Norton S. Taichman

Human neutrophils secrete vascular endothelial growth factor.

Vascular endothelial growth factor (VEGF) is a multifunctional cytokine that plays a pivotal role in mediating neovascularization as well as other endothelial cell alterations during inflammation. In this study, we demonstrate that human neutrophils are a source of VEGF. We observed that isolated blood neutrophils released VEGF in response to different stimuli and we demonstrated by immunohistochemistry that neutrophils infiltrating inflamed tissues contain VEGF. These results indicate that neutrophil‐derived VEGF may be instrumental in regulating vascular responses during acute and chronic inflammation. J. Leukoc. Biol. 62: 397–400; 1997.

Leukotoxic activity in different strains of the bacterium Actinobacillus actinomycetemcomitans isolated from juvenile periodontitis in man

Abstract Type culture strains and dental isolates (from juvenile periodontitis) of Act. actinomycetemcomitans (Aa) were tested for their ability to kill human polymorphonuclear leukocytes (PMN) in vitro. The majority of Aa, as well as sonic extracts prepared from these organisms, rapidly destroyed PMNs, shown by the extracellular release of lactate dehydrogenase from PMNs and by degenerative ultrastructural alterations. The leukotoxic properties of Aa could be modulated by serum: normal human sera enhanced killing but juvenile periodontitis or various rabbit anti-Aa sera neutralized toxic activity. Whereas toxic and non-toxic strains of Aa shared common antigens, immunologic analyses revealed a unique antigen in sonic extracts of leukotoxic organisms. Thus Aa-derived leukotoxin may be an aetiologic vector in juvenile periodontitis.

Identification and expression of the Actinobacillus actinomycetemcomitans leukotoxin gene.

The leukotoxin produced by the oral bacterium Actinobacillus actinomycetemcomitans has been implicated in the pathogenesis of juvenile periodontitis. In order to elucidate the structure of the leukotoxin, molecular cloning of the leukotoxin gene was carried out. A DNA library of A. actinomycetemcomitans, strain JP2, was constructed by partial digestion of genomic DNA with Sau3AI and ligation of 0.5 to 5.0 kilobase pair fragments into the Bam HI site of the plasmid vector pENN-vrf. After transformation into E. coli RR1 (lambda cI857), the clones were screened for the production of A. actinomycetemcomitans leukotoxin with polyclonal antibody. Six immunoreactive clones were identified. The clones expressed proteins which ranged from 21-80 kilodaltons, and the clone designated pII-2, producing the largest protein was selected for further study. Antibodies eluted from immobilized pII-2 protein also recognized the native A. actinomycetemcomitans leukotoxin molecule indicating that both molecules shared at least one epitope. DNA sequence analysis demonstrated that there are regions of significant amino acid sequence homology between the cloned A. actinomycetemcomitans leukotoxin and two other cytolysins, Escherichia coli alpha-hemolysin and Pasteurella haemolytica leukotoxin, suggesting that a family of cytolysins may exist which share a common mechanism of killing but vary in their target cell specificity.

Structure and function of the B and D genes of the Actinobacillus actinomycetemcomitans leukotoxin complex

The Actinobacillus actinomycetemcomitans leukotoxin gene complex, consisting of four genes, has been cloned and the sequence of the AaLtC and AaLtA genes reported. The present paper details the sequences of the AaLtB and AaLtD genes which, like AaLtC and AaLTA, are also homologues of genes found in other cytolytic toxin complexes of several other Gram-negative bacterial pathogens. When tested in a recombinant expression system, the AaLtB and/or AaLtD genes are required for the translocation and insertion of the A. actinomycetemcomitans leukotoxin (AaLtA) into the cell membrane of Escherichia coli.

Interaction of inflammatory cells and oral bacteria: Release of lysosomal hydrolases from rabbit polymorphonuclear leukocytes exposed to Gram-positive plaque bacteria

Abstract 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.

Activation of the complement system by some Gram-positive oral bacteria

Abstract Actinomyces viscosus 19246, T 14 V and T 14 AV, Streptococcus mutans and Streptococcus sanguis consumed complement in vitro . Complement (C) profile analysis revealed that C 4 and C 3–9 were consumed concomitantly in unadsorbed human serum. In serum from which naturally occurring agglutinating antibodies had been removed, the same microorganisms caused C 3–9 consumption in the absence of a demonstrable loss of C 4 activity. Congenitally C 4 -deficient guinea-pig serum (C 4 D) supported a similar consumption of C 3–9 . The Gram-positive plaque microorganisms tested activated serum complement by the classical as well as the alternate pathways. Dental plaque microorganisms may cause a similar activation of gingival crevicular fluid complement in vivo , thus resulting in complement-mediated inflammatory processes.

