Chemokine degradation by the Group A streptococcal serine proteinase ScpC can be reconstituted in vitro and requires two separate domains

2009 ◽  
Vol 422 (3) ◽  
pp. 533-542 ◽  
Author(s):  
Andrea Fritzer ◽  
Birgit Noiges ◽  
Daniela Schweiger ◽  
Angelika Rek ◽  
Andreas J. Kungl ◽  
...  
Keyword(s):  
Catalytic Domain ◽  
Serine Proteinase ◽  
Interferon Γ ◽  
Catalytic Triad ◽  
Terminal Region ◽  
Dependent Manner ◽  
Human Pathogens ◽  
Platelet Factor ◽  
Monocyte Chemoattractant Protein 1

Streptococcus pyogenes is one of the most common human pathogens and possesses diverse mechanisms to evade the human immune defence. One example of its immune evasion is the degradation of the chemokine IL (interleukin)-8 by ScpC, a serine proteinase that prevents the recruitment of neutrophils to an infection site. By applying the ANTIGENome technology and using human serum antibodies, we identified Spy0416, annotated as ScpC, as a prominent antigen that induces protective immune responses in animals. We demonstrate here for the first time that the recombinant form of Spy0416 is capable of IL-8 degradation in vitro in a concentration- and time-dependent manner. Mutations in the conserved amino acid residues of the catalytic triad of Spy0416 completely abolished in vitro activity. However, the isolated predicted proteinase domain does not exhibit IL-8-degrading activity, but is dependent on the presence of the C-terminal region of Spy0416. Binding to IL-8 is mainly mediated by the catalytic domain. However, the C-terminal region modulates substrate binding, indicating that the proteolytic activity is amenable to regulation via the non-catalytic regions. The specificity for human substrates is not restricted to IL-8, since we also detected in vitro protease activity for another CXC chemokine GRO-α (growth-related oncogene α), but not for NAP-2 (neutrophil-activating protein 2), SDF (stromal-cell-derived factor)-1α, PF-4 (platelet factor 4), I-TAC (interferon-γ-inducible T-cell α-chemoattractant), IP-10 (interferon-γ-inducible protein 10) and MCP-1 (monocyte chemoattractant protein 1). The degradation of two human CXC chemokines in vitro, the high sequence conservation, the immunogenicity of the protein in humans and the shown protection in animal studies suggest that Spy0416 is a promising vaccine candidate for the prevention of infections by S. pyogenes.

scholarly journals Effect ofTityus serrulatusvenom on cytokine production and the activity of murine macrophages

2002 ◽  
Vol 11 (1) ◽  
pp. 23-31 ◽  
Author(s):  
Vera L. Petricevich
Keyword(s):  
Cytokine Production ◽  
Peritoneal Macrophages ◽  
Interferon Γ ◽  
Enzyme Linked Immunosorbent Assay ◽  
No Production ◽  
Dependent Manner ◽  
Immune Functions ◽  
Macrophage Activity ◽  
Ifn Γ

The purpose of this study was to investigate the effects ofTityus serrulatusvenom (TSV) on murine peritoneal macrophages evaluated in terms of activation. The effects of crude TSV were analysed by detection of cytokines, oxygen intermediate metabolites (H2O2) and nitric oxide (NO) in supernatants of peritoneal macrophages. Several functional bioassays were employed including anin vitromodel for envenomating: cytotoxicity of TSV was assessed using the lyses percentage. Tumor necrosis factor (TNF) activity was assayed by measuring its cytotoxic activity on L-929 cells, and interleukin-6 (IL-6) and interferon-γ (IFN-γ) were assayed by enzyme-linked immunosorbent assay, whereas NO levels were detected by Griess colorimetric reactions in culture supernatant of macrophages incubated with TSV and subsequently exposed to either lipopolysaccharide or IFN-γ. Incubation of macrophages with TSV increased production of IL-6 and IFN-γ in a dose-dependent manner. TNF production was not detected in supernatants treated with TSV at any concentration. The increase in IL-6 secretion was not associated with concentration-dependent cytoxicity of TSV on these cells. These data suggest that the cytotoxicity does not appear to be the main cause of an increased cytokine production by these cells. Although NO is an important effector molecule in macrophage microbicidal activity, the inducing potential of the test compounds for its release was found to be very moderate, ranging from 125 to 800 mM. Interestingly, NO levels of peritoneal macrophages were increased after IFN-γ. Moreover, NO production had an apparent effect on macrophage activity. The results obtained here also shown that the TSV induces an important elevation in H2O2release. These results combined with NO production suggest that TSV possesses significant immunomodulatory activities capable of stimulating immune functionsin vitro.

