Phosphorylation of (Ser 291) in the linker insert of Syk negatively regulates ITAM signaling in platelets.

Receptor-induced tyrosine phosphorylation of spleen tyrosine kinase (Syk) has been studied extensively in hematopoietic cells. Metabolic mapping and high-resolution mass spectrometry, however, indicate that one of the most frequently detected phosphorylation sites encompassed S297 (S291 in mice) located within the linker B region of Syk. It has been reported that Protein kinase C (PKC) phosphorylates Syk S297, thus influencing Syk activity. However, conflicting studies suggest that this phosphorylation enhances as well as reduces Syk activity. To clarify the function of this site, we generated Syk S291A knock-in mice. We used platelets as a model system as they possess Glycoprotein VI (GPVI), a receptor containing an immunoreceptor tyrosine-based activation motif (ITAM) which transduces signals through Syk. Our analysis of the homozygous mice indicated that the knock-in platelets express only one isoform of Syk, while the wild-type expresses two isoforms at 69 and 66 kDa. When the GPVI receptor was activated with collagen-related peptide (CRP), we observed an increase in functional responses and phosphorylations in Syk S291A platelets. This potentiation did not occur with AYPGKF or 2-MeSADP, although they also activate PKC isoforms. Although there was potentiation of platelet functional responses, there was no difference in tail bleeding times. However, the time to occlusion in the FeCl3 injury model was enhanced. These data indicate that the effects of Syk S291 phosphorylation represent a significant outcome on platelet activation and signaling in vitro but also reveals its multifaceted nature demonstrated by the differential effects on physiological responses in vivo.

What is the context Spleen tyrosine kinase (Syk) is present a number of cells and important in controlling the functions of various cells and organs.Syk is known to exist in two isoforms Syk L (long form or Syk A) and Syk S (short form or Syk B).It is known that phosphorylation events regulate Syk activation and activity.In several inflammatory disease conditions, Syk mutants are known to play a role.Phosphorylation of the Syk residue Serine 291 is known to occur, but its function in the regulation of Syk activation or activity is not known.What is new In this study, we generated a mutant mouse Syk S291A, which cannot be phosphorylated on serine residue. We evaluated the function of platelets isolated from these mice and compared them to platelets isolated from wild type littermates.We observed that the mutation in Syk L unexpectedly caused Syk S to disappear from a number of tissues.Platelet functions are enhanced in mutant mouse platelets compared to those from wild-type mice.What is the impact These studies enhance our understanding of the impact of Serine 291 phosphorylation on the function of Syk in platelets.

Association between toxoplasmosis and autoimmune rheumatic diseases in Egyptian patients.

PURPOSE: To explore the association between T. gondii and autoimmune rheumatic diseases (ARDs). METHODS: This study involved 82 patients with ARDs: 44 rheumatoid arthritis (RA), 28 systemic lupus erythematosus (SLE), and 10 systemic sclerosis (SSc) and 61 age- and sex-matched controls. Sociodemographic, clinical, and laboratory data were collected, and disease activity was assessed. Exposure to toxoplasmosis risk factors was investigated. Serological tests for anti-Toxoplasma IgM and IgG antibodies were assessed using ELISA. RESULTS: In SLE patients, a significant difference of T. gondii IgM versus controls was detected (P=.03). In RA and SLE patients, T. gondii IgG showed a significant difference versus controls (34 (77.3%) P=.001 and 18 (64.3%) P=.03, respectively). There was no significant difference in SSc versus controls. Fetal congenital anomalies displayed a significant difference in IgM seropositive compared to seronegative patients (P=.04). Cat exposure showed a significant difference between IgM and IgG seropositive versus seronegative patients (12 (80.0%) P=.02 and 34 (59.6) P=.04, respectively). There was no significant difference in seropositive patients regarding history of abortion, neuro-psychiatric manifestations, disease activity parameters (ESR, CRP), or different regimens of medications. CONCLUSION: Toxoplasma IgM seropositivity is associated with SLE patients. T. gondii IgG seropositivity is associated with both RA and SLE patients. However, Toxoplasma seropositivity had no association with SSc patients. An association between fetal congenital anomalies and IgM seropositivity was demonstrated. A linkage between cat exposure as a risk factor and toxoplasmosis was suggested among ARD patiants. Exploration of impact of toxoplasmosis on ARDs is a necessity through randomized controlled trials.

