apyrase and Anemia--Sickle-Cell

Topics: Adenosine Triphosphate; Anemia, Sickle Cell; Apyrase; Cations; Cells, Cultured; Erythrocytes; Humans; Hydrogen-Ion Concentration; Hypoxia; Probenecid; Reticulocytes; Suramin

Invariant natural killer T (iNKT) cells are activated at sites of local tissue injury, or globally during vaso-occlusive episodes of sickle cell disease (SCD). Tissue damage stimulates production of CD1d-restricted lipid antigens that activate iNKT cells to produce Th1- and Th2-type cytokines. Here, we show that circulating iNKT cells in SCD patients express elevated levels of the ectonucleoside triphosphate diphosphosphohydrolase, CD39, as well the adenosine A2A receptor (A2AR). We also investigated the effects of stimulating cultured human iNKT cells on the expression of genes involved in the regulation of purinergic signaling. iNKT cell stimulation caused induction of ADORA2A, P2RX7, CD38, CD39, ENPP1, CD73, PANX1, and ENT1. Transcription of ADA, which degrades adenosine, was reduced. Induction of CD39 mRNA was associated with increased ecto-ATPase activity on iNKT cells that was blocked by POM1. Exposure of iNKT cells to A2AR agonists during stimulation reduced production of IFN-γ and enhanced production of IL-13 and CD39. Based on these findings, we define "purinergic Th2-type cytokine bias" as an antiinflammatory purinergic response to iNKT cell stimulation resulting from changes in the transcription of several genes involved in purine release, extracellular metabolism, and signaling.

Topics: 5'-Nucleotidase; ADP-ribosyl Cyclase 1; Anemia, Sickle Cell; Antigens, CD1d; Apyrase; Connexins; Cytokines; Equilibrative Nucleoside Transporter 1; GPI-Linked Proteins; Humans; Immunity, Innate; Interleukin-13; Natural Killer T-Cells; Nerve Tissue Proteins; Phosphoric Diester Hydrolases; Purinergic Agents; Purines; Pyrophosphatases; Receptor, Adenosine A2A; Receptors, Purinergic P2X7; Signal Transduction; Transcription Factors

Sickle cell anemia (SCA) is a hemoglobinopathy characterized by hemolysis and vaso-occlusions caused by rigidly distorted red blood cells. Sickle cell crisis is associated with extracellular release of nucleotides and platelets, which are critical mediators of hemostasis participating actively in purinergic thromboregulatory enzymes system.This study aimed to investigate the activities of purinergic system ecto-enzymes present on the platelet surface as well as CD39 and CD73 expressions on platelets of SCA treated patients. Fifteen SCA treated patients and 30 health subjects (control group) were selected. Ecto-nucleoside triphosphate diphosphohydrolase (E-NTPDase), ecto-5'-nucleotidase (E-5'-NT) and ecto-adenosine deaminase (E-ADA) activities were measured in platelets isolated from these individuals. Results demonstrated an increase of 41 % in the E-NTPDase for ATP hydrolysis, 52% for ADP hydrolysis and 60 % in the E-ADA activity in SCA patients (P<0.05); however, a two folds decrease in the CD39 expression in platelets was observed in the same group (P<0.01). The increased E-NTPDase activity could be a compensatory mechanism associated with the low expression of CD39 in platelets. Besides, alteration of these enzymes activities suggests that the purinergic system could be involved in the thromboregulatory process in SCA patients.

Topics: 5'-Nucleotidase; Adenosine Deaminase; Adenosine Diphosphate; Adenosine Monophosphate; Adenosine Triphosphate; Adolescent; Adult; Anemia, Sickle Cell; Antigens, CD; Apyrase; Blood Platelets; Case-Control Studies; Female; Flow Cytometry; GPI-Linked Proteins; Humans; Male; Young Adult