Effect of SR121566A, a Potent GP IIb-IIIa Antagonist, on the HIT Serum/heparin-Induced Platelet Mediated Activation of Human Endothelial Cells

1998 ◽  
Vol 80 (08) ◽  
pp. 326-331 ◽  
Author(s):  
Pierre Savi ◽  
Walter Jeske ◽  
Jeanine Walenga ◽  
Jean-Marc Herbert
Keyword(s):  
Endothelial Cells ◽  
Platelet Aggregation ◽  
Umbilical Vein ◽  
Common Adverse Effect ◽  
Surface Protein ◽  
Heparin Therapy ◽  
Serotonin Release ◽  
Dependent Manner ◽  
Platelet Factor

SummaryHeparin-induced thrombocytopenia (HIT) is a common adverse effect of heparin therapy that carries a risk of serious thrombotic events. This condition is caused by platelet aggregation, which is mediated by anti-heparin/platelet factor 4 antibodies. Sera from patients with HIT in the presence of platelets, induced the expression of E-selectin, VCAM, ICAM-1 and tissue factor and the release of IL1β, IL6, TNFα and PAI-1 by human umbilical vein endothelial cells (HUVECs) in vitro and initiated platelet adhesion to activated HUVECs. These effects which occurred in a time-dependent manner were significant in the first 1-2 h of incubation and reached a maximum after 6 to 9 h. The GP IIb-IIIa receptor antagonist SR121566A which has been shown to block platelet aggregation induced by a wide variety of agonists including HIT serum/heparin, reduced in a dose-dependent manner the HIT serum/heparin-induced, platelet mediated expression and release of the above mentioned proteins. The IC50 for inhibition of HIT serum/ heparin-induced platelet dependent HUVEC activation by SR121566A was approximately 10-20 nM. ADP, but not serotonin release, also appeared to be involved as apyrase and ATPγS blocked platelet-dependent, HIT serum/heparin-induced cell surface protein expression and cytokine release by HUVECs. Increased platelet adherence to HIT serum/heparin-activated HUVECs was inhibited by SR121566A and, to a lesser extent, by apyrase and ATPγS, showing that platelet activation and release was at the origin of the HIT serum/heparin-induced expression of these proteins by HUVECs.Thus, sera from patients with HIT induced the expression of adhesive and coagulation proteins and the release of cytokines by HUVECs through the activation of platelets which occurred in a GP IIb-IIIa-dependent manner, a process that could be selectively blocked by SR121566A.

scholarly journals Rapid Purification and Procoagulant and Platelet Aggregating Activities of Rhombeobin: A Thrombin-Like/Gyroxin-Like Enzyme fromLachesis muta rhombeataSnake Venom

2013 ◽  
Vol 2013 ◽  
pp. 1-12 ◽  
Author(s):  
Frank Denis Torres-Huaco ◽  
Cláudio C. Werneck ◽  
Cristina Pontes Vicente ◽  
Talita Vassequi-Silva ◽  
Ana Cláudia Coelho Nery-Diez ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Proteinase Inhibitors ◽  
Ex Vivo ◽  
Serine Proteinase ◽  
Dependent Manner ◽  
Amidolytic Activity ◽  
Venom Component ◽  
Purification Method ◽  
Rapid Purification

