Polymicrobial Synergy Stimulates Porphyromonas Gingivalis Survival And Gingipain Expression In A Multispecies Subgingival Community

2021 ◽  
Author(s):  
Julia R. Davies ◽  
Trupti Kad ◽  
Jessica Neilands ◽  
Bertil Kinnby ◽  
Zdenka Prgomet ◽  
...  
Keyword(s):  
Proteolytic Activity ◽  
Fusobacterium Nucleatum ◽  
Extracellular Proteins ◽  
Periodontal Pocket ◽  
Wild Type ◽  
Culture Techniques ◽  
Survival And Development ◽  
Protease Substrate ◽  
2D Gel ◽  
Coated Surfaces

Abstract Background Dysbiosis in subgingival microbial communities, resulting from increased inflammatory transudate from the gingival tissues, is an important factor in initiation and development of periodontitis. Dysbiotic communities are characterized by increased numbers of bacteria that exploit the serum-like transudate for nutrients, giving rise to a proteolytic community phenotype. Here we investigate the contribution of interactions between members of a sub-gingival community to survival and development of virulence in a serum environment - modelling that in the subgingival pocket. Methods Growth and proteolytic activity of three P. gingivalis strains in nutrient-rich broth or a serum environment were assessed using A600 and a fluorescent protease substrate, respectively. Adherence of P. gingivalis strains to serum-coated surfaces was studied with confocal microscopy and 2D-gel electrophoresis of bacterial supernatants used to investigate extracellular proteins. A model multi-species sub-gingival community containing Fusobacterium nucleatum, Streptococcus constellatus, Parvimonas micra with wild type or isogenic mutants was then created and growth and proteolytic activity in serum assessed as above. Community composition over time was monitored using culture techniques and qPCR. Results The P. gingivalis strains showed different growth rates in nutrient-rich broth related to the level of proteolytic activity (largely gingipains) in the cultures. Despite being able to adhere to serum-coated surfaces, none of the strains was able to grow alone in a serum environment. In the subgingival consortium however, all the included species were able to grow in the serum environment and the community adopted a proteolytic phenotype. Inclusion of P. gingivalis strains lacking gingipains in the consortium revealed that the ability of the community to grow was largely due to Rgp gingipain. Conclusions In the multi-species consortium, growth was facilitated by the wild-type and Rgp-expressing strains of P. gingivalis, suggesting that Rgp is involved in delivery of nutrients to the whole community through degradation of complex serum substrates. Whereas they are constitutively expressed by P. gingivalis in nutrient-rich broth, gingipain expression in the model periodontal pocket environment (serum) appears to be orchestrated through signaling to P. gingivalis from other members of the community, a phenomenon which can then promote growth of the whole community.

scholarly journals Polymicrobial synergy stimulates Porphyromonas gingivalis survival and gingipain expression in a multi-species subgingival community

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Julia R. Davies ◽  
Trupti Kad ◽  
Jessica Neilands ◽  
Bertil Kinnby ◽  
Zdenka Prgomet ◽  
...  
Keyword(s):  
Proteolytic Activity ◽  
Porphyromonas Gingivalis ◽  
Fusobacterium Nucleatum ◽  
Extracellular Proteins ◽  
Nutrient Broth ◽  
Periodontal Pocket ◽  
Wild Type ◽  
Culture Techniques ◽  
Community Growth ◽  
Coated Surfaces

