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Endometriosis's pathophysiology remains incompletely understood, with evidence pointing towards a dysregulated immune response. Regulatory T (Treg) cells, pivotal in maintaining self-tolerance, may facilitate the survival of ectopic endometrial cells within the abdominal cavity, thereby contributing to endometriosis development. This study aimed to assess the prevalence of CD39+CD73+ suppressor Treg cell subsets in the peripheral blood of endometriosis patients. This research focuses on the pivotal role of regulatory T-cells (Tregs), which are essential for maintaining immune tolerance and preventing autoimmune diseases. A case-control study was conducted, including 32 women diagnosed with endometriosis and 22 control subjects. The frequency of peripheral blood CD39+CD73+ suppressor Treg cells was quantified using flow cytometry. No significant differences were observed in the frequency of CD3+CD4+CD25High cells (Median [M]: 10.1; Interquartile Range [IQR]: 6.32‒18.3 vs. M: 9.72; IQR: 6.22-19.8) or CD3+CD4+CD25HighCD39+Foxp3+ cells (M: 31.1; IQR: 19.7-44.0 vs. M: 30.55; IQR: 18.5-45.5) between controls and patients. However, a significantly lower frequency of CD3+CD4+CD25HighCD39+CD73+ cells was observed in the endometriosis group compared to controls (M: 1.98; IQR: 0.0377-3.17 vs. M: 2.25; IQR: 0.50-4.08; p = 0.0483), suggesting a reduction in systemic immune tolerance among these patients. This finding highlights the potential role of CD39 and CD73 expression on Treg cells as biomarkers for assessing disease severity and progression. Furthermore, elucidating the mechanisms driving these alterations may unveil new therapeutic strategies to restore immune equilibrium and mitigate endometriosis symptoms., Competing Interests: Declaration of competing interest The authors declare no conflicts of interest., (Copyright © 2024. Published by Elsevier España, S.L.U.)

Immune related endonucleases have recently been described as potential therapeutic targets and predictors of response to treatment with immune checkpoint inhibitors (ICI). The aim is to evaluate the association between the expression of 5 biomarkers involved in the immune response (CD73, CD39, VISTA, Arl4d and Cytohesin-3) in parallel with the more common ICI-predictive markers, PD-L1 expression and Tumor Mutation Burden (TMB) with response to ICI therapy in an advanced non-small cell lung cancer (NSCLC) cohort., Methods: Patients with advanced NSCLC treated with ICI single agent were divided into responders and non-responders according to RECIST v1.1 and duration of response (DOR) criteria. Immunohistochemistry was performed on pretreatment tumor tissue samples for PD-L1, CD73, CD39, VISTA, Arl4d, and Cytohesin-3 expression. TMB was estimated with NEOplus v2 RUO (NEO New Oncology GmbH) hybrid capture next generation sequencing assay. Resistance mutations in STK11/KEAP1 and positive predictive mutations in ARID1A/POLE were also evaluated., Results: Included were 56 patients who were treated with ICI single agent. The median progression-free and overall survival for the whole cohort was 3.0 (95% CI, 2.4-3.6) and 15 (95% CI, 9.7-20.2) months, respectively. The distribution of CD73 in tumor cells and CD39, VISTA, Arl4d and Cytohesin-3 expression in immune cells were not different between responders and non-responders. Also, PD-L1 and TMB were not predictive for response. The frequency of STK11, KEAP1 and ARID1A mutations was low and only observed in the non-responder group., Conclusion: Separate and combined expression of 5 biomarkers involved in the immune response (CD73, CD39, VISTA, Arl4d, and Cytohesin-3) was not associated with response in our cohort of advanced NSCLC patients receiving single agent ICI. To confirm our findings the analysis of independent larger cohorts is warranted., (Copyright © 2021 The Authors. Published by Elsevier GmbH.. All rights reserved.)

Detecting the entire repertoire of tumor-specific reactive tumor-infiltrating lymphocytes (TILs) is essential for investigating their immunological functions in the tumor microenvironment. Current in vitro assays identifying tumor-specific functional activation measure the upregulation of surface molecules, de novo production of antitumor cytokines, or mobilization of cytotoxic granules following recognition of tumor-antigens, yet there is no widely adopted standard method. Here we established an enhanced, yet simple, method for identifying simultaneously CD8 + and CD4 + tumor-specific reactive TILs in vitro , using a combination of widely known and available flow cytometry assays. By combining the detection of intracellular CD137 and de novo production of TNF and IFNγ after recognition of naturally-presented tumor antigens, we demonstrate that a larger fraction of tumor-specific and reactive CD8 + TILs can be detected in vitro compared to commonly used assays. This assay revealed multiple polyfunctionality-based clusters of both CD4 + and CD8 + tumor-specific reactive TILs. In situ , the combined detection of TNFRSF9 , TNF , and IFNG identified most of the tumor-specific reactive TIL repertoire. In conclusion, we describe a straightforward method for efficient identification of the tumor-specific reactive TIL repertoire in vitro , which can be rapidly adopted in most cancer immunology laboratories., Competing Interests: MD has received honoraria for lectures from Roche and Novartis (past two years). IMS has received honoraria for consultancies and lectures from Novartis, Roche, Merck, and Bristol-Myers Squibb; a restricted research grant from Novartis; and financial support for attending symposia from Bristol-Myers Squibb, Merck, Novartis, Pfizer, and Roche. HDR is currently an employee at Novo Nordisk. The remaining 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 © 2021 Draghi, Chamberlain, Khan, Papp, Lauss, Soraggi, Radic, Presti, Harbst, Gokuldass, Kverneland, Nielsen, Westergaard, Andersen, Csabai, Jönsson, Szallasi, Svane and Donia.)

UVR and immunosuppression are major risk factors for cutaneous squamous cell carcinoma (cSCC). Regulatory T cells promote cSCC carcinogenesis, and in other solid tumors, infiltrating regulatory T cells and CD8 + T cells express ectonucleoside triphosphate diphosphohydrolase 1 (ENTPD1) (also known as CD39), an ectoenzyme that catalyzes the rate-limiting step in converting extracellular adenosine triphosphate (ATP) to extracellular adenosine (ADO). We previously showed that extracellular purine nucleotides influence DNA damage repair. In this study, we investigate whether DNA damage repair is modulated through purinergic signaling in cSCC. We found increased ENTPD1 expression on T cells within cSCCs when compared with the expression on T cells from blood or nonlesional skin, and accordingly, concentrations of derivative extracellular adenosine diphosphate (ADP), adenosine monophosphate (AMP), and ADO are increased in tumors compared with those in normal skin. Importantly, ENTPD1 expression is significantly higher in human cSCCs that metastasize than in those that are nonmetastatic. We also identify in a mouse model that ENTPD1 expression is induced by UVR in an IL-27-dependent manner. Finally, increased extracellular ADO is shown to downregulate the expression of NAP1L2, a nucleosome assembly protein we show to be important for DNA damage repair secondary to UVR. Together, these data suggest a role for ENTPD1 expression on skin-resident T cells to regulate DNA damage repair through purinergic signaling to promote skin carcinogenesis and metastasis., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Flow cytometry is a useful ancillary tool for the diagnosis of nodal B cell lymphomas. Well-established antigens have diagnostic limitations. This study aimed to assess the expression of CD71, CD81, CD44 and CD39 by flow cytometry in B cell lymphomas. Expression of these 4 antigens was queried in 185 samples with a diagnosis of a B cell lymphoma according to a histological examination of the lymph node and the World Health Organization (WHO) classification (follicular lymphoma [FL, n  = 96], diffuse large B cell lymphoma/High grade B cell lymphoma [DLBCL/HGBH, n  = 48], marginal zone lymphoma/lymphoplasmacytic lymphoma [MZL/LPL, n  = 14], chronic lymphocytic leukemia/small lymphocytic lymphoma [CLL, n  = 10], mantle cell lymphoma [MCL, n  = 11], Burkitt lymphoma [BL, n  = 4] and other [ n  = 2]). CD81 was bright and CD44 was dim in germinal center-derived malignancies, particularly aggressive lymphomas (BL and CD10-positive DLBCL/HGBL). CD81 was very dim in CLL. CD71 was bright in aggressive lymphomas (DLBCL/HGBL and BL). CD39 was bright in CD10-negative DLBCL. CD71 appeared valuable in the differential diagnosis between indolent and aggressive lymphomas, CD39 between CD10-negative DLBCL and MZL/LPL and CD81 between MCL and CLL. To conclude, we report the expression of CD71, CD81, CD44 and CD39 by FC in B cell lymphomas. Further studies will have to determine the value they add to specific FC panels.

