[Main non-state actors and progress of actions in global malaria elimination programme: a review].

With the joint efforts of countries and global non-state actors, great achievements have been made in the global malaria control programme; however, malaria remains a serious threat to human health. As the global leader for combating malaria, WHO formulated The Global Technical Strategy for Malaria 2016-2030, and the Global Malaria Programme, under the leadership of WHO, is responsible for implementing 5 key projects to achieve the goal proposed in The Global Technical Strategy for Malaria 2016-2030. In addition, the Global Fund, the U.S. President's Malaria Initiative and Bill & Melinda Gates Foundation also play an important role in global malaria elimination programme. This review describes the currently main non-state actors participating in the global malaria elimination programme, and calls for the enhanced inter-actor coordination and close collaboration with state governments to achieve the great goal of malaria elimination in the world.

Clinical efficacy of ticagrelor combined with aspirin in patients with coronary heart disease angina pectoris and its effects on NT-ProBNP and CK-MB levels.

OBJECTIVE: This study aims to explore the clinical efficacy of ticagrelor combined with aspirin in patients with coronary heart disease angina pectoris and the effects on N terminal pro B type natriuretic peptide (NT-ProBNP) and creatine kinase-MB (CK-MB) levels. PATIENTS AND METHODS: A total of 150 patients with coronary heart disease angina pectoris were prospectively analyzed in this study. These patients were admitted to Huaiyin Hospital of Huai'an City from February 2017 to February 2019. The patients were divided into control group and research group according to different treatment methods. The following indicators before and after treatment were observed: therapeutic efficacy, prevalence of adverse reactions, duration and frequency of angina attack, NT-ProBNP and CK-MB levels. Receiver operating characteristic (ROC) curve was used to analyze the predictive value of NT-ProBNP and CK-MB for the curative effect of coronary heart disease angina pectoris. RESULTS: The total effective rate in the research group was higher than that in the control group (p<0.05). The prevalence of adverse reactions in the research group was lower than that in the control group (p<0.05). The duration and frequency of seizures of the two groups after treatment were lower than those before treatment. The duration and frequency of seizures in the research group were lower than those in the control group (p<0.05). The physiological function, physical pain, vital energy score and general health status in the research group were higher than those in the control group (p<0.05). The NT-ProBNP and CK-MB levels in both groups after treatment were decreased. CONCLUSION: Ticagrelor combined with aspirin has definite therapeutic effect on patients with coronary heart disease angina pectoris, with low prevalence of adverse reactions. It can significantly reduce the levels of NT-ProBNP and CK-MB, which is worthy of promotion.

Investigation of the value of miR-21 in the diagnosis of early stage HCC and its prognosis: a meta-analysis.

The diagnostic and prognostic value of miR-21 has been examined for hepatocellular carcinoma (HCC), with inconsistent results. Present meta-analysis summarized the diagnostic accuracy and the predictive role for survival of miR-21 in patients with HCC. All eligible studies were searched using PubMed, EMBASE, and Chinese National Knowledge Infrastructure (CNKI) databases up to October 2014. For the diagnostic meta-analysis, the indices of miR-21 in the diagnosis of HCC were pooled using bivariate random-effect approach models. For the prognostic meta-analysis, data were synthesized with a random effect model, and the hazard ratio (HR) or odd ratio (OR) with its 95% confidence interval (95%CI) was used as the effect size estimate. Ten studies dealing with HCC were included. The overall pooled results for sensitivity, specificity, and the area under the curve (AUC) for the diagnostic meta-analysis (five studies) were 74.0 (95%CI = 61.0-85.0), 78.0 (95%CI = 67.0-86.0), and 0.83 (95%CI = 0.80-0.86), respectively. The combined data for the prognostic meta-analysis (seven studies) suggested that miR-21 overexpression in HCC correlated with poor overall survival [HR = 1.19 (95%CI = 0.44-1.94)], and higher miR-21 expression was associated with tumor, node, metastases (TNM) stage [OR = 0.34 (95%CI = 0.13-0.91)]. We concluded that miR-21 might be complementary to alpha fetal protein in HCC diagnosis, and might serve as an attractive estimator of HCC. We also demonstrated that miR-21 overexpression was associated with HCC TNM stage and with poor survival. As our study was limited, additional prospective studies are needed to validate these results.

Detachment-induced upregulation of XIAP and cIAP2 delays anoikis of intestinal epithelial cells.

