Cell Adenosine Deaminase Research Articles

Relationship between the outcomes of intensive phase therapy in patients with newly diagnosed infiltrative pulmonary tuberculosis and activity of purine metabolism enzymes as well as CD3+CD8+ lymphocyte level

Monitoring activity of inflammatory process and lymphocyte subsets can help assess the effectiveness of intensive phase therapy (IPT) already in the early stages of treatment. The goal is to evaluate changes in the concentration and activity of enzymes associated with purine metabolism and peripheral blood lymphocyte subset composition, and to determine their relationship with IPT effectiveness in patients with newly diagnosed infiltrative pulmonary tuberculosis (IPTb). Materials and methods. In 141 IPTb patients, the IPT data were presented as follows: “significant improvement” (SI) — disappearance of intoxication symptoms, abacillation, closure of decay cavities; “less pronounced improvement” (LMI) — eliminated symptoms of intoxication, abacillation, pronounced resorption of focal and infiltrative changes, reduction of decay cavities. We assessed the activity of adenosine deaminase in blood serum (eADA-1, 2), mononuclear cells and neutrophils, the concentration of blood serum ecto-5'-nucleotidase (eHT5E), CD26 (DPPV) in blood serum (s, soluble form) and mononuclear cells (m, membrane form), subpopulation composition. Results. Patients exhibit increased concentrations of eNT5E, mCD26 (DPPIV) and eADA-2 activity, and decreased intracellular ADA-1 activity. In the “LMI” group, after IPT, an increased sCD26 (DPPV) level was noted. The groups differed in lymphocyte counts and percentage of CD3+CD8+ cells. eADA-2 activity was higher in the LMI group and increased after IPT, in contrast to comparison group. mCD26 (DPPIV) concentrations are higher in PD patients before therapy and after IPT. Conclusion. Thus, the outcome of IPT in IPTb patients is associated with altered T-lymphocyte populations and severity of the inflammatory process. Studying the activity of membrane and soluble eADA-2, CD26 (DPPIV) and percentage of CD3+CD8+ T-lymphocytes in the early stages of therapy can provide the necessary information for correcting personalized pathogenetic therapy of patients with newly diagnosed IPTb.

Inhibition of LPS-stimulated ecto-adenosine deaminase attenuates endothelial cell activation

Vascular inflammation is an important factor in the pathophysiology of cardiovascular diseases, such as atherosclerosis. Changes in the extracellular nucleotide and in particular adenosine catabolism may alter a chronic inflammation and endothelial activation. This study aimed to evaluate the relation between vascular ecto-adenosine deaminase (eADA) activity and endothelial activation in humans and to analyze the effects of LPS-mediated inflammation on this activity as well as mechanisms of its increase. Moreover, we investigated a therapeutic potential of ADA inhibition by deoxycofromycin (dCF) for endothelial activation.We demonstrated a positive correlation of vascular eADA activity and ADA1 mRNA expression with endothelial activation parameters in humans with atherosclerosis. The activation of vascular eADA was also observed under LPS stimulation in vivo along with endothelial activation, an increase in markers of inflammation and alterations in the lipid profile of a rat model. Ex vivo and in vitro studies on human specimen demonstrated that at an early stage of vascular pathology, eADA activity originated from activated endothelial cells, while at later stages also from an inflammatory infiltrate. We proposed that LPS-stimulated increase in endothelial adenosine deaminase activity could be a result of IL-6/JAK/STAT pathway activation, since the lack of IL-6 in mice was associated with lower vascular and plasma eADA activities. Furthermore, the inhibitors of JAK/STAT pathway decreased LPS-stimulated adenosine deaminase activity in endothelial cells. We demonstrated that cell surface eADA activity could be additionally regulated by transcytosis pathways, as exocytosis inhibitors including lipid raft inhibitor, methyl-β-cyclodextrin decreased LPS-induced eADA activity. This suggests that cholesterol-dependent protein externalization mediated by lipid rafts could be an important factor in the eADA increase. Moreover, endocytosis inhibitors and exocytosis activators increased this activity on the cell surface. Furthermore, the inhibition of adenosine deaminase in endothelial cells in vitro attenuated LPS-mediated IL-6 release and soluble ICAM-1 and VCAM-1 concentration in the incubation medium through the restoration of the extracellular adenosine pool and adenosine receptor-dependent pathways.This study demonstrated that the vascular endothelial eADA activity remains under control of inflammatory mediators acting through JAK/STAT pathway that could be further modified by dyslipidemic-dependent exocytosis and transcytosis pathways. Inhibition of eADA blocked endothelial activation suggesting a crucial role of this enzyme in the control of vascular inflammation. This supports the concept of eADA targeted vascular protection therapy.