Adoptive T cell therapy (ACT) using ex vivo-expanded autologous tumor-infiltrating lymphocytes (TILs) can mediate complete regression of certain human cancers. The impact of TIL phenotypes on clinical success of TIL-ACT is currently unclear. Using high-dimensional analysis of human ACT products, we identified a memory-progenitor CD39-negative stem-like phenotype (CD39 - CD69 - ) associated with complete cancer regression and TIL persistence and a terminally differentiated CD39-positive state (CD39 + CD69 + ) associated with poor TIL persistence. Most antitumor neoantigen-reactive TILs were found in the differentiated CD39 + state. However, ACT responders retained a pool of CD39 - stem-like neoantigen-specific TILs that was lacking in ACT nonresponders. Tumor-reactive stem-like TILs were capable of self-renewal, expansion, persistence, and superior antitumor response in vivo. These data suggest that TIL subsets mediating ACT response are distinct from TIL subsets enriched for antitumor reactivity., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Platelets play central role in thrombosis and haemostasis. Platelets store adenine nucleotides in their dense granules, which are released upon agonist-stimulation. Level of these nucleotides in extracellular fluid is regulated by activities of ectonucleotidases such as ectonucleoside triphosphate diphosphohydrolase-1 (CD39) and ecto-5'-nucleotidase (CD73) expressed on platelet surface. Here we demonstrate that, expression of surface-bound ectonucleotidases rose significantly in platelets, concomitant with upregulation of their enzymatic activities, when cells were stimulated with thrombin. Interestingly, inhibition of CD73 in thrombin-treated platelets led to enhanced tyrosine phosphorylation of proteins and rise in intracellular free calcium, [Ca 2+ ] i , thus signifying the inhibitory role of the ectonucleotidase on agonist-mediated platelet signaling., Competing Interests: Declaration of competing interest Authors declare no conflict of interest., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Background and Aims: The intestinal microbiota is closely associated with resident memory lymphocytes in mucosal tissue. We sought to understand how acquired cellular and humoral immunity to the microbiota differ in health versus inflammatory bowel disease [IBD]., Methods: Resident memory T cells [Trm] in colonic biopsies and local antibody responses to intraepithelial microbes were analysed. Systemic antigen-specific immune T and B cell memory to a panel of commensal microbes was assessed., Results: Systemically, healthy blood showed CD4 and occasional CD8 memory T cell responses to selected intestinal bacteria, but few memory B cell responses. In IBD, CD8 memory T cell responses decreased although B cell responses and circulating plasmablasts increased. Possibly secondary to loss of systemic CD8 T cell responses in IBD, dramatically reduced numbers of mucosal CD8+ Trm and γδ T cells were observed. IgA responses to intraepithelial bacteria were increased. Colonic Trm expressed CD39 and CD73 ectonucleotidases, characteristic of regulatory T cells. Cytokines/factors required for Trm differentiation were identified, and in vitro-generated Trm expressed regulatory T cell function via CD39. Cognate interaction between T cells and dendritic cells induced T-bet expression in dendritic cells, a key mechanism in regulating cell-mediated mucosal responses., Conclusions: A previously unrecognised imbalance exists between cellular and humoral immunity to the microbiota in IBD, with loss of mucosal T cell-mediated barrier immunity and uncontrolled antibody responses. Regulatory function of Trm may explain their association with intestinal health. Promoting Trm and their interaction with dendritic cells, rather than immunosuppression, may reinforce tissue immunity, improve barrier function, and prevent B cell dysfunction in microbiota-associated disease and IBD aetiology., (© European Crohn’s and Colitis Organisation 2019.)

During the blood feeding, sand fly females inject saliva containing immunomodulatory and anti-haemostatic molecules into their vertebrate hosts. The saliva composition is species-specific, likely due to an adaptation to particular haemostatic pathways of their preferred host. Research on sand fly saliva is limited to the representatives of two best-studied genera, Phlebotomus and Lutzomyia. Although the members of the genus Sergentomyia are highly abundant in many areas in the Old World, their role in human disease transmission remains uncertain. Most Sergentomyia spp. preferentially attack various species of reptiles, but feeding on warm-blooded vertebrates, including humans and domestic animals, has been repeatedly described, especially for Sergentomyia schwetzi, of which salivary gland transcriptome and proteome is analyzed in the current study. Illumina RNA sequencing and de novo assembly of the reads and their annotation revealed 17,293 sequences homologous to other arthropods' proteins. In the sialome, all proteins typical for sand fly saliva were identified-antigen 5-related, lufaxin, yellow-related, PpSP15-like, D7-related, ParSP25-like, and silk proteins, as well as less frequent salivary proteins included 71kDa-like, ParSP80-like, SP16-like, and ParSP17-like proteins. Salivary enzymes include apyrase, hyaluronidase, endonuclease, amylase, lipase A2, adenosine deaminase, pyrophosphatase, 5'nucleotidase, and ribonuclease. Proteomics analysis of salivary glands identified 631 proteins, 81 of which are likely secreted into the saliva. We also compared two S. schwetzi lineages derived from the same origin. These lineages were adapted for over 40 generations for blood feeding either on mice (S-M) or geckos (S-G), two vertebrate hosts with different haemostatic mechanisms. Altogether, 20 and 40 annotated salivary transcripts were up-regulated in the S-M and S-G lineage, respectively. Proteomic comparison revealed ten salivary proteins more abundant in the lineage S-M, whereas 66 salivary proteins were enriched in the lineage S-G. No difference between lineages was found for apyrase activity; contrarily the hyaluronidase activity was significantly higher in the lineage feeding on mice., Competing Interests: The authors have declared that no competing interests exist.

In the context of adoptive T cell transfer (ACT) for cancer treatment, it is crucial to generate in vitro large amounts of tumor-specific CD8 T cells with high potential to persist in vivo . PD-1, Tim3, and CD39 have been proposed as markers of tumor-specific tumor-infiltrating CD8 T lymphocytes (CD8 TILs). However, these molecules are highly expressed by terminally differentiated exhausted CD8 T cells (Tex) that lack proliferation potential. Therefore, optimized strategies to isolate tumor-specific TILs with high proliferative potential, such as Tcf1+ precursor exhausted T cells (Tpe) are needed to improve in vivo persistence of ACT. Here we aimed at defining cell surface markers that would unequivocally identify Types for precision cell sorting increasing the purity of tumor-specific PD-1+ Tcf1+ Tpe from total TILs. Transcriptomic analysis of Tpe vs. Tex CD8 TIL subsets from B16 tumors and primary human melanoma tumors revealed that Tpes are enriched in Slamf6 and lack Entpd1 and Havcr2 expression, which encode Slamf6, CD39, and Tim3 cell surface proteins, respectively. Indeed, we observed by flow cytometry that CD39- Tim3- Slamf6+ PD-1+ cells yielded maximum enrichment for tumor specific PD-1+ Tcf1+ OT1 TILs in B16.OVA tumors. Moreover, this population showed higher re-expansion capacity upon an acute infection recall response compared to the CD39+ counterparts or bulk PD-1+ TILs. Hence, we report an enhanced sorting strategy (CD39- Tim3- Slamf6+ PD-1+) of Tpes. In conclusion, we show that optimization of CD8 TIL cell sorting strategy is a viable approach to improve recall capacity and in vivo persistence of transferred cells in the context of ACT., (Copyright © 2020 Martinez-Usatorre, Carmona, Godfroid, Yacoub Maroun, Labiano and Romero.)

Ecto-nucleoside triphosphate diphosphohydrolase1 (NTPDase1, CD39) is a major ectonucleotidase that hydrolyzes proinflammatory ATP via ADP to AMP, which is subsequently converted by ecto-5'-nucleotidase (CD73) to immunosuppressive adenosine. Activation of CD39 has potential for treating inflammatory diseases, while inhibition was suggested as a novel strategy for the immunotherapy of cancer. In the present study, we developed a selective and highly sensitive capillary electrophoresis (CE) assay using a novel fluorescent CD39 substrate, a fluorescein-labelled ATP (PSB-170621A) that is converted to its AMP derivative. To accelerate the assays, a two-directional (forward and reverse) CE system was implemented using 96-well plates, which is suitable for the screening of compound libraries (Z'-factor: 0.7). The detection limits for the forward and reverse operation were 11.7 and 2.00 pM, respectively, indicating a large enhancement in sensitivity as compared to previous methods (e.g. malachite-green assay: 1 000 000-fold, CE-UV assay: 500 000-fold, fluorescence polarization immunoassay: 12 500-fold). Enzyme kinetic studies at human CD39 revealed a Km value of 19.6 μM, and a kcat value of 119 × 10-3 s-1 for PSB-170621A, which shows similar substrate properties as ATP (11.4 μM and 82.5 × 10-3 s-1). The compound displayed similar properties at rat and mouse CD39. Subsequent docking studies into a homology model of human CD39 revealed a hydrophobic pocket that accommodates the fluorescein tag. PSB-170621A was found to be preferably hydrolyzed by CD39 as compared to other ectonucleotidases. The new assay was validated by performing inhibition assays with several standard CD39 inhibitors yielding results that were consonant with data using the natural substrates.

