Search

Background: Autoimmune diseases tend to cluster in the same individual or in families. Four types of autoimmune polyglandular syndromes (APS) have been described based on the combination of endocri...

Background Autoantibodies against-phospholipase A2 receptor (PLA2R) are specific markers of idiopathic membranous nephropathy (iMN). Enzyme-linked immunosorbent assay (ELISA) is becoming the preferred method in many laboratories for the determination of anti-PLA2R antibodies, because it provides quantitative results, and is not prone to subjective interpretation, as is the case with indirect immunofluorescence assay. Methods The purpose of our study was to determine the diagnostic performance of serum PLA2R antibodies detected by commercially available ELISA in a large Italian multicenter cohort of patients with biopsy-proven iMN and in patients with other renal diseases, with special focus on evaluating the optimal cut-off value to discriminate positive and negative results. A total of 495 consecutive patients were recruited. Renal biopsies were performed in all patients, and blood samples were taken before the initiation of immunosuppressive treatment. Results According to the clinical diagnosis and to kidney biopsy, 126 patients were diagnosed with iMN and 369 had other non-membranous nephropathies. Anti-PLA2R autoantibodies were detected using a commercial anti-PLA2R ELISA. At a cut-off value of 20 relative units (RU)/ml indicated by the manufacturer for positive classification, sensitivity was 61.1% and specificity 99.7%. At a cut-off value of 14 RU/ml indicated by the manufacturer for borderline results, sensitivity was 63.5% and specificity remained the same (99.7%). At a cut-off of 2.7 RU/ml, selected as the optimal cut-off on the basis of ROC curve analysis, sensitivity was 83.3% and specificity 95.1%. The best overall efficiency of the test was observed at 2.7 RU/ml; however, the highest positive likelihood ratio and diagnostic odds ratio were achieved at 14 RU/ml. A cut-off threshold higher than 14 RU/ml or lower than 2.7 RU/ml entailed worse test performance. Conclusion Depending on the clinical use (early diagnosis or as a support to confirm clinical diagnosis), nephrologists may take advantage of this evidence by choosing the most convenient cut-off. However, renal biopsy remains mandatory for the definitive diagnosis of iMN and for the assessment of disease severity.

Objective In patients with familial combined hyperlipidemia (FCHL), without metabolic syndrome (MS), occurrence of non-alcoholic fatty liver disease (NAFLD) is related to a specific pro-inflammatory profile, influenced by genetic traits, involved in oxidative stress and adipokine secretion. Among FCHL or MS patients, hyperactivity of the ligand–receptor for advanced glycation-end-products (RAGE) pathway, as reflected by inadequate protective response by the endogenous secretory (es)RAGE, in concert with genetic predisposition, may identify those with NAFLD even before and regardless of MS. Methods We cross-sectionally compared 60 patients with vs. 50 without NAFLD. Each group included patients with FCHL alone, MS alone, and FCHL plus MS. Results NAFLD patients had significantly lower plasma esRAGE, IL-10 and adiponectin, and higher CD40 ligand, endogenous thrombin potential and oxidized LDL. The effects of MS plus FCHL were additive. The genotypic cluster including LOX-1 IVS4-14A plus ADIPO 45GG and 256 GT/GG plus IL-10 10-1082G, together with higher esRAGE levels highly discriminate FCHL and MS patients not developing NAFLD. Conclusions Among FCHL or MS patients, noncarriers of the protective genotypic cluster, with lower esRAGE and higher degree of atherothrombotic abnormalities coincide with the diagnosis of NAFLD. This suggests an interplay between genotype, adipokine secretion, oxidative stress and platelet/coagulative activation, accelerating NAFLD occurrence as a proxy for cardiovascular disease.