Detachment of normal epithelial cells from the extracellular matrix triggers apoptosis, a phenomenon called anoikis. Conversely, carcinoma cells tend to be relatively more anoikis-resistant than their normal counterparts, and this increased resistance represents a critical feature of the malignant phenotype. Mechanisms that control susceptibility and resistance to anoikis are not fully understood. It is now known that detachment of non-malignant epithelial cells triggers both pro- and antiapoptotic signals, and it is the balance between these signals and the duration of detachment that determine further fate of the cells. Detachment-induced antiapoptotic events delay anoikis and if cells reattach relatively soon after detachment they survive. Direct regulators of apoptosis responsible for this delay of anoikis are unknown. We found that detachment of non-malignant intestinal epithelial cells triggers upregulation of inhibitors of apoptosis protein (IAP) family, such as X-chromosome-linked inhibitor of apoptosis protein and cellular inhibitor of apoptosis-2 (cIAP2). We demonstrated that this upregulation requires detachment-dependent activation of the transcription factor nuclear factor-kappaB. We further observed that various IAP antagonists accelerate anoikis, indicating that upregulation of the IAPs delays detachment-triggered apoptosis. We conclude that the IAPs are important regulators of the balance between detachment-triggered life and death signals. Perhaps, not by coincidence, these proteins are often upregulated in carcinomas, tumors composed of cells that tend to be anoikis-resistant.

Activation of extracellular signal-related protein kinases 1 and 2 of the mitogen-activated protein kinase family by lipopolysaccharide requires plasma in neutrophils from adults and newborns.

Neutrophils exposed to low concentrations of gram-negative lipopolysaccharide (LPS) become primed and have an increased oxidative response to a second stimulus (e.g., formyl-methionyl-leucyl-phenylalanine [fMLP]). In studies aimed at understanding newborn sepsis, we have shown that neutrophils of newborns are not primed in response to LPS. To further understand the processes involved in LPS-mediated priming of neutrophils, we explored the role of extracellular signal-related protein kinases (ERK 1 and 2) of the mitogen-activated protein kinase family. We found that LPS activated ERK 1 and 2 in cells of both adults and newborns and that activation was plasma dependent (maximal at > or =5%) through LPS-binding protein. Although fibronectin in plasma is required for LPS-mediated priming of neutrophils of adults assessed by fMLP-triggered oxidative burst, it was not required for LPS-mediated activation of ERK 1 and 2. LPS-mediated activation was dose and time dependent; maximal activation occurred with approximately 5 ng of LPS per ml and at 10 to 40 min. We used the inhibitor PD 98059 to study the role of ERK 1 and 2 in the LPS-primed fMLP-triggered oxidative burst. While Western blotting showed that 100 microM PD 98059 completely inhibited LPS-mediated ERK activation, oxidative response to fMLP by a chemiluminescence assay revealed that the same concentration inhibited the LPS-primed oxidative burst by only 40%. We conclude that in neutrophils, LPS-mediated activation of ERK 1 and 2 requires plasma and that this activation is not dependent on fibronectin. In addition, we found that the ERK pathway is not responsible for the lack of LPS priming in neutrophils of newborns but may be required for 40% of the LPS-primed fMLP-triggered oxidative burst in cells of adults.

Diverse effects of neutrophil integrin occupation on respiratory burst activation.

Integrin occupation can alter the function of neutrophils (PMN), but the mechanism(s) involved is still unclear. This study demonstrated that the occupation of PMN integrins (especially those of the beta(3) subfamily) strongly enhances TNF stimulation of the respiratory burst but down-regulates that induced by PMA, fMLP, Con A, and serum treated zymosan. Treatment of PMN with genistein, staurosporine, and wortmannin, inhibitors of tyrosine kinases, protein kinase C, and phosphotidylinostol 3-kinase (PI 3-kinase) respectively, completely blocked the TNF-stimulated respiratory burst in PMN. Genistein and wortmannin enhanced the PMA-stimulated respiratory burst but only in cells adherent to RGD peptide. These findings suggest that PMN integrins (beta(3) subfamily) can generate signals that regulate the PMN agonist responses, probably through the activities of tyrosine kinases and PI 3-kinase.

Beta2 integrin-dependent phosphorylation of protein-tyrosine kinase Pyk2 stimulated by tumor necrosis factor alpha and fMLP in human neutrophils adherent to fibrinogen.

Tumor necrosis factor alpha and fMLP can activate a broad range of cellular functions in neutrophils adherent to biological surfaces. These functions are mediated by integrins and involve the activation of tyrosine kinases. Here, we report that Pyk2, a member of the focal adhesion kinase family, was present in human neutrophils and was rapidly phosphorylated and activated following tumor necrosis factor alpha and fMLP stimulation in an adhesion-dependent manner. Tyrosine phosphorylation of Pyk2 was attenuated by beta2 integrin blocking with specific antibodies. The tyrosine phosphorylation of Pyk2 was downstream of protein kinases Lyn, Syk and protein kinase C and cytoskeletal organization. The activation of Pyk2 may play a role in adhesion/cytoskeleton-associated neutrophils function.

