CLINICAL SIGNIFICANCE OF STREPTOCOCCUS MEMBERS IN DEVELOPING PERIODONTITIS

Bacteria of the genus Streptococcus are one of the most numerous and diverse representatives in the normal biocenosis of human organs and systems particularly being abundant as obligatory inhabitants of the oral cavity. All streptococci are divided into six groups: S.mitis, S.anginosus, S.salivarius, S.mutans, S.bovis and S.pyogenic groups, among which their certain number may potentially participate in the infectious process of developing periodontitis. Owing to the presence of a wide range of adhesion, invasion and colonization factors, they are capable of performing a protective function such as, e.g., colonization resistance, but they may also cause formation of a pathological process in the tooth tissues and dento-facial system. The most prominent adhesion factors are antigens I / II (Ag I / II), fibronectin, collagen, laminin, fibrinogen binding proteins, serine-rich glycoproteins, pili, protein M, proteases, C5a peptidases, and the presence of a tooth capsule. Among the complex of proteolytic enzymes, it is important to note that streptococci contain enzymes hyaluronidase and lyase, which cleave the β1,4 bond between N-acetylglucosamine and d-glucuronic acid as the components of hyaluronic acid being a part of the connective tissues. The members of the S.anginosus group are able to release chondroitin sulfatase, which destroys chondroitin sulfates as specific components in cartilage, ligaments and other connective tissue structures. The enzymes noted contribute to a deeper spread of microorganisms in host tissues. Pathological processes associated with the development of periodontitis comprise a complex problem, wherein several important elements take part, including an infectious agent, a macroorganismal response in the form of nonspecific and adaptive immunity, as well as involvement of anti-inflammatory components. A great body of studies in research literature are dedicated to describe to participation of the members within the "red, orange and green" complexes as the principal components in developing periodontitis. Whereas the "yellow" and the "purple" complex plays a more protective role by acting as antagonists while interacting with periodontopathogens, but it should not be ruled out a potential participation for some representatives, particularly S. intermedius, S. gordonii, Actinomyces odontolyticus, Actinomyces naeslundii in developing periodontal disease. Altogether, it poses a problem, which may be solved solely based on a multidisciplinary approach by inviting not only dentists and bacteriologists but also researchers of other specialties. Here we review the studies found in international and national data bases such as Scopus, Web of Science, Springer, RINC.

Protection of β2GPI Deficient Mice from Thrombosis May Reflect a Platelet Activation Defect