Background: Regulatory T cells (Tregs) play a major role in tumor escape from immunosurveillance by suppressing effector cells. The number of Tregs is increased in tumor sites and peripheral blood of breast cancer patients. However, the data regarding phenotypic and functional heterogeneity of Treg subpopulations in breast cancer are limited. The present study aimed to investigate the number and suppressive potential of Tregs that possess natural naïve-(N nTregs), effector/memory-like (EM nTregs), and Tr1-like phenotypes in breast cancer patients and healthy women., Methods: The study included 10 HW and 17 primary breast cancer patients. Numbers of CD4 + CD25 + FoxP3 + CD45RA + N nTregs, CD4 + CD25 + FoxP3 + CD45RA - EM nTregs, and CD4 + IL-4 - IL-10 + Tr1 subsets and the expression of CTLA-4, CD39, GITR, LAP, and IL-35 by these Treg subsets were measured in freshly obtained peripheral blood by flow cytometry., Results: Herein, we demonstrate that the percentages of N nTregs, EM nTregs, CD25 + and FoxP3 + Tr1 cells are elevated in the peripheral blood of breast cancer patients, but do not correlate with cancer stages. Nevertheless, the frequency of CD25 + Tr1 cells was associated with nodal involvement, while the number of EM nTregs correlated with clinical outcome. The expression of CTLA-4 and IL-35 by all assessed Treg subsets was increased throughout all tumor stages (I-III)., Conclusions: Collectively, the current study shows phenotypic alterations in suppressive receptors of Treg subsets, suggesting that breast cancer patients have increased activity of N nTregs, EM nTregs and Tr1 cells; and EM nTregs and CD25 + Tr1 cells represent prospective markers for assessing disease prognosis.

Asthma is a chronic disease of airway inflammation due to excessive T helper cell type 2 (Th2) response. Present treatment based on inhalation of synthetic glucocorticoids can only control Th2-driven chronic eosinophilic inflammation, but cannot change the immune tolerance of the body to external allergens. Regulatory T cells (Tregs) are the main negative regulatory cells of the immune response. Tregs play a great role in regulating allergic, autoimmune, graft-versus-host responses, and other immune responses. In this review, we will discuss the classification and biological characteristics, the established immunomodulatory mechanisms, and the characteristics of induced differentiation of Tregs. We will also discuss the progress of Tregs in the field of asthma. We believe that further studies on the regulatory mechanisms of Tregs will provide better treatments and control strategies for asthma.

Several proteins such as membrane-associated ectonucleotidases: ecto-5'-nucleotidase (E5NT/CD73) and ectonucleoside triphosphate diphosphohydrolase 1 (ENTPD1/CD39), and intracellular heme oxygenase-1 (HO-1) may contribute to protection from inflammation-related diseases such as calcific aortic valve stenosis (CAS). Accurate quantification of these proteins could contribute to better understanding of the disease mechanisms and identification of biomarkers. This report presents development and validation of quantification method for E5NT/CD73, ENTPD1/CD39 and HO-1. The multiplexed targeted proteomic assay involved antibody-free, multiple-enzyme digestion, filter-assisted sample preparation (MED-FASP) strategy and a nanoflow liquid chromatography/mass spectrometry under multiple reaction monitoring mode (LC-MRM/MS). The method developed presented high sensitivity (LLOQ of 5 pg/mL for each of the analytes) and accuracy that ranged from 92.0% to 107.0%, and was successfully applied for the absolute quantification of HO-1, CD39 and CD73 proteins in homogenates of human calcified and non-calcified valves. The absolute CD39 and CD73 concentrations were lower in calcified aortic valves (as compared to non-stenotic ones) and were found to be: 1.16 ± 0.39 vs. 3.15 ± 0.37 pmol/mg protein and 1.94 ± 0.21 vs. 2.39 ± 0.39 pmol/mg protein, respectively, while the quantity of HO-1 was elevated in calcified valves (10.72 ± 1.18 vs. 4.28 ± 0.42 amol/mg protein). These results were consistent but more reproducible as compared to immunoassays. In conclusion, multiplexed quantification of HO-1, CD39 and CD73 proteins by LC-MRM/MS works well in challenging human tissues such as aortic valves. This analysis confirmed the relevance of these proteins in pathogenesis of CAS and could be extended to other biomedical investigations., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Various forms of immunotherapy, such as checkpoint blockade immunotherapy, are proving to be effective at restoring T cell-mediated immune responses that can lead to marked and sustained clinical responses, but only in some patients and cancer types 1-4 . Patients and tumours may respond unpredictably to immunotherapy partly owing to heterogeneity of the immune composition and phenotypic profiles of tumour-infiltrating lymphocytes (TILs) within individual tumours and between patients 5,6 . Although there is evidence that tumour-mutation-derived neoantigen-specific T cells play a role in tumour control 2,4,7-10 , in most cases the antigen specificities of phenotypically diverse tumour-infiltrating T cells are largely unknown. Here we show that human lung and colorectal cancer CD8 + TILs can not only be specific for tumour antigens (for example, neoantigens), but also recognize a wide range of epitopes unrelated to cancer (such as those from Epstein-Barr virus, human cytomegalovirus or influenza virus). We found that these bystander CD8 + TILs have diverse phenotypes that overlap with tumour-specific cells, but lack CD39 expression. In colorectal and lung tumours, the absence of CD39 in CD8 + TILs defines populations that lack hallmarks of chronic antigen stimulation at the tumour site, supporting their classification as bystanders. Expression of CD39 varied markedly between patients, with some patients having predominantly CD39 - CD8 + TILs. Furthermore, frequencies of CD39 expression among CD8 + TILs correlated with several important clinical parameters, such as the mutation status of lung tumour epidermal growth factor receptors. Our results demonstrate that not all tumour-infiltrating T cells are specific for tumour antigens, and suggest that measuring CD39 expression could be a straightforward way to quantify or isolate bystander T cells.

Extracellular adenosine, generated via the concerted action of CD39 and CD73, contributes to T-cell differentiation and function. Adenosine concentrations are furthermore influenced by adenosine deaminase binding protein CD26. Because aberrant T-cell phenotypes had been reported in anti-neutrophil cytoplasmic auto-antibody (ANCA)-associated vasculitis (AAV) patients, an impaired expression of these molecules on T-cells of AAV patients was hypothesized in the present study. While in AAV patients (n = 29) CD26 was increased on CD4 + lymphocytes, CD39 and CD73 were generally reduced on patients' T-cells. In CD4 + cells significant differences in CD73 expression were confined to memory CD45RA - cells, while in CD4 - lymphocytes differences were significant in both naïve CD45RA + and memory CD45RA - cells. The percentage of CD4 - CD73 + cells correlated with micro-RNA (miR)-31 expression, a putative regulator of factor inhibiting hypoxia-inducible factor 1 alpha (FIH-1), inversely with serum C-reactive protein (CRP) and positively with estimated glomerular filtration rate (eGFR). No correlation with disease activity, duration, and ANCA profile was found. It remains to be assessed if a decreased CD73 and CD39 expression underlies functional impairment of lymphocytes in AAV patients. Likewise, the relations between frequencies of CD4 - CD73 + cells and serum CRP or eGFR require further functional elucidation.

A novel amphiphilic imidazolium-based probe containing a dansyl fluorophore and a long cetyl chain has been developed for ATP recognition. The probe forms self-assembled micelle-like aggregates at low concentration in its aqueous solution and can selectively recognize ATP among other bioactive anions with a significant enhancement in fluorescence emission.

