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Biomedical subjects

R R Arnold

Publications and source records attributed to R R Arnold.

At least 19 recordsLinked to original sources

Influence of immunization on Porphyromonas gingivalis colonization and invasion in the mouse chamber model.

The effects of immunization with invasive or noninvasive Porphyromonas (Bacteroides) gingivalis strains on the pathogenesis of infection in a mouse chamber model were examined. BALB/c mice were immunized by a single injection of heat-killed P. gingivalis invasive strain A7436 or W83 or noninvasive strain 33277, HG405, or 381 directly into subcutaneous chambers. P. gingivalis-specific antibody was detected in chamber fluid 21 days postimmunization, and mice were subsequently challenged by injection of exponential-phase P. gingivalis into chambers. Immunization with A7436 or W83 followed by challenge with A7436 protected mice against secondary abscess formation and death; however, P. gingivalis persisted in chambers for up to 14 days postchallenge. Immunization with noninvasive strain 33277, HG405, or 381 followed by challenge with invasive strain A7436 or W83 protected mice against secondary lesion formation and death. P. gingivalis was cultured from 33277- or HG405-immunized and nonimmunized animals to day 14. All P. gingivalis strains induced an immunoglobulin G response, as measured by an enzyme-linked immunosorbent assay and Western immunoblotting of P. gingivalis whole-cell and outer membrane protein preparations. Western blot analyses indicated that sera from mice immunized with different invasive and noninvasive strains recognized common P. gingivalis antigens. In summary, immunization with invasive P. gingivalis A7436 and W83 or noninvasive P. gingivalis 33277, HG405, and 381 protected mice from secondary lesion formation and death after challenge with invasive P. gingivalis A7436 or W83. P. gingivalis-specific antibody did not, however, inhibit the colonization of P. gingivalis within chambers.

Animals

Functional differences in human neutrophils isolated pre- and post-prandially.

Activated polymorphonuclear leukocytes have been associated with neoplasia, atherogenesis and reperfusion injury. Since some of these conditions are also correlated with dietary fat, we examined the functional characteristics of leukocytes isolated from subjects before and after consumption of a lipid-rich meal. There was up to 2-fold greater superoxide generation in response to agonists in leukocytes obtained post-prandially; the maximum increase was observed about 4 h after eating and followed the peak (2-4 h) in serum triglycerides. Neutrophils isolated post-prandially also exhibited impaired chemotaxis and defective bacterial killing, but normal phagocytosis. These findings provide a new variable that should be considered in studies of leukocytes.

Bacteroides

A novel mouse model to study the virulence of and host response to Porphyromonas (Bacteroides) gingivalis.

We describe here the development of a mouse subcutaneous chamber model that allows for the examination of host-parasite interactions as well as the determination of gross pathology with Porphyromonas (Bacteroides) gingivalis challenge. When inoculated into stainless-steel chambers implanted subcutaneously in female BALB/c mice, P. gingivalis W83, W50, and A7436 (10(8) to 10(10) CFU) caused cachexia, ruffling, general erythema and phlegmonous, ulcerated, necrotic lesions, and death. P. gingivalis W50/BEI, HG405, and 33277 (10(10) CFU) produced localized abscesses in the mouse chamber model with rejection of chambers at the injection site. Analysis of chamber fluid from 33277-, HG405-, and W50/BEI-infected mice by cytocentrifugation revealed inflammatory cell debris, polymorphonuclear leukocytes, and high numbers of dead bacteria. In contrast, fluid from A7436-, W50-, and W83-infected mice revealed infiltration predominantly of polymorphonuclear leukocytes and live bacteria. Bacteria were found primarily associated with polymorphonuclear leukocytes in the fluid from W50-, HG405-, and W83-infected mice but not from A7436-infected mice. Viable isolates were recoverable from the chamber fluid through day 3 for W50/BEI, day 5 for 33277, day 6 for HG405, day 7 for W50, day 14 for W83, and day 26 for A7436. All strains induced a systemic immunoglobulin G response in serum and chamber fluid samples. The use of this model will allow us to examine the virulence of P. gingivalis as defined by the interaction of host response to localized infection with P. gingivalis.

Animals

Phagocytosis of virulent Porphyromonas gingivalis by human polymorphonuclear leukocytes requires specific immunoglobulin G.

