Bacterial Community Modifies Host Genetics Effect on Early Childhood Caries.

By age 5, approximately one-fifth of children have early childhood caries (ECC). Both the oral microbiome and host genetics are thought to influence susceptibility. Whether the oral microbiome modifies genetic susceptibility to ECC has not been tested. We test whether the salivary bacteriome modifies the association of a polygenic score (PGS, a score derived from genomic data that summarizes genetic susceptibility to disease) for primary tooth decay on ECC in the Center for Oral Health Research in Appalachia 2 longitudinal birth cohort. Children were genotyped using the Illumina Multi-Ethnic Genotyping Array and underwent annual dental examinations. We constructed a PGS for primary tooth decay using weights from an independent, genome-wide association meta-analysis. Using Poisson regression, we tested for associations between the PGS (high versus low) and ECC incidence, adjusting for demographic characteristics (n = 783). An incidence-density sampled subset of the cohort (n = 138) had salivary bacteriome data at 24 mo of age. We tested for effect modification of the PGS on ECC case status by salivary bacterial community state type (CST). By 60 mo, 20.69% of children had ECC. High PGS was not associated with an increased rate of ECC (incidence rate ratio, 1.09; 95% confidence interval [CI], 0.83-1.42). However, having a cariogenic salivary bacterial CST at 24 mo was associated with ECC (odds ratio [OR], 7.48; 95% CI, 3.06-18.26), which was robust to PGS adjustment. An interaction existed between the salivary bacterial CST and the PGS on the multiplicative scale (P = 0.04). The PGS was associated with ECC (OR, 4.83; 95% CI, 1.29-18.17) only among individuals with a noncariogenic salivary bacterial CST (n = 70). Genetic causes of caries may be harder to detect when not accounting for cariogenic oral microbiomes. As certain salivary bacterial CSTs increased ECC risk across genetic risk strata, preventing colonization of cariogenic microbiomes would be universally beneficial.

Plaque Microbiome in Caries-Active and Caries-Free Teeth by Dentition.

OBJECTIVE: Describe associations between dental caries and dental plaque microbiome, by dentition and family membership. METHODS: This cross-sectional analysis included 584 participants in the Center for Oral Health Research in Appalachia Cohort 1 (COHRA1). We sequenced the 16S ribosomal RNA gene (V4 region) of frozen supragingival plaque, collected 10 y prior, from 185 caries-active (enamel and dentinal) and 565 caries-free (no lesions) teeth using the Illumina MiSeq platform. Sequences were filtered using the R DADA2 package and assigned taxonomy using the Human Oral Microbiome Database. RESULTS: Microbiomes of caries-active and caries-free teeth were most similar in primary dentition and least similar in permanent dentition, but caries-active teeth were significantly less diverse than caries-free teeth in all dentition types. Streptococcus mutans had greater relative abundance in caries-active than caries-free teeth in all dentition types (P < 0.01), as did Veillonella dispar in primary and mixed dentition (P < 0.01). Fusobacterium sp. HMT 203 had significantly higher relative abundance in caries-free than caries-active teeth in all dentition types (P < 0.01). In a linear mixed model adjusted for confounders, the relative abundance of S. mutans was significantly greater in plaque from caries-active than caries-free teeth (P < 0.001), and the relative abundance of Fusobacterium sp. HMT 203 was significantly lower in plaque from caries-active than caries-free teeth (P < 0.001). Adding an effect for family improved model fit for Fusobacterium sp. HMT 203 but notS. mutans. CONCLUSIONS: The diversity of supragingival plaque composition from caries-active and caries-free teeth changed with dentition, but S. mutans was positively and Fusobacterium sp. HMT 203 was negatively associated with caries regardless of dentition. There was a strong effect of family on the associations of Fusobacterium sp. HMT 203 with the caries-free state, but this was not true for S. mutans and the caries-active state. KNOWLEDGE TRANSFER STATEMENT: Patients' and dentists' concerns about transmission of bacteria within families causing caries should be tempered by the evidence that some shared bacteria may contribute to good oral health.