Src-family kinase-p53/ Lyn p56 plays an important role in TNF-alpha-stimulated production of O2- by human neutrophils adherent to fibrinogen.

Stimulation of neutrophil function by TNFalpha is largely dependent on beta2 integrins. It has also been proposed that src-family kinases are involved in this process. However, the functions of src-like kinases in human neutrophils still remain to be determined. In the present study, we used the new src-family kinase specific inhibitor PP1 [4-Amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine] to investigate the role src-kinases play in TNFalpha stimulation of neutrophil function. Our results demonstrated that, in neutrophils adherent to fibrinogen, PP1 inhibited TNFalpha-stimulated superoxide production and protein tyrosine phosphorylation in a dose-dependent manner. In in vitro kinase assays, PP1 profoundly inhibited the activation of p53/56lyn but not p59hck or p72syk. Only slight inhibition was found of p58c-fgr. These data indicate that p53/56lyn plays an important role in TNFalpha-mediated stimulation of PMN function.

Antibody-induced engagement of beta2 integrins in human neutrophils causes a rapid redistribution of cytoskeletal proteins, Src-family tyrosine kinases, and p72syk that precedes de novo actin polymerization.

Beta2 integrins mediate rearrangement of the cytoskeleton and activation of selective cell functions in neutrophils. To elucidate early events following beta2 integrin ligation, we analyzed redistribution of cytoskeletal and signaling proteins as a consequence of antibody-induced integrin clustering. Incubation of neutrophils on surface-bound anti-beta2 subunit antibodies induced a very rapid (within 1 min) redistribution of the cytoskeletal proteins talin, alpha-actinin, and paxillin, and the tyrosine kinases p58(c-fgr), p53/56(lyn), and p72(syk) to a cell fraction insoluble in Triton X-100. Cytoskeletal and signaling proteins redistribution preceded de novo actin polymerization because cytochalasin B did not inhibit this redistribution. Antibody engagement of all the three distinct beta2 integrins (CD11a, CD11b, CD11c) expressed by neutrophils induced redistribution of cytoskeletal proteins and tyrosine kinases. Several tyrosine phosphorylated proteins were also rapidly redistributed as a consequence of beta2 integrin engagement and this was not affected by blocking de novo actin polymerization with cytochalasin B. In addition, genistein, an inhibitor of tyrosine kinase activities which strongly reduced protein tyrosine phosphorylation, had no effect on redistribution of cytoskeletal proteins, Src-family kinases, and p72(syk). These findings suggest that integrin-dependent cytoskeleton rearrangement in neutrophils occurs in at least two distinct steps and nucleation of some cytoskeletal proteins together with tyrosine kinases precedes rearrangement of the actin-based cytoskeleton and tyrosine kinases activation. On the basis of these and previous findings we propose a model explaining mechanisms of integrin signaling in neutrophils.

Signaling by adhesion in human neutrophils: activation of the p72syk tyrosine kinase and formation of protein complexes containing p72syk and Src family kinases in neutrophils spreading over fibrinogen.

Src family kinases are implicated in signaling by integrins in polymorphonuclear neutrophils (PMN). To identify proteins present in complexes with Src family kinases, we subjected p58(c-fgr) or p53/56(lyn) immunoprecipitates from Triton X-100 lysates of PMN incubated on fibrinogen-coated surfaces to in vitro kinase assays. Assays on p58(c-fgr) or p53/56(lyn) immunoprecipitates from Triton lysates of PMN induced to spread over fibrinogen in response to TNF-alpha showed that several proteins form complexes with Src family kinases and can be phosphorylated in vitro. Immunoblot analysis showed that the p72(syk) tyrosine kinase is one of these proteins. Formation of protein complexes containing Src family kinases and p72(syk) required PMN spreading because p72(syk) was not detected in p58(c-fgr) or p53/56(lyn) immunoprecipitates from PMN, which were stimulated with TNF-alpha, in suspension. In addition, induction of PMN spreading with Mn2+ in the absence of TNF-alpha promoted the formation of protein complexes containing Src family kinases and p72(syk). We also found that p72(syk)-autophosphorylating kinase activity was enhanced, and a fraction of p72(syk) was translocated to a Triton-insoluble cytoskeletal fraction in PMN induced to spread over fibrinogen by TNF-alpha or Mn2+. In vitro kinase assays on CD18 (beta 2 integrin subunit) immunoprecipitates from Triton lysates of spread PMN showed that several proteins formed complexes with CD18 and could be phosphorylated in vitro. Immunoblot analysis of CD18 immunoprecipitates allowed us to identify p72(syk) as one of these proteins. These findings show that PMN spreading is accompanied by activation of p72(syk) and formation of multimolecular complexes in which Src family kinases and p72(syk) colocalize.