No studies to date clearly define the interactions between Porphyromonas gingivalis and human peripheral blood polymorphonuclear leukocytes (PMN), nor has a protective role for antibody to P. gingivalis been defined. Using a fluorochrome phagocytosis microassay, we investigated PMN phagocytosis and killing of P. gingivalis as a function of P. gingivalis-specific antibody. Sera from a nonimmune rabbit and a healthy human subject were not opsonic for virulent P. gingivalis A7436, W83, and HG405; phagocytosis of these strains (but not 33277) required opsonization with hyperimmune antiserum (RaPg). Diluting RaPg with a constant complement source decreased proportionally the number of P. gingivalis A7436 cells phagocytosed per phagocytic PMN. Enriching for the immunoglobulin G fraction of RAPg A7436 enriched for opsonic activity toward A7436. An opsonic evaluation of 18 serum samples from adult periodontitis patients revealed that only 3 adult periodontitis sera of 17 with elevated immunoglobulin G to P. gingivalis A7436 were opsonic for A7436 and, moreover, that the serum sample with the highest enzyme-linked immunosorbent assay titer was most opsonic (patient 1). However, the opsonic activity of serum from patient 1 was qualitatively and not just quantitatively different from that of the nonopsonic human sera (but was less effective opsonin than RaPg). Strain variability was observed in resistance of P. gingivalis to phagocytosis, and opsonization was strain specific for some, but not all, strains tested. An evaluation of killing of A7436 revealed that serum killing and extracellular killing of P. gingivalis were less effective alone when compared with intracellular PMN killing alone.

Adult

Antibody-dependent alternate pathway of complement activation in opsonophagocytosis of Porphyromonas gingivalis.

It has been suggested that the ability of Porphyromonas gingivalis to proteolyse complement, as well as its production of a capsule, contributes to resistance to phagocytosis by polymorphonuclear leukocytes. In this report, the opsonic role of serum complement and its activation pathways were investigated, using individual sera heat treated or depleted of factors B, C2, and C1q and the divalent cations Mg2+ and Ca2+. A fluorochrome microassay was used to quantitate phagocytosis of P. gingivalis A7436 by human polymorphonuclear leukocytes. Heat treatment of rabbit antiserum to P. gingivalis (RaPg) (56 degrees C, 30 min) resulted in a reduction in phagocytosis from 100% to 55% +/- 5%, while heat treatment of chronic adult periodontal disease serum abrogated phagocytosis. The heat-labile activity of RaPg was fully restored with MgEGTA-chelated rabbit serum but not EDTA- or EGTA-chelated rabbit serum. The addition of serum depleted of factor B but not C2 or C1q restored most of the heat-labile activity; however, the factor B-depleted serum was suspect, due to low-level opsonization of zymosan (inhibitable by EDTA but not MgEGTA). Adding C1q at 80 micrograms/ml to serum depleted of C1q restored much but not all of the activity lost through heat treatment or through depletion of C1q. A large part of opsonic activity with C2- and C1q-depleted sera was enhanced by the addition of 4 x 10(-3) M Mg2+. The data indicate that although opsonophagocytosis of P. gingivalis A7436 is dependent on the classical complement pathway, a significant contribution is made by an antibody-dependent alternate pathway.

Animals

Defective neutrophil function in an insulin-dependent diabetes mellitus patients. A case report.

The objective of this study was to evaluate the polymorphonuclear leukocyte (PMN) function in a poorly controlled adult insulin-dependent diabetic patient (IDDM) with severe recurrent periodontitis, while describing the microbiological and clinical findings. Chemotaxis, superoxide production, and phagocytosis and killing of Porphyromonas (Bacteroides) gingivalis by the IDDM PMN were evaluated 1 week before treatment relative to a healthy, matched control. These analyses revealed a significant (P less than .05) depression in the number of IDDM PMNs migrating along an FMLP gradient (Boyden chamber assay). In addition, a significant (P less than .05) enhancement of IDDM PMN superoxide production in response to opsonized zymosan (cytochrome C reduction) was observed. Phagocytosis and killing (fluorochrome phagocytosis assay) by IDDM PMN of two P. gingivalis strains was also impaired significantly (P less than .05). The subgingival microflora contained significant levels of P. gingivalis, Actinobacillus actinomycetemcomitans, Eikenella corrodens, and Peptostreptococcus micros. Periodontal treatment consisted of extraction of hopeless teeth, scaling and root planing and 3 weeks of Augmentin therapy. The antibiotic therapy resulted in unrecoverable numbers of the putative pathogens and a reduction in both gingival inflammation and disease progression. The IDDM healing response to previous surgical treatment and extractions was poor, presumably due to a marked thrombocytopenia (91 x 10(3) platelets/mm3).

Bacteroides

Bactericidal effect of lactoferrin on Legionella pneumophila: effect of the physiological state of the organism.