Abstract Background Dysbiosis in subgingival microbial communities, resulting from increased inflammatory transudate from the gingival tissues, is an important factor in initiation and development of periodontitis. Dysbiotic communities are characterized by increased numbers of bacteria that exploit the serum-like transudate for nutrients, giving rise to a proteolytic community phenotype. Here we investigate the contribution of interactions between members of a sub-gingival community to survival and development of virulence in a serum environment—modelling that in the subgingival pocket. Methods Growth and proteolytic activity of three Porphyromonas gingivalis strains in nutrient broth or a serum environment were assessed using A600 and a fluorescent protease substrate, respectively. Adherence of P. gingivalis strains to serum-coated surfaces was studied with confocal microscopy and 2D-gel electrophoresis of bacterial supernatants used to investigate extracellular proteins. A model multi-species sub-gingival community containing Fusobacterium nucleatum, Streptococcus constellatus, Parvimonas micra with wild type or isogenic mutants of P. gingivalis was then created and growth and proteolytic activity in serum assessed as above. Community composition over time was monitored using culture techniques and qPCR. Results The P. gingivalis strains showed different growth rates in nutrient broth related to the level of proteolytic activity (largely gingipains) in the cultures. Despite being able to adhere to serum-coated surfaces, none of the strains was able to grow alone in a serum environment. Together in the subgingival consortium however, all the included species were able to grow in the serum environment and the community adopted a proteolytic phenotype. Inclusion of P. gingivalis strains lacking gingipains in the consortium revealed that community growth was facilitated by Rgp gingipain from P. gingivalis. Conclusions In the multi-species consortium, growth was facilitated by the wild-type and Rgp-expressing strains of P. gingivalis, suggesting that Rgp is involved in delivery of nutrients to the whole community through degradation of complex protein substrates in serum. Whereas they are constitutively expressed by P. gingivalis in nutrient broth, gingipain expression in the model periodontal pocket environment (serum) appeared to be orchestrated through signaling to P. gingivalis from other members of the community, a phenomenon which then promoted growth of the whole community.

scholarly journals Mutational Analysis of Ocriplasmin to Reduce Proteolytic and Autolytic Activity in Pichia pastoris

2020 ◽  
Vol 22 (1) ◽  
Author(s):  
Roghayyeh Baghban ◽  
Safar Farajnia ◽  
Younes Ghasemi ◽  
Reyhaneh Hoseinpoor ◽  
Azam Safary ◽  
...  
Keyword(s):  
Pichia Pastoris ◽  
Proteolytic Activity ◽  
Mutational Analysis ◽  
Expression System ◽  
Catalytic Efficiency ◽  
Site Directed Mutagenesis ◽  
Wild Type ◽  
Vitreomacular Adhesion ◽  
Autolytic Activity

Abstract Background Ocriplasmin (Jetrea) is using for the treatment of symptomatic vitreomacular adhesion. This enzyme undergoes rapid inactivation and limited activity duration as a result of its autolytic nature after injection within the eye. Moreover, the proteolytic activity can cause photoreceptor damage, which may result in visual impairment in more serious cases. Results The present research aimed to reduce the disadvantages of ocriplasmin using site-directed mutagenesis. To reduce the autolytic activity of ocriplasmin in the first variant, lysine 156 changed to glutamic acid and, in the second variant for the proteolytic activity reduction, alanine 59 mutated to threonine. The third variant contained both mutations. Expression of wild type and three mutant variants of ocriplasmin constructs were done in the Pichia pastoris expression system. The mutant variants were analyzed in silico and in vitro and compared to the wild type. The kinetic parameters of ocriplasmin variants showed both variants with K156E substitution were more resistant to autolytic degradation than wild-type. These variants also exhibited reduced Kcat and Vmax values. An increase in their Km values, leading to a decreased catalytic efficiency (the Kcat/Km ratio) of autolytic and mixed variants. Moreover, in the variant with A59T mutation, Kcat and Vmax values have reduced compared to wild type. The mix variants showed the most increase in Km value (almost 2-fold) as well as reduced enzymatic affinity to the substrate. Thus, the results indicated that combined mutations at the ocriplasmin sequence were more effective compared with single mutations. Conclusions The results indicated such variants represent valuable tools for the investigation of therapeutic strategies aiming at the non-surgical resolution of vitreomacular adhesion.

scholarly journals Role of FAD-I in Fusobacterial Interspecies Interaction and Biofilm Formation

2020 ◽  
Vol 8 (1) ◽  
pp. 70 ◽  
Author(s):  
Bhumika Shokeen ◽  
Jane Park ◽  
Emily Duong ◽  
Sonam Rambhia ◽  
Manash Paul ◽  
...  
Keyword(s):  
Biofilm Formation ◽  
Fusobacterium Nucleatum ◽  
Interspecies Interaction ◽  
Wild Type ◽  
Oral Epithelial Cells ◽  
Small Proteins ◽  
Mutant Strains ◽  
Oral Epithelial ◽  
Wild Type Parent

