CD39 and control of cellular immune responses

CD39 is the cell surface-located prototypic member of the ecto-nucleoside triphosphate diphosphohydrolase (E-NTPDase) family. Biological actions of CD39 are a consequence (at least in part) of the regulated phosphohydrolytic activity on extracellular nucleotides. This ecto-enzymatic cascade in tandem with CD73 (ecto-5–nucleotidase) also generates adenosine and has major effects on both P2 and adenosine receptor signalling. Despite the early recognition of CD39 as a B lymphocyte activation marker, little is known of the role of CD39 in humoral or cellular immune responses. There is preliminary evidence to suggest that CD39 may impact upon antibody affinity maturation. Pericellular nucleotide/nucleoside fluxes caused by dendritic cell expressed CD39 are also involved in the recruitment, activation and polarization of naïve T cells. We have recently explored the patterns of CD39 expression and the functional role of this ecto-nucleotidase within quiescent and activated T cell subsets. Our data indicate that CD39, together with CD73, efficiently distinguishes T regulatory cells (Treg) from other resting or activated T cells in mice (and humans). Furthermore, CD39 serves as an integral component of the suppressive machinery of Treg, acting, at least in part, through the modulation of pericellular levels of adenosine. We have also shown that the coordinated regulation of CD39/CD73 expression and of the adenosine receptor A2A activates an immunoinhibitory loop that differentially regulates Th1 and Th2 responses. The in vivo relevance of this network is manifest in the phenotype of Cd39-null mice that spontaneously develop features of autoimmune diseases associated with Th1 immune deviation. These data indicate the potential of CD39 and modulated purinergic signalling in the co-ordination of immunoregulatory functions of dendritic and Treg cells. Our findings also suggest novel therapeutic strategies for immune-mediated diseases.     

The rational simplification of a recombinant cocktail vaccine to control the parasitic nematode Teladorsagia circumcincta

Using data from five independent vaccine trials, which employed a subunit cocktail vaccine containing eight recombinant proteins to protect sheep against Teladorsagia circumcincta, a strategy was developed to simplify antigen complexity of the vaccine. A meta-analysis of data from these five trials demonstrated statistically significant reductions in cumulative faecal egg count and worm burden in vaccinated sheep when compared with those which had received adjuvant only (P = 0.009 and P < 0.0001, respectively). Relationships between antigen-specific antibody levels, antibody avidity and parasitological parameters of efficacy were analysed for each of the eight proteins in these trials. Of these, the strongest correlations between percentage reduction in cumulative faecal egg count and avidity were obtained for the vaccine antigen T. circumcincta apyrase-1 (Tci-APY-1) in relation to either total antigen-specific IgG or IgG1 in sera (P = 0.019 and P = 0.030, respectively). In addition, IgG and IgA within the serum and abomasal mucus of control (parasite challenged) lambs strongly recognised Tci-APY-1 and T. circumcincta metalloproteinase-1 (Tci-MEP-1) but only weakly bound the other six antigens, indicating Tci-APY-1 and Tci-MEP-1 are most effectively recognised by the parasite-induced antibody response. On the basis of these findings, a two-protein vaccine comprising Tci-APY-1 and Tci-MEP-1 was tested in a direct comparison with the original eight-component vaccine. A further group was immunised with Tci-MEP-1 in combination with a mutated form of Tci-APY-1 (mTci-APY-1), which had no enzymatic activity. Across the trial, the mean faecal egg count levels of the eight-antigen recipients were lower than those of the adjuvant only control group (P = 0.013) and the mean FEC of the mTci-APY-1 and Tci-MEP-1 recipients was lower, although not statistically significantly, than that of the adjuvant-only control group (P = 0.093). Mean cumulative faecal egg count levels were reduced by 43% in lambs immunised with mTci-APY-1 plus Tci-MEP-1 compared with the controls (P = 0.079).     

ATPase and Adpase activities in synovial membrane of equine metacarpophalangeal joint

ATPase and ADPase activities capable of hydrolyzing nucleoside di- and triphosphates in the presence of Ca2+ are present in synovial membrane of metacarpophalangeal joint mainly associated to membrane fractions. These hydrolytic activities have been considered involved in the inflammatory process where ATP and ADP are inflammatory mediators while adenosine counteracts this effect. Both, subcellular localization and kinetic properties of these nucleotidase activities, suggest that could correspond to single enzyme called ATP-diphosphohydrolase or apyrase. The comparison of the activity on ATP-Ca and ADP-Ca from normal and pathological equine synovial membrane did not show significant differences either in the subcellular fraction distribution or in the enrichment of each subcellular fraction. Neither differences on 5'-nucleotidase activity present in the microsomal fraction were observed.     

