Exhaustion of CD39-Expressing CD8+ T Cells in Crohn's Disease Is Linked to Clinical Outcome.

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.

Microenvironment tailors nTreg structure and function.

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, TGFß, 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.

Post-thyroidectomy hypothyroidism increases the expression and activity of ectonucleotidases in platelets: Possible involvement of reactive oxygen species.

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.

Aberrant circulating levels of purinergic signaling markers are associated with several key aspects of peripheral atherosclerosis and thrombosis.

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.

Evaluation of CD4+ CD25+/- CD39+ T-cell populations in peripheral blood of patients following kidney transplantation and during acute allograft rejection.

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.

Decrease of serum adenine nucleotide hydrolysis in an irritant contact dermatitis mice model: potential P2X7R involvement.

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.

Loss of ectonucleotidases from the coronary vascular bed after ischemia-reperfusion in isolated rat heart.

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.

Evidence of platelet activation at medically used hypothermia and mechanistic data indicating ADP as a key mediator and therapeutic target.

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.

Hematological indices and activity of NTPDase and cholinesterase enzymes in rats exposed to cadmium and treated with N-acetylcysteine.

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.

Role of CD39 in COVID-19 Severity: Dysregulation of Purinergic Signaling and Thromboinflammation.

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 P2Y12 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.

Characterization of Regulatory T Cells in Preterm and Term Infants.

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+highFoxp3+, CD39+ Tregs, HLA-DR+ Tregs and the expression of Foxp3+ in CD4+CD25+highFoxp3 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+highFoxp3+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.

CD39 positive regulatory T cell frequency as a biomarker of treatment response to methotrexate in rheumatoid arthritis.

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.

Enzymes that hydrolyze adenine nucleotides of patients with hypercholesterolemia and inflammatory processes.

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.

CD39/NTPDase-1 activity and expression in normal leukocytes.

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.

Lipopolysaccharide alters nucleotidase activities from lymphocytes and serum of rats.

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.

Expression of Ecto-nucleoside Triphosphate Diphosphohydrolases-2 and -3 in the Enteric Nervous System Affects Inflammation in Experimental Colitis and Crohn's Disease.

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.

Immune phenotypes predict survival in patients with glioblastoma multiforme.

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.

NTPDase and 5'-nucleotidase as inflammatory markers in cattle naturally infected by Eurytrema coelomaticum.

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.

Subcellular localization and properties of adenosine diphosphatase activity in human polymorphonuclear leukocytes.

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.

Heightened Expression of CD39 by Regulatory T Lymphocytes Is Associated with Therapeutic Remission in Inflammatory Bowel Disease.

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.