Regulatory T cells (Tregs) have been suggested to play a role in the pathogenesis of atopic dermatitis (AD). However, alterations in the ability of Tregs remain to be determined. To investigate the expression of various surface receptors on CD4(+)CD25(high) regulatory T cells and to investigate their capacity for inhibiting the proliferation of CD4(+) CD25(-) effector T cells (Teffs). Peripheral blood samples were obtained from 15 patients with severe atopic dermatitis (AD) and 20 control subjects. FACs was then carried out to analyze the expression levels of FoxP3, CD152 (CTLA-4), CD39, CD73, CD223 (LAG-3), CCR4, CCR5, and CCR10 on Tregs. The proliferative responses of Teffs were assessed in the absence or presence of autologous Tregs and the TGF-β1 and IL-10 levels in the culture supernatant and sera were detected by enzyme-linked immunosorbent assay (ELISA). The CD152, CD39, CD73, CCR4, and CCR5 expression levels on Tregs were higher in patients with severe AD than in the controls. Tregs showed an attenuated suppressive function of the proliferation of autologous Teffs in severe AD. The concentrations of IL-10 and TGF-β in the culture supernatants of Tregs were lower in the AD group than in the control. The attenuated ability of Tregs to suppress Teff proliferation may be responsible for the autoimmune reaction of severe AD.

CD39/ectonucleoside triphosphate diphosphohydrolase-1 (ENTPD1) is a cell surface-located, rate-limiting enzyme in the generation of adenosine, and plays a crucial role in tumor development. We examined co-expression of CD39 and CD8in gastric cancer (GC) and showed that the expression of CD39 and CD8 increased significantly in tumor tissues compared to paired peritumor tissues. The expression of tumoral CD39 (tCD39), but not tumoral CD8 (tCD8), was related to overall survival. Furthermore, the CD39(+)/CD8(+) ratio was associated with poor prognosis in resected GC patients. Taken together, our data indicate that highCD39 expression and high tCD39(+)/CD8(+) ratio in GC is a predictor of poor prognosis for GC patients after radical resection. Moreover, CD39 could serve as a potential target for cancer immunotherapy.

Polysaccharide A (PSA) derived from the human commensal Bacteroides fragilis is a symbiosis factor that stimulates immunologic development within mammalian hosts. PSA rebalances skewed systemic T helper responses and promotes T regulatory cells (Tregs). However, PSA-mediated induction of Foxp3 in humans has not been reported. In mice, PSA-generated Foxp3(+) Tregs dampen Th17 activity thereby facilitating bacterial intestinal colonization while the increased presence and function of these regulatory cells may guard against pathological organ-specific inflammation in hosts. We herein demonstrate that PSA induces expression of Foxp3 along with CD39 among naïve CD4 T cells in vitro while promoting IL-10 secretion. PSA-activated dendritic cells are essential for the mediation of this regulatory response. When cultured with isolated Foxp3(+) Tregs, PSA enriched Foxp3 expression, enhanced the frequency of CD39(+)HLA-DR(+) cells, and increased suppressive function as measured by decreased TNFα expression by LPS-stimulated monocytes. Our findings are the first to demonstrate in vitro induction of human CD4(+)Foxp3(+) T cells and enhanced suppressive function of circulating Foxp3(+) Tregs by a human commensal bacterial symbiotic factor. Use of PSA for the treatment of human autoimmune diseases, in particular multiple sclerosis and inflammatory bowel disease, may represent a new paradigm in the approach to treating autoimmune disease.

Background: Sepsis encompasses two phases, the 'hyper'-reactive phase and the 'hypo'-reactive phase. The initial inflammatory stage is quickly counterbalanced by an anti-inflammatory response, which compromises the immune system, leading to immune suppression. Regulatory T cells (Tregs) have been implicated in the pathogenesis of sepsis by inducing immunosuppression; however, the role of CD39(+) Tregs in the process of sepsis is uncertain. This study investigated the dynamic levels of CD39(+) Tregs and their phenotypic change in sepsis., Methods: Fourteen patients with systemic inflammatory response syndrome (SIRS), 42 patients with sepsis, and 14 healthy controls were enrolled. Sequential blood samples were used to analyze the numbers of CD39(+) Tregs and their phenotypic changes. Survival at 28 days was used to evaluate the capacity of CD39(+) Treg levels to predict mortality in sepsis patients., Results: Sepsis patients displayed a high percentage (3.13%, 1.46%, and 0.35%, respectively) and mean fluorescence intensity (MFI) (59.65, 29.7, and 24.3, respectively) of CD39(+) Tregs compared with SIRS patients and healthy subjects. High-level expression of CD39(+) Tregs was correlated with the severity of sepsis, which was reflected by the sepsis-related organ failure assessment score (r=0.322 and r=0.31, respectively). In addition, the expression of CD39(+) Tregs was associated with survival of sepsis patients (p<0.01). By receiver-operating characteristic (ROC) curve analysis, the percentage and MFI of CD39(+) Tregs showed similar sensitivities and specificities to predict mortality (74.2% and 85.1%, and 73.9% and 84.1%, respectively). Using Kaplan-Meier curves to assess the impact of CD39(+) Tregs percentage and MFI on overall survival, we found that a high CD39(+) Tregs percentage (p<0.001; >4.1%) and MFI (p<0.001; >49.2) were significantly associated with mortality. Phenotypically, CD39(+) Tregs from sepsis patients showed high expression of CD38 and PD-1 (p<0.01 and p<0.01 respectively)., Conclusions: Increased expression of CD39(+) Tregs was associated with a poor prognosis for sepsis patients, which suggests that CD39(+) Treg levels could be used as a biomarker to predict the outcome of sepsis patients., (Copyright © 2014 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Phosphohydrolysis of extracellular ATP and ADP is an essential step in purinergic signaling that regulates key pathophysiological processes, such as those linked to inflammation. Classically, this reaction has been known to occur in the pericellular milieu catalyzed by membrane bound cellular ecto-nucleotidases, which can be released in the form of both soluble ecto-enzymes as well as being associated with exosomes. Circulating ecto-nucleoside triphosphate diphosphohydrolase 1 (NTPDase 1/CD39) and adenylate kinase 1 (AK1) activities have been shown to be present in plasma. However, other ecto-nucleotidases have not been characterized in depth. An in vitro ADPase assay was developed to probe the ecto-enzymes responsible for the ecto-nucleotidase activity in human platelet-free plasma, in combination with various specific biochemical inhibitors. Identities of ecto-nucleotidases were further characterized by chromatography, immunoblotting, and flow cytometry of circulating exosomes. We noted that microparticle-bound E-NTPDases and soluble AK1 constitute the highest levels of ecto-nucleotidase activity in human plasma. All four cell membrane expressed E-NTPDases are also found in circulating microparticles in human plasma, inclusive of: CD39, NTPDase 2 (CD39L1), NTPDase 3 (CD39L3), and NTPDase 8. CD39 family members and other ecto-nucleotidases are found on distinct microparticle populations. A significant proportion of the microparticle-associated ecto-nucleotidase activity is sensitive to POM6, inferring the presence of NTPDases, either -2 or/and -3. We have refined ADPase assays of human plasma from healthy volunteers and have found that CD39, NTPDases 2, 3, and 8 to be associated with circulating microparticles, whereas soluble AK1 is present in human plasma. These ecto-enzymes constitute the bulk circulating ADPase activity, suggesting a broader implication of CD39 family and other ecto-enzymes in the regulation of extracellular nucleotide metabolism.

Tolerance established by host-commensal interactions regulates host immunity at both local mucosal and systemic levels. The intestinal commensal strain Bacteroides fragilis elicits immune tolerance, at least in part, via the expression capsular polysaccharide A (PSA). How such niche-specific commensal microbial elements regulate extra-intestinal immune responses, as in the brain, remains largely unknown. We have recently shown that oral treatment with PSA suppresses neuro-inflammation elicited during experimental autoimmune encephalomyelitis (EAE), an animal model for multiple sclerosis. This protection is dependent upon the expansion of immune-regulatory CD4 T cells (Treg) expressing CD39, an ectonucleotidase. Here, we further show that CD39 modulation of purinergic signals enhances migratory phenotypes of both total CD4 T cells and Foxp3(+) CD4 Tregs at central nervous system (CNS) lymphoid-draining sites in EAE in vivo and promotes their migration in vitro. These changes are noted during PSA treatment, which leads to heightened accumulation of CD39(+) CD4 Tregs in the CNS. Deficiency of CD39 abrogates accumulation of Treg during EAE, and is accompanied by elevated Th1/Th17 signals in the CNS and in gut-associated lymphoid tissues. Our results demonstrate that immune-modulatory commensal bacterial products impact the migratory patterns of CD4 Treg during CNS autoimmunity via the regulation of CD39. These observations provide clues as to how intestinal commensal microbiome is able to modulate Treg functions and impact host immunity in the distal site.