Adenosine Deaminase (ADA) deficiency is an autosomal recessive variant of severe combined immunodeficiency (SCID) caused by systemic accumulation of ADA substrates. Neurological and behavioral abnormalities observed in ADA-SCID patients surviving after stem cell transplantation or gene therapy represent an unresolved enigma in the field. We found significant neurological and cognitive alterations in untreated ADA-SCID patients as well as in two groups of patients after short- and long-term enzyme replacement therapy with PEG-ADA. These included motor dysfunction, EEG alterations, sensorineural hypoacusia, white matter and ventricular alterations in MRI as well as a low mental development index or IQ. Ada-deficient mice were significantly less active and showed anxiety-like behavior. Molecular and metabolic analyses showed that this phenotype coincides with metabolic alterations and aberrant adenosine receptor signaling. PEG-ADA treatment corrected metabolic adenosine-based alterations, but not cellular and signaling defects, indicating an intrinsic nature of the neurological and behavioral phenotype in ADA deficiency.

Adenosine deaminase (ADA) deficiency is a rare, autosomal-recessive systemic metabolic disease characterized by severe combined immunodeficiency (SCID). The treatment of choice for ADA-deficient SCID (ADA-SCID) is hematopoietic stem cell transplant from an HLA-matched sibling donor, although

Thiols play a fundamental role in cell biology, biochemistry and pharmacology. Altered thiol levels in body fluids are linked to specific pathological conditions. Glutathione is the most abundant intracellular low-molecular-mass thiol, playing an essential role in protecting cells from toxic species; other relevant thiol-containing compounds are homocysteine (Hcy), cysteine (Cys), cysteinylglycine (CysGly). Plasma aminothiols can be bound to proteins but they also occur free in the disulfide (symmetrical and mixed) and in the reduced forms. The simultaneous determination of these aminothiols, their precursor and metabolites is a useful tool in studying oxidative stress, metabolic and redox regulation. Many capillary electrophoresis methods have been proposed for this purpose, the aim of the present review is to support researchers in the choice of suitable methods for the determination of thiols in body fluids evaluating the different approaches and technologies proposed from the literature.

We tested whether rat liver preservation performed by machine perfusion (MP) at 20 degrees C can enhance the functional integrity of steatotic livers versus simple cold storage. We also compared MP at 20 degrees C with hypothermic MP at 8 degrees C, and 4 degrees C. Obese and lean male Zucker rats were used as liver donors. MP was performed for 6 hours with a glucose and N-acetylcysteine-supplemented Krebs-Henseleit solution. Both MP and cold storage preserved livers were reperfused with Krebs-Henseleit solution (2 hours at 37 degrees C). MP at 4 degrees C and 8 degrees C reduced the fatty liver necrosis compared with cold storage but we further protected the organs using MP at 20 degrees C. Necrosis did not differ in livers from lean animals submitted to the different procedures; the enzymes released in steatotic livers preserved by MP at 20 degrees C were similar to those showed in nonsteatotic organs. The adenosine triphosphate/adenosine diphosphate ratio and bile production were higher and the oxidative stress and biliary enzymes were lower in steatotic livers preserved by MP at 20 degrees C as compared with cold storage. In livers from lean rats, the adenosine triphosphate/adenosine diphosphate ratio appears better conserved by MP at 20 degrees C as compared with cold storage. In steatotic livers preserved by cold storage, a 2-fold increase in tumor necrosis factor-alpha levels and caspase-3 activity was observed as compared with organs preserved by MP at 20 degrees C. These data are substantiated by better morphology, higher glycogen content, and lower reactive oxygen species production by sinusoidal cells in steatotic liver submitted to MP at 20 degrees C versus cold storage. MP at 20 degrees C improves cell survival and leads to a marked improvement in hepatic preservation of steatotic livers as compared with cold storage.

