Interaction of inflammatory cells and oral bacteria: release of lysosomal hydrolases from rabbit polymorphonuclear leukocytes exposed to gram-positive plaque bacteria.

The purpose was to ascertain whether Gram-positive bacteria derived from dental plaque could stimulate the release of lysosomal enzymes from rabbit peritoneal exudate polymorphonuclear leukocytes (PMNs). Microorganisms which were capable of inducing periodontal syndromes in experimental animals and of synthesizing extracellular polysaccharides were capable of triggering hydrolase release from PMNs. On the other hand, bacteria which did not produce extracellular polysaccharides under the conditions employed were relatively inactive in inducing a PMN-release response. Lysosome release resulting from bacterial-PMN interactions may be one important mechanism in the pathogenesis of gingivitis and periodontal disease.     

Suicide gene therapy for premalignant disease: a new strategy for the treatment of intraepithelial neoplasia

The potential of gene therapy to treat premalignant disease or recurrent cancer has not been investigated. The goal of the present investigation was to explore the efficacy of pro-drug-mediated, suicide gene therapy as a strategy to treat incipient neoplasia in stratified squamous epithelium. To test this strategy, a tissue model of premalignancy was generated by mixing normal human keratinocytes (NHK) that express the bacterial cytosine deaminase gene (CD) with premalignant keratinocytes which have been genetically marked with the bacterial gene for beta-galactosidase (II-4-beta-gal) in skin-like organotypic cultures. Preliminary studies in monolayer cultures demonstrated that CD-transduced NHK (NHK/CD) efficiently expressed the transgene and deaminated the pro-drug 5-fluorocytosine (5FC) to the toxic product 5-fluorouracil (5FU). The capacity of NHK/CD to kill II-4-beta-gal cells through bystander effect was assayed in both submerged culture and in the organotypic model of premalignancy. In submerged cultures, it was found that CD-mediated killing of II-4-beta-gal cells did not require cell-cell contact and that the LD(50) of 5FC for efficient bystander killing of II-4-beta-gal was 0.5 mM. When this concentration of pro-drug was used in organotypic cultures, a significant number of dysplastic II-4-beta-gal cells were eliminated from the tissue. Bystander killing of II-4-beta-gal cells was related to the number of NHK/CD present. These findings demonstrated that potentially malignant keratinocytes could be eliminated from a dysplastic tissue through activation of pro-drug and killing of adjacent cells through the bystander effect. By establishing an in vitro model to eliminate premalignant cells using suicide gene therapy, these studies provide a new approach for the treatment of incipient cancer as it develops, thereby preventing invasive disease.     

Interaction of inflammatory cells and oral microorganisms. VIII. Detection of leukotoxic activity of a plaque-derived gram-negative microorganism.

In the present study we identified a gram-negative anaerobic rod referred to as Y4 which was cytotoxic for human polymorphonuclear leukocytes. Y4 was isolated from dental plaque of a patient with juvenile periodontitis and presented most of the taxonomic characteristics of Actinobacillus species. Under experimental conditions, viable Y4 were cytotoxic for human peripheral blood polymorphonuclear leukocytes in serum-free cultures. Cytotoxicity was dependent on bacterial concentrations and was enhanced in the presence of a fresh or heat-inactivated (56 degrees C, 30 min) autologous serum. Leukotoxicity was independent of phagocytosis. Y4 leukotoxic effect was abolished when bacteria were heat treated (56 degrees C, 30 min) or when incubations were carried out at 4 degrees C instead of at 37 degrees C. The leukotoxicity was monitored by electron microscopy and biochemically by measuring lactate dehydrogenase indicator of cell viability. No cytotoxic effects of Y4 on human mononuclear cells, chicken fibroblasts, or mouse macrophages were detected under the conditions studied. Polymorphonuclear leukocytes may play an important role in the host defense against bacteria in periodontal disease. The cytotoxic effect of Y4 for polymorphonuclear leukocytes presented in this study is the first report of a direct offensive microbial vector in a plaque-derived microorganism and may prove to be relevant in the pathogenesis of juvenile periodontitis.     

The assessment of stress,depression, and inflammation as a collective risk factor for periodontal diseases: a systematic review

Clinical Oral Investigations - The purpose of this review was to provide a novel perspective utilizing an assessment of biomarkers to evaluate the impact of stress-related disorders on the...     

