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Background & Aims: Exhaustion of CD8 T cells has been suggested to inform different clinical outcomes in Crohn's disease, but detailed analyses are lacking. This study aimed to identify the role of exhaustion on a single-cell level and identify relevant CD8 T cell populations in Crohn's disease., Methods: Blood and intestinal tissue from 58 patients with Crohn's disease (active disease or remission) were assessed for CD8 T cell expression of exhaustion markers and their cytokine profile by highly multiplexed flow and mass cytometry. Key disease-associated subsets were sorted and analyzed by RNA sequencing. CD39 inhibition assays were performed in vitro., Results: Activated CD39 + and CD39 + PD-1 + CD8 T cell subsets expressing multiple exhaustion markers were enriched at low frequency in active Crohn's disease. Their cytokine production capacity was inversely linked to the Harvey-Bradshaw Index. Subset-level protein and transcriptome profiling revealed co-existence of effector and exhaustion programs in CD39 + and CD39 + PD-1 + CD8 T cells, with CD39 + cells likely originating from the intestine. CD39 enzymatic activity controlled T cell cytokine production. Importantly, transcriptional exhaustion signatures were enriched in remission in CD39-expressing subsets with up-regulation of TOX. Subset-level transcriptomics revealed a CD39-related gene module that is associated with the clinical course., Conclusions: These data showed a role for the exhaustion of peripheral CD39-expressing CD8 T cell subsets in Crohn's disease. Their low frequency illustrated the utility of single-cell cytometry methods for identification of relevant immune populations. Importantly, the link of their exhaustion status to the clinical activity and their specific gene signatures have implications for exhaustion-based personalized medicine approaches., (Copyright © 2022 AGA Institute. Published by Elsevier Inc. All rights reserved.)

CD39/NTPDase1 has emerged as an important molecule that contributes to maintain inflammatory and coagulatory homeostasis. Various studies have hypothesized the possible role of CD39 in COVID-19 pathophysiology since no confirmatory data shed light in this regard. Therefore, we aimed to quantify CD39 expression on COVID-19 patients exploring its association with severity clinical parameters and ICU admission, while unraveling the role of purinergic signaling on thromboinflammation in COVID-19 patients. We selected a prospective cohort of patients hospitalized due to severe COVID-19 pneumonia (n=75), a historical cohort of Influenza A pneumonia patients (n=18) and sex/age-matched healthy controls (n=30). CD39 was overexpressed in COVID-19 patients' plasma and immune cell subsets and related to hypoxemia. Plasma soluble form of CD39 (sCD39) was related to length of hospital stay and independently associated with intensive care unit admission (adjusted odds ratio 1.04, 95%CI 1.0-1.08, p=0.038), with a net reclassification index of 0.229 (0.118-0.287; p=0.036). COVID-19 patients showed extracellular accumulation of adenosine nucleotides (ATP and ADP), resulting in systemic inflammation and pro-coagulant state, as a consequence of purinergic pathway dysregulation. Interestingly, we found that COVID-19 plasma caused platelet activation, which was successfully blocked by the P2Y 12 receptor inhibitor, ticagrelor. Therefore, sCD39 is suggested as a promising biomarker for COVID-19 severity. As a conclusion, our study indicates that CD39 overexpression in COVID-19 patients could be indicating purinergic signaling dysregulation, which might be at the basis of COVID-19 thromboinflammation disorder., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Díaz-García, García-Tovar, Alfaro, Zamarrón, Mangas, Galera, Ruíz-Hernández, Solé-Violán, Rodríguez-Gallego, Van-Den-Rym, Pérez-de-Diego, Nanwani-Nanwani, López-Collazo, García-Rio and Cubillos-Zapata.)

Natural regulatory T cells (nTregs) ensure the control of self-tolerance and are currently used in clinical trials to alleviate autoimmune diseases and graft-versus-host disease after hematopoietic stem cell transfer. Based on CD39/CD26 markers, blood nTreg analysis revealed the presence of five different cell subsets, each representing a distinct stage of maturation. Ex vivo added microenvironmental factors, including IL-2, TGFβ, and PGE2, direct the conversion from naive precursor to immature memory and finally from immature to mature memory cells, the latest being a no-return stage. Phenotypic and genetic characteristics of the subsets illustrate the structural parental maturation between subsets, which further correlates with the expression of regulatory factors. Regarding nTreg functional plasticity, both maturation stage and microenvironmental cytokines condition nTreg activities, which include blockade of autoreactive immune cells by cell-cell contact, Th17 and IL-10 Tr1-like activities, or activation of TCR-stimulating dendritic cell tolerization. Importantly, blood nTreg CD39/CD26 profile remained constant over a 2-y period in healthy persons but varied from person to person. Preliminary data on patients with autoimmune diseases or acute myelogenous leukemia illustrate the potential use of the nTreg CD39/CD26 profile as a blood biomarker to monitor chronic inflammatory diseases. Finally, we confirmed that naive conventional CD4 T cells, TCR-stimulated under a tolerogenic conditioned medium, could be ex vivo reprogrammed to FOXP3 lineage Tregs, and further found that these cells were exclusively committed to suppressive function under all microenvironmental contexts., Competing Interests: Conflict of interest statement: D.Z. is a cofounder of Neovacs, and R.C. is an employee of eBioscience., (Copyright © 2019 the Author(s). Published by PNAS.)

Our study aimed to study regulatory T cells (Tregs) and their expression of CD45RA, HLA-DR, and CD39 in preterm and full-term infants. In an observational study, we used a three-color flow cytometry for determination of Tregs and their expression of CD45RA, HLA-DR, and CD39 in preterm and full-term infants. The percentages of CD4 + CD25 +high Foxp3 + , CD39 + Tregs, HLA-DR + Tregs and the expression of Foxp3 + in CD4 + CD25 +high Foxp3 Tregs cells were significantly lower in neonates when compared to healthy adult controls. The levels of naïve resting Tregs (CD45RA + Tregs) were significantly higher in neonates than controls. The percentages of CD4 + CD25 +high Foxp3 + Tregs, total CD4 + CD25 + and CD4 + CD25 +high were significantly higher in preterm infants when compared to the full-term group. Moreover, CD45RA + Tregs were significantly higher in preterm than in term infants. We found significant inverse correlations between the gestational age and the levels of both Tregs (r = - 0.395, p = 0.017) and CD45RA + Tregs (r = - 0.422, p = 0.010). Relative to full-term, the frequencies, and phenotypes of Tregs were affected by prematurity. A larger longitudinal study with a sufficient number of newborns is needed to investigate the Treg pool of term and preterm infants thoroughly and to explore the association between the Treg pool and clinical variables.