Introduction Antiphospholipid syndrome (APS) is an autoimmune disorder caused by "antiphospholipid" antibodies (aPL) directed against β2-glycoprotein I (β2GPI). Although β2GPI is proposed to have both anti- and procoagulant properties in vitro, its physiological role in coagulation in vivo is not well understood. Previous studies have shown that β2GPI deficient mice display impaired thrombin generation but failed to demonstrate an anticoagulant role in vivo. Recent findings from our laboratory demonstrated that β2GPI-deficient mice (APOH -/-) developed by CRISPR/Cas9 had shown delayed time to thrombosis as evidenced by prolonged clotting times in carotid artery occlusion models. However, no mechanism was identified for the delayed time to thrombosis phenotype. Methods Venous thrombosis by complete occlusion of the IVC was performed as described previously (Wrobleski et al., 2011). In brief, an abdominal incision was made on anesthetized mice to visualize IVC. The activated partial thromboplastin time (aPTT); prothrombin time (PT) and Tissue factor-induced thrombin generation time (TGT) were performed as described previously (Stavrou et al., 2014). Protein C activity was performed according to manufacturer's recommendations (Chromogenix Coamatic® Protein C, Diapharma). To assess platelet activation, platelets were isolated under resting conditions using retro-orbital blood and the final washed platelet pellet was resuspended in Tyrodes solution. Platelets were stimulated with 0.25 U/ml thrombin and incubated with 1 µL of CD62P-FITC or Oregon Green conjugated-fibrinogen for 30 minutes. Platelets were fixed with 100 μL 2% formalin, and quantification of platelet surface P-selectin and fibrinogen binding to activated platelet GPIIb/IIIa was performed by flow cytometry (Accuri Flow Cytometer, BD Biosciences). For mouse tail vein bleeding time assays, mice were anesthetized and the tail transected approximately 5 mm from the tip. The tail was then placed in a 50 ml falcon tube containing saline pre-warmed to 37°C. The time to cessation of bleeding was determined visually. All experiments were performed on 8-12 week old mice. Results aPTT and PT results for APOH +/+ and APOH -/- mice were 41.82 ± 1.174 and 41.38 ± 2.026 seconds, and 14.55 ± 2.262 and 11.30 ± 0.578 seconds, respectively (NS). After IVC ligation, thrombi in APOH -/- mice had a mean weight of 16.94 ± 1.782 mg compared to 27.69 ± 1.725 mg in APOH +/+ mice littermates (P = 0.0002, Figure A). Thrombin generation induced by either tissue factor or aPPT reagent showed peak thrombin generation at 15 min with no difference between APOH +/+ and APOH -/- mice. Likewise, no significant differences were observed in percent Protein C activity levels between APOH +/+ (8.058 ± 1.433) and APOH -/- (6.453 ± 0.924). Stimulation with 0.25 U/ml thrombin resulted in significantly greater expression of P-selectin) on platelets of APOH +/+ (161.3 ± 30.62 MFI) compared to those from APOH -/- mice (54.47 ± 9.721 MFI) (P= 0.0101;Figure 1B). Likewise, there was significantly greater fibrinogen binding to stimulated platelets from APOH +/+ (129.7 ± 32.21 MFI) compared to APOH-/- mice (35.30 ± 2.144 MFI) with P = 0.0111 (Figure 1C). Finally, consistent with platelet activation studies, tail vein bleeding times were mildly, but significantly prolonged in APOH -/- mice (192.3 ± 45.86 sec) compared to APOH+/+ mice (95.14 ± 9.582 sec) with P = 0.. Conclusion The effects of β2GPI, if any, on coagulation processes is controversial, and has not been thoroughly studied in β2GPI deficient animals. The results presented here suggest that the smaller thrombi that form in mice deficient in β2GPI following IVC occlusion may reflect a mild platelet function defect. This hypothesis is supported by diminished P-selectin expression and fibrinogen binding in response to 0.25 U/ml thrombin by platelets from β2GPI deficient mice compared to wild-type littermates. Moreover, tail vein bleeding times were mildly prolonged in β2GPI deficient mice. Taken together, these studies suggest that β2GPI may actually contribute to hemostasis by supporting platelet responses to low concentrations of thrombin. Additional studies are needed to characterize these effects in detail. Figure 1 Figure 1. Disclosures McCrae: Sanofi, Novartis, Alexion, and Johnson & Johnson: Consultancy, Honoraria; Dova, Novartis, Rigel, and Sanofi Genzyme: Consultancy.

Mimetics of the Arg-Gly-Asp Sequence: Synthesis and Studies of the Antiaggregative Properties

Abstract— It is known that the Arg-Gly-Asp sequence in the fibrinogen molecule is key in binding to the receptors on the surface of platelets. We searched for the compounds which were able to inhibit the binding and synthesized the following analogs of this sequence: 2-acetoxybenzoyl-Arg-βAla-Asp, 4-piperidinecarbonyl-βAla-Asp, and 4-aminobezoyl-βAla-Asp. These compounds were shown to inhibit the platelets aggregation in a different degree. The 2-acetoxybenzoyl-Arg-βAla-Asp analog demonstrated the highest inhibitory activity. A decrease in the expression of the CD62p and CD63 markers on platelets was also found after the action of the Arg-Gly-Asp analogs, confirming the ability of these compounds to block the fibrinogen binding sites for the GP IIb/IIIa glycoprotein receptors.