Adenosine deaminase (ADA) deficiency is caused by a purine metabolic dysfunction, leading to severe combined immunodeficiency (SCID) and multiple organ damage. To investigate the efficacy of ex vivo gene therapy with self-inactivating lentiviral vectors (LVs) in correcting this complex phenotype, we used an ADA(-/-) mouse model characterized by early postnatal lethality. LV-mediated ADA gene transfer into bone marrow cells combined with low-dose irradiation rescued mice from lethality and restored their growth, as did transplantation of wild-type bone marrow. Mixed chimerism with multilineage engraftment of transduced cells was detected in the long term in animals that underwent transplantation. ADA activity was normalized in lymphocytes and partially corrected in red blood cells (RBCs), resulting in full metabolic detoxification and prevention of severe pulmonary insufficiency. Moreover, gene therapy restored normal lymphoid differentiation and immune functions, including antigen-specific antibody production. Similar degrees of detoxification and immune reconstitution were obtained in mice treated early after birth or after 1 month of enzyme-replacement therapy, mimicking 2 potential applications for ADA-SCID. Overall, this study demonstrates the efficacy of LV gene transfer in correcting both the immunological and metabolic phenotypes of ADA-SCID and supports the future clinical use of this approach. Adenosine deaminase (ADA) deficiency is caused by a purine metabolic dysfunction, leading to severe combined immunodeficiency (SCID) and multiple organ damage. To investigate the efficacy of ex vivo gene therapy with self-inactivating lentiviral vectors (LVs) in correcting this complex phenotype, we used an ADA(-/-) mouse model characterized by early postnatal lethality. LV-mediated ADA gene transfer into bone marrow cells combined with low-dose irradiation rescued mice from lethality and restored their growth, as did transplantation of wild-type bone marrow. Mixed chimerism with multilineage engraftment of transduced cells was detected in the long term in animals that underwent transplantation. ADA activity was normalized in lymphocytes and partially corrected in red blood cells (RBCs), resulting in full metabolic detoxification and prevention of severe pulmonary insufficiency. Moreover, gene therapy restored normal lymphoid differentiation and immune functions, including antigen-specific antibody production. Similar degrees of detoxification and immune reconstitution were obtained in mice treated early after birth or after 1 month of enzyme-replacement therapy, mimicking 2 potential applications for ADA-SCID. Overall, this study demonstrates the efficacy of LV gene transfer in correcting both the immunological and metabolic phenotypes of ADA-SCID and supports the future clinical use of this approach.

B-cell chronic lymphocytic leukemia (B-CLL) is an adult-onset highly heterogeneous malignancy characterized by a cells resistance to apoptosis rather than to highly proliferative cells. In previous research, we evidenced an imbalance of purine metabolism in B-CLL cells. Since the extracellular adenosine has been proved to induce apoptosis via A2b receptor, enzymes involved in adenosine metabolism could play an important role in apoptosis resistance of B-CLL cells. We prepared a microarray chip for the analysis of 50 selected genes that could be of interest in B-CLL: enzymes of purine de-novo, salvage and catabolic pathway, oxidative stress enzymes, and apoptotis-related proteins. Preliminary results identify many genes of purine metabolism that exhibit low or high expression, while genes involved in signal transduction and apoptosis exhibit lower alterations even if of remarkable interest. This application of microarray technique seems promising and at least a subset of these genes will be valid candidates for further studies.

Apoptosis and necrosis coexist in ischemia-reperfusion (I/R) injury following organ transplant. During experimental liver transplant we evidenced a deep alteration in energy and antioxidant status. The activity of purine catabolic enzymes was also altered. Caspase-3 (C-3), protein tyrosine phosphatase (PTP) showed significative alterations that lead to DNA fragmentation. These findings could be of interest in new potential strategy to prevent and treat I/R injury.