Carbon nanomaterial combined with aptamer has been developed as an efficient bioanalytical method in sensor design. Herein, depending on carbon nanoparticle (cCNP)-enhanced fluorescence anisotropy (FA), a novel aptamer-based sensor (aptasensor) enabling signal-amplification and real-time detection of apyrase is reported. The foundation of our sensor design based on ATP-aptamer(P) can be adsorbed on the surface of cCNPs, resulting in the increase of FA due to the mass of cCNPs, and P-ATP complex has weak binding ability to cCNPs with minimal change of FA. Apyrase, being an integral membrane protein, can hydrolyze ATP and make P-ATP complex disassemble, and thus lead to the increasing of FA. Therefore, this approach is demonstrated to be a novel candidate for the detection of apyrase, with high sensitivity and selectivity. The linear dynamic range for the concentrations of apyrase is between 0.1 and 0.5 U/μL along with a detection limit of 0.05 U/μL. Furthermore, these results indicated that our design is a flexible and sensitive method for biomolecule analysis, which makes it promising for practical biomolecule analyses., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Objective: We previously reported the coexistence, within cultured mesenchymal stem cells (MSCs) from human synovial membrane, of single-cell-derived clonal cell populations with distinct differentiation potency. The aim of this study was to investigate marker sets for prospective purification of functionally distinct MSC subsets., Methods: Cells were enzymatically released from human synovium and culture expanded. Phenotype analysis was performed by flow cytometry using combinations of MSC markers. Sorting was carried out using the FACS DiVA cell sorter. Sorted cell populations were assessed for clonogenicity, kinetics of growth, cell senescence and chondro-osteogenic potency., Results: During culture expansion, the co-localization of CD39 within the CD73(+) cell population identified a small cell subset that was maintained from passage 1 (P1) up to at least P12 in all donors tested. The CD73(+)CD39(+) cell subset displayed higher expression levels of Sox9 and Runx2 and a significantly greater chondro-osteogenic potency than the CD73(+)CD39(-) cell subset. In contrast, it was less clonogenic and proliferative. There was no difference in cell senescence between the sorted MSC subsets and the parental MSCs. Notably, there were no detectable differences in chondro-osteogenic potency between the CD73(+)CD39(-) and CD73(+)CD39(+) cell subsets purified from fresh synovial cell populations., Conclusion: Our findings indicate that the combination of CD73 and CD39 allows the prospective purification from culture-expanded heterogeneous synovial MSC populations of a distinct MSC subset with greater chondro-osteogenic potency. We anticipate that such an approach will enhance the consistency of cell-based therapeutic protocols for the repair of osteochondral defects.

The interactions taking place between mother and embryo have been the focus of detailed studies in recent years, where pregnancy is considered as an in vivo transplant. The immune systems of the mother and the embryo together establish a condition of tolerance, which lasts throughout the pregnancy. Alongside immunogenetic components, a contribution is provided by the ectoenzyme network, a chain of surface molecules mainly operating in closed environments and potentially providing inhibitory or activator signals. One of the soluble products of the ectoenzyme network with immunosuppressory potential is adenosine, a purine nucleoside that plays multiple roles in almost all tissues and organs. The hypothesis behind the work was studied in patients with recurrent pregnancy loss (RPL), an event which remains unexplained in over 50 percent of cases. To this aim, we analyzed the expression of CD39 (ectonucleoside triphosphate diphosphohydrolase 1, ENTPD1) and CD73 (ecto-5’-nucleotidase, NT5E), the main pathway for adenosine generation, in samples obtained from women with RPL. The study included the evaluation of the expression of TNF-alpha (a pro-inflammatory cytokine) and of an alternative pathway of adenosine generation run by CD38 (ADP-ribosyl cyclase/cyclic ADP-ribose hydrolase) and PC-1 (ectonucleotide pyrophosphatase/phosphodiesterase 1, ENPP1). The results of this study highlight the existence of a network of surface enzymes expressed at the maternal/fetal interface and addressed to the production of adenosine. Perturbation of this network may induce a rescue pathway driven by CD38 and ENPP1. Ectoenzyme and inflammation may be considered now key elements in orchestrating the events leading to the interruption of pregnancy in the RPL sample analyzed and at the same potentially becoming therapeutic targets.

An imbalance of immunosuppressive and cytotoxic cells plays an important role in inhibiting the anti-tumor immune response of the tumor-bearing host. The purpose of this study was to elucidate the involvement of immunosuppressive cells, such as regulatory T cells and CD163+ M2 macrophages as well as cytotoxic cells, such as granulysin-bearing cells and TIA-1+ cells in cutaneous angiosarcoma (AS) by immunohistochemical staining. In addition we evaluated the potencies of bisphosphonate, which was previously reported to suppress the expression of matrix metalloproteinase 9 (MMP-9), as a supportive therapy for AS together with docetaxel in 6 cases of cutaneous AS. These findings suggest that a high number of immunosuppressive cells might be related to the prognosis of AS, and that a combination of docetaxel with bisphosphonate risedronate sodium might be effective for MMP-9-expressing AS.

HTLV-1 infection is associated with several inflammatory disorders, including the neurodegenerative condition HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). It is unclear why a minority of infected subjects develops HAM/TSP. CD4⁺ T cells are the main target of infection and play a pivotal role in regulating immunity to HTLV and are hypothesized to participate in the pathogenesis of HAM/TSP. The CD39 ectonucleotidase receptor is expressed on CD4⁺ T cells and based on co-expression with CD25, marks T cells with distinct regulatory (CD39⁺CD25⁺) and effector (CD39⁺CD25⁻) function. Here, we investigated the expression of CD39 on CD4⁺ T cells from a cohort of HAM/TSP patients, HTLV-1 asymptomatic carriers (AC), and matched uninfected controls. The frequency of CD39⁺ CD4⁺ T cells was increased in HTLV-1 infected patients, regardless of clinical status. More importantly, the proportion of the immunostimulatory CD39⁺CD25⁻ CD4⁺ T-cell subset was significantly elevated in HAM/TSP patients as compared to AC and phenotypically had lower levels of the immunoinhibitory receptor, PD-1. We saw no difference in the frequency of CD39⁺CD25⁺ regulatory (Treg) cells between AC and HAM/TSP patients. However, these cells transition from being anergic to displaying a polyfunctional cytokine response following HTLV-1 infection. CD39⁻CD25⁺ T cell subsets predominantly secreted the inflammatory cytokine IL-17. We found that HAM/TSP patients had significantly fewer numbers of IL-17 secreting CD4⁺ T cells compared to uninfected controls. Taken together, we show that the expression of CD39 is upregulated on CD4⁺ T cells HAM/TSP patients. This upregulation may play a role in the development of the proinflammatory milieu through pathways both distinct and separate among the different CD39 T cell subsets. CD39 upregulation may therefore serve as a surrogate diagnostic marker of progression and could potentially be a target for interventions to reduce the development of HAM/TSP.

Nucleoside triphosphate diphosphohydrolase (NTPDase) activity was recently characterized in Leishmania (Viannia) braziliensis promastigotes (Lb), and an antigenic conserved domain (r82-121) from the specific NTPDase 1 isoform was identified. In this work, mouse polyclonal antibodies produced against two synthetic peptides derived from this domain (LbB1LJ, r82-103; LbB2LJ, r102-121) were used. The anti-LbB1LJ or anti-LbB2LJ antibodies were immobilized on protein A-sepharose and immunoprecipitated the NTPDase 1 of 48 kDa and depleted approximately 40% of the phosphohydrolytic activity from detergent-homogenized Lb preparation. Ultrastructural immunocytochemical microscopy identified the NTPDase 1 on the parasite surface and in its subcellular cytoplasmic vesicles, mitochondria, kinetoplast and nucleus. The ATPase and ADPase activities of detergent-homogenized Lb preparation were partially inhibited by anti-LbB1LJ antibody (43-79%), which was more effective than that inhibition (18-47%) by anti-LbB2LJ antibody. In addition, the immune serum anti-LbB1LJ (67%) or anti-LbB2LJ (33%) was cytotoxic, significantly reducing the promastigotes growth in vitro. The results appoint the conserved domain from the L. braziliensis NTPDase as an important target for inhibitor design and the potential application of these biomolecules in experimental protocols of disease control., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Intrinsic immunosuppression is a major obstacle for successful cancer therapy. The mechanisms for the induction and regulation of immunosuppression in humans are ill defined. A microenvironmental component that might prevent antitumor immunity is the presence of dying tumor cells, which are abundant following conventional cancer ablation methods such as chemo- or radiotherapy. Shedding of apoptotic debris and/or secretion of factors to the tumor bed or draining lymph nodes thus might have a profound impact on professional phagocytes, such as DCs, and subsequent priming of lymphocytes. Here, we exposed human DCs to supernatants of live, apoptotic, or necrotic human breast cancer cells and cocultured them with autologous T cells. Priming with apoptotic debris prevented DCs from establishing cytotoxicity toward live human tumor cells by inducing a Treg-cell population, defined by coexpression of CD39 and CD69. Immunosuppression via Treg cells was transferable and required the release of sphingosine-1-phosphate (S1P) from apoptotic cells, acting via S1P receptor 4 on DCs to induce IL-27 secretion. We propose that CD69 expression on CD39(+) Treg cells enables them to interact with CD73-expressing CD8(+) T cells to generate adenosine, thereby suppressing cytotoxicity. These findings aid the understanding of how dying tumor cells limit antitumor immunity., (© 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Both T helper interleukin 17 (IL-17)-producing cells (Th17 cells) and regulatory T cells (Tregs) have been found to be increased in human tuberculous pleural effusion (TPE); however, the possible interaction between Th17 cells and Tregs in TPE remains to be elucidated. The objective of the present study was to investigate the distribution of Th17 cells in relation to Tregs, as well as the mechanism of Tregs in regulating generation and differentiation of Th17 cells in TPE. In the present study, the numbers of Th17 cells and Tregs in TPE and blood were determined by flow cytometry. The regulation and mechanism of CD39(+) Tregs on generation and differentiation of Th17 cells were explored. Our data demonstrated that the numbers of Th17 cells and CD39(+) Tregs were both increased in TPE compared with blood. Th17 cell numbers were correlated negatively with Tregs in TPE but not in blood. When naïve CD4(+) T cells were cultured with CD39(+) Tregs, Th17 cell numbers decreased as CD39(+) Treg numbers increased, and the addition of the anti-latency-associated peptide monoclonal antibody to the coculture reversed the inhibitory effect exerted by CD39(+) Tregs. This study shows that Th17/Treg imbalance exists in TPE and that pleural CD39(+) Tregs inhibit generation and differentiation of Th17 cells via a latency-associated peptide-dependent mechanism.