RadD, a major adhesin of oral fusobacteria, is part of a four-gene operon encoding the small lipoprotein FAD-I and two currently uncharacterized small proteins encoded by the rapA and rapB genes. Previously, we described a role for FAD-I in the induction of human B-defensin 2 (hBD2) upon contact with oral epithelial cells. Here, we investigated potential roles for fad-I, rapA, and rapB in interspecies interaction and biofilm formation. Gene inactivation mutants were generated for each of these genes in the nucleatum and polymorphum subspecies of Fusobacterium nucleatum and characterized for their adherence to partner species, biofilm formation, and operon transcription. Binding to Streptococcus gordonii was increased in all mutant strains with Δfad-I having the most significant effect. This increased adherence was directly proportional to elevated radD transcript levels and resulted in significantly different architecture and height of the biofilms formed by Δfad-I and S. gordonii compared to the wild-type parent. In conclusion, FAD-I is important for fusobacterial interspecies interaction as its lack leads to increased production of the RadD adhesin suggesting a role of FAD-I in its regulation. This regulatory effect does not require the presence of functional RadD.

scholarly journals Interleukin-6 Deficiency Increases Inflammatory Bone Destruction

2001 ◽  
Vol 69 (2) ◽  
pp. 744-750 ◽  
Author(s):  
Khaled Balto ◽  
Hajime Sasaki ◽  
Philip Stashenko
Keyword(s):  
Bone Resorption ◽  
Interleukin 1 ◽  
Bone Destruction ◽  
Fusobacterium Nucleatum ◽  
Wild Type ◽  
Periapical Lesions ◽  
Elevated Expression ◽  
Pulp Exposure ◽  
Anti Inflammatory

ABSTRACT Periapical bone destruction occurs as a consequence of pulpal infection. In previous studies, we showed that interleukin-1 (IL-1) is the primary stimulator of bone destruction in this model. IL-6 is a pleiotropic cytokine that is induced in these infections and has both pro- and anti-inflammatory activities. In the present study, we determined the role of IL-6 in regulating IL-1 expression and bone resorption. The first molars of IL-6 knockouts (IL-6−/−) and wild-type mice were subjected to surgical pulp exposure and infection with a mixture of four common pulpal pathogens, includingPrevotella intermedia, Fusobacterium nucleatum,Peptostreptococcus micros, and Streptococcus intermedius. Mice were killed after 21 days, and bone destruction and cytokine expression were determined. Surprisingly, bone destruction was significantly increased in IL-6−/− mice versus that in wild-type mice (by 30%; P < 0.001). In a second experiment, the effects of chronic (IL-6−/−) IL-6 deficiency and short-term IL-6 deficiency induced by in vivo antibody neutralization were determined. Both IL-6−/− (30%;P < 0.001) and anti-IL-6 antibody-treated mice (40%;P < 0.05) exhibited increased periapical bone resorption, compared to wild-type controls. The increased bone resorption in IL-6-deficient animals correlated with increases in osteoclast numbers, as well as with elevated expression of bone-resorptive cytokines IL-1α and IL-1β, in periapical lesions and with decreased expression of the anti-inflammatory cytokine IL-10. These data demonstrate that endogenous IL-6 expression has significant anti-inflammatory effects in modulating infection-stimulated bone destruction in vivo.

Oxidative Stress Induces Binding of FANCD2 to STAT5 and Facilitates STAT5-Dependent Survival Signals.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 33-33 ◽  
Author(s):  
Grover Bagby ◽  
Winifred Keeble ◽  
Tara Koretsky ◽  
Dylan Zodrow ◽  
Richard Jove ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Ground State ◽  
Mutant Cell ◽  
Extracellular Proteins ◽  
Wild Type ◽  
Direct Role ◽  
Normal Cells ◽  
Jak2 Inhibitor ◽  
Stat Signaling ◽  
Mutant Cells