We report a rapid purification method using one-step chromatography of SVSP Rhombeobin (LMR-47) fromLachesis muta rhombeatavenom and its procoagulant activities and effects on platelet aggregation. The venom was fractionated by a single chromatographic step in RP-HPLC on a C8 Discovery BIO Wide Pore, showing high degree of molecular homogeneity with molecular mass of 47035.49 Da. Rhombeobin showed amidolytic activity upon BAρNA, with a broad optimum pH (7–10) and was stable in solution up to 60°C. The amidolytic activity was inhibited by serine proteinase inhibitors and reducing agents, but not chelating agents. Rhombeobin showed high coagulant activity on mice plasma and bovine fibrinogen. The deduced amino acid sequence of Rhombeobin showed homology with other SVSPs, especially with LM-TL (L. m. muta) and Gyroxin (C. d. terrificus). Rhombeobin acts,in vitro, as a strong procoagulant enzyme on mice citrated plasma, shortening the APTT and PT tests in adose-dependent manner. The protein showed, “ex vivo”, a strong defibrinogenating effect with 1 µg/animal. Lower doses activated the intrinsic and extrinsic coagulation pathways and impaired the platelet aggregation induced by ADP. Thus, this is the first report of a venom component that produces a venom-induced consumptive coagulopathy (VICC).

scholarly journals ClyJ Is a Novel Pneumococcal Chimeric Lysin with a Cysteine- and Histidine-Dependent Amidohydrolase/Peptidase Catalytic Domain

2019 ◽  
Vol 63 (4) ◽  
Author(s):  
Hang Yang ◽  
Yujing Gong ◽  
Huaidong Zhang ◽  
Irina Etobayeva ◽  
Paulina Miernikiewicz ◽  
...  
Keyword(s):  
Catalytic Domain ◽  
High Specificity ◽  
Multidrug Resistant ◽  
Penicillin G ◽  
Dependent Manner ◽  
Pneumococcal Infections ◽  
Promising Alternative ◽  
Content Type ◽  
Conventional Antibiotics

ABSTRACT Streptococcus pneumoniae is one of the leading pathogens that cause a variety of mucosal and invasive infections. With the increased emergence of multidrug-resistant S. pneumoniae, new antimicrobials with mechanisms of action different from conventional antibiotics are urgently needed. In this study, we identified a putative lysin (gp20) encoded by the Streptococcus phage SPSL1 using the LytA autolysin as a template. Molecular dissection of gp20 revealed a binding domain (GPB) containing choline-binding repeats (CBRs) that are high specificity for S. pneumoniae. By fusing GPB to the CHAP (cysteine, histidine-dependent amidohydrolase/peptidase) catalytic domain of the PlyC lysin, we constructed a novel chimeric lysin, ClyJ, with improved activity to the pneumococcal Cpl-1 lysin. No resistance was observed in S. pneumoniae strains after exposure to incrementally doubling concentrations of ClyJ for 8 continuous days in vitro. In a mouse bacteremia model using penicillin G as a control, a single intraperitoneal injection of ClyJ improved the survival rate of lethal S. pneumoniae-infected mice in a dose-dependent manner. Given its high lytic activity and safety profile, ClyJ may represent a promising alternative to combat pneumococcal infections.

Effects of reactive oxygen and nitrogen metabolites on MCP-1-induced monocyte chemotactic activity in vitro

1999 ◽  
Vol 277 (3) ◽  
pp. L543-L549 ◽  
Author(s):  
Etsuro Sato ◽  
Keith L. Simpson ◽  
Matthew B. Grisham ◽  
Sekiya Koyama ◽  
Richard A. Robbins
Keyword(s):  
Protein Function ◽  
Inhibitory Effect ◽  
Chemotactic Activity ◽  
Dependent Manner ◽  
No Donor ◽  
Monocyte Chemoattractant Protein 1 ◽  
Dependent Inhibition ◽  
Monocyte Chemotaxis ◽  
Dose Dependent