Signaling mediated by purines is a widespread mechanism of cell-cell communication related to vasomotor responses and the control of platelet function in the vascular system. However, little is known about the involvement of this signaling as well as the role of reactive oxygen species (ROS) in the development of hypothyroidism. Therefore, the present study investigates changes in the purinergic system, including enzyme activities and expression in platelets, and oxidative profiles in patients with post-thyroidectomy hypothyroidism. The nucleoside triphosphate diphosphohydrolase 1 (NTPDase/CD39) expression in patients increased by 40%, and the adenosine triphosphate (ATP) or adenosine diphosphate (ADP) hydrolyzing activity increased by 82% and 70%, respectively. The activities of ecto-5´-nucleotidase and adenosine deaminase (ADA) also significantly enhanced (39% and 52%, respectively), which correlates with a 45% decrease in adenosine concentration. Furthermore, these patients demonstrated an increased production of ROS (42%), thiobarbituric acid reactive substances (TBARS) (115%), carbonyl protein (30%) and a decreased glutathione S-transferase (GST) activity (20%). This study demonstrates that hypothyroidism interferes with adenine nucleoside and nucleotide hydrolysis and this is correlated with oxidative stress, which might be responsible for the increase in ADA activity. This increase causes rapid adenosine deamination, which can generate a decrease in their concentration in the systemic circulation, which can be associated with the development of vascular complications.

Aim: Nearly one-third of patients with rheumatoid arthritis (RA) do not respond to Methotrexate (MTX), the first-line therapy in RA. CD39, an ectonucleotidase highly expressed on regulatory T cells (Tregs), is responsible for production of adenosine, an important anti-inflammatory mediator of MTX action. Higher expression of CD39 on Tregs improves their suppressive capacity. Therefore, we aimed to study the role of CD39 + Treg frequency as a biomarker for MTX treatment response in RA., Methods: Patients with active RA who were naive to disease-modifying anti-rheumatic drugs were enrolled. Frequencies of CD39 + Tregs (CD4 + CD25 + FoxP3 + CD39 + cells) and CD4 + CD25 + CD39 + cells were determined by flow cytometry in peripheral blood before the start of therapy. After 4 months of MTX monotherapy, patients were classified into responders (European League Against Rheumatism [EULAR] good/moderate response) and non-responders (EULAR no response). All samples were genotyped for single nucleotide polymorphisms (SNPs) rs11188513 and rs7071836 in the ENTPD1 (CD39) gene., Results: After 4 months of MTX monotherapy, 54 patients were classified as responders and 16 as non-responders. The baseline CD39 + Treg and CD4 + CD25 + CD39 + cell frequencies were significantly higher in the responder group as compared with the non-responder group (P < 0.05 and P < 0.01, respectively). AA genotype at SNP rs7071836 was associated with poor response to MTX (P < 0.05; odds ratio = 5.67; 95% CI = 1.12-28.75)., Conclusion: Higher frequencies of CD39 + Tregs and CD4 + CD25 + CD39 + cells in the peripheral blood are associated with response to MTX in RA and hence, these could be considered as potential biomarkers for prediction of response to MTX treatment., (© 2018 Asia Pacific League of Associations for Rheumatology and John Wiley & Sons Australia, Ltd.)

Objective: Recent studies have suggested that the enteric nervous system can modulate gut immunity. Ecto-nucleoside triphosphate diphosphohydrolases [E-NTPDases] regulate purinergic signalling by sequential phosphohydrolysis of pro-inflammatory extracellular adenosine 5'-triphosphate [ATP]. Herein, we test the hypothesis that E-NTPDases modulate gut inflammation via neuro-immune crosstalk., Design: We determined expression patterns of NTPDase2 and NTPDase3 in murine and human colon. Experimental colitis was induced by dextran sodium sulphate [DSS] in genetically engineered mice deficient in NTPDase2 or NTPDase3. We compared plasma adenosine diphosphatase [ADPase] activity from Crohn's patients and healthy controls, and linked the enzyme activity to Crohn's disease activity., Results: NTPDase2 and -3 were chiefly expressed in cells of the enteric nervous system in both murine and human colon. When compared with wild type, DSS-induced colitis was exacerbated in Entpd2, and to a lesser extent, Entpd3 null mice as measured by disease activity score and histology, and marked anaemia was seen in both. Colonic macrophages isolated from Entpd2 null mice displayed a pro-inflammatory phenotype compared with wild type. In human plasma, Crohn's patients had decreases in ADPase activity when compared with healthy controls. The drop in ADPase activity was likely associated with changes in NTPDase2 and -3, as suggested by inhibitor studies, and were correlated with Crohn's disease activity., Conclusions: NTPDase2 and -3 are ecto-enzymes expressed in the enteric nervous system. Both enzymes confer protection against gut inflammation in experimental colitis and exhibit alterations in Crohn's disease. These observations suggest that purinergic signalling modulated by E-NTPDases governs neuro-immune interactions that are relevant in Crohn's disease., (Copyright © 2017 European Crohn’s and Colitis Organisation (ECCO). Published by Oxford University Press. All rights reserved. For permissions, please email: journals.permissions@oup.com)