Expression of Adenosine A2B Receptor and Adenosine Deaminase in Rabbit Gastric Mucosa ECL Cells.

Adenosine is readily available to the glandular epithelium of the stomach. Formed continuously in intracellular and extracellular locations, it is notably produced from ATP released in enteric cotransmission. Adenosine analogs modulate chloride secretion in gastric glands and activate acid secretion in isolated parietal cells through A2B adenosine receptor (A2BR) binding. A functional link between surface A2BR and adenosine deaminase (ADA) was found in parietal cells, but whether this connection is a general feature of gastric mucosa cells is unknown. Here we examine whether A2BR is expressed at the membrane of histamine-producing enterochromaffin-like (ECL) cells, the major endocrine cell type in the oxyntic mucosa, and if so, whether it has a vicinity relationship with ADA. We used a highly homogeneous population of rabbit ECL cells (size 7.5–10 µm) after purification by elutriation centrifugation. The surface expression of A2BR and ADA proteins was assessed by flow cytometry and confocal microscopy. Our findings demonstrate that A2BR and ADA are partially coexpressed at the gastric ECL cell surface and that A2BR is functional, with regard to binding of adenosine analogs and adenylate cyclase activation. The physiological relevance of A2BR and ADA association in regulating histamine release is yet to be explained.

The Novel Missense Mutation of GATA1 Caused Red Cell Adenosine Deaminase Overproduction Associated with Congenital Hemolytic Anemia

Red cell adenosine deaminase (ADA) overproduction (OMIM 102730) is a rare form of congenital hemolytic anemia. To date, only four independent families have been reported. Recently, we examined the red cell enzyme activities of an 18-year-old male Japanese patient with congenital hemolytic anemia, and diagnosed a new case of ADA overproduction. His ADA activity was measured as 39.7 IU/gHb, representing an over 30-fold elevation of the normal mean value. The patient’s mother also showed a high red cell ADA, 7.40 IU/gHb, and the father had normal ADA activity. To elucidate the molecular basis of the elevated ADA activity, we performed a target-captured sequencing focusing on the 67 congenital-anemia related genes. The results showed that there was no structural mutation of ADA gene, and that the proband had a novel missense mutation of GATA1, c.920G>A, p.R307H. The proband was hemizygous, and the mother was heterozygous for the mutation. Subsequently, we examined a previously reported case of ADA overproduction, and identified the identical missense mutation of GATA1. These two cases had clinical similarities, such as low birth weight with hypospadias, splenomegaly, and slightly decreased platelet counts, suggesting that these cases could be categorized as X-linked anemia with or without neutropenia and/or platelet abnormality (XLANP, OMIM#300835). The previous case showed a rare blood type, Lu(a-b-) and a low beta/alpha globin synthetic ratio, whereas the present case depicted abnormal red cell morphology, such as stomatocytosis and target cells. Taken together, the missense mutation of GATA1 might cause the aberrant expression of erythroid-genes, inducing a short life-span of red cells. DisclosuresNo relevant conflicts of interest to declare.