Novel de novo synthesized phosphate carrier compound ABA-PEG20k-Pi20 suppresses collagenase production in Enterococcus faecalis and prevents colonic anastomotic leak in an experimental model.

BACKGROUND: Previous work has demonstrated that anastomotic leak can be caused by collagenolytic bacteria such as Enterococcus faecalis via an effect on wound collagen. In humans, E. faecalis is the organism cultured most commonly from a leaking anastomosis, and is not routinely eliminated by standard oral or intravenous antibiotics. Novel strategies are needed to contain the virulence of this pathogen when present on anastomotic tissues. METHODS: Polyphosphorylated polymer ABA-PEG20k-Pi20 was tested in mice for its ability to prevent anastomotic leak caused by collagenolytic E. faecalis. The study design included a distal colonic resection and anastomosis followed by introduction of E. faecalis to anastomotic tissues via enema. Mice were assigned randomly to receive either ABA-PEG20-Pi20 or its unphosphorylated precursor ABA-PEG20k in their drinking water. The development of anastomotic leak was determined after the animals had been killed. RESULTS: Overnight incubation of two different E. faecalis collagenolytic strains with 2 mmol/l of ABA-PEG20k-Pi20 led to near complete inhibition of collagenase production (from 21 000 to 1000 and from 68 000 to 5000 units; P < 0·001; 6 samples per group) without suppressing bacterial growth. In mice drinking 1 per cent ABA-PEG20k-Pi20, the phosphate concentration in the distal colonic mucosa increased twofold and leak rates decreased from eight of 15 to three of 15 animals (P < 0·001). In mice drinking ABA-PEG20k-Pi20, the percentage of collagenolytic colonies among E. faecalis populations present at anastomotic tissue sites was decreased by 6-4800-fold (P = 0·008; 5 animals). CONCLUSION: These data indicate that oral intake of ABA-PEG20k-Pi20 may be an effective agent to contain the virulence of E. faecalis and may prevent anastomotic leak caused by this organism. Clinical relevance Progress in understanding the pathogenesis of anastomotic leak continues to point to intestinal bacteria as key causative agents. The presence of pathogens such as Enterococcus faecalis that predominate on anastomotic tissues despite antibiotic use, coupled with their ability to produce collagenase, appears to alter the process of healing that leads to leakage. Further antibiotic administration may seem logical, but carries the unwanted risk of eliminating the normal microbiome, which functions competitively to exclude and suppress the virulence of pathogens such as E. faecalis. Therefore, non-antibiotic strategies that can suppress the production of collagenase by E. faecalis without affecting its growth, or potentially normal beneficial microbiota, may have unique advantages. The findings of this study demonstrate that drinking a phosphate-based polymer can achieve the goal of preventing anastomotic leak by suppressing collagenase production in E. faecalis without affecting its growth.

Association of localized Ca2+ gradients with redistribution of glycoprotein IIb-IIIa and F-actin in activated human blood platelets.

We monitored the intracellular distribution of ionized free Ca2+ concentration ([Ca2+]i) in individual human platelets by digital imaging fluorescence microscopy with fura 2 during platelet activation induced by surface contact or a soluble platelet agonist (thrombin). Contact of platelets with glass resulted in pseudopod formation and spreading, accompanied by a nonuniform rise in [Ca2+]i. The rise in [Ca2+]i was maximal during pseudopod formation. Locally elevated [Ca2+]i was frequently found in pseudopodia and at the edge and core of spread platelets. This pattern was faithfully duplicated by the local pattern of distribution of the cytoskeletal components F-actin, gelsolin, and surface glycoproteins (GP) IIb-IIIa but not by calmodulin. Platelets stimulated by thrombin also showed an inhomogeneous rise in [Ca2+]i, which was well correlated with the staining of F-actin and GPIIb-IIIa. Cytochalasin D, an inhibitor of actin polymerization, inhibited the inhomogeneous increase or redistribution of F-actin and GPIIb-IIIa but did not inhibit the rise in mean [Ca2+]i. These observations suggest that a localized change in [Ca2+]i may be associated with cytoskeletal reorganization and redistribution of GPIIb-IIIa in activated platelets.