Aim: Regulatory T cells (Treg) are important in mediating immune tolerance and outcomes of allotransplantation. CD4 + CD25 + CD39 + co-expression identifies memory Treg; CD4 + CD25 - CD39 + memory T effectors. We sought to determine CD4 + CD25 +/- CD39 + expression from the peripheral blood of patients with end stage renal failure, following transplantation and during episodes of acute cellular rejection., Methods: CD4 + T cells were isolated from peripheral blood leucocytes and analysed for CD25 and CD39 expression by flow cytometry. Treg suppressive function was measured by suppression of autologous effector T-cell proliferation by Treg in co-culture., Results: CD4 + CD25 +/- CD39 + T-cell subsets were tracked longitudinally in the peripheral blood of 17 patients following renal transplantation. Patients with acute T-cell-mediated rejection diagnosed on biopsy had reduced CD4 + CD25 + CD39 + mTreg (P < 0.05) and CD4 + CD25 - CD39 + mTeff (P < 0.01) cells compared with non-rejecting patients. CD4 + CD25 + CD39 + mTreg (P < 0.05) and CD4 + CD25 - CD39 + mTeff (P = 0.057) were reduced in long-term transplant patients (>1 year) compared with non-immunosuppressed controls. Interestingly, remaining CD4 + CD25 + CD39 + mTreg in the stable transplant patients displayed more potent suppressive capacity compared with non-immunosuppressed controls (83.2% ± 3.1% vs 45.7% ± 8.0%, nTeff:Treg ratio 8:1, P < 0.01)., Conclusion: CD4 + CD25 + CD39 + mTreg and CD4 + CD25 - CD39 + mTeff in peripheral blood can be tracked in renal transplant patients. Acute cellular rejection was accompanied by reduced mTreg and mTeff. Determining changes in these T-cell subsets may help to identify patients with, or at high risk of, renal allograft rejection., (© 2016 Asian Pacific Society of Nephrology.)

The aim of this study was to evaluate seric NTPDase and 5'nucleotidase activities of cattle naturally infected by Eurytrema coelomanticum, as well as to correlate them to histopathological lesions in the pancreas and the degree of parasitism. Blood samples and pancreas of 51 bovines were collected on a slaughterhouse in Southern Brazil: 33 from cattle naturally infected by E. coelomanticum (the Group A), and 18 from uninfected animals (the Group B). Infected animals showed an average of 532 parasites per pancreas. In the pancreatic histology, ducts displayed hyperplasia, stenosis, proliferation of fibrous tissue, and interstitial inflammatory infiltration of lymphocytes. The serum from infected animals showed an increase in NTPDase activity when ATP was used as substrate (P<0.001). For the ADP substrate, there was no difference between groups regarding NTPDase activity (P=0.37), as well as 5'-nucleotidase activity (P=0.27). Correlating NTPDase activity (ATP substrate) with the degree of histopathological lesions (rho=0.66, P<0.001) and the parasitic load on the pancreas (rho=0.65, P<0.001), a positive correlation was observed. Similar results were found between the degree of histopathological lesions and NTPDase activity (ADP substrate; rho=0.29, P=0.03), and 5'nucleotidase activity (rho=0.35, P=0.01). Based on the results of NTPDase and 5'nucleotidase enzymes in cattle naturally infected by E. coleomanticum, it is possible to suggest that these enzymes are involved in the modulation of inflammation, and they can act as markers of inflammatory response., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Background: Glioblastoma multiforme (GBM), a common primary malignant brain tumor, rarely disseminates beyond the central nervous system and has a very bad prognosis. The current study aimed at the analysis of immunological control in individual patients with GBM., Methods: Immune phenotypes and plasma biomarkers of GBM patients were determined at the time of diagnosis using flow cytometry and ELISA, respectively., Results: Using descriptive statistics, we found that immune anomalies were distinct in individual patients. Defined marker profiles proved highly relevant for survival. A remarkable relation between activated NK cells and improved survival in GBM patients was in contrast to increased CD39 and IL-10 in patients with a detrimental course and very short survival. Recursive partitioning analysis (RPA) and Cox proportional hazards models substantiated the relevance of absolute numbers of CD8 cells and low numbers of CD39 cells for better survival., Conclusions: Defined alterations of the immune system may guide the course of disease in patients with GBM and may be prognostically valuable for longitudinal studies or can be applied for immune intervention.

The adenosinergic pathway converting endogenous ATP to adenosine (ADO) is a major immunosuppressive pathway in cancer. Emerging data indicate that plasma small extracellular vesicles (sEV) express CD39 and CD73 and produce ADO. Using a noninvasive, highly sensitive newly developed assay, metabolism of N 6 -etheno-labeled eATP, eADP or eAMP by ecto-nucleotidases on the external surface of sEV was measured using high pressure liquid chromatography with fluorescence detection. Ecto-nucleotidase activity in sEV isolated from plasma of randomly selected cancer patients and healthy donors (HDs) was compared. Relative to sEV of HDs, sEV from the plasma of melanoma patients metabolized eATP to eADP and eAMP to eADO with significantly greater efficiency. Activities of both CD39 and CD73 were elevated, as determined by the use of pharmacologic inhibitors selective for these enzymes. In contrast, metabolic activity of CD39 and CD73 on sEV isolated from plasma of patients with head and neck cancer was comparable to that of HDs, suggesting that the activity of ecto-nucleotidases on sEV may differ depending on the cancer type or cancer stage. The N 6 -etheno-purine assay measuring contributions of ecto-nucleotidases residing on the surface of sEV to the extracellular ATP to ADO pathway can discriminate cancer patients from HDs, differentiate among different cancer types, and potentially identify patients most likely to benefit from anti-adenosinergic therapy designed to inhibit the adenosine-mediated immune suppression.

Background: To evaluate whether changes in expression of CD39 by regulatory T lymphocytes (Treg) impact treatment response in inflammatory bowel disease. To then define the biological role of expression of CD39 on Treg in an animal model of colitis., Methods: A prospective study of consecutive patients commencing anti-tumor necrosis factor therapy with infliximab (IFX) or adalimumab (ADA), who were then followed for 12 months. Treatment responses were defined both symptomatically and by endoscopy showing mucosal healing. Peripheral blood Tregs were quantified by flow cytometry. Functional importance of CD39 expression by Treg was determined in an adoptive T-cell transfer model of colitis., Results: Forty-seven patients (ulcerative colitis, n = 22; Crohn's disease, n = 25) were recruited; 16 patients were complete responders and 13 nonresponders to anti-tumor necrosis factor. CD39 expression by Treg was lower in active inflammatory bowel disease and increased significantly after treatment in responders (CD39Treg/total Treg; 8% at baseline to 22.5% at late time point, P < 0.001). Responders were more likely to have therapeutic drug levels and in multivariate analysis therapeutic drug levels were associated with higher expression of CD39 by FoxP3 Treg and lower frequencies of interleukin 17A expressing cells. Tregs with genetic deletion of CD39 exhibit decrements in potential to suppress intestinal inflammation in a murine (CD45RB) T-cell transfer model of colitis in vivo, when compared with wild-type Treg., Conclusions: Increased expression of CD39 by peripheral blood Treg is observed in the setting of clinical and endoscopic remission in inflammatory bowel disease. Deficiency of CD39 expression by Treg can be linked to inability to suppress experimental colitis.