Association between toxoplasmosis and autoimmune rheumatic diseases in Egyptian patients / Asociación entre toxoplasmosis y enfermedades reumáticas autoinmunes en pacientes egipcios

Purpose: To explore the association between T. gondii and autoimmune rheumatic diseases (ARDs). Methods: This study involved 82 patients with ARDs: 44 rheumatoid arthritis (RA), 28 systemic lupus erythematosus (SLE), and 10 systemic sclerosis (SSc) and 61 age- and sex-matched controls. Sociodemographic, clinical, and laboratory data were collected, and disease activity was assessed. Exposure to toxoplasmosis risk factors was investigated. Serological tests for anti-Toxoplasma IgM and IgG antibodies were assessed using ELISA. Results: In SLE patients, a significant difference of T. gondii IgM versus controls was detected (P=.03). In RA and SLE patients, T. gondii IgG showed a significant difference versus controls (34 (77.3%) P=.001 and 18 (64.3%) P=.03, respectively). There was no significant difference in SSc versus controls. Fetal congenital anomalies displayed a significant difference in IgM seropositive compared to seronegative patients (P=.04). Cat exposure showed a significant difference between IgM and IgG seropositive versus seronegative patients (12 (80.0%) P=.02 and 34 (59.6) P=.04, respectively). There was no significant difference in seropositive patients regarding history of abortion, neuro-psychiatric manifestations, disease activity parameters (ESR, CRP), or different regimens of medications. Conclusion: Toxoplasma IgM seropositivity is associated with SLE patients. T. gondii IgG seropositivity is associated with both RA and SLE patients. However, Toxoplasma seropositivity had no association with SSc patients. An association between fetal congenital anomalies and IgM seropositivity was demonstrated. A linkage between cat exposure as a risk factor and toxoplasmosis was suggested among ARD patiants. Exploration of impact of toxoplasmosis on ARDs is a necessity through randomized controlled trials.(AU)

Propósito: Explorar la asociación entre Toxoplasma gondii y enfermedades reumáticas autoinmunes (ERA).Métodos: Este estudio involucró a 82 pacientes con ERA: 44 con artritis reumatoide (AR), 28 con lupus eritematoso sistémico (LES) y 10 con esclerosis sistémica (SSc); y 61 controles emparejados por edad y sexo. Se recopilaron datos sociodemográficos, clínicos y de laboratorio, y se evaluó la actividad de la enfermedad. Se indagó exposición a factores de riesgo de toxoplasmosis. Las pruebas serológicas de anticuerpos IgM e IgG antitoxoplasma se evaluaron mediante ELISA.Resultados: En pacientes con LES se detectó una diferencia significativa de T. gondii IgM vs. controles (p = 0,03). En pacientes con AR y LES, T. gondii IgG mostró una diferencia significativa frente a los controles (34 [77,3%] p = 0,001 y 18 [64,3%] p = 0,03, respectivamente). No hay diferencia significativa en SSc vs. controles. Las anomalías congénitas fetales mostraron una diferencia significativa en los pacientes seropositivos para IgM en comparación con los pacientes seronegativos (p = 0,04). La exposición a los gatos mostró una diferencia significativa entre los pacientes seropositivos para IgM e IgG frente a los seronegativos (12 [80%] p = 0,02 y 34 [59,6] p = 0,04, respectivamente). No hubo diferencias significativas en pacientes seropositivos con respecto a antecedentes de aborto, manifestaciones neuropsiquiátricas, parámetros de actividad de la enfermedad (ESR, CRP) o diferentes regímenes de medicamentos.(AU)

IL-11 induces NLRP3 inflammasome activation in monocytes and inflammatory cell migration to the central nervous system.

