggesting that these tumors may share a common B cell lineage.

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.

Monoclonal antibodies defining serologically distinct HLA-D/DR related Ia-like antigens in man

Four monoclonal antisera-identifying antigens with the identical tissue distribution and molecular weight of previously described Ia-like antigens were characterized. Two of these antisera, I-1 and I-2, identified antigens expressed on the HLA-D/DR positive cells from all HLA heterozygous individuals. Further characterization on homozygous typing cells (HTCs) demonstrated that I-2 was not reactive with most Dw7 and Dw11 HTCs. Monoclonal antisera, termed I-LR1 and I-LR2, defined polymorphic Ia-like antigens that demonstrated restricted expression on cells from HLA heterozygous individuals. Antigen I-LR1 was expressed on cells from 60% of HLA heterozygotes and its reactivity with HTCs did not conform to any previously described monotypic or supertypic HLA-D/DR pattern. In contrast, I-LR2 was expressed on 40% of HLA heterozygotes and identified only HLA-DR3, 5 and 6 HTCs. Studies of families with HLA recombinants permitted the demonstration that the I-LR1 and I-LR2 antigens are tightly linked to the HLA-D/DR locus. These experiments permit the direct demonstration by immunoprecipitation, linkage studies, and MHC recombinant families that the p29,34 complex in man is closely linked to or is within the HLA-D/DR locus. These studies suggest that the human Ia-like antigens are more heterogeneous than previously demonstrated and that monoclonal antisera will be useful in further defining the structural, genetic, and functional characteristics of these molecules.

Expression of bone-resorptive and regulatory cytokines in murine periapical inflammation

Periapical bone destruction earlier was shown to be mediated primarily by interleukin (IL)-1alpha in a rat model. The production and action of IL-1alpha is in turn potentially modulated by a network of cytokines, which are produced by infiltrating T-helper type 1 (Th1) and type 2 (Th2) lymphocytes, and resident connective tissue cells within the lesion. This study was designed to examine the kinetics of expression of 10 cytokines in experimentally induced murine periapical lesions, including bone-resorptive [IL-1alpha, tumour necrosis factor alpha (TNFalpha), IL-6, IL-11], Th1-type [IL-2, IL-12, interferon-gamma (IFNgamma)] and Th2-type (IL-4, IL-6, IL-10, IL-13) mediators. Cytokine mRNA expression was assessed qualitatively by reverse transcription-polymerase chain reaction, and cytokine proteins quantified by enzyme-linked immunosorbent assay. IL-1alpha and TNFalpha protein and mRNA were highly expressed, beginning on day 7, and increased to day 28. IL-6 increased to day 14 and then declined, whereas the expression of IL-11 was not modulated by pulp exposure. Most of the Th1-type cytokines, including IL-2, IL-12, and IFNgamma, showed an increase in mRNA and/or protein expression in periapical lesions after pulpal exposure; the expression of Th2-type cytokines was similarly increased, but had declined at the latest time-point (day 28), suggesting possible inhibition by Th1-type mediators. Significant correlations were observed between levels of IL-1alpha and Th1-derived pro-inflammatory mediators IL-2, IL-12, TNFalpha, and IFNgamma. There was a lack of correlation between IL-1alpha and Th2-type anti-inflammatory mediators, including IL-4, -6, and -10. These results indicate that a cytokine network is activated in the periapex in response to bacterial infection, and that Th1-modulated pro-inflammatory pathways may predominate during periapical bone destruction.

Serotonin Regulates Osteoclast Differentiation Through Its Transporter

5‐HTT mediates antidepressant‐sensitive clearance of 5‐HT after its release into neural synapses. We found increased expression of 5‐HTT in RANKL‐induced osteoclast‐like cells. Fluoxetine, an inhibitor of 5‐HTT, reduced osteoclast differentiation but not activation. Reserpine, an inhibitor of 5‐HT intracellular transport, potentiated differentiation. These results indicate a role for 5‐HTT in osteoclast function and suggest that commonly used antidepressive agents may affect bone mass.