Extracellular adenosine 5'-triphosphate (ATP) has significant effects on a variety of pathological conditions and it is the main physiological agonist of P2X7 purinergic receptor (P2X7R). It is known that ATP acting via purinergic receptors plays a relevant role on skin inflammation, and P2X7R is required to neutrophil recruitment in a mice model of irritant contact dermatitis (ICD).The present study investigated the effects of chemical irritant croton oil (CrO) upon ATP, ADP, and AMP hydrolysis in mice blood serum, and the potential involvement of P2X7R. The topical application CrO induced a decrease on soluble ATP/ADPase activities (~50 %), and the treatment with the selective P2X7R antagonist, A438079, reversed these effects to control level. Furthermore, we showed that CrO decreased cellular viability (52.6 % ± 3.9) in relation to the control and caused necrosis in keratinocytes (PI positive cells). The necrosis induced by CrO was prevented by the pre-treatment with the selective P2X7R antagonist A438079. The results presented herein suggest that CrO exerts an inhibitory effect on the activity of ATPDase in mouse serum, reinforcing the idea that ICD has a pathogenic mechanism dependent of CD39. Furthermore, it is tempting to suggest that P2X7R may act as a controller of the extracellular levels of ATP.

Rationale: Purinergic signaling plays an important role in inflammation and vascular integrity, but little is known about purinergic mechanisms during the pathogenesis of atherosclerosis in humans., Objective: The objective of this study is to study markers of purinergic signaling in a cohort of patients with peripheral artery disease., Methods and Results: Plasma ATP and ADP levels and serum nucleoside triphosphate diphosphohydrolase-1 (NTPDase1/CD39) and ecto-5'-nucleotidase/CD73 activities were measured in 226 patients with stable peripheral artery disease admitted for nonurgent invasive imaging and treatment. The major findings were that ATP, ADP, and CD73 values were higher in atherosclerotic patients than in controls without clinically evident peripheral artery disease (P<0.0001). Low CD39 activity was associated with disease progression (P=0.01). In multivariable linear regression models, high CD73 activity was associated with chronic hypoxia (P=0.001). Statin use was associated with lower ADP (P=0.041) and tended to associate with higher CD73 (P=0.054), while lower ATP was associated with the use of angiotensin receptor blockers (P=0.015)., Conclusions: Purinergic signaling plays an important role in peripheral artery disease progression. Elevated levels of circulating ATP and ADP are especially associated with atherosclerotic diseases of younger age and smoking. The antithrombotic and anti-inflammatory effects of statins may partly be explained by their ability to lower ADP. We suggest that the prothrombotic nature of smoking could be a cause of elevated ADP, and this may explain why cardiovascular patients who smoke benefit from platelet P2Y12 receptor antagonists more than their nonsmoking peers., (© 2015 American Heart Association, Inc.)

Background: Ectonucleotidase plays an important role in the regulation of cardiac function by controlling extracellular levels of adenine nucleotides and adenosine. To determine the influence of ischemia-reperfusion injury on ectonucleotidase activity in coronary vascular bed, we compared the metabolic profile of adenine nucleotides during the coronary circulation in pre- and post-ischemic heart., Methods: Langendorff-perfused rat hearts were used to assess the intracoronary metabolism of adenine nucleotides. The effects of ischemia on the adenine nucleotide metabolism were examined after 30 min of ischemia and 30 min of reperfusion. Adenine nucleotide metabolites were measured by high performance liquid chromatography., Results: ATP, ADP and AMP were rapidly metabolized to adenosine and inosine during the coronary circulation. After ischemia, ectonucleotidase activity of the coronary vascular bed was significantly decreased. In addition, the perfusate from the ischemic heart contained a considerable amount of enzymes degrading ATP, AMP and adenosine. Immunoblot analysis revealed that the perfusate from the ischemic heart dominantly contained ectonucleoside triphosphate diphosphohydrolase 1, and, to a lesser extent, ecto-5'-nucleotidase. The leakage of nucleotide metabolizing enzymes from the coronary vascular bed by ischemia-reperfusion was more remarkable in aged rats, in which post-ischemic cardiac dysfunction was more serious., Conclusion: Ectonucleotidases were liberated from the coronary vascular bed by ischemia-reperfusion, resulting in an overall decrease in ectonucleotidase activity in the post-ischemic coronary vascular bed. These results suggest that decreased ectonucleotidase activity by ischemia may exacerbate subsequent reperfusion injury, and that levels of circulating ectonucleotidase may reflect the severity of ischemic vascular injury.

Background: We have previously demonstrated that CD133 and CD39 are expressed by hematopoietic stem cells (HSC), which are mobilized after liver injury and target sites of injury, limit vascular inflammation, and boost hepatic regeneration. Plasma microparticles (MP) expressing CD39 can block endothelial activation. Here, we tested whether CD133 MP might be shed in a CD39-dependent manner in a model of liver injury and could potentially serve as biomarkers of liver failure in the clinic., Methods: Wild-type and Cd39-null mice were subjected to acetaminophen-induced liver injury. Mice were sacrificed and plasma MP were isolated by ultracentrifugation. HSC and CD133 MP levels were analyzed by fluorescence-activated cell sorting. Patients were enrolled with acute (n=5) and acute on chronic (n=5) liver injury with matched controls (n=7). Blood was collected at admission and plasma CD133 and CD39 MP subsets were analyzed by fluorescence-activated cell sorting., Results: HSC and CD133 MP levels were significantly increased only in the plasma of wild-type mice with acetaminophen hepatotoxicity (P<0.05). No increases in CD133 MP were noted in Cd39-null mice. Plasma MP increases were observed in patients with liver injury. These MP were characterized by significantly higher levels of CD39 (P<0.05)., Conclusions: HSC and plasma CD133 MP levels increase in a CD39-dependent manner during experimental acute liver injury. Increased levels of CD39 MP are differentially noted in patients with liver injury. Further research is needed to determine whether MP fluxes are secondary to pathophysiologic insults to the liver or might reflect compensatory responses.