Lytic effects of Actinobacillus actinomycetemcomitans leukotoxin on human neutrophil cytoplasts

Actinobacillus actinomycetemcomitans leukotoxin lysed human neutrophil cytoplasts. The reaction was associated with a rapid influx of extracellular calcium, a collapse in membrane potential, release of lactate dehydrogenase, and overt disintegration of the plasma membrane. These functional and structural alterations in the plasmalemma of neutrophil cytoplasts reinforce the hypothesis that A. actinomycetemcomitans leukotoxin acts as a pore‐forming, membranolytic agent and indicate that neutrophil cytoplasts are useful tools in studying the biology of membrane‐active toxins.

Electron microscopic study of the interaction of oral microorganisms with polymorphonuclear leukocytes.

Abstract A variety of Gram-positive plaque bacteria can trigger lysosomal enzyme release from rabbit polymorphonuclear leukocytes (PMNs) in vitro . This electron microscopic study was undertaken to assess the correlation between phagocytic activity by PMNs and lysosome release to Streptococcus mutons 6715, Streptococcus sanguis G9B, and Actinomyces viscosus T14. PMN phagocytic activity was quantitated by determining the percentage of cells showing evidence of phagocytosis (phagocytic index) and the number of bacteria within the PMNs (avidity index). High phagocytic activity was accompanied by vigorous PMN lysosomal enzyme release. On the other hand, when PMNs exhibited low phagocytic activity lysosomal enzyme release was minimal. For example, Streptococcus mutans grown in brain-heart infusion broth did not induce significant lysosome release or demonstrable phagocytic activity by PMNs. In contrast, Strep. mutans grown in BHI containing sucrose or pre-incubated with anti- Strep. mutans antibodies acted as potent triggers of lysosome release and PMN phagocytosis. Our results indicate that various species of oral Gram-positive bacteria trigger different responses from PMNs and that phagocytic activity of PMNs correlates well with the ability of these microorganisms to stimulate lysosomal enzyme release.

Role of salivary vascular endothelial growth factor (VEGF) in palatal mucosal wound healing.

The mucosa of alimentary tract heals more rapidly than cutaneous wounds. The underlying mechanisms of this enhanced healing have not been completely elucidated. Constant exposure to salivary growth factors has been shown to play a critical role in mucosal homeostasis and tissue repair. Angiogenesis also has an essential role in successful wound repair. One of the main angiogenic growth factors, vascular endothelial growth factor (VEGF), has a pleiotropic role in tissue repair via neovascularization, reepithelialization, and regulation of extracellular matrix. We have previously reported a critical role for salivary VEGF in bowel adaptation after small bowel resection. We hypothesize that salivary VEGF is an essential stimulus for oral mucosal tissue repair, and use the murine palatal wound model to test our hypothesis. In a loss‐of‐function experiment, we removed the primary source of VEGF production through selective submandibular gland (SMG) sialoadenectomy in a murine model and observed the effects on wound closure and neovascularization. We then performed a selective loss‐of‐function experiment using the protein VEGF‐Trap to inhibit salivary VEGF. In a gain‐of‐function experiment, we supplemented oral VEGF following SMG sialoadenectomy. After SMG sialoadenectomy, there was significant reduction in salivary VEGF level, wound closure, and vessel density. Lower levels of salivary VEGF were correlated with impaired neovascularization and reepithelialization. The selective blockade of VEGF using VEGF‐Trap resulted in a similar impairment in wound healing and neovascularization. The sole supplementation of oral VEGF after SMG sialoadenectomy rescued the impaired wound healing phenotype and restored neovascularization to normal levels. These data show a novel role for salivary‐VEGF in mucosal wound healing, and provide a basis for the development of novel therapeutics aimed at augmenting wound repair of the oral mucosa, as well as wounds at other sites in the alimentary tract.

Chapter 23. Current Concepts in Periodontal Disease

Publisher Summary This chapter highlights the mechanisms thought to be involved in the etiology and pathogenesis of chronic periodontitis. The etiology of periodontal disease has been definitively linked to the deposition and accumulation of microbial colonies on the crowns of teeth in the region of the in gingival sulcus, where the gingiva or gum meets the tooth. Bacterial aggregates adhere onto the tooth surface in a soft, sticky mass called “supragingival plaque.” The chapter outlines that plaque in some way triggers and sustains an inflammatory response in the underlying gingival tissues. During its earliest stages, periodontal disease is confined to the marginal gingival. Diseased gingival tissues exhibit all the clinical and microscopic features characteristic of a chronic inflammatory process. An exudate, rich in plasma and tissue-derived proteins and white cells, drains from the affected gingiva into the crevice. This is the so-called “crevicular” or “sulcular exudate,” which can be detected throughout the course of periodontal disease.