Adenosine is known to be associated with effects such as inhibition of immune response, coronary vasodilation, stimulation of angiogenesis, and inhibition of inflammatory reactions. Some authors suggest that adenosine may also have similar functions in tumor tissues. Tissue levels of adenosine are under close regulation by different enzymes acting at different levels. Adenosine is produced from AMP by the action of 5'-nucleotidase (5'-NT) and is converted back into AMP by adenosine kinase (AK) or into inosine by adenosine deaminase (ADA). Inosine is converted into purine catabolites by purine nucleoside phosphorylase (PNP), whereas AMP is converted into ADP and ATP by adenylate kinase (MK). The aim of this study was to analyze the activities of the above enzymes in fragments of neoplastic and apparently normal mucosa, obtained less than 5 cm and at least 10 cm from tumors, in 40 patients with colorectal cancer. The results showed much higher activities of ADA, AK, 5'-NT, and PNP in tumor tissue than in neighboring mucosa (p > 0.01 for ADA, AK, and PNP; p > 0.05 for 5'-NT), suggesting that the activities of purine metabolizing enzymes increase to cope with accelerated purine metabolism in cancerous tissue. The simultaneous increase in ADA and 5'-NT activities might be a physiological attempt by cancer cells to provide more substrate to accelerate salvage pathway activity.

The relationships between high-energy phosphate levels, oxidative insult and mechanical function represent a key point in heart transplantation and related post-ischemic functional recovery. We evaluated myocardial purine compounds and glutathione antioxidant defence mechanism during 19 heart transplant operations. Heart biopsies were taken before harvesting on beating heart (t1), at the end of cold static preservation (t2) and 30 min after implantation and reperfusion (t3); perchloric extracts of the tissue were analyzed by capillary electrophoresis (CE). Correlation analyses were performed with hemodynamic parameters evaluated 90 min after aortic declamping (T90), 6 h following admission in intensive care unit (T6A) and 1 d post-operation (D1). We evidenced that AMP levels measured at T1 negatively correlate with both cardiac index (CI) and oxygen delivery index (DO2I) evaluated at T6A, respectively. The same behavior was evident plotting IMP levels measured at T3 with CI and DO2I evaluated at D1. After t2 the nucleotide/(nucleoside + base) ratio was in positive correlation with hemodynamic parameters at T6A. Energy charge and GSH/GSSG ratio measured before harvesting were in positive correlation with DO2I evaluated at T90. The present research shows that despite the complexity of the high-energy phosphate metabolism and that of the events associated to a clinical heart transplantation, there are some parameters that, besides reflecting the degree of myocardial preservation, also represents predictive parameters for the following organ functional recovery. It also suggests that heart preservation strategies should carefully take into account the sub-optimal nature of the donor heart at the time of procurement, through a broad spectrum of purine compound and glutathione antioxidant system measurements.

Myocardial and endothelial damage is still a widely debated problem during the ischemia–reperfusion sequence in heart surgery. We evaluated myocardial purine metabolites, antioxidant defense mechanisms, oxidative status and endothelial dysfunction markers in 14 patients undergoing coronary artery by-pass graft (CABG). Heart biopsies were taken before aortic cross-clamping (t1), before clamp removal (t2) and 30 min after reperfusion (t3); perchloric extracts of the tissue were analyzed for glutathione, NAD, nucleotide nucleoside and base content by capillary electrophoresis (CE). In plasma samples from the coronary sinus we evaluated: nitrate and nitrite concentrations by CE, plasma glutathione peroxidase (plGPx) by ELISA, endothelin-1 (ET-1) by RIA and reactive oxygen metabolites (ROM) by colorimetric assay. During the ischemic period (t2) we observed a reduction in cellular NAD and GSH levels, as well as nitrate, nitrite and plGPx. ATP and GTP levels decreased and their catabolic products AMP, GMP, IMP, adenosine, inosine and hypoxanthine accumulated. The energy charge, ATP/ADP ratio, and nucleotide/(nucleoside + base) ratios decreased. At t3, levels of plasma ET-1 increased and monophosphate nucleotides tended to return to basal values. The energy charge did not increase but the nucleotide/(nucleoside + nucleobase) ratio recovered to some extent. Levels of nitrates plus nitrites continued to decrease. No significant variation in ROM levels was observed. Our data indicate that oxidative stress and endothelial damage are major events during CABG, overwhelming the scavenging capacity of the myocyte and preventing restoration of the normal energy balance for 30 min after reperfusion. The AMP deaminase pathway leading to IMP production is active during ischemia and adenosine is not the main compound derived from ATP break-down in the human heart. The possible role of extracorporeal circulation is also discussed.