The present study aimed to investigate the influence of N-acetylcysteine (NAC) on cadmium (Cd) poisoning by evaluating Cd concentration in tissues, hematological indices as well as the activity of NTPDase, acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) enzymes of rats exposed to Cd and co-treated with NAC. For this purpose, the rats received Cd (2 mg/kg) and NAC (150 mg/kg) by gavage every other day for 30 days. Animals were divided into four groups (n = 6-8): control/saline, NAC, Cd, and Cd/NAC. Cd exposure increased Cd concentration in plasma, spleen and thymus, and NAC co-treatment modulated this augment in both lymphoid organs. Cd exposure reduced red blood cell count, hemoglobin content and hematocrit value. Cd intoxication caused a decrease in total white blood cell count. NAC treatment per se caused an increase in lymphocyte and a decrease in neutrophil counts. On contrary, Cd exposure caused a decrease in lymphocyte and an increase in neutrophil and monocyte counts. NAC reversed or ameliorated the hematological impairments caused by Cd poisoning. There were no significant alterations in the NTPDase activity in lymphocytes of rats treated with Cd and/or NAC. Cd caused a decrease in the activities of lymphocyte AChE, whole blood AChE and serum BChE. However, NAC co-treatment was inefficient in counteracting the negative effect of Cd in the cholinesterase activities. The present investigation provides ex vivo evidence supporting the hypothesis that Cd induces immunotoxicity by interacting with the lymphoid organs, altering hematological parameters and inhibiting peripheral cholinesterase activity. Also, it highlights the possibility to use NAC as adjuvant against toxicological conditions.

Objective: Hypothermia is used in various clinical settings to inhibit ischemia-related organ damage. However, prothrombotic effects have been described as potential side effects. This study aimed to elucidate the mechanism of hypothermia-induced platelet activation and subsequent prothrombotic events and to develop preventative pharmacological strategies applicable during clinically used hypothermia., Methods and Results: Platelet function was investigated ex vivo and in vivo at clinically used hypothermia (28°C/18°C). Hypothermic mice demonstrated increased expression of platelet activation marker P-selectin, platelet-leukocyte aggregate formation, and thrombocytopenia. Intravital microscopy of FeCl(3)-injured murine mesenteric arteries revealed increased platelet thrombus formation with hypothermia. Ex vivo flow chamber experiments indicated increased platelet-fibrinogen adhesion under hypothermia. We show that hypothermia results in reduced ADP hydrolysis via reduction of CD39 (E-NTPDase1) activity, resulting in increased levels of ADP and subsequent augmented primary and secondary platelet activation. In vivo administration of ADP receptor P(2)Y(12) antagonists and recombinant soluble CD39 prevented hypothermia-induced thrombus formation and thrombocytopenia, respectively., Conclusions: The platelet agonist ADP plays a key role in hypothermia-induced platelet activation. Inhibition of receptor binding or hydrolysis of ADP has the potential to protect platelets against hypothermia-induced activation. Our findings provide a rational basis for further evaluation of novel antithrombotic strategies in clinically applied hypothermia.

Circadian rhythms represent an important mechanism to prepare the organism for environmental variations. ATP, ADP, AMP, and adenosine can act as extracellular messengers in a range of biological processes and are metabolized by a number of enzymes, including NTPDases and 5'-nucleotidase. In the present study the authors report that ATPase and ADPase activities present 24-h temporal variations that peak during dark (activity) span. These findings suggest that this enzymatic temporal pattern in blood serum might be important for the normal physiology and function of the organism through the maintenance of extracellular nucleotides at physiological levels.

Background: Uterine cervical neoplasia is an important worldwide malignancy sometimes associated with thrombosis. Ectonucleotidases are membrane-bound enzymes which participate in thromboregulation by hydrolyzing adenine nucleotides in the extracellular medium. In this sense, we aimed to investigate their activity in patients with uterine cervical neoplasia., Methods: We evaluated NTPDase and 5'-nucleotidase activities from patients previously treated for uterine cervical neoplasia with either conization or radiotherapy (RTX). These patients were divided into four groups: two conization groups (I and II) and two RTX groups (III and IV), which were further divided based on the amount of time that had passed since the conclusion of their treatment, where groups I and III were extended-remission-period groups (patients with 1 to 5 years elapsed after the conclusion of treatment), and groups II and IV were recently treated patients (treated up to three months before)., Results: For both conization and RTX groups, ATP and ADP hydrolysis decreased in the extended-remission groups when compared to the control and recently treated groups. On the other hand, AMP hydrolysis was decreased in all the treated groups (both conization and RTX) compared to the control. CD39 expression was decreased in extended-remission groups (I and III) when compared to the other groups., Conclusions: NTPDase protects against platelet aggregation and 5'-nucleotidase is more involved in the control of adenosine formation., (2010 Elsevier Masson SAS. All rights reserved.)

Influence of DL-homocysteine thiolactone loading (100 mg/kg by intragastric administration for 28 days) on enzymes activity of adenylic nucleotide and adenosine metabolism in the blood serum, platelets and liver of rats was investigated. The relation between revealed disturbance and platelet hyper-reactivity was estimated. It was established, that apyrase and 5'-nucleotidase activities decreased and adenosine deaminase activity increased in platelets of the rats with hyperhomocysteinemia (HHC). HHC also interrupted adenosine production in the blood serum and liver in rats. Under this condition the platelet sensitivity to ADP-stimulation was significantly increased. Vitamin-microelement complex decreased HHC-induced disorder of adenosine metabolism and prevented platelet hyper-reactivity formation. In vitro homocysteine inhibited platelet hydrolysis of ADP and AMP in a dose-dependent manner and this effect reduced in the presence of hydrogen sulfide donor NaHS.