Development of a capillary electrophoresis method for analyzing adenosine deaminase and purine nucleoside phosphorylase and its application in inhibitor screening

A novel capillary electrophoresis (CE) method was developed for simultaneous analysis of adenosine deaminase (ADA) and purine nucleoside phosphorylase (PNP) in red blood cells (RBCs). The developed method considered and took advantage of the natural conversion from the ADA product, inosine to hypoxanthine. The transformation ratio was introduced for ADA and PNP analysis to obtain more reliable results. After optimizing the enzymatic incubation and electrophoresis separation conditions, the determined activities of ADA and PNP in 12 human RBCs were 0.237–0.833 U/ml and 9.013–10.453 U/ml packed cells, respectively. The analysis of ADA in mice RBCs indicated that there was an apparent activity difference between healthy and hepatoma mice. In addition, the proposed method was also successfully applied in the inhibitor screening from nine traditional Chinese medicines, and data showed that ADA activities were strongly inhibited by Rhizoma Chuanxiong and Angelica sinensis. The inhibition effect of Angelica sinensis on ADA is first reported here and could also inhibit PNP activity.

Elevated Red Cell Reduced Glutathione Is a Novel Biomarker of Diamond-Blackfan Anemia

Diamond-Blackfan anemia (DBA) is a rare congenital red cell aplasia characterized by congenital anomalies and predisposition to cancer. Recent observation disclosed that heterogeneous mutations in ribosomal protein (RP) genes are present in approximately 50% of patients, suggesting that diagnosis should be made by clinical phenotypes such as age, hematological findings or positive family history. Although elevated activity of red cell adenosine deaminase (eADA) has been utilized as a useful biomarker for differential diagnosis of DBA, approximately 20% of DBA patients are eADA-negative. Recent observations suggested that ribosomal haploinsufficiency increases oxidative stress, leading to p53 gene activation and premature death of erythroid cells. We hypothesized that reduced glutathione (GSH), an essential antioxidant of erythroid cells, might be upregulated in red cells of DBA subjects. In order to test this hypothesis, we examined red cell GSH as well as eADA of 22 patients in 18 DBA families, in whom we had identified gene mutations in RPS19, RPL5, RPL11, RPS10, RPS17 or RPS35a. All except one DBA patients showed elevated GSH (>88.6 mg/dl RBC, M+SD), whereas 17 out of 22 patients exhibited elevated eADA (>2.31 IU/g Hb, M+3SD). We also examined 14 unaffected members of the DBA families, with 1 out of 14 subjects showing elevated GSH and none showing elevated eADA. We performed linear discriminant analysis between DBA and non-DBA subjects with both eADA and GSH using the Support Vector Machine (SVM) from 36 subjects, and successfully obtained a formula to discriminate DBA from unaffected subjects: 0.937*eADA+0.0702*GSH-7.9044 >0. By using this formula, all DBA examined can be diagnosed and unaffected family members can be excluded. Since approximately 50% of clinically diagnosed DBA cases have no causative RP gene mutations, the combined assessment of eADA and GSH might be quite useful for biochemical diagnosis of DBA. DisclosuresNo relevant conflicts of interest to declare.