TMB-8 and dibucaine induce tyrosine phosphorylation and dephosphorylation of a common set of proteins in platelets.

Dibucaine and 8-(N,N-diethylamino)octyl 3,4,5-trimethoxybenzoate (TMB-8), which are local anesthetics, affect diverse functions of many cell types. For example, platelet aggregation is inhibited by both, and both cause changes in platelet morphology and structure. Little is known of the mechanisms. We found that both dibucaine (0.125-0.5 mM) and TMB-8 (0.25-1.0 mM) induced rapid tyrosine phosphorylation of several platelet proteins (160, 70-75, and 40 kDa) and dephosphorylation of a 62- to 64-kDa protein detectable by a specific antiphosphotyrosine monoclonal antibody (4G10). Platelet aggregation induced by alpha-thrombin (10 nM) was inhibited by the local anesthetics in approximately the same dose range. Neither dibucaine nor TMB-8 induced activation of protein kinase C (PKC) or myosin light-chain kinase. Their activation was not essential for tyrosine phosphorylation induced by local anesthetics. However, an increase in tyrosine phosphorylation of several proteins (95-130 kDa) induced by alpha-thrombin (10 nM) was inhibited by dibucaine (0.5 mM) or TMB-8 (0.5 mM). Furthermore, when dibucaine (0.5 mM) was added 1 min after addition of alpha-thrombin (10 nM), disaggregation was paralleled to dephosphorylation of many proteins, including those mentioned. Tyrosine phosphorylation and dephosphorylation of specific proteins may account for some of the diverse effects of local anesthetics on platelets and other cells. Addition of TMB-8 (0.5 mM) or dibucaine (0.5 mM) also inhibited activation of PKC, induced by alpha-thrombin (10nM), suggesting that some of the inhibitory effects of dibucaine or TMB-8 may be due to inhibitory effects of local anesthetics on PKC.

Spatial distribution and temporal change in cytosolic pH and [Ca2+] in resting and activated single human platelets.

In human platelets, a rapid rise in cytoplasmic Ca2+ and slower rise in cytoplasmic pH follow stimulation by thrombin. With the Ca2+ probe Fura-2 and the pH probe SNARF-1 for digitized fluorescence microscopy, we studied simultaneously the distribution and changes with time of [pH]i and [Ca2+]i in individual human platelets. In platelets coloaded with both probes, the probes had no detectable fluorescence at each other's excitation wavelength. The monovalent cation ionophore, nigericin (2 microM), produced a homogeneous rise in pH but no change in [Ca2+]. Platelets, in contact with glass, spread and developed an irregular, apparently mutually independent rise in both [Ca2+] and pH. Stimulation of platelets by thrombin 1.0 U/ml elevated [Ca2+]i and produced slow alkalinization without initial acidification. Replacement of extracellular Na+ by choline abolished thrombin-induced alkalinization, but had no effect on thrombin-induced [Ca2+]i elevation. ADP 10 microM caused a rapid rise of [Ca2+]i and transient alkalinization. Most stimulated platelets developed a gradient in pH, that was highest in the center. ADP and thrombin caused oscillation of [Ca2+]i but not of [pH]i. We conclude that alkalinization in stimulated platelets, presumably involving Na+/H+ antiport, is not essential for the rise of [Ca2+]i that may accompany it.

c-myb affects intracellular calcium handling in vascular smooth muscle cells.