Objectives: The objective of this work was to evaluate the effect of different glucose levels on the ATP, ADP and AMP hydrolysis in the platelets of diabetic, hypertensive and diabetic/hypertensive participants., Methods: The activities of the enzymes NTPDase (ATP and ADP hydrolysis) and 5'-nucleotidase (AMP hydrolysis), and CD39 expression were analyzed in human blood platelets of diabetic (DM-2), hypertensive (HT) and diabetic/hypertensive (DM-2/HT) patients. To evaluate the interference of glucose and fructose in NTPDase and 5'-nucleotidase activities, experiments were performed with glucose, fructose and mannitol concentrations ranging from 5 to 30 mM in platelet-rich plasma (PRP). Pre-incubation times of 10, 120 min and 24h were used., Results: NTPDase and 5'-nucleotidase activities increased with increasing glucose and fructose concentrations (P<0.001) and the different times of pre-incubation did not interfere in ectonucleotidases activities (P>0.5). NTPDase and 5'-nucleotidase activities demonstrated a positive correlation between serum glucose levels and ATP and ADP hydrolysis in DM-2 and DM-2/HT patients. CD39 expression demonstrated that DM-2, HT and DM-2/HT groups presented a significant increase when compared to the control group (P<0.004)., Conclusion: The hydrolysis of adenine nucleotides is enhanced in platelets of patients with diabetes and hypertension. We observed that an increasing glucose concentration had a direct effect on ATP, ADP and AMP hydrolysis. Furthermore, CD39 expression was enhanced in all patients groups, indicating that these enzyme activities are related with diabetes and hypertension.

The activity of NTPDase (EC 3.6.1.5, apyrase, CD39) was verified in platelets from patients with increasing cholesterol levels. A possible association between cholesterol levels and inflammatory markers, such as oxidized low-density lipoprotein, highly sensitive C-reactive protein and oxidized low-density lipoprotein autoantibodies, was also investigated. Lipid peroxidation was estimated by measurement of thiobarbituric acid reactive substances in serum. The following groups were studied: group I, < 150 mg.dL(-1) cholesterol; group II, 151-200 mg.dL(-1) cholesterol; group III, 201-250 mg.dL(-1) cholesterol; and group IV, > 251 mg.dL(-1) cholesterol. The results demonstrated that both ATP hydrolysis and ADP hydrolysis were enhanced as a function of cholesterol level. Low-density lipoprotein levels increased concomitantly with total cholesterol levels. Triglyceride levels were increased in the groups with total cholesterol above 251 mg.dL(-1). Oxidized low-density lipoprotein levels were elevated in groups II, III, and IV. Highly sensitive C-reactive protein was elevated in the group with cholesterol levels higher than 251 mg.dL(-1). Oxidized low-density lipoprotein autoantibodies were elevated in groups III and IV. Thiobarbituric acid reactive substance content was enhanced as a function of cholesterol level. In summary, hypercholesterolemia is associated with enhancement of inflammatory response, oxidative stress, and ATP and ADP hydrolysis. The increased ATP and ADP hydrolysis in group IV was confirmed by an increase in CD39 expression on its surface. The increase in CD39 activity is possibly related to a compensatory response to the inflammatory and pro-oxidative state associated with hypercholesterolemia.

ATP exerts a proinflammatory role and induces cytokine release by acting at P2X(7) receptors. The product of ATP hydrolysis is the nucleoside adenosine, an important immunomodulator. The main source of extracellular adenosine is the hydrolysis of extracellular ATP by a group of ecto-enzymes: ENTPDase family, NPP family and ecto-5'-nucleotidase. Considering the role of ATP and adenosine in inflammatory processes, we investigated the effect of lipopolysaccharide on ectonucleotidases activities and expression in lymphocytes from mesenteric lymph nodes and serum of rats, in order to better understand the involvement of extracellular nucleotide hydrolysis in an endotoxemia model. We observed significant changes on nucleotidase activities from lymphocytes and serum of rats after in vitro and in vivo exposure to LPS. In vitro results have shown an increase on nucleotide hydrolysis in lymphocytes and a decrease on the enzyme activity of NPP in blood serum. In vivo, we observed an increase on nucleotide hydrolysis in lymphocytes and a decrease in the hydrolysis of all nucleotides tested in blood serum. After 24 and 48 h of LPS treatment, there was a reduction in NTPDase1, 2, 3 and ecto-5'-nucleotidase transcripts. These results suggest that there is a time-dependent enhancement of extracellular nucleotides metabolism in lymphocytes and blood serum after the induction of an endotoxemic model. The changes observed suggest that these enzymes can act in the regulation of extracellular nucleosides and nucleotides in a model able to trigger inflammatory process.

Introduction: CD39/NTPDase-1 is a cell surface enzyme expressed on leukocytes and endothelial cells that metabolizes ATP to ADP and AMP. CD39 is expressed on numerous different types of normal leukocytes, but details of its expression have not been determined previously., Methods: We examined CD39 expression and activity in leukocytes isolated from healthy volunteers. Expression of CD39 on leukocytes was measured by FACS and activity of CD39 in lymphocytes and neutrophils was determined by an enzymatic radio-TLC assay., Results: We established that CD39 is expressed on neutrophils, lymphocytes, and monocytes. The enzyme is found on >90% of monocytes, neutrophils, and B-lymphocytes, and 6% of T-lymphocytes and natural killer cells. Per cell density of expression varied, with the highest expression on monocytes and B-lymphocytes. ATPase and ADPase activities were highest on B-lymphocytes, lower on neutrophils, lowest on T-lymphocytes. The ratio of ADPase:ATPase activity was 1.8 for neutrophils and B-lymphocytes and 1.4 for T-lymphocytes. Hypertensive volunteers had lower levels of CD39 on their T-lymphocytes and NK cells. No correlation between age, gender, ethnic background, or cholesterol level and CD39 expression was observed., Conclusions: We conclude that CD39 activity and expression are present to varying degrees on all leukocytes types examined. Differences between leukocyte types should be considered when examining CD39 in disease states.

Background and Methods: The in vitro effect of the nonsteroidal anti-inflammatory drug, acetylsalicylic acid (ASA), on the extracellular adenine nucleotide hydrolysis by intact rat blood platelets was studied., Results: Our results demonstrate that aspirin, at final concentrations of 2.0 and 3.0 mM, inhibits ATP extracellular hydrolysis in vitro by approximately 17% and 21%, respectively. Aspirin, at a final concentration of 3.0 mM, also inhibited in vitro extracellular ADP hydrolysis by approximately 41%. The same concentrations of this drug, however, did not alter AMP hydrolysis by intact rat blood platelets under similar assay conditions. The kinetic analysis demonstrated that the inhibition of ADP and ATP hydrolysis by aspirin in rat platelets is of the uncompetitive type., Conclusion: In this study, we demonstrated an inhibitory effect of ASA upon E-NTPDase 3 activity of platelets from adult rats and discussed the significance of our findings.