Abstract Fanconi anemia (FA) cells are hypersensitive to oxidative stress and exhibit aberrant STAT activation responses to defined extracellular proteins but whether these abnormalities are linked is unclear. Because oxidative stress is known to induce STAT activation, we hypothesized that proper STAT signaling responses in normal cells exposed to H2O2 require intact FA proteins. In fact, we found that FA-C, FA-G, and FA-D2 cells (fibroblasts) showed a significant increase in apoptosis after H2O2-exposure compared to retrovirally-complemented cells. H2O2 induced higher phospho-STAT5 (P-STAT5) expression in complemented cells than in mutant cells. Conversely, mutant cells expressed higher levels of P-STAT3 in both the ground state and after H2O2-induction than complemented cells. Aberrant STAT activation in FA mutant cells was shown to be both nucleus- and JAK2 kinase-dependent. Only low levels of STAT3 and STAT5 were induced in both mutant and complemented cytoplasts and AG490 (a Jak2 inhibitor) significantly suppressed H2O2-induced STAT5 responses. Seeking a direct role of FANCD2 in regulating proper STAT activation responses to H2O2, we carried out immunoprecipitation experiments (with an antibody to the N-terminal fragment of FANCD2) using PD20, a FA-D2 mutant cell line, and FANCD2 complemented PD20. In FANCD2-complemented and normal cells, anti-FANCD2 antibody immunoprecipitated STAT5. However, in mutant cells the same antibody immunoprecipitated STAT3, not STAT5. Thus, mutant (truncated) FANCD2 preferentially binds to and may activate STAT3 in the ground state. In fact, wild type FANCD2 also binds aberrantly to STAT3 in HSC536 (FA-C lymphoblasts) indicating that FANCC may influence the function of wild type FANCD2 and that binding of wild type FANCD2 to STAT3 does not require FANCD2 ubiquitinylation (FANCD2 is not ubiquitinylated in FA-C). Suspecting that in H2O2-exposed cells STAT5 signaling pathways lead to survival while STAT3 pathways lead to apoptosis, we transduced constitutively active mutants (*) of STATs 3 and 5 in mutant D2 and complemented cells. STAT3* increased apoptotic responses to H2O2 in complemented FA-D2 cells and STAT5* decreased apoptotic responses in H2O2-induced FA-D2 cells. In addition, the STAT5 inducible anti-apoptotic gene Bcl-XL was induced in H2O2-exposed complemented FA-D2 cells but not in FA-D2 cells. We conclude that FANCD2 functions to promote survival by ordering proper STAT signaling responses to oxidative stress and that this function of FANCD2 depends in part upon FA-C. We propose that FA cells are hypersensitive to oxidative stress in part because of imbalanced STAT signal transduction responses.

Activity and Regulation of Factor VIIa Analogs with Increased Potency at the Endothelial Cell Surface.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1751-1751
Author(s):  
Samit Ghosh ◽  
Mirella Ezban ◽  
Egon Persson ◽  
Ulla Hedner ◽  
Usha Pendurthi ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Cell Surface ◽  
Proteolytic Activity ◽  
Thrombin Generation ◽  
Factor Viia ◽  
Factor Xa ◽  
Factor Ix ◽  
Factor X ◽  
Wild Type