Lactoferrin has been previously shown to be bactericidal for Legionella pneumophila. The current study showed that CaCl2, Mg(NO3)2, and MgCl2, but not NaCl, blocked killing. Activity was pH dependent with the greatest activity at 5.0. Sensitivity of the organism was dramatically affected by the growth conditions. Log phase 12 h, broth-grown cells were most sensitive, with older cultures becoming more resistant. Plate-grown cells were completely resistant. Lactoferrin binding, as detected by immunofluorescence microscopy, was temperature dependent (no binding at 4 degrees C), but was independent of killing.

Calcium Chloride

Superior leukocyte separation with a discontinuous one-step Ficoll-Hypaque gradient for the isolation of human neutrophils.

A discontinuous gradient system composed of two commercially available Ficoll-Hypaque mixtures (Mono-Poly resolving medium (MPRM) and Histopaque 1.077) is described for the purification of mononuclear and polymorphonuclear leukocytes from human blood. Like the original one-step Hypaque-Ficoll procedure (Ferrante and Thong, 1978), a single centrifugation at 500 X g for 30 min resulted in the formation of two distinct leukocyte fractions. In contrast to MPRM alone, the discontinuous system (MPRM-HP) was capable of resolving leukocyte fractions from blood volumes as small as 1 ml with excellent purity and yield. MPRM-HP was also compatible with a wider range of anticoagulants and permitted fractionation of specimens resistant to MPRM.

Adult

Killing of Actinobacillus actinomycetemcomitans by human lactoferrin.

Actinobacillus actinomycetemcomitans is a fastidious, facultative gram-negative rod associated with endocarditis, certain forms of periodontal disease, and other focal infections. Human neutrophils have demonstrated bactericidal activity against A. actinomycetemcomitans, and much of the oxygen-dependent killing has been attributed to the myeloperoxidase-H2O2-halide system. However, the contribution of other neutrophil components to killing activity is obscure. Lactoferrin, an iron-binding glycoprotein, is a major constituent of neutrophil-specific granules and is also found in mucosal secretions. In this report, we show that human lactoferrin is bactericidal for A. actinomycetemcomitans. Killing activity required an unsaturated (iron- and anion-free) molecule that produced a 2-log decrease in viability within 120 min at 37 degrees C at a concentration of 1.9 microM. Besides exhibiting concentration dependence, killing kinetics were affected by minor variations in temperature and pH. Magnesium, a divalent cation thought to stabilize lipopolysaccharide interactions on the surface of gram-negative organisms, enhanced lactoferrin killing of A. actinomycetemcomitans, while other cations, such as potassium and calcium, had no effect. Our data suggest that lactoferrin contributes to killing of A. actinomycetemcomitans by human neutrophils and that it may also play a significant role in innate secretory defense against this potential periodontopathogen.

Actinobacillus

Protein kinase C inhibition by sphingoid long-chain bases: effects on secretion in human neutrophils.

Sphingoid long-chain bases (sphinganine and sphingosine) have recently been shown to inhibit protein kinase C both in vitro [Y. Hannun et al. (1986) J. Biol. Chem. 261, 12604-12609] and in intact human neutrophils, in which they block activation of the superoxide-generating respiratory burst [E. Wilson et al. (1986) J. Biol. Chem. 261, 12616-12623]. In the present study we have used sphingosine to investigate the pathways for agonist-induced secretion of neutrophil granule contents. Induction of secretion of the specific granule component lactoferrin by a variety of agonists [phorbol 12-myristate-13-acetate (PMA), formyl-methionyl-leucyl-phenylalanine (fMLP), and calcium ionophore A23187] was completely inhibited by sphingosine with an ED50 of 6 to 10 microM. PMA-induced secretion of lysozyme (present in both the azurophilic and specific granules) was completely blocked with an ED50 of 10 microM, whereas fMLP-induced secretion was only about 50% inhibited. Secretion of the azurophilic granule proteins beta-glucuronidase and myeloperoxidase was activated by fMLP and A23187, but not by PMA, and was not affected by sphingosine. The use of A23187 in the presence of sphingosine allowed differentiation between calcium activation of protein kinase C-dependent versus-independent pathways. The effect of sphingosine was not mediated by neutralizing intracellular acidic compartments, since treatment of neutrophils with inhibitory concentrations of sphingosine did not significantly alter the uptake of labeled methylamine. We conclude that at least two mechanisms participate in the regulation of specific and azurophilic granule secretion, respectively: a protein kinase C-dependent pathway and a calcium-dependent pathway which does not involve protein kinase C.

Calcimycin

Separation of human IgA1 and IgA2 using jacalin-agarose chromatography.