Platelet binding to polymerizing fibrin is avidity driven and requires activated αIIbβ3 but not fibrin cross-linking

The molecular basis of platelet-fibrin interactions remains poorly understood despite the predominance of fibrin in thrombi. We have studied the interaction of platelets with polymerizing fibrin by adding thrombin to washed platelets in the presence of the peptide RGDW, which inhibits the initial platelet aggregation mediated by fibrinogen binding to αIIbβ3 but leaves intact a delayed increase in light transmission (delayed wave; DW) as platelets interact with the polymerizing fibrin. The DW was absent in platelets from a patient with Glanzmann thrombasthenia, indicating a requirement for αIIbβ3. The DW required αIIbb3 activation and it was inhibited by the αIIbβ3 antagonists eptifibatide and the monoclonal antibody (mAb) 7E3, but only at much higher concentrations than needed to inhibit platelet aggregation initiated by a thrombin receptor activating peptide (T6). Surface plasmon resonance and scanning electron microscopy studies both supported fibrin having greater avidity for αIIbβ3 than fibrinogen rather than greater affinity, consistent with fibrin’s multivalency. mAb 10E5, a potent inhibitor of T6-induced platelet aggregation, did not inhibit the DW, suggesting that fibrin differs from fibrinogen in its mechanism of binding. Inhibition of factor XIII–mediated fibrin cross-linking by >95% reduced the DW by only 32%. Clot retraction showed a pattern of inhibition similar to that of the DW. We conclude that activated αIIbβ3 is the primary mediator of platelet-fibrin interactions leading to clot retraction, and that the interaction is avidity driven, does not require fibrin cross-linking, and is mediated by a mechanism that differs subtly from that of the interaction of αIIbβ3 with fibrinogen.

First Report of Antimicrobial Susceptibility and Virulence Gene Characterization Associated with Staphylococcus aureus Carriage in Healthy Camels from Tunisia

A total of 318 nasal and rectal swabs were collected from 159 apparently healthy camels (Camelus dromedarius) randomly selected from five regions in southern and central Tunisia and screened for Staphylococcus aureus carriage. Staphylococcus spp. were recovered from 152 of 159 camels studied (95.6%) and in total 258 swabs (81%) were positive. Among these isolates, 16 were coagulase positive Staphylococcus (CoPS) (6.2%) and were characterized by biochemical and molecular tests as S. aureus. These were isolated from 14 camels (8.8%) with co-carriage in nasal and rectal mucosa by two camels. All S. aureus isolates recovered were methicillin-susceptible Staphylococcus aureus (MSSA) and were characterized by spa typing and PFGE. Three different spa types were recovered: t729, t4013 and a spa type newly registered as t19687, which was the most common. PFGE analysis revealed seven different patterns and these were characterized by MLST, which revealed five different sequence types (ST6, ST88, ST3583 and two new sequences, ST6504 and ST6506). All isolates harbored different virulence genes, including hld, encoding delta hemolysin; lukE–lukD, encoding bicomponent leukotoxin LukE–LukD; the clfB gene, encoding clumping factor B; the laminin gene, encoding laminin-binding protein; and cap8, encoding capsule type 8. Fifteen isolates harbored hemolysin beta (hlb) and fourteen encoded hemolysin alpha (hla) and hemolysin G2 (hlgv). Adhesin factors, including clfA and fnbB, were detected in five and four isolates respectively. Binding proteins, including collagen (cbp) and elastin-binding protein (ebp), were detected in two S. aureus isolates while fibrinogen-binding protein (fib) was identified in four isolates. This study provides the first set of genotyping data on the population structure and presence of toxin genes of S. aureus strains in Tunisian camels.

Helicobacter cyclurae sp. Nov., Isolated From Endangered Blue Iguanas (Cyclura lewisi)

Blue iguanas (Cyclura lewisi) are endangered reptiles found only on Grand Cayman. Previously, DNA for a novel Helicobacter species GCBI1 was detected in sick and dead iguanas. In the current study, fecal and cloacal swab samples were obtained from 25 iguanas. Through molecular and microbiological techniques, a novel Helicobacter species was cultured from feces and characterized, for whom we propose the name Helicobacter cyclurae. This novel helicobacter had a prevalence of 56% by PCR and 20% by culture in samples analyzed. The type strain MIT 16-1353 was catalase, oxidase, and gamma-glutamyl transpeptidase positive. By electron microscopy, H. cyclurae has a curved rod morphology and a single sheathed polar flagellum. Phylogenetic analysis using 16S rRNA, gyrB, and hsp60 indicated that these strains were most closely related to Helicobacter sp. 12502256-12 previously isolated from lizards. H. cyclurae has a 1.91-Mb genome with a GC content of 33.37%. There were 1,969 genes with four notable virulence genes: high temperature requirement-A protein-secreted serine protease, gamma-glutamyl transpeptidase, fibronectin/fibrinogen binding protein, and neutrophil-activating protein. Whole-genome phylogeny, average nucleotide identity, and digital DNA–DNA hybridization analysis confirmed that H. cyclurae is a novel species, and the first helicobacter cultured and characterized from blue iguanas.