RPS29 is Mutated in a Multi-Case Diamond Blackfan Anemia Family

AbstractAbstract ▪511▪This icon denotes a clinically relevant abstractDiamond Blackfan anemia (DBA) is an inherited bone marrow failure syndrome (IBMFS) characterized by red blood cell aplasia, variable physical anomalies, and increased risk of leukemia, myelodysplastic syndrome, lymphoma, and certain solid tumors, including osteosarcoma. DBA has been considered to be a disorder of ribosomal biogenesis because approximately 50% of cases are due to a mutation or deletion in 1 of 9 ribosomal protein genes (RPS19, RPS24, RPS17, RPL35A, RPL5, RPL11, RPS7, RPS10, or RPS26). However, mutations in GATA1, a hematopoietic transcription factor, have recently been reported to also cause DBA. DBA is inherited in an autosomal dominant manner, but de novo germline mutations have also been reported.Our IBMFS cohort study conducts detailed clinical evaluations and medical record review of patients with DBA and their family members. We evaluated a large family with DBA in which mutation testing for the 10 known genes was negative. The male proband had steroid-responsive anemia as a child; he was in remission until he was treated with chemotherapy for squamous cell lung cancer at age 55 years. His healthy sister is an obligate carrier because her daughter was diagnosed with DBA as a child; her daughter had steroid-responsive anemia. The proband's maternal 1st cousin had steroid-responsive DBA as a child which relapsed during pregnancy. Her 3 children (the proband's 2nd cousins) had DBA. One died due to complications of transfusion-related iron overload, one had successful hematopoietic stem cell transplantation for steroid-refractory and transfusion-dependent anemia at age 26 years, and one has been off treatment and in remission for over 20 years. Except for the proband with lung cancer and a transfusion-dependent individual, all affected individuals had elevated red blood cell adenosine deaminase consistent with DBA.We performed whole-exome sequencing on the 5 clinically affected individuals (a male proband, his affected first cousin, 2 affected second cousins, and an affected niece), his obligate carrier sister, and the unaffected father of the proband’s niece. Genomic DNA was used to create an enriched multiplexed sequencing library (Nimblegen v2); this was followed by paired-end sequencing using an Illumina HiSeq™. We detected a total of 229,024 exonic nucleotide variants across this family, including 2,484 uncommon (minor allele frequency <5%) non-synonymous amino-acid substitutions. After removing variants that were present in publically available databases (1000Genomes, ESP, Kaviar, and dbSNP) and applying quality control filters, 731 candidate variants remained. We then evaluated these variants for autosomal dominant inheritance in this family. Remarkably, there was only 1 nonsynonymous variant present in all 5 affected individuals and in the obligate carrier, but absent from the unaffected non-obligate carrier parent. This nonsynonymous variant was present in exon 2 of the ribosomal protein S29 (RPS29) gene (chromosome 14q). This region of the RPS29 protein is very highly evolutionarily conserved. Bioinformatic analyses suggested that this variant is highly likely to affect protein function. SIFT, Polyphen 2, and Condel algorithms all predict this variant to be deleterious. RPS29 gene encodes the 40S ribosomal protein S29, which is a component of the small 40S ribosomal subunit and is important for ribosomal RNA processing and ribosome biogenesis. In addition, Zebrafish models of RPS29 mutations have significant defects in hematopoietic stem cell and red blood cell development. We are currently sequencing RPS29 in additional patients with DBA. In summary, whole-exome sequencing has allowed us to uncover another human DBA gene, RPS29, which will likely lead to improved understanding of this complex disorder. Disclosures:No relevant conflicts of interest to declare.

Mononuclear cell adenosine deaminase and CD26/dipeptidylpeptidase-IV activities are sensitive markers of reperfusion during percutaneous transluminal angioplasty

During ischaemia, the extracellular level of adenosine increases, which has cytotoxic effects. In endothelium, cell surface adenosine deaminase (ADA) complexing CD26 is coordinately induced during ischaemia as part of an adaptative response by eliminating adenosine. We examined whether a similar mechanism exists for mononuclear cells. We studied mononuclear cell surface ADA (MCADA) and dipeptidyl-peptidase IV activity (DPPIV) of membrane CD26 during percutaneous transluminal coronary angioplasty (PTCA) as a model of ischaemia–reperfusion. Enzymatic activities were compared with levels of ischaemia-modified albumin (IMA), a marker of ischaemia-reperfusion. Methods and resultsPatients (15 men and 5 women) with non-ST segment elevation acute coronary syndrome related to a stenosis of proximal left anterior descending artery were prospectively included before revascularization. MCADA, DPPIV and IMA were measured before PTCA (T0) then 15 (T15) and 120 (T120) minutes after reperfusion. Fifteen healthy control subjects were enrolled.At T0, MCADA and IMA levels were higher in patients than in controls. MCADA decreased at T15 (median, IQR: 8.2 [7.6–9.8] IU) relative to T0 (11.25 [10–13.5] IU, p<0.01) and remained low at T120. DPPIV decreased at T15 (0.9 [0.7–1.1] AU) relative to T0 (1.05 [0.99–1.48] AU; p<0.01) and remained low at T120. IMA level increased only at T120. MCADA and DPPIV were correlated. Our findings are that MCADA and DPPIV decreased rapidly after angioplasty, suggesting that both catalysts are early markers of reperfusion. ConclusionMCADA and DPPIV are sensitive and early markers of ischaemia–reperfusion process during PTCA.