Apyrase Activity and Platelet Aggregation Inhibitors in the Tick Ornithodoros Savignyi (Acari: Argasidae)

Ticks are ectoparasites that cause considerable damage to their hosts while feeding. The feeding process is facilitated by anti-haemostatic factors present in the tick saliva. Apyrase (ATP diphosphohydrolase, EC 3.6.1.5) is a platelet aggregation inhibitor found in most haematophagous organisms studied. The present study describes the identification and characterization of such an activity in the tick Ornithodoros savignyi. The enzyme conformed to many properties common to apyrases. These included a low substrate specificity, dependence on bivalent metal ions for activity and insensitivity to the classical ATPase inhibitors. Heat denaturation studies, pH optima and similar effects of inhibitors on the enzyme's ATP and ADP hydrolysing activities supported its classification as an apyrase. Salivary gland extracts inhibited the platelet aggregation induced by ADP, collagen and thrombin and disaggregated aggregated platelets. The results suggest the presence of two or more anti-platelet factors present in the salivary glands of this tick species.     

双向BN/SDS-PAGE分离鉴定痢疾杆菌福氏5型M90T株毒力相关膜蛋白复合物

通过蓝色非变性凝胶电泳(BN-PAGE)比较痢疾杆菌福氏5型野生株M90T和大质粒缺失株M90T△T的膜蛋白复合物,发现一个野生株特有的复合物,其分子量的为290 kD,命名为M90T-290.通过第二向SDS-PAGE分离M90T-290得到6个蛋白亚基,质谱鉴定为:一个由大质粒编码的毒力蛋白Apyrase(ATP-二磷酸水解酶)和5个染色体编码的蛋白,这些蛋白可能以膜复合物的形式影响毒力蛋白IcsA的单极性分布和痢疾杆菌在细胞间的扩散.这个新发现的毒力相关膜复合物在痢疾杆菌的致病过程中可能发挥重要作用.     

Apyrase and 5′-nucleotidase Activities in Synaptosomes from the Cerebral Cortex of Rats Experimentally Demyelinated with Ethidium Bromide and Treated with Interferon-β

Apyrase and 5′-nucleotidase activities were analyzed in an ethidium bromide (EB) demyelinating model associated with interferon-β (IFN-β). The animals were divided in groups: I, control (saline); II, saline and IFN-β; III, EB and IV, EB and IFN-β. After 7, 15 and 30 days the animals (n=5) were sacrificed and the cerebral cortex was removed for synaptosome preparation and enzymatic assays. Apyrase activity using ATP as substrate increased in groups II, III and IV (P<0.001) after 7 days and in groups III and IV (P<0.001) after 15 days. Using ADP as substrate, an activation of this enzyme was observed in group III (P<0.05) after seven and 15 days. The 5′-nucleotidase activity increased in group III (P<0.05) after 7 days and in groups II, III and IV (P<0.001) after 15 days. After 30 days treatment, no significant alteration was observed in enzyme activities. Results showed that apyrase and 5′-nucleotidase activities are altered in demyelination events and that IFN-β was able to regulate the adenine nucleotide hydrolysis.     

Effects of phenylalanine and phenylpyruvate on ATP-ADP hydrolysis by rat blood serum

The nucleotide (ATP-ADP)/nucleoside (adenosine) ratio in the circulation can modulate the processes of vasoconstriction, vasodilatation and platelet aggregation. The main objective of the present study with rat blood serum was to evaluate the possibility of changes in nucleotide hydrolysis by phenylalanine (Phe) and phenylpyruvate (PP), the levels of which could increase in the circulation of individuals with phenylketonuria. Results demonstrated that Phe in the range 1.0-5.0 mM inhibited the ADP hydrolysis by rat serum. The effect of inhibition by Phe on ATP hydrolysis appeared only at a concentration of 5.0 mM. PP had no significant effect upon nucleotide hydrolysis. Kinetic analysis indicated that the inhibition of ADP and ATP hydrolysis by Phe in rat blood serum is uncompetitive. Conversely, Phe and PP did not affect the hydrolysis of p-nitrophenyl-5'-TMP by rat serum.     

Endogenous extracellular purine nucleotides redirect α2-adrenoceptor signaling

Many receptors coupled to inhibitory G_o/G_i-type G proteins often also produce stimulatory signals like Ca²⁺ mobilisation. When expressed in CHO cells the α₂-adrenoceptor subtypes α_2A, α_2B and α_2C mobilised Ca²⁺. These responses were strongly reduced by the P_2Y-purinoceptor antagonist suramin. A large proportion of the total pool of purine nucleotides was found extracellularly. Removal of extracellular nucleotides with apyrase or by constant perfusion had a similar effect as suramin. These treatments did not affect the α₂-adrenoceptor-mediated inhibition of cAMP production. This indicates that cells may be primed or their signaling pathways redirected towards Ca²⁺ mobilisation by `autocrine' release of nucleotides.