vhp Is a Fibrinogen-Binding Protein Related to vWbp in Staphylococcus aureus

The life-threatening diseases caused by multidrug-resistant Staphylococcus aureus strains are a worldwide medical problem due to treatment limitations and the lack of an effective vaccine. The ability of S. aureus to coat itself with a protective fibrinogen (Fg)/fibrin shield allows the organism to survive in blood and to disseminate and cause invasive diseases.

Mapping of the fibrinogen-binding site on the staphylocoagulase C-terminal repeat region

The N-terminus of S. aureus staphylocoagulase (SC) triggers activation of host prothrombin (ProT), and the SCProT* complex cleaves host fibrinogen (Fbg) to form fibrin (Fbn) deposits, a hallmark of SC-positive endocarditis. The C-terminal domain of the prototypical Newman D2 Tager 104 SC contains 1 pseudo-repeat (PR) and 7 repeats (R1R7) that bind Fbg/Fbn Fragment D (Frag D). This work defines affinities and stoichiometries of Frag D binding to single- and multi-repeat C-terminal constructs, using fluorescence equilibrium binding, NMR titration, Ala scanning, and native PAGE. Constructs containing PR and each single repeat bound Frag D with KD ~50 130 nM and a 1:1 stoichiometry, indicating a conserved binding site shared between PR and each repeat. NMR titration of PR-R7 with Frag D revealed that residues 22-49, bridging PR and R7, constituted the minimal peptide (MP) required for binding, corroborated by Ala scanning, and binding of labeled MP to Frag D. MP alignment with the PR-repeat and inter-repeat junctions identified conserved residues critical for binding. Labeled PR-(R1R7) bound Frag D with KD ~7 32 nM and stoichiometry of 1:5; and PR-R1R2R3, PR-R1R6R7, PR-R3R4R7, and PR-R3R6R7 competed with PR-(R1R7) for Frag D binding, with a 1:3 stoichiometry and KD ~7 42 nM. These findings are consistent with binding at the PR-R junctions with modest inter-repeat sequence variability. Circular dichroism of PR-R7 and PR-(R1R7) suggested a largely disordered structure and conformational flexibility, allowing binding of multiple fibrin(ogen) molecules. This property facilitates pathogen localization on host fibrin networks.

In Vitro Antiplatelet Activity of Mulberroside C through the Up-Regulation of Cyclic Nucleotide Signaling Pathways and Down-Regulation of Phosphoproteins

Physiological agonists trigger signaling cascades, called “inside-out signaling”, and activated platelets facilitate adhesion, shape change, granule release, and structural change of glycoprotein IIb/IIIa (αIIb/β3). Activated αIIb/β3 interacts with fibrinogen and begins second signaling cascades called “outside-in signaling”. These two signaling pathways can lead to hemostasis or thrombosis. Thrombosis can occur in arterial and venous blood vessels and is a major medical problem. Platelet-mediated thrombosis is a major cause of cardiovascular disease (CVD). Therefore, controlling platelet activity is important for platelet-mediated thrombosis and cardiovascular diseases. In this study, focus on Morus alba Linn, a popular medicinal plant, to inhibit the function of platelets and found the containing component mulberroside C. We examine the effect of mulberroside C on the regulation of phosphoproteins, platelet-activating factors, and binding molecules. Agonist-induced human platelet aggregation is dose-dependently inhibited by mulberroside C without cytotoxicity, and it decreased Ca2+ mobilization and p-selectin expression through the upregulation of inositol 1, 4, 5-triphosphate receptor I (Ser1756), and downregulation of extracellular signal-regulated kinase (ERK). In addition, mulberroside C inhibited thromboxane A2 production, fibrinogen binding, and clot retraction. Our results show antiplatelet effects and antithrombus formation of mulberroside C in human platelets. Thus, we confirm that mulberroside C could be a potential phytochemical for the prevention of thrombosis-mediated CVDs.