Expression of Drosophila Adenosine Deaminase in Immune Cells during Inflammatory Response

Extra-cellular adenosine is an important regulator of inflammatory responses. It is generated from released ATP by a cascade of ectoenzymes and degraded by adenosine deaminase (ADA). There are two types of enzymes with ADA activity: ADA1 and ADGF/ADA2. ADA2 activity originates from macrophages and dendritic cells and is associated with inflammatory responses in humans and rats. Drosophila possesses a family of six ADGF proteins with ADGF-A being the main regulator of extra-cellular adenosine during larval stages. Herein we present the generation of a GFP reporter for ADGF-A expression by a precise replacement of the ADGF-A coding sequence with GFP using homologous recombination. We show that the reporter is specifically expressed in aggregating hemocytes (Drosophila immune cells) forming melanotic capsules; a characteristic of inflammatory response. Our vital reporter thus confirms ADA expression in sites of inflammation in vivo and demonstrates that the requirement for ADA activity during inflammatory response is evolutionary conserved from insects to vertebrates. Our results also suggest that ADA activity is achieved specifically within sites of inflammation by an uncharacterized post-transcriptional regulation based mechanism. Utilizing various mutants that induce melanotic capsule formation and also a real immune challenge provided by parasitic wasps, we show that the acute expression of the ADGF-A protein is not driven by one specific signaling cascade but is rather associated with the behavior of immune cells during the general inflammatory response. Connecting the exclusive expression of ADGF-A within sites of inflammation, as presented here, with the release of energy stores when the ADGF-A activity is absent, suggests that extra-cellular adenosine may function as a signal for energy allocation during immune response and that ADGF-A/ADA2 expression in such sites of inflammation may regulate this role.

Mononuclear Cell Adenosine Deaminase in Patients with Chronic Renal Failure

Infections are considered as an important complication of uremia. The cause of immunosuppression in patients with renal failure is not exactly localized. Defect in the mononuclear cells with disturbance related to adenosine metabolism was presumed to be an important contributor to immune dysfunction in patients with renal failure. The study was carried out on 30 patients with chronic renal failure on regular hemodialysis (HD) (group 1), 30 patients with chronic renal impairment in predialysis periods (group 2), and 30 healthy control subjects (group 3). The complete medical history of all the patients, with respect to clinical examination, echocardiography, and nerve conduction studies was obtained. The mononuclear cell adenosine (MCAD) level, and mononuclear cell adenosine deaminase (MCADA) activity were measured. The MCAD level was higher in group 1 than in group 2, and was significantly higher in both groups than in the control group. However, the MCADA activity was found to be significantly lower in group 1 than in group 2, and was considerably lower in both groups when compared with the control group. The MCAD was found to be positively correlated to the history of infection and negatively correlated to the ejection fraction in groups 1 and 2. There was a positive correlation between MCAD and the presence of neuropathy in group 1, but not in group 2. The MCADA activity was found to be low in patients with chronic renal failure, which further decreased with HD. This condition may lead to high level of MCAD, which is related to the development of infection, neuropathy, and myocardial depression in those patients.