Monoclonal antibodies to leukotoxin of Actinobacillus actinomycetemcomitans

Hybridoma cell lines which produce monoclonal antibodies to a leukotoxin from Actinobacillus actinomycetemcomitans were prepared. The monoclonal antibodies were selected for their ability to neutralize the cytotoxic activity of the leukotoxin and recognize the toxin on nitrocellulose blots. The antibodies belonged to either the immunoglobulin G1 (IgG1) or IgG2 subclass and differed in their ability to bind to the leukotoxin on nitrocellulose blots. However, only slight differences in neutralization titers were observed. Use of the monoclonal antibodies revealed that polymyxin B-extracted or osmotic shock-released leukotoxin could be separated into several high-molecular-weight polypeptides by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Immunoblot analysis with the monoclonal antibodies also demonstrated that the leukotoxin was present in eight oral strains of A. actinomycetemcomitans that had been previously classified by a biological assay as leukotoxic. The availability of these monoclonal antibodies should facilitate and expand studies concerning the role of the leukotoxin in the pathogenicity of A. actinomycetemcomitans.     

Annexin II expressed by osteoblasts and endothelial cells regulates stem cell adhesion, homing, and engraftment following transplantation

Differentiation of hematopoietic stem cells (HSCs) after birth is largely restricted to the bone marrow cavity, where HSCs are associated closely with osteoblasts (OBs). How OBs localize HSCs to the endosteal niche remains unclear. To explore adhesive interactions between HSCs and OBs, a cell blot analysis was used that revealed 2 major bands that corresponded to monomers and multimers of annexin II (Anxa2). Immunohistochemistry revealed that OBs and marrow endothelial cells express Anxa2 at high levels. Function-blocking studies confirmed that Anxa2 mediates HSC adhesion mainly via the N-terminal portion of the Anxa2 peptide. Adhesion of HSCs to OBs derived from Anxa2-deficient animals (Anxa2(-/-)) was significantly impaired compared with OBs obtained from wild-type animals (Anxa2(+/+)). Moreover, fewer HSCs were found in the marrow of Anxa2(-/-) versus Anxa2(+/+) animals. Short-term lodging, engraftment, and survival of irradiated mice with whole marrow cells were substantially inhibited by N-terminal peptide fragments of Anxa2 or anti-Anxa2 antibodies. Similar findings were noted in long-term competitive repopulation studies. Collectively, these findings reveal that Anxa2 regulates HSC homing and binding to the bone marrow microenvironment and suggest that Anxa2 is crucial for determining the bone marrow niche of HSCs.     

Tissue- and stratum-specific expression of the human involucrin promoter in transgenic mice.

Involucrin is a marker of keratinocyte terminal differentiation and is expressed only in the suprabasal layers of stratified squamous epithelium. In a previous study with various cell types in culture, we noted that expression of the putative human involucrin promoter was keratinocyte specific. To determine if this promoter is sufficient to direct expression to the suprabasal cells of stratified squamous epithelia in vivo, we have now generated transgenic mouse lines harboring the involucrin promoter sequences linked to a beta-galactosidase reporter gene. In the resulting lines, beta-galactosidase was expressed in the suprabasal compartment of stratified squamous epithelia and in hair follicles in a tissue-specific manner. In the palate, distinct vertical stacks of beta-galactosidase-expressing cells were present, suggesting movement of clonally derived cells through the epithelium. The involucrin gene has a single intron upstream of the translational start site, and removal of this intron did not affect tissue- or stratum-specific expression. These results show that the 3.7-kb involucrin upstream sequences contain all the information necessary for a high level of tissue- and stratum-specific expression.     

Impact of proteoglycan‐4 and parathyroid hormone on articular cartilage

Proteoglycan‐4 (Prg4) protects synovial joints from arthropathic changes by mechanisms that are incompletely understood. Parathyroid hormone (PTH), known for its anabolic actions in bone, increases Prg4 expression and has been reported to inhibit articular cartilage degeneration in arthropathic joints. To investigate the effect of Prg4 and PTH on articular cartilage, 16‐week‐old Prg4 mutant and wild‐type mice were treated with intermittent PTH (1–34) or vehicle control daily for six weeks. Analyses included histology of the knee joint, micro‐CT of the distal femur, and serum biochemical analysis of type II collagen fragments (CTX‐II). Compared to wild‐type littermates, Prg4 mutant mice had an acellular layer of material lining the surfaces of the articular cartilage and menisci, increased articular cartilage degradation, increased serum CTX‐II concentrations, decreased articular chondrocyte apoptosis, increased synovium SDF‐1 expression, and irregularly contoured subchondral bone. PTH‐treated Prg4 mutant mice developed a secondary deposit overlaying the acellular layer of material lining the joint surfaces, but PTH‐treatment did not alter signs of articular cartilage degeneration in Prg4 mutant mice. The increased joint SDF‐1 levels and irregular subchondral bone found in Prg4 mutant mice introduce novel candidate mechanisms by which Prg4 protects articular cartilage. © 2012 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 31: 183–190, 2013