CD4(+)CD25(+)Foxp3(+) regulatory T cells (Tregs) produce immunosuppressive adenosine by degradation of adenosine triphosphate (ATP) by the ectonucleotidases CD39 and CD73. In this sequence of events, ATP is not only the substrate for generation of adenosine but it also activates the immunosuppressive functions of Tregs. To compare the effects of ATP on IL-10-deficient (IL-10(-/-)) Tregs with wild-type (wt) Tregs, we incubated both types of Tregs with ATP and assessed their phenotype and function. We show that IL-10(-/-) Tregs failed to become activated by ATP and were impaired in adenosine production. As a consequence, IL-10(-/-) Tregs were unable to block adherence of effector T cells to the endothelium in vitro. When testing the signaling of the ATP receptor P2X(7) in IL-10(-/-) Tregs, we recorded no elevation of intracellular calcium after engagement of P2X(7) receptors, as compared with wt Tregs, thus indicating that IL-10(-/-) Tregs fail to react normally to ATP and display impaired adenosine production, which explains their inability to suppress contact hypersensitivity responses. Therefore, when using IL-10(-/-) Tregs in different disease models, one has to take into account that adenosine production is abrogated and reduced suppressive effects may not be exclusively attributable to the lack of IL-10 production.

There are conflicting data about the frequency and role of regulatory T cells (Tregs) during the course of HIV infection. Peripheral blood of a large cohort of HIV-infected patients (n = 131) at different stages of disease, including 15 long-term nonprogressors and 21 elite controllers, was analyzed to determine the frequency and phenotype of Tregs, defined as CD4(+), CD25(high), CD127(low), FoxP3(high) cells. A significantly increased relative frequency of Tregs within the CD4(+) compartment of HIV(+) patients compared to that of healthy controls (P < 0.0001) was observed. Additionally, the relative frequency of Tregs directly correlated with HIV viral load and inversely with CD4(+) counts. However, the absolute Treg number was reduced in HIV-infected patients versus healthy controls (P < 0.0001), with the exception of elite controllers (P > 0.05). The loss of absolute Treg numbers coincided with rising markers of immune activation (P < 0.0006). The initiation of antiviral therapy significantly increased absolute Treg numbers (P < 0.0031). We find that the expression of CD39, a newly defined ectonucleotidase with immunomodulatory functions on Tregs, correlated with progressive HIV disease, HIV viral load, and immune activation. Of note, when tested in peripheral blood mononuclear cells of healthy volunteers, the in vitro capacity to suppress T-cell proliferation was limited to CD4(+), CD25(high), CD39(+) T cells. Interestingly, Tregs of elite controllers exhibited not only the highest expression of CCR5, CTLA-4, and ICOS but also the lowest level of CD39. The data presented here reconcile the seemingly contradictory results of previous studies looking at Tregs in HIV and highlight the complexity of Treg-mediated immunoregulation during human viral infections.

The P2X7 receptor is an extracellular ATP-gated cation channel critical in inflammation and immunity, and can be up-regulated by IFN-γ and LPS. This study aimed to examine the effect of TGF-β1 on the up-regulation of P2X7 function and expression in leukemic THP-1 monocytes differentiated with IFN-γ and LPS. Cell-surface molecules including P2X7 were examined by immunofluorescence staining. Total P2X7 protein and mRNA was assessed by immunoblotting and RT-PCR respectively. P2X7 function was evaluated by ATP-induced cation dye uptake measurements. Cell-surface P2X7 was present on THP-1 cells differentiated for 3days with IFN-γ and LPS but not on undifferentiated THP-1 cells. ATP induced ethidium(+) uptake into differentiated but not undifferentiated THP-1 cells, and the P2X7 antagonist, KN-62, impaired ATP-induced ethidium(+) uptake. Co-incubation of cells with TGF-β1 plus IFN-γ and LPS prevented the up-regulation of P2X7 expression and ATP-induced ethidium(+) uptake in a concentration-dependent fashion with a maximum effect at 5ng/ml and with an IC(50) of ~0.4ng/ml. Moreover, ATP-induced YO-PRO-1(2+) uptake and IL-1β release were abrogated in cells co-incubated with TGF-β1. TGF-β1 also abrogated the amount of total P2X7 protein and mRNA induced by IFN-γ and LPS. Finally, TGF-β1 prevented the up-regulation of cell-surface CD86, but not CD14 and MHC class II, by IFN-γ and LPS. These results indicate that TGF-β1 prevents the up-regulation of P2X7 function and expression by IFN-γ and LPS in THP-1 monocytes. This suggests that TGF-β1 may limit P2X7-mediated processes in inflammation and immunity., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Background: During extracorporeal photophoresis (ECP), peripheral blood mononuclear cells are treated with DNA-intercalating agents and irradiated with ultraviolet light. This procedure exerts immunosuppressive effects, most likely mediated by regulatory T cells (Treg). However, the underlying mechanisms are not clear yet. In our study, we investigated the effect of ECP on frequency and function of Treg in the peripheral blood of patients suffering from graft-versus-host disease., Methods: Whole blood samples from graft-versus-host disease patients were taken before and after the ECP treatment on 2 consecutive days. Phenotypical analysis of changes in distinct leukocyte subsets within the peripheral blood of patients and healthy controls was performed by means of flow cytometry. Functional analysis of the Treg population after magnetic bead isolation was performed using conventional suppression assays, and adenosine was detected by means of high pressure liquid chromatography and Lanzetta assays., Results: We show that the frequency of CD4/CD25/FoxP3 Treg in the peripheral blood increases after each cycle of ECP and also in the course of treatment. The suppressive capacity of Treg after ECP was increased compared with that of Treg before ECP, although not reaching the suppression levels obtained with Treg from healthy controls. Furthermore, we show that ECP stimulates the CD39-mediated production of adenosine by Treg, which substantially reduces the T-cell proliferation in in vitro suppression assays., Conclusion: Our data indicate that ECP stimulates the conversion of ATP to adenosine by the ectonucleotiodase CD39, which acts as a novel soluble immunosuppressive reagent mediating immunosuppression of Treg.

Human regulatory T cells (Treg) have been variously defined as CD4(+)CD25(+), CD4(+)CD25(high) or CD4(+)CD25(high)FOXP3(+) cells which are responsible for maintaining peripheral tolerance. Their isolation from human peripheral blood or tissues depends on the expression level of CD25(IL-2Ralpha) - a surface marker which is also expressed on activated effector helper T cells. CD39, a cell surface associated ectonucleotidase, can be used to purify Treg with strong suppressor functions. The CD4(+)CD39(+) T cells catalyze cleavage of adenosine triphosphate (ATP) to adenosine monophosphate (AMP), which is then further cleaved to adenosine. CD4(+)CD39(+) T cells largely overlap with CD4(+)CD25(high)FOXP3(+) but not CD4(+)CD25(+) T cell subset, and mediate equally potent immune suppression. Thus, CD39 surface marker can be successfully used for routine isolation of functionally-active human Treg from the peripheral blood of healthy donors or patients with cancer for studies of their role in health and disease.