Peroxynitrite, an oxidant generated by the interaction between superoxide and nitric oxide (NO), can nitrate tyrosine residues, resulting in compromised protein function. Monocyte chemoattractant protein-1 (MCP-1) is a chemokine that attracts monocytes and has a tyrosine residue critical for function. We hypothesized that peroxynitrite would alter MCP-1 activity. Peroxynitrite attenuated MCP-1-induced monocyte chemotactic activity (MCA) in a dose-dependent manner ( P < 0.05) but did not attenuate leukotriene B4 or complement-activated serum MCA. The reducing agents dithionite, deferoxamine, and dithiothreitol reversed the MCA inhibition by peroxynitrite, and exogenous l-tyrosine abrogated the inhibition by peroxynitrite. PAPA-NONOate, an NO donor, or superoxide generated by xanthine and xanthine oxidase did not show an inhibitory effect on MCA induced by MCP-1. The peroxynitrite generator 3-morpholinosydnonimine caused a concentration-dependent inhibition of MCA by MCP-1. Peroxynitrite reduced MCP-1 binding to monocytes and resulted in nitrotyrosine formation. These findings are consistent with nitration of tyrosine by peroxynitrite, with subsequent inhibition of MCP-1 binding to monocytes, and suggest that peroxynitrite may play a role in regulation of MCP-1-induced monocyte chemotaxis.

scholarly journals Molecular and Biochemical Characterization of a Cathepsin B-Like Protease Family Unique to Trypanosoma congolense

2008 ◽  
Vol 7 (4) ◽  
pp. 684-697 ◽  
Author(s):  
Carlos Mendoza-Palomares ◽  
Nicolas Biteau ◽  
Christiane Giroud ◽  
Virginie Coustou ◽  
Theresa Coetzer ◽  
...  
Keyword(s):  
Cathepsin B ◽  
Biochemical Characterization ◽  
Catalytic Domain ◽  
Expression Profiles ◽  
Specific Inhibitor ◽  
Cysteine Proteases ◽  
Catalytic Triad ◽  
Trypanosoma Congolense

ABSTRACT Cysteine proteases have been shown to be essential virulence factors and drug targets in trypanosomatids and an attractive antidisease vaccine candidate for Trypanosoma congolense. Here, we describe an important amplification of genes encoding cathepsin B-like proteases unique to T. congolense. More than 13 different genes were identified, whereas only one or two highly homologous genes have been identified in other trypanosomatids. These proteases grouped into three evolutionary clusters: TcoCBc1 to TcoCBc5 and TcoCBc6, which possess the classical catalytic triad (Cys, His, and Asn), and TcoCBs7 to TcoCBs13, which contains an unusual catalytic site (Ser, Xaa, and Asn). Expression profiles showed that members of the TcoCBc1 to TcoCBc5 and the TcoCBs7 to TcoCBs13 groups are expressed mainly in bloodstream forms and localize in the lysosomal compartment. The expression of recombinant representatives of each group (TcoCB1, TcoCB6, and TcoCB12) as proenzymes showed that TcoCBc1 and TcoCBc6 are able to autocatalyze their maturation 21 and 31 residues, respectively, upstream of the predicted start of the catalytic domain. Both displayed a carboxydipeptidase function, while only TcoCBc1 behaved as an endopeptidase. TcoCBc1 exhibited biochemical differences regarding inhibitor sensitivity compared to that of other cathepsin B-like proteases. Recombinant pro-TcoCBs12 did not automature in vitro, and the pepsin-matured enzyme was inactive in tests with cathepsin B fluorogenic substrates. In vivo inhibition studies using CA074Me (a cell-permeable cathepsin B-specific inhibitor) demonstrated that TcoCB are involved in lysosomal protein degradation essential for survival in bloodstream form. Furthermore, TcoCBc1 elicited an important immune response in experimentally infected cattle. We propose this family of proteins as a potential therapeutic target and as a plausible antigen for T. congolense diagnosis.

scholarly journals Phosphorylation of Tet3 by cdk5 is critical for robust activation of BRN2 during neuronal differentiation

2019 ◽  
Vol 48 (3) ◽  
pp. 1225-1238 ◽  
Author(s):  
Vinay Kumar Rao ◽  
Adusumalli Swarnaseetha ◽  
Guo-Hong Tham ◽  
Wei-Qi Lin ◽  
Bin-Bin Han ◽  
...  
Keyword(s):  
Neuronal Differentiation ◽  
Knockout Mouse ◽  
Catalytic Domain ◽  
Dynamic Balance ◽  
Embryonic Stem ◽  
Specific Gene ◽  
Dependent Manner ◽  
Wild Type ◽  
Expression Of Genes