Adenosine Deaminase Activity in Cats Infected with Feline Immunodeficiency Virus

Adenosine deaminase (ADA), an enzyme involved in purine metabolism, has been shown to be of clinical importance in several diseases in humans. To investigate whether ADA is of any clinical significance in cats, plasma adenosine deaminase (P-ADA) and T cell adenosine deaminase (T-ADA) activities were measured in feline immunodeficiency virus (FIV) negative and positive cats. The AIDS-related complex (ARC) group showed a significant elevation in P-ADA activity compared to the asymptomatic carrier (AC), and FIV-negative groups (P<0.005). T-ADA activity was significantly elevated in FIV-positive cats compared to the FIV-negative group (P<0.05) and this elevation was attributed to the increase in the ARC group (P<0.01). A correlation was found between P-ADA and T-ADA activities in the FIV-negative group. T-ADA activity and CD4(+)cell number showed a strong negative correlation in FIV-positive cats (P<0.0005). CD4(+) cell numbers were significantly reduced in the ARC group compared to the healthy controls (P<0.005). Our results showed that T-ADA is increased in FIV-positive cats during the ARC stage. These results also suggest that ADA may be an indicator of T cell activation in the ARC stage of FIV infection.

Autoantibodies against tumour suppressor protein p53 in pleural effusions of patients with tuberculosis pleurisy

Autoantibodies against the p53 proteins (p53 Abs) can be detected in the serum, ascites, saliva and pleural effusions of various malignant patients. It is suggested that p53 Abs in pleural effusions might have some value for tumour diagnosis, prognosis or monitoring. The present study investigated the prevalence of p53 Abs in the pleural effusions of 90 patients with various diseases. Patients with suspicious pleural effusions in chest film received thoracocentesis and their pleural effusions were collected. The presence of p53 Abs in effusion was detected by immunoblotting. Differences of p53 Abs with respect to the patient's age, gender, white blood cell count, lactate dehydrogenase, total proteins and adenosine deaminase scores were calculated by chi2-test. p53 Abs were detected in 14.4% (13/90) of our patients, with prevalences of 10.5% (6/57) and 21.2% (7/33) among patients with benign and malignant diseases, respectively. Notably, 16.1% (5/31) of patients with tuberculosis pleurisy were positive for p53 Abs. These five patients had no history of cancer and, so far, have had no manifestations related to tumorigenesis. As far as we know, this is the first report regarding the detection of p53 Abs in pleural effusions from patients with tuberculosis pleurisy.

Kinetic study of adenosine concentrations and the expression of adenosine deaminase in mononuclear cells during hemodialysis

We previously showed that high intralymphocytic adenosine (Ado) concentrations are found in hemodialyzed patients due to the reduced activity of mononuclear cell adenosine deaminase (MCADA). These abnormalities contribute to the immune defect observed in HD patients. The kinetics of these abnormalities and the causes of the low MCADA activity, however, have not been investigated. Here, we addressed this question. Since interferon gamma (IFNgamma) partially modulates MCADA, we also evaluated the effect of IFNgamma on MCADA activity in vitro. The study included 12 patients (eight men and four women) who were observed from the first to the 36th hemodialysis (HD) session, and eight healthy subjects (controls). MCADA activity and Ado concentrations were normal before the first HD session. Ado concentrations progressively increased from the first (10.5 +/- 3.1 pmol/10(7) cells) to the fourth session (26.7 +/- 3 pmol/10(7) cells), before stabilizing at a high level. MCADA activity increased transiently until the second session (2.2 +/- 0.5 IU/10(7) cells before HD vs. 2.8 +/- 0.6 IU/10(7)cells), and then decreased and stabilized at a low level (1.0 +/- 0.5 IU/10(7)cells). The amount of MCADA mRNA transiently increased until the third session (mRNA MCADA/mRNA beta-actin: 0.6 +/- 0.2 vs. 0.8 +/- 0.2), and then decreased to 0.3 +/- 0.1 at the 36th session. MCADA activity underwent a dose-dependent increase after IFNgamma stimulation. HD affects the transcription of the gene encoding MCADA after just three HD sessions, leading to decreased MCADA activity and increased plasma concentration of Ado.

Diamond Blackfan anaemia in the UK: clinical and genetic heterogeneity.