Forkhead box P3 (FoxP3) is a transcription factor whose expression characterizes regulatory T cells (T(reg)), but it is also present on activated T cells, thus hindering correct T(reg) identification. Using classical markers for T(reg) recognition, discordant results were found in terms of T(reg) expansion during active tuberculosis (TB) disease. Recently CD39 has been shown to be an accurate marker for T(reg) detection. The objectives of this study were: (i) to identify T(reg) expressing CD39 in patients with TB and to compare the results with those obtained by the standard phenotypic markers; (ii) to evaluate if T(reg) are expanded in vitro by exogenous interleukin (IL)-2 or by antigen-specific stimulation; and (iii) to characterize T(reg) function on the modulation of antigen-specific responses. We enrolled 13 patients with pulmonary TB and 12 healthy controls. T(reg) were evaluated by flow cytometry ex vivo and after antigen-specific in vitro stimulation using CD25, FoxP3, CD127 and CD39 markers. Results indicate that CD39(+) cells within the CD4(+)CD25(high) cells have T(reg) properties (absence of interferon-gamma production and transforming growth factor-beta1 release upon stimulation). Ex vivo analysis did not show significant differences between TB patients and controls of T(reg) by classical or novel markers. In contrast, a significantly higher percentage of T(reg) was found in TB patients after antigen-specific stimulation both in the presence or absence of IL-2. Depletion of CD39(+) T(reg) increased RD1-specific responses significantly. In conclusion, CD39 is an appropriate marker for T(reg) identification in TB. These results can be useful for future studies to monitor Mycobacterium tuberculosis-specific response during TB.

For lack of simple inexpensive early detection methods for Shigella spp. and enteroinvasive Escherichia coli (EIEC), bacillary dysentery remains a major cause of childhood mortality and morbidity in India and other developing countries. Rapid stool testing for apyrase, a specific periplasmic enzyme essential for the pathogen's intracellular spread, may provide a solution. We have developed a whole-cell colorimetric pyrophosphate hydrolysis assay based on cheap, stable, and locally available reagents. An innovative filtration-cum-inoculation step eliminates interfering stool solids and ensures sufficient bacterial growth and apyrase expression in 6 to 7 h at 37 degrees C. In a limited double-blind study of 57 clinical isolates of common enterobacteria, the test showed 100% sensitivity and 80% specificity for Shigella spp. and EIEC. Requiring only widely available equipment and inexpensive consumables, this affordable test is readily adaptable for determining antibiograms and for surveillance of food and water samples for the presence of Shigella and EIEC.

Purpose: To determine the effects of LY294002, a phosphatidylinositol 3-kinase inhibitor, on suppressing experimental retinal neovascularization in an animal model of ischemic retinopathy., Methods: The effect of LY294002 on the survival of RF/6A cells stimulated by vascular endothelial growth factor (VEGF) was investigated colorimetrically. The inhibitory activity of LY294002 on the migration of cells stimulated with VEGF was measured by cell counting. C57BL/6N mice at postnatal day (P) 7 were exposed to 75 +/- 2% oxygen for 5 days (P7-P11) and then returned to room air for 5 days (P12-P17) to induce retinal neovascularization. Beginning on P12, mice received daily intraperitoneal injections of LY294002 or dimethyl sulfoxide and phosphate-buffered saline (control) through P17. Retinal neovascularization was examined by adenosine diphosphatase staining after 5 days in room air and was quantitated histologically by counting the neovascular endothelial cell nuclei anterior to the inner limiting membrane., Results: LY294002 significantly inhibited VEGF-induced survival and migration. LY294002-treated and control animals demonstrated no perfusion regions in the posterior retina. Retinas from control mice at P17 contained neovascular tufts at the junction between the perfused and nonperfused retina. The tufts contained numerous neovascular nuclei. Retinas from mice treated with LY294002 demonstrated a significant reduction in neovascular cell nuclei compared with control mice., Conclusions: LY294002 significantly inhibits retinal neovascularization in a mouse model of retinal neovascularization.

Distribution of two enzymes involved in the ectonucleotidase enzyme chain, ecto-nucleoside triphosphate diphosphohydrolase1 (NTPDase1) and ecto-5'-nucleotidase, was assessed by immunohistochemistry in the rat hippocampus. Obtained results have shown co-expression of the enzymes in the hippocampal region, as well as wide and strikingly similar cellular distribution. Both enzymes were expressed at the surface of pyramidal neurons in the CA1 and CA2 sections, while cells in the CA3 section were faintly stained. The granule cell layer of the dentate gyrus was moderately stained for NTPDase1, as well as for ecto-5'-nucleotidase. Glial association for ecto-5'-nucleotidase was also observed, and fiber tracts were intensively stained for both enzymes. This is the first comparative study of NTPDase1 and ecto-5'-nucleotidase distribution in the rat hippocampus. Obtained results suggest that the broad overlapping distribution of these enzymes in neurons and glial cells reflects the functional importance of ectonucleotidase actions in the nervous system.

Acute lung injury (ALI), such as that which occurs with mechanical ventilation, contributes to morbidity and mortality of critical illness. Nonetheless, in many instances, ALI resolves spontaneously through unknown mechanisms. Therefore, we hypothesized the presence of innate adaptive pathways to protect the lungs during mechanical ventilation. In this study, we used ventilator-induced lung injury as a model to identify endogenous mechanisms of lung protection. Initial in vitro studies revealed that supernatants from stretch-induced injury contained a stable factor which diminished endothelial leakage. This factor was subsequently identified as adenosine. Additional studies in vivo revealed prominent increases in pulmonary adenosine levels with mechanical ventilation. Because ectoapyrase (CD39) and ecto-5'-nucleotidase (CD73) are rate limiting for extracellular adenosine generation, we examined their contribution to ALI. In fact, both pulmonary CD39 and CD73 are induced by mechanical ventilation. Moreover, we observed pressure- and time-dependent increases in pulmonary edema and inflammation in ventilated cd39(-/-) mice. Similarly, pharmacological inhibition or targeted gene deletion of cd73 was associated with increased symptom severity of ventilator-induced ALI. Reconstitution of cd39(-/-) or cd73(-/-) mice with soluble apyrase or 5'-nucleotidase, respectively, reversed such increases. In addition, ALI was significantly attenuated and survival improved after i.p. treatment of wild-type mice with soluble apyrase or 5'-nucleotidase. Taken together, these data reveal a previously unrecognized role for CD39 and CD73 in lung protection and suggest treatment with their soluble compounds as a therapeutic strategy for noninfectious ALI.

Background: Calcific aortic valve disease is a common condition and is associated with inflammatory changes and expression of osteoblast-like cell phenotypes, but the cellular mechanisms are unclear. Recent studies identified extracellular ATP and P2Y receptor cascade as important regulators of bone remodeling, whereas its breakdown product, adenosine, is known to have anti-inflammatory properties. We hypothesize that extracellular ATP and adenosine have important roles in regulating osteoblast differentiation in human valve interstitial cells, and that this can be a potential target for therapy. Method and Results- Primary cultures of human valve interstitial cells (ICs) treated for 21 days with osteogenic media, ATP, and ATP-gamma-S (a stable agonist of the P2Y receptor) revealed a significant increase in alkaline phosphatase (ALP) (an osteoblast marker) activity and expression as measured using spectrophotometric assay and immunocytochemistry staining. Valve ICs treated with adenosine alone did not cause an increase in ALP activity; however, adenosine treatment decreased the ALP activity and expression induced by osteogenic media after 21 days of incubation. In addition, atorvastatin inhibited the activity of ALP induced by ATP in human valve ICs, and enzyme studies revealed that atorvastatin upregulated the breakdown of extracellular ATP into adenosine in human valve ICs after 24-hour treatment., Conclusions: These findings identify a novel role for extracellular nucleotides in inducing osteoblast differentiation in human valve ICs in vitro and provide a potential therapeutic target for preventing the disease progression.

The aim of this study was to obtain experimental evidence that phlebotomine saliva is actually ingested during the carbohydrate ingestion phase (before and after blood digestion). The ingestion of carbohydrate was simulated as it occurs in the field by offering the insects balls of cotton soaked in sucrose, sucrose crystals or orange juice cells. The results obtained here showed that ingestion occurred under each condition investigated, as indicated by the presence of apyrase, an enzyme used as a marker to detect saliva in the insect gut and/or carbohydrate sources. Saliva ingestion by phlebotomine during the carbohydrate ingestion phase is important to explain how it could promote starch digestion and to trigger Leishmania promastigotes to follow a differentiation pathway as proposed previously by some authors.