The objective of this study is to examine IL-11-induced mechanisms of inflammatory cell migration to the central nervous system (CNS). We report that IL-11 is produced at highest frequency by myeloid cells among the peripheral blood mononuclear cell (PBMC) subsets. Patients with relapsing-remitting multiple sclerosis (RRMS) have an increased frequency of IL-11+ monocytes, IL-11+ and IL-11R+ CD4+ lymphocytes, and IL-11R+ neutrophils in comparison to matched healthy controls. IL-11+ and granulocyte-macrophage colony-stimulating factor (GM-CSF)+ monocytes, CD4+ lymphocytes, and neutrophils accumulate in the cerebrospinal fluid (CSF). The effect of IL-11 in-vitro stimulation, examined using single-cell RNA sequencing, revealed the highest number of differentially expressed genes in classical monocytes, including up-regulated NFKB1, NLRP3, and IL1B. All CD4+ cell subsets had increased expression of S100A8/9 alarmin genes involved in NLRP3 inflammasome activation. In IL-11R+-sorted cells from the CSF, classical and intermediate monocytes significantly up-regulated the expression of multiple NLRP3 inflammasome-related genes, including complement, IL18, and migratory genes (VEGFA/B) in comparison to blood-derived cells. Therapeutic targeting of this pathway with αIL-11 mAb in mice with RR experimental autoimmune encephalomyelitis (EAE) decreased clinical scores, CNS inflammatory infiltrates, and demyelination. αIL-11 mAb treatment decreased the numbers of NFκBp65+, NLRP3+, and IL-1ß+ monocytes in the CNS of mice with EAE. The results suggest that IL-11/IL-11R signaling in monocytes represents a therapeutic target in RRMS.

Fluconazole as Schistosoma mansoni cytochrome P450 inhibitor: In vivo murine experimental study.

Schistosomiasis is a chronic disease caused by blood flukes of the Schistosoma spp. New approaches against this morbid infection are needed. In this study, we investigated fluconazole (FLZ) as an inhibitor of Schistosoma mansoni cytochrome P450 (S. mansoni CYP450) enzyme at different life cycle stages. We compared FLZ (10 mg/kg for two days) effects when administrated early 5 days post-infection (dpi) (Early I) and 21 dpi (Early II) versus late administration 60 dpi on S. mansoni CYP450 gene expression. These different FLZ treatment regimens were evaluated in experimentally infected mice with S. mansoni. This study showed that administration of FLZ, whether early or late during schistosomal infection, resulted in significant inhibition of S. mansoni CYP450 expression in the adult stage (P < 0.001). Early exposure to FLZ during the first week of infection significantly decreased the number of schistosomula that reached the adult stage compared to the infected control group and resulted in significant inhibition of S. mansoni CYP450 expression (P < 0.001) in the adult stage. In the Early I group, the fewest number of eggs per liver tissue gram was recorded. Our data suggested that FLZ is a S. mansoni CYP450 gene expression inhibitor with greater effect on schistosomula stages.

Therapeutic Effect of Anti-CD52 Monoclonal Antibody in Multiple Sclerosis and Its Animal Models Is Mediated via T Regulatory Cells.

The objective of this study is to determine the mechanism of action of anti-CD52 mAb treatment in patients with relapsing-remitting multiple sclerosis (RRMS). Experimental autoimmune encephalomyelitis (EAE), an animal model of the disease, was used to address the role of T regulatory cells (Tregs) in the anti-CD52 mAb-induced suppression of the disease. In vitro studies on PBMCs from RRMS patients and matched healthy controls determined the effect of IL-7 on the expansion of CD4+CD25+CD127- Tregs and induction of their suppressive phenotype. This study using EAE animal models of MS has shown that mouse anti-CD52 mAb suppression of clinical disease was augmented by coadministration of IL-7 and partially reversed by anti-IL-7 mAb. In vitro human studies showed that IL-7 induced expansion of CD4+CD25+CD127- Tregs and increased their FOXP3, GITIR, CD46, CTLA-4, granzyme B, and perforin expression. Anti-CD52 mAb treatment of mice with relapsing-remitting EAE induced expansion of Foxp3+CD4+ Tregs and the suppression of IL-17A+CD4+ and IFN-γ+CD4+ cells in peripheral immune organs and CNS infiltrates. The effect was detected immediately after the treatment and maintained over long-term follow-up. Foxp3+CD4+ Treg-mediated suppression of IL-17A+CD4+ and IFN-γ+CD4+ cells in the spinal cord infiltrates was reversed after inducible Foxp3 depletion. Our results demonstrated that the therapeutic effect of U.S. Food and Drug Administration-approved anti-CD52 mAb is dependent on the presence of Tregs.