Recent studies have demonstrated the role of adenosine (ADO) in sickle-cell anemia (SCA). ADO is produced by CD39 and CD73 and converted to inosine by adenosine deaminase (ADA). We evaluated the effects of hydroxycarbamide (HU) treatment on the modulation of adenosine levels in SCA patients. The expressions of CD39, CD73, and CD26 were evaluated by flow cytometry on blood cells in 15 HU-treated and 17 untreated patients and 10 healthy individuals. RNA was extracted from monocytes, and ADA gene expression was quantified by real-time PCR. ADA activity was also evaluated. We found that ADA transcripts were two times higher in monocytes of HU-treated patients, compared with untreated (P = 0.039). Monocytes of HU-treated patients expressed CD26, while monocytes of controls and untreated patients did not (P = 0.023). In treated patients, a lower percentage of T lymphocytes expressed CD39 compared with untreated (P = 0.003), and the percentage of T regulatory (Treg) cells was reduced in the treated group compared with untreated (P = 0.017) and controls (P = 0.0009). Besides, HU-treated patients displayed increased ADA activity, compared with untreated. Our results indicate a novel mechanism of action of HU mediated by the reduction of adenosine levels and its effects on pathophysiological processes in SCA.

Topics: 5'-Nucleotidase; Adenosine; Adenosine Deaminase; Adolescent; Adult; Anemia, Sickle Cell; Antigens, CD; Antisickling Agents; Apyrase; Blood Cells; Case-Control Studies; Child; Dipeptidyl Peptidase 4; Gene Expression Regulation, Enzymologic; GPI-Linked Proteins; Humans; Hydroxyurea; Metabolic Networks and Pathways; Middle Aged; Young Adult

Phosphatase enzymes cleave an inorganic phosphate from a substrate. Phosphatase enzyme histochemistry followed by flat-embedding in glycol methacrylate is extremely useful in studying retinal and choroidal vascular development and loss, since only viable blood vessels have these enzyme activities. Sites of occlusion and remodeling can be identified and analysed, resulting in new insights into the cause of occlusion. The phosphatase activities are elevated in neovascularization making possible high resolution analysis of neovascularization, the feeder vessels, and the retinal milieu in which angiogenesis occurs. Adenosine diphosphatase (ADPase) catalyzes ADP to an inorganic phosphate plus adenosine monophosphate, preventing accumulation of ADP, one of the most potent stimuli for platelet aggregation. The ADPase technique can be used in any species but this report highlights its use in dog and human retinas. The ADPase technique has yielded important insights into vaso-occlusive and vasoproliferative processes in retinopathy of prematurity, sickle cell and diabetic retinopathies. The alkaline phosphatase flatembedding technique is useful in evaluating dog, cat, and human choroidal vasculatures. It has permitted quantification of the loss of choriocapillaris in diabetic choroidopathy and of the RPE and choriocapillaris in geographic atrophy and exudative age-related macular degeneration.

Topics: Adult; Aged, 80 and over; Anemia, Sickle Cell; Animals; Apyrase; Cats; Choroid; Diabetic Retinopathy; Dogs; Endothelial Cells; Histocytochemistry; Humans; Retinal Vessels

To examine the sickle cell retina in dual perspective (vascular patterns en bloc and structure in serial sections) to gain new insights into sickle cell retinopathy.. We analyzed the retinas of two patients with sickle cell disease (a 54-year-old patient with hemoglobin SC, heterozygous for the S and C mutation in the beta chain of the globin gene, and a 20-month-old patient with sickle cell anemia [SS], homozygous for the S mutation) using the previously described adenosine diphosphatase flat-embedding technique.. The dual-perspective analysis afforded by our technique revealed that the primary site of occlusions was located at the precapillary level. An unusual neovascular formation, the hairpin loop, was observed in both patients' retinas and appeared to result from recanalization of the wall of an occluded vessel. Many autoinfarcted pre-retinal neovascular formations were observed in the older SC patient. Two patent preretinal formations were studied in detail and their evolution appeared to be influenced by mechanical factors. The vessels appeared to have been extruded from the retina, perhaps owing to hydrostatic pressure secondary to downstream occlusions. Small pigmented lesions consisting of retinal pigment epithelial cells ensheathing channels that resembled autoinfarcted vessels were found in the eye of the SC patient.. This study illustrates unusual morphological features of intraretinal and preretinal neovascularization and of chorioretinal lesions in sickle cell retinopathy and suggests alternative mechanisms for their formation.

Topics: Adult; Anemia, Sickle Cell; Apyrase; Choroid Diseases; Electron Probe Microanalysis; Female; Humans; Infant; Male; Middle Aged; Retinal Artery Occlusion; Retinal Diseases; Retinal Neovascularization; Retinal Vessels; Tissue Embedding