Photodynamic treatment of endodontic polymicrobial infection in vitro

We investigated the photodynamic effects of methylene blue on multispecies root canal biofilms comprising Actinomyces israelii, Fusobacterium nucleatum subspecies nucleatum, Porphyromonas gingivalis, and Prevotella intermedia in experimentally infected root canals of extracted human teeth in vitro. The 4 test microorganisms were detected in root canals by using DNA probes. Scanning electron microscopy showed the presence of biofilms in root canals before therapy. Root canal systems were incubated with methylene blue (25 microg/mL) for 10 minutes followed by exposure to red light at 665 nm with an energy fluence of 30 J/cm(2). Light was delivered from a diode laser via a 250-microm diameter polymethyl methacrylate optical fiber that uniformly distributed light over 360 degrees. Photodynamic therapy (PDT) achieved up to 80% reduction of colony-forming unit counts. We concluded that PDT can be an effective adjunct to standard endodontic antimicrobial treatment when the PDT parameters are optimized.

IL-10, But Not IL-4, Suppresses Infection-Stimulated Bone Resorption In Vivo

Periapical bone resorption occurs following infection of the dental pulp and is mediated mainly by IL-1α in the murine model. The production and activity of IL-1α is modulated by a network of regulatory cytokines, including those produced by Th1 (pro-inflammatory) and Th2 (anti-inflammatory) subset T cells. This study was designed to assess the functional role of the Th2-type cytokines IL-4 and IL-10 in infection-stimulated bone resorption in vivo. The dental pulps of the first molars were exposed and infected with a mixture of four common endodontic pathogens, and bone destruction was determined by micro-computed tomography at sacrifice on day 21. The results demonstrate that IL-10−/− mice had significantly greater infection-stimulated bone resorption in vivo compared with wild-type mice (p < 0.001), whereas IL-4−/− exhibited no increased resorption. IL-10−/− had markedly elevated IL-1α production within periapical inflammatory tissues (>10-fold) compared with wild type (p < 0.01), whereas IL-4−/− exhibited decreased IL-1α production (p < 0.05). IL-10 also suppressed IL-1α production by macrophages in a dose-dependent fashion in vitro, whereas IL-4 had weak and variable effects. We conclude that IL-10, but not IL-4, is an important endogenous suppressor of infection-stimulated bone resorption in vivo, likely acting via inhibition of IL-1α expression.

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.

Quantification of Periapical Bone Destruction in Mice by Micro-computed Tomography

Bacterial infections of the dental pulp result in tissue destruction and periapical bone resorption. The availability of genetically engineered mouse strains is a major advantage in the use of this model system for studies of periapical pathogenesis. The main limitation of the mouse model is its small size, and the necessity for laborious histologic analyses to quantify periapical bone destruction. In the present study, we evaluated the use of a new technology, high-resolution micro-computed tomography (micro-CT), for the rapid and non-invasive quantification of periapical bone destruction. Periapical lesions were induced in the lower first molars of mice by exposing the pulp to the oral environment. Mandibles were harvested on day 21 after pulp exposure, and were subjected to micro-CT analysis, with 17-μm-thick radiographic sections. Samples were then decalcified, embedded, and sectioned for histology. The cross-sectional area of periapical lesions was determined by image analysis of corresponding micro-CT and histologic sections. The results showed a highly significant correlation between micro-CT and histology (p < 0.0001), with mean differences of 4.1% (range, 0.9 to 7.2%) between the two methods. The mean error associated with image analysis was 4.9% for images obtained by both micro-CT and histology. The variability of replicate (n = 5) independent micro-CT determinations was 3.4%, less than that associated with the image analysis error. These results demonstrate that micro-CT imaging is a rapid, reproducible, and non-invasive method, that gives results that are closely comparable with those obtained by histology. Micro-CT appears to have utility for the accurate quantification of changes in bone architecture in small biological specimens.