The protooncogene c-myb is responsible for elevating intracellular calcium concentration ([Ca2+]i) at the G1/S interface in vascular smooth muscle cells (VSMC). However, the molecular components of this pathway are undefined, and the biological effects of increased levels of divalent cation are unknown. We have demonstrated that growth-arrested c-myb-transfected VSMC, compared with wild type VSMC, exhibit a fourfold increased number of insulin-like growth factor I (IGF-I) receptors, increased amount of secreted IGF-I activity, and a twofold increased level of [Ca2+]. The c-myb transfected cells, compared with wild type cells, also possess a twofold increased rate of calcium influx and a twofold decreased rate of calcium efflux. The elevated calcium influx rate of transfected cells is decreased to that of wild type cells with IGF-I neutralizing antibody, whereas the decreased calcium efflux rate of transfected cells is increased to that of wild type cells with antisense c-myb oligonucleotides. Proliferating wild type VSMC exhibit an increased calcium influx rate in late G1, which is dependent on production of augmented amounts of IGF-I activity but not increased levels of IGF-I receptors. The wild type VSMC also show a decreased calcium efflux rate at the same point in the cell cycle, which is dependent on expression of c-myb. The treatment of wild type cells with antisense c-myb or IGF-I receptor oligonucleotides induces a late G1 block in cell proliferation, which can be overcome by exposure to the calcium ionophore, 4-bromo-A-27318, in amounts sufficient to raise [Ca2+]i to levels observed at the G1/S interface. We conclude that IGF-I/IGF-I receptors and c-myb are involved in control of [Ca2+]i at the G1/S interface by separately regulating the rates of calcium influx and efflux and that elevated levels of divalent cation are necessary for progression of VSMC into the S phase of the cell cycle.

Participation of calpain in protein-tyrosine phosphorylation and dephosphorylation in human blood platelets.

The possible role of calpains in protein-tyrosine phosphorylation in platelets was examined by the use of the cell-permeant calpain inhibitor calpeptin. In platelets stimulated by 1 U/mL thrombin, protein-tyrosine phosphorylation was maximal after 2 minutes and was followed by protein-tyrosine dephosphorylation. Calpeptin (30 mumol/L) or vanadate (2 mmol/L) enhanced protein-tyrosine phosphorylation and delayed protein-tyrosine dephosphorylation. The effects of these two compounds were not additive. We also observed proteolysis of pp60src and autoproteolysis of mu-calpain. Cleavage of the former was significantly slower than that of the latter and slower than protein-tyrosine dephosphorylation. The activity of protein-tyrosine phosphatase in the platelet lysate was transiently increased to 190% by addition of Ca2+. Ca(2+)-dependent activation of protein-tyrosine phosphatase was not observed in the presence of leupeptin. Those observations suggest that platelet calpains may be involved in modulation of protein-tyrosine phosphorylation through activation of protein-tyrosine phosphatase rather than through the inactivation of pp60src, a mechanism that was previously suggested.

Calpain-catalyzed cleavage and subcellular relocation of protein phosphotyrosine phosphatase 1B (PTP-1B) in human platelets.

The non-transmembrane phosphotyrosine phosphatase 1B (PTP-1B) is an abundant enzyme, normally localized to the cytosolic face of the endoplasmic reticulum via a C-terminal targeting sequence. We have found that agonist-induced platelet activation results in proteolytic cleavage of PTP-1B at a site upstream from this targeting sequence, causing subcellular relocation of its catalytic domain from membranes to the cytosol. PTP-1B cleavage is catalyzed by the calcium-dependent neutral protease calpain and is a general feature of platelet agonist-induced aggregation. Moreover, PTP-1B cleavage correlates with the transition from reversible to irreversible platelet aggregation in platelet-rich plasma. Engagement of gpIIb-IIIa is necessary for inducing PTP-1B cleavage, suggesting that integrins regulate tyrosine phosphatases as well as tyrosine kinases. PTP-1B cleavage is accompanied by a 2-fold stimulation of its enzymatic activity, as measured by immune complex phosphatase assay, and correlates with discrete changes in the pattern of tyrosyl phosphorylation. Cleavage and subcellular relocation of PTP-1B represents a novel mechanism for altering tyrosyl phosphorylation that may have important physiological implications in cell types other than platelets.

pp60src is an endogenous substrate for calpain in human blood platelets.