Abstract Tet3 regulates the dynamic balance between 5-methylcyotsine (5mC) and 5-hydroxymethylcytosine (5hmC) in DNA during brain development and homeostasis. However, it remains unclear how its functions are modulated in a context-dependent manner during neuronal differentiation. Here, we show that cyclin-dependent kinase 5 (cdk5) phosphorylates Tet3 at the highly conserved serine 1310 and 1379 residues within its catalytic domain, changing its in vitro dioxygenase activity. Interestingly, when stably expressed in Tet1, 2, 3 triple-knockout mouse embryonic stem cells (ESCs), wild-type Tet3 induces higher level of 5hmC and concomitant expression of genes associated with neurogenesis whereas phosphor-mutant (S1310A/S1379A) Tet3 causes elevated 5hmC and expression of genes that are linked to metabolic processes. Consistent with this observation, Tet3-knockout mouse ESCs rescued with wild-type Tet3 have higher level of 5hmC at the promoter of neuron-specific gene BRN2 when compared to cells that expressed phosphor-mutant Tet3. Wild-type and phosphor-mutant Tet3 also exhibit differential binding affinity to histone variant H2A.Z. The differential 5hmC enrichment and H2A.Z occupancy at BRN2 promoter is correlated with higher gene expression and more efficient neuronal differentiation of ESCs that expressed wild-type Tet3. Taken together, our results suggest that cdk5-mediated phosphorylation of Tet3 is required for robust activation of neuronal differentiation program.

scholarly journals Comprehensive analysis of inflammatory markers in chronic thromboembolic pulmonary hypertension patients

2014 ◽  
Vol 44 (4) ◽  
pp. 951-962 ◽  
Author(s):  
Diana Zabini ◽  
Akos Heinemann ◽  
Vasile Foris ◽  
Chandran Nagaraj ◽  
Patrick Nierlich ◽  
...  
Keyword(s):  
Pulmonary Hypertension ◽  
Chronic Thromboembolic Pulmonary Hypertension ◽  
Serum Levels ◽  
Interferon Γ ◽  
Walking Distance ◽  
Healthy Controls ◽  
Monocyte Chemoattractant Protein 1 ◽  
Inflammatory Protein ◽  
Thromboembolic Pulmonary Hypertension

Chronic thromboembolic pulmonary hypertension (CTEPH) is associated with chronic inflammation but the pathological mechanisms are largely unknown. Our study aimed to simultaneously profile a broad range of cytokines in the supernatant of pulmonary endarterectomy (PEA) surgical material, as well as prospectively in patients with CTEPH to investigate whether circulating cytokines are associated with haemodynamic and physical characteristics of CTEPH patients.Herein, we show that PEA specimens revealed a significant upregulation of interleukin (IL)-6, monocyte chemoattractant protein-1, interferon-γ-induced protein-10 (IP)-10, macrophage inflammatory protein (MIP)1α and RANTES compared to lung tissue from healthy controls.In prospectively collected serum, levels of IL-6, IL-8, IP-10, monokine induced by interferon-γ (MIG) and MIP1α were significantly elevated in CTEPH patients compared to age- and sex-matched healthy controls. In serum of idiopathic pulmonary arterial hypertension (IPAH) patients, only IP-10 and MIG were significantly increased. In CTEPH but not in IPAH, IP-10 was negatively correlated with cardiac index, 6-min walking distance and carbon monoxide diffusion capacity. In vitro, IP-10 significantly increased migration of freshly isolated adventitial fibroblasts.Our study is the first to show that IP-10 secretion is associated with poor pulmonary haemodynamics and physical capacity in CTEPH and might be involved in the pathological mechanism of PEA tissue formation.

scholarly journals CXCL10/Gamma Interferon-Inducible Protein 10-Mediated Protection against Leishmania amazonensis Infection in Mice