ADP induces platelet aggregation in human whole blood and platelet-rich plasma (PRP). ATP induces aggregation in whole blood only; this involves leukocytes and is mediated by ADP. Here we studied ATP- and ADP-induced aggregation in patients with raised leukocyte counts (mean 46.2x10(3) leukocytes/microl). Platelet aggregation was measured by platelet counting. ATP, ADP and metabolites were measured by HPLC. Aggregation to ADP (1-10 microM) and ATP (10-100 microM) was markedly reduced, but to ATP (1000 microM) was enhanced (all p<0.001). Aggregation to ADP in PRP was normal. Increasing the leukocyte count in normal blood reproduced the findings in the patients. Adding leukocytes (either MNLs or PMNLs) to normal PRP enabled a response to ATP and caused marked inhibition of ADP-induced aggregation. Breakdown of ATP or ADP to AMP and adenosine in leukocyte-rich plasma was rapid (t1/2=4 min) and far higher than in cell-free plasma or PRP. With ATP there was also formation of ADP, maximal at 4 min. The presence of the ectonucleotidase NTPDase1 (CD39) was demonstrated on MNLs (all of the monocytes and a proportion of the lymphocytes) and all PMNLs by flow cytometry. We conclude that leukocytes provide a means of dephosphorylating ATP which enables ATP-induced aggregation via conversion to ADP, but also convert ADP to AMP and adenosine. Platelet aggregation extent is a balance between these activities, and high white cell counts influence this balance.

Introduction: CD39 (NTPDase1), an endothelial cell membrane glycoprotein, is the predominant ATP diphosphohydrolase (ATPDase) in vascular endothelium. It hydrolyses both triphosphonucleosides and diphosphonucleosides at comparable rates, thus terminating platelet aggregation and recruitment responses to ADP and other platelet agonists. This occurs even when nitric oxide (NO) formation and prostacyclin production are inhibited. Thus, CD39 represents the main control system for platelet reactivity. Reduced or deficient local ecto-nucleotidase activity may predispose to development of vascular disease. Based on data in animal models and in vitro, CD39 constitutes a new therapeutic modality for vascular disease with a novel and unique mode of action., Materials and Methods: Lymphocytes were isolated from 46 patients with angiographically proven coronary artery disease (CAD) as well as from matched healthy control subjects. Ectonucleotidase ADPase and ATPase activities (prototypical for the ATPDase activity of endothelial cells) were measured using established radio-TLC procedures., Results and Discussion: In the patients, a decreased ratio of ADPase to ATPase activities (from 1.26 to 1.04) was observed despite increases in both ADPase and ATPase activities. Coronary artery disease was the only independent predictor of a difference in the ADPase/ATPase activity ratio by multivariate linear regression analysis (P=0.0035). This altered ADPase/ATPase activity ratio in patients may represent a reduction in endogenous defense systems against platelet-driven thrombotic events. These data may identify a population of patients with excessive platelet reactivity in their circulation. Increased generation of prothrombotic ADP in these patients implies a potential benefit from therapeutic intervention with soluble forms of CD39.

Schistosoma mansoni, a human parasite that constitutes a major health problem in developing countries, escapes from host defenses and survives in the human bloodstream. Here, we report the cloning of a S. mansoni ATP-diphosphohydrolase ortholog (SmATPDase1). Southern blots indicated that in S. mansoni it is a single-copy gene. RT-PCR revealed that SmATPDase1 is expressed in five stages of the parasite life cycle, namely cercaria, schistosomula, adults, eggs, and miracidia. Using confocal microscopy, SmATPDase1 protein was immunolocalized on the external surface in all stages, except eggs, being conspicuously present in adults. ATPDase, which is present on the outer surface of endothelial cells lining human blood vessels, has been implicated in thromboregulation by promoting ADP hydrolysis and inhibition of platelet aggregation. The presence of an ATPDase ortholog on the surface of S. mansoni suggests that the enzyme might play a role in the escape from host defenses that would involve platelet activation.

Evidence suggests that Pseudomonas aeruginosa stromal keratitis and corneal perforation (susceptibility) is a CD4(+) T cell-regulated inflammatory response following experimental P. aeruginosa infection. This study examined the role of Langerhans cells (LC) and the B7/CD28 costimulatory pathway in P. aeruginosa-infected cornea and the contribution of costimulatory signaling by this pathway to disease pathology. After bacterial challenge, the number of LC infiltrating the central cornea was compared in susceptible C57BL/6 (B6) vs resistant (cornea heals) BALB/c mice. LC were more numerous at 1 and 6 days postinfection (p.i.), but were similar at 4 days p.i., in susceptible vs resistant mice. Mature, B7 positive-stained LC in the cornea and pseudomonas Ag-associated LC in draining cervical lymph nodes also were increased significantly p.i. in susceptible mice. To test the relevance of these data, B6 mice were treated systemically and subconjunctivally with neutralizing B7 (B7-1/B7-2) mAbs. Treatment decreased corneal disease severity and reduced significantly the number of B7-positive cells as well as the recruitment and activation of CD4(+) T cells in the cornea. IFN-gamma mRNA levels also were decreased significantly in the cornea and in draining cervical lymph nodes of mAb-treated mice. When CD28(-/-) animals were tested, they exhibited a less severe disease response (no corneal perforation) than wild-type B6 mice and had a significantly lower delayed-type hypersensitivity response to heat-killed pseudomonas Ag. These results support a critical role for B7/CD28 costimulation in susceptibility to P. aeruginosa ocular infection.

Extracellular nucleotides are ubiquitous extracellular mediators that interact with and activate nucleotide type 2 (P2) receptors. These receptors initiate a wide variety of signalling pathways that appear important for functional associations between neurons and glial cells and for the regulation of blood flow, haemostatic and inflammatory reactions in the brain. Ectonucleotidases are extracellular nucleotide-metabolizing enzymes that modulate P2 receptor-mediated signalling by the regulated hydrolysis of these agonists. A considerable number of ectoenzyme species with partially overlapping substrate and tissue distributions have been described. Major candidates for expression in the brain are members of the ecto-nucleoside triphosphate diphosphohydrolase (E-NTPDase or CD39) family. The production of cd39-/- mice and specific reagents have enabled us to analyse the specific cellular distribution of NTPDase1 (CD39), the prototype member of the enzyme family, in the mouse brain. Using monospecific antibodies and enzyme histochemical staining, we have identified NTPDase1 as a major ectonucleotidase associated with both microglia and the endothelial and smooth muscle cells of the vasculature. NTPDase1 is not expressed by neurons and astrocytes. Additional unidentified ectonucleotidase functional activity is observed at lower levels throughout the brain parenchyma. NTPDase1 may regulate P2 receptor-mediated functions of microglia as well as influence nucleotide signalling between neurons or astrocytes that are associated with multiple microglial ramifications. The expression of NTPDase1 by cerebrovascular endothelial and smooth muscle cells also suggests involvement in the regulation of blood flow and thrombogenesis.

We localised three important enzymes histochemically in placental trophoblasts from women who gave birth to dichorionic discordant twins, in which the co-twin was affected by foetal growth restriction (FGR). The enzymes studied were adenosine diphosphate-degrading enzyme (ADP-degrading enzyme, plasma membrane enzyme), cytochrome c oxidase (mitochondrial enzyme), and glucose-6-phosphatase (endoplasmic reticular enzyme). We compared these enzyme activities and their distribution patterns among placentas of the smaller (FGR) co-twin, larger co-twin, pre-eclamptic singleton with FGR, and normal singletons with birth weight of appropriate for their gestational ages. In FGR co-twin placentas, the intensity and localisation pattern of these three enzymes did not differ from those seen in the larger co-twin and normal singleton placentas. Decreased ADP-degrading activity and cytochrome c oxidase negative mitochondria, which were characteristic features of pre-eclamptic trophoblasts, were not observed in FGR co-twin placentas. These observations indicated that, in the FGR co-twin, enzyme-histochemically detectable trophoblastic cell dysfunction may be absent, or if present, less prominent, compared with pre-eclamptic FGR. We previously reported that placental trophoblasts from singleton idiopathic FGR also showed no reduction in these enzyme activities. In mechanism and pathophysiology, FGR in dichorionic discordant twins may be quite different from pre-eclamptic FGR, but somewhat resembles idiopathic FGR, though all three disorders lead to placental insufficiency, resulting in limited foetal growth.