Richard Niederman

Human platelet myosin. I. Purification by a rapid method applicable to other nonmuscle cells

Abstract Using refinements of established procedures and a new two buffer gel filtration method, it is possible to prepare human platelet myosin which by gel electrophoresis is free from actin and over 95% pure. This method with minor modifications is useful for the purification of myosin from several other nonmuscle cells.

Short-chain Carboxylic Acid Concentration in Human Gingival Crevicular Fluid

Short-chain carboxylic acids (e.g., lactic acid, propionic acid, butyric acid) are metabolic by-products of bacterial metabolism which can accumulate in the gingival crevice. It is of no small consequence, therefore, that 1- to 5-mM concentrations of these acids exhibit significant biological activity, including the ability to alter cell proliferation and gene expression in cells of importance to the periodontium. This communication reports on the in vivo concentrations of propionic and butyric acid in the gingival crevices of periodontal subjects with severe and mild disease. The results indicated that severely diseased subjects exhibited a > 10-fold increase in the mM concentration of these acids when compared with mildly diseased subjects (mean propionic acid - severe = 9.5 ± 1.8 mM, and mild = 0.8 ± 0.3 mM; mean butyric acid - severe = 2.6 ± 0.4 mM, and mild = 0.2 ± 0.04 mM). These differences (mean ± SE) were significant (p < 0.0001). The propionic and butyric acid concentrations were below detection limits in healthy sites of mildly diseased subjects. The propionic and butyric acid concentrations also associated significantly with clinical measures of disease severity (e.g., pocket depth, attachment level) and inflammation (e.g., subgingival temperature, % of sites bleeding when probed), and with the total microbial load (all p < 0.05). Taken together, these data suggest that short-chain carboxylic acids play a mediating role in periodontal disease pathogenesis.

Cadherin-Mediated Adhesion Is Required for Normal Growth Regulation of Human Gingival Epithelial Cells

The cadherins are a family of cell membrane proteins that mediate calcium-dependent cell-cell adhesion. E-cadherin is required for the formation, differentiation, polarization and stratification of epithelia; P-cadherin is also expressed on many epithelia. We report here the first study of cadherin expression in immortalized human gingival epithelial cells (IHGK) and examine the role of cadherins in growth regulation of these cells. We found that the IHGK cells are similar to normal gingival epithelial cells in their cadherin expression and density-dependent inhibition of growth. The IHGK cells proliferate more rapidly at low calcium concentration (0.15 mM) than at physiological concentrations of calcium (1.8 mM) and magnesium (0.65 mM; Ca/Mg medium) suggesting that calcium is required for density-dependent regulation of proliferation. To evaluate the possibility that cadherin function is required for contact inhibition in these cells, we grew them in Ca/Mg medium in the presence of adhesion-blocking anti-cadherin monoclonal antibodies. At anti-E-cadherin concentrations sufficient to disrupt cell-cell adhesion, the proliferation of the IHGK cells was similar to that observed in medium containing 0.2 mM EDTA. Anti-P-cadherin had a much weaker effect on cell proliferation than anti-E-cadherin, and cells grown in medium containing both antibodies grew at intermediate rates. The increased proliferation of the IHGK cells in either low calcium medium or Ca/Mg medium containing adhesion-blocking anti-cadherin antibodies suggests that cadherin-mediated adhesion is required for density-dependent regulation of growth of these cells.

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-β1-6-glucotriosyl-β1-3-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 < 0.01). PGG glucan-treated animals also had less soft tissue destruction, as indicated by decreased pulpal necrosis. Only 3.3% of 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. These findings support the concept that a biological response modifier which enhances endogenous antibacterial mechanisms in neutrophils can decrease infection-stimulated alveolar bone and soft tissue destruction in vivo.

Tradition-based Dental Care and Evidence-based Dental Care

T radition-based dental care and evidence-based dental care offer complementary paradigms for clinical Tdecision-making. Tradition-based care emphasizes the primacy of knowledge, experience, and intuition in the exercise of good clinical judgment. Evidence-based care emphasizes the integration of good judgment with the best available evidence and the patients values in the making of clinical decisions (Haynes et al., 1996; Sackett et al., 1996). Evidence-based care therefore requires a new set of tools and rules. The new tools are the Internet-linked computer databases. The new rules facilitate an unbiased, rapid identification, critical appraisal, and clinical implementation of important clinical data found in these databases. In this communication, we highlight some of the limitations of tradition-based care, and highlight some of the benefits of evidence-based care.