Alterations of enzyme activities involved in adenine nucleotide hydrolysis have been reported in spinal cord and blood serum after repeated restraint stress. On the other hand, no effect was observed in the spinal cord of rats after acute stress. In the present study, we investigated the effect of acute stress on the hydrolysis of adenine nucleotides in rat blood serum. Adult male Wistar rats were submitted to 1-h restraint stress and were sacrificed at 0, 6, 24 and 48 h. Increased ATP and ADP hydrolysis were observed in the blood serum of stressed rats 24 h after stress (58% and 54%, respectively, when compared to controls). On the other hand, the AMP hydrolysis was increased after 6 h (68% when compared to controls) and at 24 h (94% when compared to controls) after stress. The results suggest that altered activity of soluble enzymes in serum may be a biochemical marker for stress situations.

Studies were carried out to investigate the activity of ATPase and ADPase in rat blood serum after strong (137Cs) on the rate of 1 Gy. Rats were examined for various postirradiation periods up to 3 months. Two-fold decreasing of serum ATPase activity was recorded within the two weeks after gamma-irradiation. The rate of 3H-ADP enzymatic hydrolysis in the serum of irradiated animals remained unchanged as compared to controls.

In the present report we demonstrate the in vitro effects of free radicals on an ecto-ATP diphosphohydrolase activity (apyrase, EC 3.6.1.5) from rat blood platelets. Rat blood platelets were exposed to an oxidant-generating system (H2O2/Fe2+/ascorbate) and the ATP diphosphohydrolase activity was inhibited. The enzyme inhibition was prevented by glutathione (GSH) and cysteine but not by trolox as a vitamin E analogue. The TBARS (thiobarbituric acid reactive substances) assay and the determination of sulphydryl groups indicate that the inhibition of the enzyme activity in resting platelets is not related to lipid peroxidation or to oxidation of sulphydryl residues. These results demonstrate the susceptibility of ATP diphosphohydrolase activity from platelets to free radicals and suggest that amino acid residues which are essential for the enzyme function are probably modified.

ATP diphosphohydrolase (EC 3.6.1.5) catalyzes the hydrolysis of diphospho- and triphosphonucleosides and is activated by divalent cations. The enzyme described in rat blood platelets hydrolyzes Ca(2+)-ATP and Ca(2+)-ADP with a high affinity for these Ca(2+)-nucleotide complexes as substrates. In the present paper, we demonstrate that free ATP or free ADP induces inhibition and kinetic alterations of the enzyme from rat blood platelets. From these results, we draw conclusions about the binding of free nucleotides to the enzyme and their action as inhibitors with respect to calcium-nucleotide complex.

In the present report we describe an apyrase (ATP diphosphohydrolase, EC 3.6.1.5) in rat blood platelets. The enzyme hydrolyses almost identically quite different nucleoside di- and triphosphates. The calcium dependence and pH requirement were the same for the hydrolysis of ATP and ADP and the apparent Km values were similar for both Ca(2+)-ATP and Ca(2+)-ADP as substrates. Ca(2+)-ATP and Ca(2+)-ADP hydrolysis could not be attributed to the combined action of different enzymes because adenylate kinase, inorganic pyrophosphatase and nonspecific phosphatases were not detected under our assay conditions. The Ca(2+)-ATPase and Ca(2+)-ADPase activity was insensitive to ATPase, adenylate kinase and alkaline phosphatase classical inhibitors, thus excluding these enzymes as contaminants. The results demonstrate that rat blood platelets contain an ATP diphosphohydrolase involved in the hydrolysis of ATP and ADP which are vasoactive and platelet active adenine nucleotides.

Salivary gland extracts of the deerfly contain a potent inhibitor of platelet aggregation, which assists the insect in obtaining a blood meal. The extract prevents platelet aggregation induced by ADP, thrombin, and collagen and inhibits fibrinogen binding to the glycoprotein IIb/IIIa receptor on platelets. The active component in deerfly salivary gland extract appears to be a protein that is comparatively more potent than the disintegrins present in viper venoms. Isolation and characterization of this protein may provide different directions in therapeutics and studies of normal platelet physiology.

With the intention to study the regulation of the availability of free arachidonic acid through the enzymes of the Lands cycle, we established a model system in homogenates of human platelets. Phospholipase A2, arachidonoyl-CoA synthetase and lysophosphatidyl acyltransferase proved to be simultaneously active and a steady turnover of arachidonic acid was the consequence. EGTA suppressed the deacylating activity that acted on endogenous membrane phospholipids and prevented eicosanoid formation from previously esterified exogenous arachidonoyl-CoA. The reacylating enzymes took part in the control of eicosanoid biosynthesis by re-esterification of liberated arachidonic acid. Blockade of the reacylation by apyrase made arachidonic acid completely available for further metabolization into 12-HETE and thereby induced an increase in the eicosanoid release.

ADPase enzyme activity was assessed in maternal and cord plasma by adding radiolabelled ADP and quantitating the degradation products. Cord plasma had sufficiently greater ADPase activity than the corresponding maternal plasma obtained ante- and post-partum. Thus, residual radiolabelled ADP was 30, 32 and 17% of total radioactivity after 30 min incubation (37 degrees C) in maternal ante-partum, maternal post-partum and cord plasmas, respectively. ADPase may act as a platelet aggregation inhibitor in the placental and fetal circulation.

Adenosine diphosphatase (ADPase) activities were studied in human polymorphonuclear leukocytes with a recently developed radio-assay. The neutrophils were homogenized in isotonic sucrose and subjected to analytical subcellular fractionation. The sucrose density gradient fractions were assayed for ADPase activity and for principal organelle marker enzymes. ADPase activity was distributed between the plasma membrane, specific granule and soluble fractions. The plasma membrane and specific granule activities had similar kinetic and inhibitor properties but the cytosolic enzyme was clearly different. Studies with the non-penetrating inhibitor diazotized sulphanilic acid and measurements of latent activity indicate that plasma membrane ADPase activity is located on the external aspect to the cell. Its possible role in inhibiting platelet aggregation is discussed. Neutrophils were isolated from control subjects, patients with chronic granulocytic leukaemia and patients in the third trimester of pregnancy. The specific activities (mU/mg protein) of ADPase activity, in contrast to those of alkaline phosphatase, were similar in all three groups. This result, together with fractionation experiments and inhibition studies strongly suggests that ADPase activity is not attributable to neutrophil alkaline phosphatase.