Activity of isoflavone biochanin A in chronic experimental toxoplasmosis: impact on inflammation.

Toxoplasma gondii is a worldwide prevalent parasite. The infection has been linked to variable inflammatory effects including neuroinflammation. Biochanin A (BCA) is an isoflavone, known for its anti-inflammatory and anti-oxidative properties. In this study, we examined the effect of BCA on the brain and liver inflammatory lesions in a murine model with chronic toxoplasmosis. Mice were divided in to six groups: non-infected control, non-infected BCA-treated, and four infected groups with Toxoplasma gondii Me49-type II cystogenic strain: infected control, BCA (50 mg/kg/day)-treated, combined BCA/cotrimoxazole-treated and cotrimoxazole (370 mg/kg/day) alone-treated. Gene expression of tumor necrosis factor (TNF)-α, interleukin (IL)-1ß, and inducible nitric oxide synthase (iNOS) was evaluated by quantitative real-time PCR in the brain and liver tissues. In the infected control group, an upregulation of TNF-α and IL-1ß mRNA expression levels was found. However, a downregulation of iNOS expression was detected in the brain of infected control mice. In both BCA- and combined-treated groups, the brain and liver tissues showed significantly reduced inflammatory lesions compared to the infected control mice with inhibited TNF-α and IL-1ß mRNA levels. The iNOS expression levels in the brain tissues of BCA group were significantly higher than the levels of the infected control group. BCA alone or combined significantly reduced T. gondii cyst count in the brain tissues. In conclusion, the anti-inflammatory activity of BCA was demonstrated in the brain tissues of mice with chronic toxoplasmosis with decreased TNF-α and IL-1ß expression levels and increased iNOS expression levels.

Schistosomes Impede ATP-Induced T Cell Apoptosis In Vitro: The Role of Ectoenzyme SmNPP5.

Schistosomes (blood flukes) can survive in the bloodstream of their hosts for many years. We hypothesize that proteins on their host-interactive surface impinge on host biochemistry to help ensure their long-term survival. Here, we focus on a surface ectoenzyme of Schistosoma mansoni, designated SmNPP5. This ~53 kDa glycoprotein is a nucleotide pyrophosphatase/phosphodiesterase that has been previously shown to: (1) cleave adenosine diphosphate (ADP) and block platelet aggregation; and (2) cleave nicotinamide adenine dinucleotide (NAD) and block NAD-induced T cell apoptosis in vitro. T cell apoptosis can additionally be driven by extracellular adenosine triphosphate (ATP). In this work, we show that adult S. mansoni parasites can inhibit this process. Further, we demonstrate that recombinant SmNPP5 alone can both cleave ATP and impede ATP-induced T cell killing. As immunomodulatory regulatory T cells (Tregs) are especially prone to the induction of these apoptotic pathways, we hypothesize that the schistosome cleavage of both NAD and ATP promotes Treg survival and this helps to create a less immunologically hostile environment for the worms in vivo.

Histopathological and ultrastructural assessment of atovaquone-proguanil hydrochloride combination in chronic murine toxoplasmosis.