Calpain is distributed ubiquitously in virtually every tissue (Croall, D. E., and DeMartino, G. N. (1991) Physiol. Rev. 71, 813-846), but its physiological role remains to be determined. The identification of its natural endogenous substrates would be of great interest. Since pp60src, a major tyrosine kinase in platelets, is known to be easily cleaved during purification from cells (Feder, D., and Bishop, J. M. (1990) J. Biol. Chem. 265, 8205-8211), we examined the possibility that it is an endogenous substrate of calpain. In the whole cell lysate from resting platelets, which was analyzed by Western blotting with monoclonal antibody 327, we found pp60src almost exclusively in a 60-kDa form, with a trace of 52-kDa form. Addition of A23187 (a calcium ionophore) or dibucaine, which are known to be activators of platelet calpain (Croall and DeMartino, 1991; Fox, J. E., Reynolds, C., Morrow, J. S., and Phillips, D. R. (1987) Blood 76, 2510-2519; Fox, J. E., Austin, C. D., Boyles, J. K., and Steffen, P. K. (1990b) J. Cell Biol. 111, 483-493), caused dose- and time-dependent cleavage of actin-binding protein and p235 protein (talin). At the same time, loss of the 60-kDa species of pp60src and generation of the 52-kDa (occasionally seen as doublets) and 47-kDa species were detected by the Western blotting. In platelets aggregated by 1 unit/ml thrombin, apparently identical cleavage products were found. The cleavage of pp60src was inhibited by calpeptin (20 microM), an inhibitor of calpain (Tsujinaka, T., Kajiwara, Y., Kambayashi, J., Sakon, M., Higuchi, N., Tanaka, T., and Mori, T. (1988) Biochem. Biophys. Res. Commun. 153, 1201-1208; Tsujinaka, T., Ariyoshi, H., Uemura, Y., Sakon, M., Kambayashi, J., and Mori, T. (1990) Life Sci. 46, 1059-1066; Fox, J. E., Clifford, C. C., and Austin, C. D. (1990) Blood 76, 2510-2519; Fox, J. E., Austin, C. D., Boyles, J. K., and Steffen, P. K. (1990) J. Cell. Biol. 111, 483-493; Fox, J. E., Austin, C. D., Clifford, C. C., and Steffen, P. K. (1991) J. Biol. Chem. 266, 13289-13295). Addition of EGTA (3 mM) to the extracellular media completely inhibited the cleavage of actin-binding protein, talin, and pp60src in response to A23187 (1 microM). Intact pp60src was distributed in both cytosolic and particulate (membrane) fractions. Cleaved species were found exclusively in the cytosolic fraction. pp60src-associated enolase kinase activity was reduced. Thus, pp60src is an endogenous substrate for calpain, the cleavage of which may have regulatory effects on the kinase.

Membrane glycoproteins and platelet cytoskeleton in immune complex-induced platelet activation.