Regulation of Src family tyrosine kinase activities in adherent human neutrophils. Evidence that reactive oxygen intermediates produced by adherent neutrophils increase the activity of the p58c-fgr and p53/56lyn tyrosine kinases.

Src family tyrosine kinases have been implicated in the adhesion-dependent activation of neutrophil functions (Yan, S. R., Fumagalli, L., and Berton, G. (1995) J. Inflamm. 45, 297-312; Lowell, C. A., Fumagalli, L., and Berton, G. (1996) J. Cell Biol. 133, 895-910). Because the activity of tyrosine kinases can be affected by oxidants, we investigated whether reactive oxygen intermediates (ROI) produced by adherent neutrophils regulate Src family kinase activities. Inhibition of ROI production by diphenylene iodonium, an inhibitor of NADPH oxidase, or degradation of H2O2 by exogenously added catalase inhibited the adhesion-stimulated activities of p58(c-fgr) and p53/56(lyn). In addition, adhesion-stimulated p58(c-fgr) and p53/56(lyn) activities were greatly reduced in neutrophils from patients with chronic granulomatous disease (CGD) that are deficient in the production of ROI. Exogenously added H2O2 increased p58(c-fgr) and p53/56(lyn) activities in nonadherent neutrophils. Although ROI regulated the activities of p58(c-fgr) and p53/56(lyn), they did not affect the redistribution of the two kinases to a Triton X-100-insoluble, cytoskeletal fraction that occurs in adherent neutrophils. Tyrosine phosphorylation of proteins in adherent, CGD neutrophils was only partially inhibited, suggesting that the full activation of p58(c-fgr) and p53/56(lyn), which depends on endogenously produced ROI, does not represent an absolute requirement for protein tyrosine phosphorylation. The adhesion-stimulated activity of the tyrosine kinase p72(syk) was not affected by catalase in normal neutrophils, and it was comparable in normal and CGD neutrophils. These findings suggest that ROI endogenously produced by adherent neutrophils regulate Src family kinases activity selectively and establish the existence of a cross-talk between reorganization of the cytoskeleton, production of ROI, and Src family tyrosine kinase activities in signaling by adhesion.

Activation of SRC family kinases in human neutrophils. Evidence that p58C-FGR and p53/56LYN redistributed to a Triton X-100-insoluble cytoskeletal fraction, also enriched in the caveolar protein caveolin, display an enhanced kinase activity.

Protein tyrosine phosphorylation is one of the signals involved in stimulation of neutrophil (PMN) functions. We found that phorbol myristate acetate (PMA) activates the src family tyrosine kinases p58c-fgr and p53/56lyn in suspended PMNs. Moreover, we found that up to about 20% of p58c-fgr and p53/56lyn redistribute to a Triton X-100-insoluble fraction after PMA stimulation, and it is this fraction of the two kinases which diplays an increased activity. These changes of p58c-fgr and p53/56lyn distribution and activity correlate with tyrosine phosphorylation of endogenous substrates. In fact, in PMA-stimulated PMNs tyrosine phosphorylated proteins are mostly recovered in a Triton-insoluble cell fraction. To separate cytoskeletal from caveolar structures, which both display Triton X-100-insolubility, we used the detergent n-octyl beta-D-glucopyranoside (OGP) which solubilises components of caveolae. We found that the caveolae marker protein, caveolin, as well as the cytoskeletal protein alph-actinin and p58c-fgr and p53/56lyn, is insoluble in OGP. These findings suggest that PMA stimulation promotes the formation of multimolecular complexes containing cytoskeletal proteins, caveolin-containing structures and src family protein tyrosine kinases. Moreover, they show that p58c-fgr and p53/56lyn associated with this multimolecular complex display an enhanced kinase activity.

Neutrophil activation by adhesion: mechanisms and pathophysiological implications.

Neutrophil adhesion plays an essential role in the formation of an inflammatory exudate. Moreover, adhesion activates selective neutrophil functions and regulates the cell response to additional stimuli. In this review we summarize the information available on adhesion molecules involved in neutrophil adhesion to endothelial cells and extracellular matrix proteins and the experimental approaches which have been developed to block neutrophil adhesion and neutrophil mediated tissue damage. We also address the mechanisms of activation of selective neutrophil functions by adhesion molecules and, in particular the mechanisms of signal transduction by neutrophil integrins. On the basis of recent results obtained in our and other laboratories we propose a model hypothesizing mechanisms of signaling by neutrophil integrins involved in regulation of selective functions.