Over one billion people worldwide are expected to have Toxoplasma gondii infection with anonymous health problems. Available therapies are ineffective for persistent chronic toxoplasmosis. So, there is an imperative need for effective therapies to eliminate chronic tissue stage. In this study, we aimed to assess the effect of a drug combination of atovaquone and proguanil hydrochloride in the treatment of experimental chronic toxoplasmosis. Fifty Swiss Webster mice were used in the study. Forty mice were infected with Me49 type II cystogenic Toxoplasma gondii strain and allocated into four groups: infected untreated (vehicle-administered), infected and treated with cotrimoxazole (CTX) 370 mg/kg/day, infected and treated with atovaquone (ATV) 100 mg/kg/day, and infected and treated with atovaquone/proguanil (ATV/PROG) 50 mg/kg/day. An additional group of uninfected mice was used as an uninfected control group. Drug treatment was initiated 8 weeks post-infection and continued for two weeks. All mice were sacrificed 12 weeks post-infection. Parasitological and histopathological parameters were assessed. Toxoplasma gondii cysts recovered from brain tissue homogenates of both infected untreated and ATV/PROG-treated groups were examined by scanning electron microscopy. Combined ATV/PROG treatment demonstrated a significant reduction of Toxoplasma gondii cyst count in brain tissue (a reduction rate of 84.87%) compared to untreated group (P < .001). Brain tissues obtained from ATV/PROG treated group showed reduction of inflammatory infiltrate and marked attenuation and deformation of recovered Toxoplasma gondii cysts. We conclude that ATV/PROG drug combination could offer a potential drug therapy for Toxoplasma gondii chronic cystic stage.

Rhinovirus and Innate Immune Function of Airway Epithelium.

Airway epithelial cells, which lines the respiratory mucosa is in direct contact with the environment. Airway epithelial cells are the primary target for rhinovirus and other inhaled pathogens. In response to rhinovirus infection, airway epithelial cells mount both pro-inflammatory responses and antiviral innate immune responses to clear the virus efficiently. Some of the antiviral responses include the expression of IFNs, endoplasmic reticulum stress induced unfolded protein response and autophagy. Airway epithelial cells also recruits other innate immune cells to establish antiviral state and resolve the inflammation in the lungs. In patients with chronic lung disease, these responses may be either defective or induced in excess leading to deficient clearing of virus and sustained inflammation. In this review, we will discuss the mechanisms underlying antiviral innate immunity and the dysregulation of some of these mechanisms in patients with chronic lung diseases.

Vitamin B6 Acquisition and Metabolism in Schistosoma mansoni.

Schistosomes are parasitic platyhelminths that currently infect >200 million people globally. The adult worms can live within the vasculature of their hosts for many years where they acquire all nutrients necessary for their survival and growth. In this work we focus on how Schistosoma mansoni parasites acquire and metabolize vitamin B6, whose active form is pyridoxal phosphate (PLP). We show here that live intravascular stage parasites (schistosomula and adult males and females) can cleave exogenous PLP to liberate pyridoxal. Of the three characterized nucleotide-metabolizing ectoenzymes expressed at the schistosome surface (SmAP, SmNPP5, and SmATPDase1), only SmAP hydrolyzes PLP. Heat-inactivated recombinant SmAP can no longer cleave PLP. Further, parasites whose SmAP gene has been suppressed by RNAi are significantly impaired in their ability to cleave PLP compared to controls. When schistosomes are incubated in murine plasma, they alter its metabolomic profile-the levels of both pyridoxal and phosphate increase over time, a finding consistent with the action of host-exposed SmAP acting on PLP. We hypothesize that SmAP-mediated dephosphorylation of PLP generates a pool of pyridoxal around the worms that can be conveniently taken in by the parasites to participate in essential, vitamin B6-driven metabolism. In addition, since host PLP-dependent enzymes play active roles in inflammatory processes, parasite-mediated cleavage of this metabolite may serve to limit parasite-damaging inflammation. In this work we also identified schistosome homologs of enzymes that are involved in intracellular vitamin B6 metabolism. These are pyridoxal kinase (SmPK) as well as pyridoxal phosphate phosphatase (SmPLP-Ph) and pyridox(am)ine 5'-phosphate oxidase (SmPNPO) and cDNAs encoding these three enzymes were cloned and sequenced. The three genes encoding these enzymes all display high relative expression in schistosomula and adult worms suggestive of robust vitamin B6 metabolism in the intravascular life stages.

Schistosomes can hydrolyze proinflammatory and prothrombotic polyphosphate (polyP) via tegumental alkaline phosphatase, SmAP.