A detailed family study was undertaken of patients notified to the UK Diamond Blackfan Anaemia (DBA) Registry. RPS19 mutations were detected in 16 of 104 families, including two patients with deletions detected by intragenic loss of heterozygosity of tightly linked polymorphisms. In two further cases, polymorphisms were used to determine the parental allele of origin of RPS19 point mutations. A review of clinical details of patients with mutations and patients in the literature having identical or equivalent mutations revealed evidence for a genotype:phenotype correlation with respect to the prevalence of physical anomalies, and the occurrence of mild or variable haematological severity. Nine of 60 patients had a known family history of DBA. Haematological abnormalities, including raised red cell adenosine deaminase activity, were found in first-degree relatives of 16 of 51 (31%) of patients not previously considered to have familial DBA. Results of both parents and any siblings were normal in only 35 of 60 (58%) of cases, who were therefore assumed to have sporadic de novo DBA. The classical inheritance pattern for DBA is autosomal dominant; however, 12 of 60 families (20%) had more than one affected child despite normal results in both parents. These results have important implications for genetic counselling, and for the selection of potential sibling bone marrow donors.

Lymphodepleting effects and safety of pentostatin for nonmyeloablative allogeneic stem-cell transplantation1

Nonmyeloablative allogeneic stem-cell transplantation (alloNST) is the focus of investigations searching for less-toxic transplantation regimens. We report studies on the kinetics of lymphodepletion and safety of pentostatin (PT) conditioning in alloNST. Patients with hematologic malignancy received mobilized blood from human leukocyte antigen-matched related (n=4) or unrelated (n=8) donors. PT 4 mg/m2 was administered on days -21, -20, and -19 and 200 cGy of total-body irradiation was administered on day -1, followed by cyclosporine A and mycophenolate mofetil. Mononuclear cell adenosine deaminase after PT was inhibited 84%. The absolute CD3+ cells decreased significantly by day -7 (49%) and CD19+ cells declined 92% by day -1. CD4+ cells were depressed more than CD8+ cells. Neutrophils and monocytes were minimally affected by PT. Median posttransplant peripheral blood chimerism on day 70 showed 95% donor leukocytes and 82.5% donor CD3 lymphocytes. PT demonstrated lymphodepleting effects and promising safety, supporting alloNST as early as 7 days after initiation of PT.

Activation of adenosine deaminase in MCF-7 cells through IGF-estrogen receptor alpha crosstalk

Adenosine deaminase (ADA) regulates cellular levels of adenosine and deoxyadenosine, and 17beta-estradiol (E(2)) induces ADA mRNA in MCF-7 human breast cancer cells. IGF-I also induces ADA gene expression in these cells, and induction of this response through IGF activation of estrogen receptor alpha (ERalpha) was further investigated. IGF and other polypeptide growth factors induce reporter gene expression in MCF-7 cells cotransfected with ERalpha expression plasmid and pADA211, a construct containing the -211 to +11 region of the ADA gene promoter which is required for high basal and E(2)-inducible activity. Deletion analysis of this promoter demonstrates that IGF activates ERalpha/Sp1 interactions with multiple GC-rich sites in the promoter and this response is abrogated in cells transfected with ERalpha containing mutations at Ser(118) or Ser(163). IGF induces both MAPK (mitogen-activated protein kinase) and PI3-K (phosphatidylinositol-3-kinase) phosphorylation cascades in MCF-7 cells; however, using a series of inhibitors and dominant negative constructs, our results show that induction of ADA by IGF activation of ERalpha/Sp1 is dependent on the MAPK signaling pathway.

Adeno-associated virus vectors for gene therapy of the hematopoietic system.