A lectin isolated from the tropical jackfruit, jacalin, previously reported to precipitate human immunoglobulin A (IgA), and conjugated to agarose was used to separate the two subclasses of IgA from secretions. Jacalin-agarose binds specifically to the D-galactose moiety of IgA1 but not to IgA2 which has a different carbohydrate content and structure. IgA2 passed through the jacalin-agarose column and was collected in the void volume. IgA1 was eluted from the lectin by 0.8 M galactose. Of a representative diluted anti-alpha chain-purified colostral IgA preparation containing 50.2 micrograms IgA1 and 55.8 micrograms IgA2, 40.3 micrograms IgA1 (80.3% of the original) and 49.6 micrograms IgA2 (88.9%) was collected following jacalin-agarose chromatography. The jacalin-purified IgA1 fraction contained 8.0% IgA2 and the IgA2 fraction contained no IgA1. In addition, the IgA1 and IgA2 fractions had naturally occurring antibody activity to a normal oral bacterium. The method is easy, reproducible and specific and has many applications to mucosal immunological investigations.

Chromatography, Agarose

Differentiation and interaction of secretory immunoglobulin A and a calcium-dependent parotid agglutinin for several bacterial strains.

Previous studies have suggested that both secretory immunoglobulin A (sIgA) and various nonimmunoglobulin salivary glycoproteins are capable of agglutinating a variety of bacteria. The present study was designed to compare the nature of the agglutinins for Streptococcus mutans and Salmonella typhimurium in parotid saliva and colostrum. S. mutans was aggregated by saliva and colostrum, whereas S. typhimurium was aggregated only by saliva as detected by a spectrophotometric method. The principal salivary agglutinin for both S. mutans and S. typhimurium was calcium dependent and could be desorbed in phosphate-buffered saline (pH 6.8). In contrast, the colostral agglutinin was calcium independent and not readily desorbed. The agglutinin activities of saliva and colostrum for S. mutans were additive, suggesting independent target sites on the bacterial surface. The agglutinin activity of colostrum was totally associated with sIgA as was suggested by blocking of the agglutinating activity with anti-alpha-chain serum and the absence of blocking with an antibody specific for salivary agglutinin. Interestingly, anti-alpha-chain serum removed all agglutinating activity from saliva, but not from the phosphate-buffered saline-desorbed agglutinin. Dialysis of parotid saliva against 0.1 M disodium EDTA eliminated the agglutinin blocking activity of anti-alpha-chain serum but not that of the antiagglutinin antibody. The ability of anti-alpha-chain serum to block agglutination of the EDTA-dialyzed saliva could be restored by the addition of calcium chloride, suggesting that sIgA and salivary agglutinin are associated through a calcium-mediated interaction. These results indicate that bacterial agglutinating activity of colostrum, as detected spectrophotometrically, is mediated by sIgA, and that of saliva is mainly dependent upon a calcium-dependent nonimmunoglobulin agglutinin. The agglutinating activities of sIgA and parotid agglutinin seem to be additive, and their calcium-dependent association may favor the enhancement of their respective activities.

Agglutination

Characterization of lactoferrin interaction with Streptococcus mutans.

Lactoferrin (LF) is an iron-binding glycoprotein common to exocrine secretions and the specific granules of neutrophils. Each molecule is capable of high-affinity coordinate-binding of two ferric ions with two bicarbonate or carbonic anions. The initial aspect of the present study was directed at determining the nature of anion involvement in LF bactericidal activity. It was found that selective anions were capable of inhibiting the expression of bactericidal activity by LF on S. mutans 10449. The ability to block LF expression was directly related to the capacity of the anion to serve as a coordinate ion in iron-binding by the transferrin molecules. These data support the hypothesis that the LF target site on the bacterial surface is anionic. There has been controversy in the literature regarding LF involvement in hydroxy radical generation. The second phase of these studies indicated that treatment of S. mutans with LF under anaerobic conditions abrogated the bactericidal effect of this molecule. LF-killing could be enhanced by the presence of thiocyanate and inhibited by catalase and lactoperoxidase; however, bovine serum albumin was equally effective as an inhibitor. The apparent requirement for oxygen in LF bactericidal effect on S. mutans is not inconsistent with a hydroxy radical mechanism.

Anaerobiosis

Hydrogen peroxide inhibits glucose metabolism and collagen synthesis in bone.

Effects of H2O2 on bone were evaluated in an organ culture system. Tibiae from chick embryos were incubated for up to 3 days in culture medium containing 0.07 to 20 mM H2O2. Glucose metabolism was monitored by measuring lactate production and oxygen consumption, and collagen synthesis was determined by hydroxylation of proline. In addition to markedly inhibiting these parameters, H2O2 also decreased bone weight and alkaline phosphatase activity. Multiple exposures to H2O2 were somewhat more effective than a single exposure. Since H2O2 inhibits bone at low concentrations in vitro, the results suggest that the potential for harmful effects of H2O2 in the oral cavity should be investigated.

Animals