To clarify the mechanism of platelet activation by immune complexes and the possible involvement of surface glycoproteins (GPs), we studied platelet activation induced by heat-aggregated IgG (HAG). We examined the effects of monoclonal antibodies (MoAbs) against GPIb, GPIIb/IIIa, and the Fc receptor on resting platelets and on platelets stimulated by HAG. HAG increased the cytosolic ionized calcium concentration ([Ca2+]i) and stimulated protein (P47 and P20) phosphorylation, phosphatidic acid (PA) synthesis, serotonin secretion, and platelet aggregation. IV.3, an anti-Fc gamma RII receptor MoAb, inhibited HAG binding to platelets and all subsequent platelet responses. Like IV.3, MoAbs against GPIIb/IIIa (Tab, 10E5, AP-3) or GPIb (AP-1, 6D1) strongly inhibited platelet activation by HAG. However, while anti-GPIIb/IIIa MoAbs inhibited binding of IV.3 and HAG to platelets, anti-GPIb MoAbs had little effect on platelet binding of IV.3 or HAG. These observations suggest a close topographical and functional association of GPIIb/IIIa with Fc gamma RII in the platelet response to HAG. Cytochalasin B, an inhibitor of actin polymerization, also inhibited platelet activation but not HAG or IV.3 binding. Measurement of the fluorescence of 7-nitrobenz-2-oxa-1,3-(NBD)-phallacidin, a specific marker for filamentous actin (F-actin), showed that both cytochalasin B and AP-1 blocked the increase of F-actin induced by HAG. The common effects of anti-GPIb MoAbs and of cytochalasin B suggest that unlike the activity of GPIIb/IIIa, the ability of anti-GPIb to inhibit the activation of platelets by immune complexes is associated with perturbation of the cytoskeleton.

Adventures in hemostasis. Desmopressin in cardiac surgery.

Desmopressin acetate (1-deamino-8-D-arginine vasopressin [DDAVP]) improves hemostasis in hemophilia A and von Willebrand's disease and in some platelet disorders. In complex cardiac operations, excluding simple coronary artery bypass graft procedures, we found that desmopressin reduced blood loss by 40% and the need for transfusion by 34%. Conflicting reports followed. Future trials should emphasize patients with excessive bleeding. A possible post-desmopressin prothrombotic state was studied after hip replacement surgery. The incidence of deep vein thrombosis associated with warfarin sodium therapy was the same as that associated with desmopressin plus warfarin therapy. No desmopressin-induced thrombotic tendency was detected. A trend toward reduced blood loss with desmopressin was not significant. During cardiac catheterization, the plasma von Willebrand factor level was correlated with hemodynamic variables, including pulmonary vascular resistance, pulmonary arterial pressure, and (inversely) with cardiac index. von Willebrand factor concentration was highest in mitral stenosis. The relationship of these factors to the response to desmopressin remains to be defined.

Association of pp60src with Triton X-100-insoluble residue in human blood platelets requires platelet aggregation and actin polymerization.

Protein-tyrosine phosphorylation during platelet activation is inhibited under conditions that inhibit platelet binding of fibrinogen and aggregation. We suggested that pp60src, a major platelet tyrosine kinase, or its protein substrates might become associated with the cytoskeleton upon platelet stimulation, and that this might be related to aggregation. By Western blotting with an anti-Src monoclonal antibody, we found time-dependent association of pp60src with the cytoskeleton (10,000 x g Triton X-100-insoluble matrix) but not the "membrane" cytoskeleton (100,000 x g Triton X-100-insoluble matrix) in platelets activated by U46619 (PGH2 analog). Cytoskeletal association and platelet aggregation were inhibited by the peptide Arg-Gly-Asp-Ser (RGDS) (but not by Arg-Gly-Glu-Ser (RGES)), by 10E5 antibody against glycoprotein (Gp) IIb/IIIa, and by EGTA. U46619-induced association of pp60src with cytoskeleton but not secretion or aggregation was inhibited by cytochalasin D (2 microM). Both cytochalasin D and RGDS inhibited "slow" tyrosine phosphorylation of platelet proteins. Association of pp60src with cytoskeleton induced by U46619 or ADP was not blocked by aspirin. Aspirin blocked epinephrine-induced association of pp60src with the cytoskeleton during a second phase of aggregation when an initial phase had occurred without shape change or secretion. Association of GpIIb/IIIa with the cytoskeleton also accompanied platelet aggregation, shape change, and actin polymerization; this was shown with anti-GpIIb and anti-GpIIIa antibodies. Association of pp60src and GpIIb/IIIa with the cytoskeleton and slow tyrosine phosphorylation are related phenomena.