Over the past 30 years, bone marrow transplantation has come to be accepted as standard curative therapy for many pathophysiologic conditions (Forman et al. 1994) including malignancies (Brenner et al. 1993) and metabolic diseases (Blaese 1993). Thus, the reconstitution of the hematopoietic system with normal allogeneic cells capable of providing long term engraftment with disease-free, physiologically functional cells has been used to treat various inherited diseases including severe combined immunodeficiency disease (SCID), lysosomal storage disorders and various hemoglobinopathies. However, difficulties in acquisition of histocompatible marrow cells for transplantation and onset of graft vs host disease has led to the continued search for better therapeutic strategies. Meanwhile, the delineation of the genetic bases of many inherited diseases and the subsequent isolation of appropriate wild-type genes promoted the feasibility of disease correction at the DNA level. To this end, much effort has focussed upon the development of both the identification of genetic strategies as well as efficient methods of delivery of therapeutic genes to appropriate target cells.KeywordsHematopoietic Progenitor CellRous Sarcoma VirusSevere Combine Immunodeficiency DiseaseNondividing CellAdenosine Deaminase DeficiencyThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Hereditary Overexpression of Adenosine Deaminase in Erythrocytes: Studies in Erythroid Cell Lines and Transgenic Mice

Overexpression of adenosine deaminase (ADA) in red blood cells is characterized by a marked, tissue-specific increase in levels of structurally normal ADA mRNA and enzymatic activity in the erythrocytes of affected individuals, leading to adenosine triphosphate (ATP) depletion and hemolytic anemia. This autosomal dominant trait is linked to the ADA gene. To investigate the molecular mechanism responsible for this disorder, we examined relative reporter gene activity using constructs containing 10.6 kb of 5' flanking sequence and 12.3 kb of the first intron of the ADA gene from the normal and mutant alleles. No differences in chloramphenicol acetyltransferase (CAT) activity were found in transient transfection experiments using erythroleukemia cell lines. Transgenic mice containing the ADA constructs expressed CAT in the appropriate tissue-specific fashion, with 10(2)- to 10(4)-fold higher activity in the thymus. However, CAT activities in erythrocytes and bone marrow of mice containing high transgene copy numbers did not differ between the normal and mutant alleles. These results indicate that the mutation responsible for ADA overexpression is unlikely to reside in the 5' and promoter regions or in the regulatory regions of the first intron. It is possible that the erythroid-specific overexpression of ADA results from a mutation at some distance from the gene or requires an interaction of a proximal mutation with more distal DNA elements.

Hereditary overexpression of adenosine deaminase in erythrocytes: studies in erythroid cell lines and transgenic mice

Overexpression of adenosine deaminase (ADA) in red blood cells is characterized by a marked, tissue-specific increase in levels of structurally normal ADA mRNA and enzymatic activity in the erythrocytes of affected individuals, leading to adenosine triphosphate (ATP) depletion and hemolytic anemia. This autosomal dominant trait is linked to the ADA gene. To investigate the molecular mechanism responsible for this disorder, we examined relative reporter gene activity using constructs containing 10.6 kb of 5′ flanking sequence and 12.3 kb of the first intron of the ADA gene from the normal and mutant alleles. No differences in chloramphenicol acetyltransferase (CAT) activity were found in transient transfection experiments using erythroleukemia cell lines. Transgenic mice containing the ADA constructs expressed CAT in the appropriate tissue-specific fashion, with 10(2)- to 10(4)-fold higher activity in the thymus. However, CAT activities in erythrocytes and bone marrow of mice containing high transgene copy numbers did not differ between the normal and mutant alleles. These results indicate that the mutation responsible for ADA overexpression is unlikely to reside in the 5′ and promoter regions or in the regulatory regions of the first intron. It is possible that the erythroid-specific overexpression of ADA results from a mutation at some distance from the gene or requires an interaction of a proximal mutation with more distal DNA elements.

Biologic relevance of elevated red cell adenosine deaminase activity in myelodysplastic syndromes and paroxysmal nocturnal hemoglobinuria.

Red cell adenosine deaminase (ADA-RBC) activity in patients with myelodysplastic syndromes and paroxysmal nocturnal hemoglobinuria is significantly increased compared to that observed in normal controls. ADA-RBC activity is not related to fetal hemoglobin concentration, but it is significantly correlated with hemoglobin concentration at diagnosis and with the degree of morphologic dysplasia in the erythroid lineage. The results of our study suggest that the observed enzymatic abnormality may constitute a non-specific manifestation of the stem cell alteration that determines these disorders.