Tumor necrosis factor triggers redistribution to a Triton X-100-insoluble, cytoskeletal fraction of beta 2 integrins, NADPH oxidase components, tyrosine phosphorylated proteins, and the protein tyrosine kinase p58fgr in human neutrophils adherent to fibrinogen.

Tumor necrosis factor (TNF) triggers cell spreading, release of granule constituents, and production of toxic oxygen derivatives in human neutrophils adherent to fibrinogen. This response requires cytoskeleton reorganization and is dependent on expression of beta 2 integrins. We analyzed distribution of distinct proteins in Triton X-100-soluble and insoluble fractions in neutrophils adherent to fibrinogen. We found that stimulation of adherent neutrophils with TNF causes the redistribution to a Triton-insoluble fraction of alpha-actinin, beta 2 integrins, and the four components whose assembly constitutes an active NADPH oxidase: the gp91-phox, p22-phox, p47-phox, and p67-phox proteins. Redistribution of these different proteins to a Triton-insoluble fraction took relatively long times and was maximal after about 30 min of stimulation with TNF. Prevention of actin polymerization with cytochalasin B hampered the TNF-induced redistribution of these proteins from a Triton-soluble to an insoluble fraction. In addition, tyrosine phosphorylated proteins and the protein tyrosine kinase p58fgr were recovered in this Triton-insoluble fraction. These findings show that stimulated, beta 2 integrin-dependent adhesion of neutrophils to fibrinogen is accompanied by redistribution to cytoskeletal structures of (1) beta 2 integrins, that is, neutrophil receptors for fibrinogen; (2) proteins involved in neutrophil effector functions, that is, components of NADPH oxidase; and (3) tyrosine phosphorylated proteins and the protein tyrosine kinase p58fgr, molecules that are potentially involved in the formation of a submembranous signaling complex.

Activation of p58c-fgr and p53/56lyn in adherent human neutrophils: evidence for a role of divalent cations in regulating neutrophil adhesion and protein tyrosine kinase activities.

Tumor necrosis factor (TNF) stimulates generation of reactive oxygen intermediates, secretion of granule constituents, and rearrangement of the cytoskeleton in neutrophils (PMN); this response requires that PMN be adherent to plasma or extracellular matrix proteins, and is dependent on beta 2 integrins. Tyrosine phosphorylation of distinct proteins [Fuortes et al., J Cell Biol 120:777-784, 1993] and activation of the protein tyrosine kinase p58c-fgr [Berton et al., J Cell Biol 126:1111-1121, 1994] were recently recognized as signals involved in beta 2 integrin-dependent responses of TNF-treated PMN. As the integrin capability to bind their ligands is regulated by divalent cations we investigated whether modulation of PMN adhesion to fibrinogen by divalent cations also affected activation of protein tyrosine kinases. In the absence of divalent cations or in the presence of Ca2+ alone, PMN did not adhere to fibrinogen in response to TNF. However, Mg2+, either alone or together with Ca2+, promoted stimulated adhesion to fibrinogen. We also found that Mn2+ promoted PMN adhesion to fibrinogen without additional stimuli. Analysis of the activity of two src family tyrosine kinases, p58c-fgr and p53/56lyn, showed that their autophosphorylating kinase activity strictly correlated with adhesion. In fact, only in the presence of Mg2+, but not in the absence of divalent cations or in the presence of Ca2+ alone, TNF increased p58c-fgr and p53/56lyn kinase activities; and this was prevented by anti-CD18 antibodies. In addition, Mn2+ strongly promoted activation of p58c-fgr and p53/56lyn without additional stimuli. Analysis of tyrosine phosphorylated proteins with anti-phosphotyrosine immunoblots showed that divalent cations regulated adhesion and protein tyrosine phosphorylation in the same fashion. Detergent extraction of proteins showed that the Mg(2+)-dependent, TNF-stimulated adhesion redistributed p58c-fgr and p53/56lyn to a Triton-insoluble fraction. In addition, analysis of p58c-fgr activity allowed us to demonstrate that the fraction of p58c-fgr which became Triton-insoluble displayed a higher kinase activity. These findings establish that PMN adhesion signals for activation of two different src family tyrosine kinases. The evidence that Mn2+, a strong promoter of integrin function, induces adhesion and activation of tyrosine kinases without additional stimuli suggest the existence of a direct link between beta 2 integrins binding to fibrinogen and activation of tyrosine kinases in neutrophils.