Abstract High doses of recombinant factor VIIa (FVIIa) have been found to bypass factor IX or factor VIII deficiency and ameliorate the bleeding problems associated with hemophilia patients with inhibitors. Recent studies show that FVIIa also acts as an effective hemostatic agent in other categories of patients, and thus has become a promising candidate for prevention and treatment of excessive bleeding associated with many other diseases/injuries. Although recombinant FVIIa has proven to be a very effective and safe drug in the treatment of bleeding episodes in hemophilia patients with inhibitors and other indications, a small fraction of patients may be refractory to FVIIa treatment. The reason for this is unclear at present, but it is possible that administration of very high pharmacological doses of FVIIa or use of genetically modified FVIIa molecules with increased potencies may circumvent the problem. The most dramatic effect on the activity (a 40-fold increase in proteolytic activity) of FVIIa was obtained by occupying the corresponding positions in thrombin/factor IXa for those positions 158, 296 and 298 of FVIIa (FVIIaDVQ). A FVIIa mutant in which the hydrophobic residue Met 298 was replaced with Gln (FVIIaQ) has 7-fold higher proteolytic activity. In the present study, we investigated the interactions of FVIIaQ and FVIIaDVQ with plasma inhibitors, tissue factor pathway inhibitor (TFPI) and antithrombin (AT) in solution and at the vascular endothelium. Both TFPI and AT/heparin inhibited the FVIIa variants more rapidly than the wild-type FVIIa in the absence of TF. In the presence of TF, TFPI, TFPI-Xa and AT/heparin inhibited FVIIa and FVIIa variants at similar rates. Although the wild-type FVIIa failed to generate significant amounts of factor Xa on unperturbed endothelial cells, FVIIa variants, particularly FVIIaDVQ, generated a substantial amount of factor Xa on unperturbed endothelium (1 nM of factor VIIa generated 0.3 ± 0.15 nM factor Xa/h whereas FVIIaQ and FVIIaDVQ generated 1.26 ± 0.1 nM/h and 9.48 ± 1.32 nM/h, respectively). Annexin V fully attenuated the FVIIa-mediated activation of factor X on unperturbed endothelial cells whereas anti-TF IgG had no effect. On stimulated HUVEC, FVIIa and FVIIa variants activated factor X at similar rates (30–40 nM/h). AT/heparin and TFPI-Xa inhibited the activity of FVIIa and FVIIa variants bound to endothelial cell TF in a similar fashion. AT inhibition of FVIIa bound to stimulated endothelial cells requires exogenous heparin. Interestingly, TFPI-Xa was found to inhibit the activities of both FVIIa and FVIIa analogs bound to unperturbed endothelial cells. Despite significant differences observed in factor Xa generation on native endothelium exposed to FVIIa and FVIIa analogs, no differences were found in thrombin generation when cells were exposed to FVIIa or FVIIa analogs under plasma mimicking conditions, probably due to limited availability of anionic phospholipids and/or putative factor Xa and Va binding sites on their cell surface. Over all, our present data suggest that although FVIIa variants may generate factor Xa on native endothelium, the resultant factor Xa does not lead to enhanced thrombin generation on native endothelium compared to FVIIa. These data should reduce potential concerns about whether the use of FVIIa variants triggers unwanted coagulation on native endothelium, and may facilitate the development of FVIIa analogs as effective therapeutic agents in near future for treatment of patients with bleeding disorders.

Phenotypic Correction of Adamts13-Deficient Mice by Early Intra-Amniotic Gene Transfer of Lentiviral Vector Encoding ADAMTS13 Genes.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 197-197
Author(s):  
Masami Niiya ◽  
Masayuki Endo ◽  
Philip W. Zoltick ◽  
Nidal E. Muvarak ◽  
David G. Motto ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Gene Transfer ◽  
Proteolytic Activity ◽  
Lentiviral Vector ◽  
Wild Type ◽  
Occlusion Time ◽  
Carboxyl Terminal ◽  
Human Wild Type

Abstract ADAMTS13, a member of A Disintegrin and Metalloprotease with ThromboSpondin type 1 repeats (ADAMTS) family, is mainly synthesized in the hepatic stellate cells, endothelial cells and megakaryocytes or platelets. It controls the sizes of von Willebrand factor (VWF) multimers by cleaving VWF at the Tyr1605-Met1606 bond. Genetic deficiency of plasma ADAMTS13 activity results in hereditary thrombotic thrombocytopenic purpura (TTP), also named Upshaw-Schülman syndrome. To develop a potential gene therapy approach and to determine the domains of ADAMTS13 required for recognition and cleavage of VWF in vivo, a self-inactivating lentiviral vector encoding human wild-type ADAMTS13 or variant truncated after the spacer domain (construct MDTCS) was administrated by intra-amniotic injection on embryonic day 8. Direct stereomicroscopy and immunofluorescent microscopic analysis revealed that the green fluorescent protein (GFP) reporter, ADAMTS13 and MDTCS were predominantly expressed in the heart, kidneys and skin. The synthesized ADAMTS13 and truncated variant were detectable in mouse plasma by immunoprecipitation and Western blot, as well as by proteolytic cleavage of FRETS-VWF73 substrate. The levels of proteolytic activity in plasma of mice expressing ADAMTS13 and MDTCS were 5 ± 7% and 60 ± 70%, respectively using normal human plasma as a standard, and this proteolytic activity persisted for at least 24 weeks in Adamts13−/−mice and 42 weeks in wild-type mice tested (the duration of observation). The mice expressing both recombinant ADAMTS13 and MDTCS showed a significantly decreased ratio of plasma VWF collagen-binding activity to antigen and a reduction in VWF multimer sizes as compared to those in the controls. Moreover, the mice expressing ADAMTS13 and MDTCS showed a significant prolongation of ferric chloride-induced carotid arterial occlusion time (9.0 ± 0.6 and 25.2 ± 3.2 min, respectively) as compared to the Adamts13−/− mice expressing GFP alone (5.6 ± 0.5 min) (p&lt;0.01). The ferric chloride-induced carotid occlusion time in Adamts13−/− mice expressing ADAMTS13 was almost identical to that in wild type mice with same genetic background (C56BL/6) (8.0 ± 0.2 min) (p&gt;0.05). The data demonstrate the correction of the prothrombotic phenotype in Adamts13−/−mice by gene transfer to the fetus by viral vectors encoding human wild type ADAMTS13 and the carboxyl terminal truncated variant (MDTCS), supporting the feasibility of developing a gene therapy based treatment for hereditary TTP. The discrepancy in the proteolytic activity of MDTCS between in vitro (Zhang P et al. Blood, 2007 in press) and in vivo in the present study suggests the potential cofactors in murine circulation that may rescue the defective proteolytic activity of the carboxyl-terminal truncated ADAMTS13 protease seen in vitro.