PROPIONIC ACID STIMULATES SUPEROXIDE GENERATION IN HUMAN NEUTROPHILS

Short‐chain carboxylic acids are the metabolic by‐products of pathogenic anaerobic bacteria and are found at sites of infection in millimolar quantities. We previously reported that propionic acid, one of the short‐chain carboxylic acids, induces an increase in intracellular Ca2+([Ca2+]i) in human neutrophils. Here we investigate the effect of propionic acid on superoxide generation in human neutrophils. Propionic acid (10mm) induced inositol 1,4,5‐trisphosphate (IP3) formation and a rapidly transient increase in [Ca2+]i, but not superoxide generation, whereas 1μm formylmethionyl‐leucyl‐phenylalanine (fMLP), a widely used neutrophil‐stimulating bacterial peptide, stimulated not only IP3formation and Ca2+mobilization but also superoxide generation. The IP3level induced by propionic acid was slightly lower than that induced by fMLP. The transient increase in [Ca2+]iinduced by propionic acid immediately returned to the basal level, whereas a sustained increase in [Ca2+]i, which was higher than the basal level, following a transient increase in [Ca2+]iwas induced by fMLP. The peak level induced by propionic acid was lower than that with fMLP. In the absence of extracellular Ca2+, thapsigargin, a potent inhibitor of endoplasmic reticulum Ca2+‐ATPase, induced an increase in [Ca2+]ieven after propionic acid stimulation, but not after fMLP. The Ca2+ionophore A23187 and thapsigargin induced superoxide generation by themselves. Propionic acid enhanced the superoxide generating effect of A23187 and thapsigargin. These results suggest that Ca2+mobilization induced by propionic acid is much weaker than that with fMLP, and propionic acid is able to generate superoxide in the presence of a Ca2+ionophore and a Ca2+influx activator.

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 nflammatory tissues of P/E−/− mice. P/E−/− mice had higher tissue levels of the bone resorptive cytokine, interleukin (IL)‐1α. Tissue levels of IL‐2, IL‐4, IL‐10, tumour necrosis factor‐α (TNF‐α) and interferon‐γ (IFN‐γ) 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.

INTRACELLULAR PH REGULATES THE PRODUCTION OF DIFFERENT OXYGEN METABOLITES IN NEUTROPHILS : EFFECTS OF ORGANIC ACIDS PRODUCED BY ANAEROBIC BACTERIA

The effects of short chain carboxylic acids (SCCA), namely succinic, butyric, and iso‐butyric, on neutrophil metabolic activation were assessed. SCCA induced a significant decrease in O2 ‐ recovery and chemlluminescent response in neutrophils activated with the diacylglycerol analog tetradecanoyl‐phorbol‐acetate (TPA). SCCA did not alter O2 consumption, H2O2 production, or the calorimetrically determined energy expenditure occurring during the metabolic burst. SCCA also induced a significant acidification of intracellular pH (pHi). These results are consistent with an increased divalent versus univalent O2 reduction performed by the NADPH oxidase at a more acidic intracellular pH.

Ammonia as a potential mediator of adult human periodontal infection: inhibition of neutrophil function.

Neutrophils (polymorphonuclear leucocytes) are the principal cell of the host defence system. Consequently, if periodontal pathogen-derived substances in the gingival crevice significantly inhibit their function, they could shift the bacterial-host balance in favour of the bacteria. The hypothesis that ammonia can inhibit neutrophil function was tested. Ammonia was specifically selected because periodontal pathogens produce substantial amounts of ammonia. The findings indicated that ammonia can inhibit neutrophil phagocytosis, degranulation and oxygen metabolism. Ammonia decreased the total number of phagocytosing polymorphonuclear neutrophils (66% of control) and also decreased degranulation (61% of control). Ammonia decreased oxygen metabolism of both resting and stimulated neutrophils (33 and 42% of control, respectively). These observations support the hypothesis that ammonia can inhibit the function of polymorphonuclear leukocytes. They suggest that the presence of ammonia in the gingival crevice may increase the risk of development of periodontal disease.

Benchmarking the Endodontic Literature on MEDLINE

The purpose of this study was to identify and quantify the endodontic literature available for clinical decision making. A search strategy based on Medical Subject Headings for endodontics was developed to examine MEDLINE. The identified articles were limited to human subjects and English. Sensitive and specific methodological search filters identified four categories of information: etiology, diagnosis, therapy, and prognosis. The results were then subdivided by year to identify trends. Between 1990 and 1998 MEDLINE identified 3,152 articles published in English on endodontics in humans. The number of articles per year (mean +/- SD) for sensitive and specific searches was etiology (28+/-10, 1+/-2), diagnosis (38+/-11, 1+/-1), therapy (59+/-15, 3+/-3), and prognosis (40+/-13, 10+/-5), respectively. The number of articles in each category increased by 1 to 3% each year. There were 150 articles/yr in endodontics in at least 120 journals cited on MEDLINE on which to base clinical decisions.