Schistosoma mansoni is a long-lived intravascular trematode parasite that can infect humans causing the chronic debilitating disease, schistosomiasis. We hypothesize that the action of host-interactive proteins found at the schistosome surface allows the worms to maintain a safe, anti-thrombotic and anti-inflammatory environment around them in the bloodstream. One such protein is the ˜60 kDa alkaline phosphatase SmAP which is known to be expressed in the outer tegument of all intravascular life stages. We demonstrate in this work that the parasites (schistosomula as well as adult males and females) can hydrolyze polyphosphate (polyP) - an anionic, linear polymer of inorganic phosphates that is produced and released by immune cells as well as by activated platelets and that induce proinflammatory and prothrombotic pathways. Purified recombinant SmAP can likewise cleave polyP and with a Km of 6.9 ±â€¯1 mM. Finally, parasites whose SmAP gene has been suppressed by RNAi are significantly impaired in their ability to hydrolyze polyP. SmAP-mediated cleavage of polyP may contribute to the armamentarium of schistosomes that promotes their survival in the hostile intravascular habitat. This is the first report of any pathogen cleaving this bioactive metabolite.

Intravascular Schistosoma mansoni Cleave the Host Immune and Hemostatic Signaling Molecule Sphingosine-1-Phosphate via Tegumental Alkaline Phosphatase.

Schistosomes are parasitic flatworms that infect the vasculature of >200 million people around the world. These long-lived parasites do not appear to provoke blood clot formation or obvious inflammation around them in vivo. Proteins expressed at the host-parasite interface (such as Schistosoma mansoni alkaline phosphatase, SmAP) are likely key to these abilities. SmAP is a glycoprotein that hydrolyses the artificial substrate p-nitrophenyl phosphate in a reaction that requires Mg2+ and at an optimal pH of 9. SmAP additionally cleaves the nucleoside monophosphates AMP, CMP, GMP, and TMP, all with a similar Km (~600-650 µM). Living adult worms, incubated in murine plasma for 1 h, alter the plasma metabolome; a decrease in sphingosine-1-phosphate (S1P) is accompanied by an increase in the levels of its component parts-sphingosine and phosphate. To test the hypothesis that schistosomes can hydrolyze S1P (and not merely recruit or activate a host plasma enzyme with this function), living intravascular life-stage parasites were incubated with commercially obtained S1P and cleavage of S1P was detected. Parasites whose SmAP gene was suppressed using RNAi were impaired in their ability to cleave S1P compared to controls. In addition, recombinant SmAP hydrolyzed S1P. Since extracellular S1P plays key roles in controlling inflammation and platelet aggregation, we hypothesize that schistosome SmAP, by degrading S1P, can regulate the level of this bioactive lipid in the environment of the parasites to control these processes in the worm's local environment. This is the first report of any parasite being able to cleave S1P.

The Essential Ectoenzyme SmNPP5 from the Human Intravascular Parasite Schistosoma mansoni is an ADPase and a Potent Inhibitor of Platelet Aggregation.

Schistosomes are intravascular parasitic platyhelminthes infecting > 200 million people globally and causing a debilitating disease, schistosomiasis. Despite the relatively large size of the adult worms and their disruption of blood flow, surprisingly, they do not appear to provoke thrombus formation around them in vivo. We hypothesize that proteins expressed at the host-parasite interface are key to this ability. Here, we functionally express an ectonucleotide pyrophosphatase/phosphodiesterase homologue, SmNPP5, that is expressed at the tegumental surface of intravascular Schistosoma mansoni. We report that SmNPP5, a known virulence factor for the worms, is a type one glycoprotein that cleaves the artificial substrate p-Nph-5'-TMP in a reaction that requires cations and at an optimal pH of 9. Using immunolocalization and enzyme activity measurements, we confirm that SmNPP5 is exclusively expressed at the host interactive surface of all intravascular life stages. SmNPP5 inhibits platelet aggregation in a dose-dependent manner, as measured by multiple electrode aggregometry (MEA) using whole blood. Inhibition is apparent when either collagen or adenosine diphosphate (ADP) is used as agonist but is lost following heat treatment of SmNPP5. Unlike its mammalian homologue, NPP5, the schistosome protein cleaves ADP and with a Km of 246 ± 34 µM. In sum, SmNPP5 is expressed in the intravascular environment where it can degrade ADP and act as an anticoagulant. In this manner, the protein likely helps limit blood clot formation around the worms in vivo to permit the parasites free movement within the vasculature.