Ectopic Expression of ADAMTS13 in Platelets As a Novel Therapeutic Approach for Arterial Thrombosis

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 491-491
Author(s):  
Brandy Pickens ◽  
Sheng-Yu Jin ◽  
Dengju Li ◽  
X. Long Zheng
Keyword(s):  
Transgenic Mice ◽  
Proteolytic Activity ◽  
Targeted Delivery ◽  
Arterial Thrombosis ◽  
Thrombus Formation ◽  
Clotting Factor ◽  
Platelet Glycoprotein ◽  
Wild Type ◽  
F1 Hybrid ◽  
Novel Therapeutic

Abstract Abstract 491 Megakaryocytes and platelets have been shown to produce ADAMTS13 and its only known substrate, von Willebrand factor (VWF). However, the role of platelet expression of ADMTS13 in modulation of thrombus formation is not known. Previous studies have shown that platelet-targeted delivery of clotting factor VIII corrects bleeding phenotype in hemophilia A mice despite of inhibitors. These results suggest that platelet-delivery of ADAMTS13 may also be efficacious for anti-arterial thrombosis and perhaps for treatment of acquired idiopathic thrombotic thrombocytopenic purpura (TTP) with inhibitors. In the present study, transgenic mice (JAX B6SJL/F1 hybrid) carrying a human full-length ADAMTS13 gene under a platelet glycoprotein 1b alpha promoter were generated. The mice were crossed with Adamts13−/− and TTP-sensitive mice (CAST/Ei) for 4 generations. Plasma and platelet ADAMTS13 protein and proteolytic activity were determined. By Western blotting and the cleavage of a fluorescein-labeled VWF73 substrate, we were able to show that human ADAMTS13 protein (∼195 kDa) and activity were present in the platelet lysate of transgenic (A13-PltTG) mice, but not in adamts13−/− mice or wild-type mice. No proteolytic activity was detected in plasma of the transgenic mice. The platelet ADAMTS13 protein was releasable upon stimulation with various concentrations of thrombin (0.1–0.5 U/ml) and collagen (2.5–10 μg/ml). The released ADAMTS13 and VWF (as a positive control) were primarily associated with platelet membrane, demonstrated by surface biotinylation. However, a small fraction of the released ADAMTS13 and VWF proteins were detected in the releasate after stimulation. Moreover, the A13-PltTG mice exhibited systemic anti-thrombotic activity, which attenuated the rate of thrombus formation in the mesenteric arterioles induced by a topical application of 10% ferric chloride. The rate of arterial thrombus formation in the transgenic mice was significantly lower than that in Adamts13−/−mice and wild-type mice in the same genetic background. We conclude that we have generated transgenic mice overexpressing human ADAMTS13 metalloprotease in platelets. The platelet expressed ADAMTS13 is releasable upon stimulation by agonists. The platelet derived ADAMTS13 is biologically functional in cleaving VWF in vitro and in vivo, which attenuate systemic arterial thrombosis after oxidative injury. Our ongoing effort is to determine the efficacy of platelet delivered ADAMTS13 as a potential novel therapeutic for acquired TTP patients with inhibitors. Disclosures: No relevant conflicts of interest to declare.