2006 ◽  
Vol 74 (12) ◽  
pp. 6769-6777 ◽  
Author(s):  
Rene E. Vasquez ◽  
Lynn Soong
Keyword(s):  
Cell Function ◽  
Gamma Interferon ◽  
Leishmania Amazonensis ◽  
Interleukin 4 ◽  
Dependent Manner ◽  
Monocyte Chemoattractant Protein 1 ◽  
Inducible Protein ◽  
Interferon Inducible Protein

ABSTRACT Leishmania amazonensis can cause progressive disease in most inbred strains of mice. We have previously shown that L. amazonensis-infected C57BL/6 mice have profound impairments in expression of proinflammatory cytokines and chemokines and in activation of antigen-specific CD4+ T cells. These impairments are independent of interleukin-4 (IL-4) but partially due to IL-10 production. The precise mechanism of pathogenesis associated with L. amazonensis infection remains largely unresolved. Since chemokines are essential mediators of leukocyte recruitment and effector cell function, we hypothesized that these molecules are important for the initiation of early responses locally and for the eventual control of the infection. In this study, we examined the roles of CXCL10/gamma interferon-inducible protein 10 (IP-10) and CCL2/monocyte chemoattractant protein 1 (MCP-1) in the activation of the macrophage effector function in vitro and their efficacy in ameliorating infection in vivo. Bone marrow-derived macrophages of both BALB/c and C57BL/6 mice were treated with increasing concentrations of recombinant chemokines prior to infection with either stationary-phase promastigotes or tissue-derived amastigotes. We found that treatment with IP-10 or MCP-1 significantly reduced parasite burdens, in a dose-dependent manner, and triggered nitric oxide production. When susceptible C57BL/6 mice were injected locally with IP-10 following L. amazonensis infection, there was a significant delay in lesion development and a reduction in parasite burdens, accompanied by 7- and 3.5-fold increases in gamma interferon and IL-12 secretion, respectively, in restimulated lymph node cells. This study confirms that IP-10 plays a protective role in promoting the reduction of intracellular parasites and thereby opens new avenues for therapeutic control of nonhealing cutaneous leishmaniasis in the New World.

scholarly journals Activation of Heterophils and Monocytes in Chicken with a Formulation Containing a Seaweed Extract from Ulva armoricana

2021 ◽  
Vol 6 (1) ◽  
pp. 1-8
Author(s):  
Bussy F
Keyword(s):  
Interleukin 1 ◽  
Interferon Γ ◽  
Protective Role ◽  
Interferon Α ◽  
Dependent Manner ◽  
Additional Layer ◽  
Transcription Profiles ◽  
Ulva Armoricana

Responsiveness to invasive pathogens, clearance via the inflammatory response, and activation of appropriate acquired responses are all coordinated by innate host defenses. We have previously demonstrated that a purified ulvan extract of Ulva armoricana is able to activate avian heterophils and monocytes in vitro and in vivo , leading to in vivo release of cytokines including interleukin 1 β (IL1β), interferon α (IFNα) and interferon γ (IFNγ), in a transient and dose-dependent manner. In this study, we used the same protocol to evaluate a formulated version of this extract, called Searup ® . Our experiments showed that a single oral administration of this product at the dose recommended for use in the farm, results in heterophils and monocytes activation. In heterophils, activation was evidenced by β-D-glucuronidase release and increased mRNA expression of IL1β, IFNα and IFNγ. In monocytes, the expression of IFNγ and inducible nitrite oxide synthase (iNOS) were also up-regulated. Finally, plasmatic NO increased significantly on day 1, decreased on day 2 and was no longer significant at day 3. A similar pattern was observed for β-D-glucuronidase and for the modifications of the transcription profiles in monocytes as well as in heterophils. The only notable exception is gene transcription of 2'-5' Oligoadenylate Synthase, which is maximal at day 2 in monocytes. Due to its protective role in virus infection, this may constitute an additional layer of protection for this class of pathogens. Together our results show that the formulated solution, Searup ® , similarly to the purified extract allow to activate monocytes and heterophils but with some variations in the cytokines profiles and may provide protection against a larger variety of pathogens.

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