Specific assays for 5'-nucleotidase, adenosine diphosphatase (ADPase) and Mg2+-dependent adenosine triphosphatase (Mg2+-ATPase) have been optimized for human lymphocytes and their subcellular localizations determined by sucrose density gradient centrifugation. 5'-Nucleotidase was localized solely to the plasma membrane of the lymphocyte. ADPase activity has been shown to have a dual localization to the plasma membrane and mitochondria, whilst Mg2+-ATPase was mainly located in the mitochondria. No [Na+,K+] activated Mg2+-dependent ATPase could be measured in these cells. We have confirmed the striking decrease in the specific activity of 5'-nucleotidase activity in lymphocytes from patients with common variable primary hypogammaglobulinaemia. In contrast, the specific activities of ADPase and Mg2+-ATPase showed no alteration in lymphocytes from the patient group when compared to controls. Thus the deficiency of ecto-5'-nucleotidase in the lymphocytes of patients with hypogammaglobulinaemia is a highly selective defect in purine metabolism.

The role of ADP as an important inducer of platelet aggregation is generally accepted. Therefore it has been postulated by many authors that the enzymatic removal of extracellular ADP from the circulation is essential to avoid platelet aggregation and thrombus formation. Here we show that erythrocytes essentially contribute to the clearance of ADP. The removal of ADP from suspensions of washed human erythrocytes was due to at least two different activities. One activity, which had already been observed by earlier workers, was identified as adenylate kinase, on the basis of the reaction products and the inhibition by adenosine(5')pentaphospho(5')adenosine (Ap5A). This enzyme was not associated with the cells and was always detectable in cell-free supernatants, indicating that the enzyme had leaked from the cells into the extracellular medium. In contrast, the second activity, which is described here for the first time, was tightly bound to the cells. The activity was not inhibited by Ap5A. The main product of the reaction was AMP, and enzyme activity depended on the presence of divalent cations. The Michaelis constant was about 28 mumol/l. This activity seemed to be an ecto-ADPase. Studies with various inhibitors revealed that degradation of ADP was not due to a non-specific phosphatase. Besides the well known ADPase on the endothelium, the ecto-activity on erythrocytes may play an important part in destroying pro-aggregatory ADP.

The properties of vascular ADP-ase were investigated in microsomal preparations of rabbit aortic cell homogenates. The enzyme was found to have a Km of 9 microM. Its activity was not affected by substrates for other purine nucleoside phosphatases nor by substrates for non-specific phosphatases. However, ATP and beta-gamma-methylene ATP inhibited the enzyme. None of sodium fluoride, ouabain, neuraminidase affected ADP-ase activity whereas oligomycin potentiated the activity. The enzyme had an SH requirement and a divalent cation requirement.

Using a recently developed radioassay, the conditions for measuring adenosine diphosphatase (ADPase) activity in human lymphocytes were optimized. Kinetic studies showed that the activity was optimal at pH 8.0 and required 0.2 mM MgCl2. The Km of the enzyme for ADP was 0.03 mM. Analytical subcellular fractionation showed that the ADPase activity was distributed between the plasma membrane and mitochondria. Studies with the non-penetrating inhibitor, diazotized sulphanilic acid, indicate that plasma membrane ADPase activity is located on the external aspect of the cell. Polymorphonuclear leucocytes were found to have about three times the ADPase activity. Lymphocytes, therefore, have the ability to generate AMP on their surface which can be further metabolized to adenosine by ecto-5' nucleotidase. A role for this system is discussed.

The ADP-hydrolyzing enzyme apyrase inhibited platelet aggregation and phosphorylation of a 40,000 dalton platelet protein (P40) induced by 1-oleoyl-2-acetyl glycerol (OAG), indicating a dependence on secreted ADP. Apyrase also enhanced OAG-induced potentiation of forskolin or prostaglandin I2 activation of cyclic AMP formation in platelets. Cyclic AMP formation induced by OAG alone could be demonstrated in the presence of a phosphodiesterase inhibitor. Elevation of cyclic AMP level inhibits platelet aggregation so that secreted ADP may be required to inhibit OAG-activated adenylate cyclase for aggregation to proceed. In contrast, apyrase only partially affected phosphorylation of P40 and aggregation induced by the tumor promoter 12-0-tetradecanoyl phorbol-13-acetate (TPA). TPA caused marked inhibition of forskolin-stimulated cyclic AMP formation. TPA inhibition of cyclic AMP formation was largely reversed by apyrase, indicating that it was mainly due to release of ADP.

A rapid radioassay was used to characterise the adenosine diphosphatase (ADPase) activities in human plasma. There was a major peak at pH 9.3, 80% of whose activity was attributable to non-specific alkaline phosphatase, with the remaining 20% probably due to a specific ADPase. There was also a small peak of ADPase activity at pH 4.0. Inhibitor and chromatographic studies showed that whilst much of this activity was attributable to non-specific acid phosphatase, there was a discrete acid ADPase. Assays of plasma ADPase activities in vascular disorders, including myocardial infarction, peripheral vascular disease and diabetes mellitus, reveal no alterations from control values. Activities of alkaline ADPase were elevated in both chronic and acute liver failure. Acid ADPase was also increased in chronic liver disease and it is suggested that alterations in ADPase activities in liver disorders may contribute to the haemostatic problems observed in these patients.

Radiolabelled adenosine diphosphate [beta 32Pi]ADP was synthesized from radioactive orthophosphate and adenosine-5'-monophosphate with dicyclohexylcarbodiimide. The purified radioactive adenosine diphosphate was used as the substrate in a rapid, direct, one-step assay for adenosine diphosphatase in human neutrophilic leukocyte homogenates. Certain kinetic properties of the enzyme activity have been determined.