Dynamics of GPIIb/IIIa-mediated platelet-platelet interactions in platelet adhesion/thrombus formation on collagen in vitro as revealed by videomicroscopy

Blood ◽  
2003 ◽  
Vol 101 (3) ◽  
pp. 929-936 ◽  
Author(s):  
Dipti Patel ◽  
Heikki Väänänen ◽  
Markéta Jiroušková ◽  
Thomas Hoffmann ◽  
Carol Bodian ◽  
...  
Keyword(s):  
Platelet Adhesion ◽  
Thrombus Formation ◽  
Wild Type Mouse ◽  
Poisson Model ◽  
Human Platelets ◽  
Wild Type ◽  
Initial Adhesion ◽  
Platelet Deposition ◽  
Coated Surfaces

Abstract The conventional description of platelet interactions with collagen-coated surfaces in vitro, based on serial static measurements, is that platelets first adhere and spread to form a monolayer and then recruit additional layers of platelets. To obtain dynamic information, we studied gravity-driven platelet deposition in vitro on purified type 1 collagen by video phase-contrast microscopy at 22°C. With untreated human and wild-type mouse platelets, soon after the initial adhesion of a small number of “vanguard” platelets, “follower” platelets attached to the spread-out vanguard platelets. Follower platelets then adhered to and spread onto nearby collagen or over the vanguard platelets. Thus, thrombi formed as a concerted process rather than as sequential processes. Treatment of human platelets with monoclonal antibody (mAb) 7E3 (anti–GPIIb/IIIa (αIIbβ3) + αVβ3) or tirofiban (anti–GPIIb/IIIa) did not prevent platelet adhesion but nearly eliminated the deposition of follower platelets onto vanguard platelets and platelet thrombi. Similar results were obtained with Glanzmann thrombasthenia platelets. Wild-type mouse platelets in the presence of mAb 1B5 (anti–GPIIb/IIIa) and platelets from β3-null mice behaved like human platelets in the presence of 7E3 or tirofiban. Deposition patterns of untreated human and wild-type mouse platelets were consistent with random distributions under a Poisson model, but those obtained with 7E3- and tirofiban-treated human platelets, 1B5-treated mouse platelets, or β3-null platelets demonstrated a more uniform deposition than predicted. Thus, in this model system, absence or blockade of GPIIb/IIIa receptors interferes with thrombus formation and alters the pattern of platelet deposition.

scholarly journals Fusobacterium nucleatum facilitates cetuximab resistance in colorectal cancer via the PI3K/AKT and JAK/STAT3 pathways

2020 ◽  
Author(s):  
Hu Han ◽  
Yan Li ◽  
Wan Qin ◽  
Lu Wang ◽  
Han Yin ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Fusobacterium Nucleatum ◽  
Therapy Resistance ◽  
Tumor Burden ◽  
Wild Type ◽  
Poor Clinical Outcome ◽  
Functional Studies ◽  
Cetuximab Therapy

AbstractInfectious pathogens contribute to about 20% of the total tumor burden. Fusobacterium nucleatum (Fn) has been associated with the initiation, progression, and therapy resistance in colorectal cancer (CRC). The over-abundance of Fn has been observed in patients with right-sided CRC than in those with left-sided CRC. While the KRAS/NRAS/BRAF wild-type status of the CRC conferred better response to cetuximab in patients with left-sided CRC than with right-sided CRC. However, treatment failure remains the leading cause of tumor relapse and poor clinical outcome in patients with CRC. Here, we have studied the association of Fn to cetuximab resistance. Our functional studies indicate that Fn facilitates resistance of CRC to cetuximab in vitro and in vivo. Moreover, Fn was found to target the PI3K/AKT and JAK/STAT3 pathways, which altered the response to cetuximab therapy. Therefore, assessing the levels and targeting Fn and the associated signaling pathways may allow modulating the treatment regimen and improve prognoses of CRC patients.

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