Literature on the behaviour of ecto-5'-nucleotidase in the course of B-cell chronic lymphocytic leukemia is briefly reviewed and aims for further researches are highlighted.

Purine nucleotide metabolism has been studied in T-lymphocyte population in healthy subjects and in AIDS bearing patients. Nucleotide content was determined by HPLC. The overall rate of purine nucleotide synthesis was measured following the incorporation of 14C-formate into the nucleotides of a T cell suspension. The authors discuss the results, which indicate interesting variations in nucleotide content and a lower nucleotide synthesis, determined by kinetic studies.

Adenosine acts as anti-inflammatory mediator on the immune system and has been described in regulatory T cell (Treg)-mediated suppression. In the absence of adenosine deaminase (ADA), adenosine and other purine metabolites accumulate, leading to severe immunodeficiency with recurrent infections (ADA-SCID). Particularly ADA-deficient patients with late-onset forms and after enzyme replacement therapy (PEG-ADA) are known to manifest immune dysregulation. Herein we provide evidence that alterations in the purine metabolism interfere with Treg function, thereby contributing to autoimmune manifestations in ADA deficiency. Tregs isolated from PEG-ADA-treated patients are reduced in number and show decreased suppressive activity, whereas they are corrected after gene therapy. Untreated murine ADA(-/-) Tregs show alterations in the plasma membrane CD39/CD73 ectonucleotidase machinery and limited suppressive activity via extracellular adenosine. PEG-ADA-treated mice developed multiple autoantibodies and hypothyroidism in contrast to mice treated with bone marrow transplantation or gene therapy. Tregs isolated from PEG-ADA-treated mice lacked suppressive activity, suggesting that this treatment interferes with Treg functionality. The alterations in the CD39/CD73 adenosinergic machinery and loss of function in ADA-deficient Tregs provide new insights into a predisposition to autoimmunity and the underlying mechanisms causing defective peripheral tolerance in ADA-SCID. Trials were registered at www.clinicaltrials.gov as NCT00598481/NCT00599781. (Blood. 2012; 119(6): 1428-1439) Adenosine acts as anti-inflammatory mediator on the immune system and has been described in regulatory T cell (Treg)-mediated suppression. In the absence of adenosine deaminase (ADA), adenosine and other purine metabolites accumulate, leading to severe immunodeficiency with recurrent infections (ADA-SCID). Particularly ADA-deficient patients with late-onset forms and after enzyme replacement therapy (PEG-ADA) are known to manifest immune dysregulation. Herein we provide evidence that alterations in the purine metabolism interfere with Treg function, thereby contributing to autoimmune manifestations in ADA deficiency. Tregs isolated from PEG-ADA-treated patients are reduced in number and show decreased suppressive activity, whereas they are corrected after gene therapy. Untreated murine ADA(-/-) Tregs show alterations in the plasma membrane CD39/CD73 ectonucleotidase machinery and limited suppressive activity via extracellular adenosine. PEG-ADA-treated mice developed multiple autoantibodies and hypothyroidism in contrast to mice treated with bone marrow transplantation or gene therapy. Tregs isolated from PEG-ADA-treated mice lacked suppressive activity, suggesting that this treatment interferes with Treg functionality. The alterations in the CD39/CD73 adenosinergic machinery and loss of function in ADA-deficient Tregs provide new insights into a predisposition to autoimmunity and the underlying mechanisms causing defective peripheral tolerance in ADA-SCID.

We studied the effect of carbamoylphosphate (CP) on L-aspartate aminotransferase (GOT) and L-alanine aminotransferase (GPT), compared to its effect on L-threonine deaminase (TD). GPT and GOT were slightly inhibited by CP, while TD was strongly inhibited. GPT and TD, but not GOT, were inactivated when preincubated with CP. Only GOT was enhanced by pyridoxal 5'-phosphate (PLP), but not when the coenzyme was preincubated with CP. When the enzymes were resolved by p-chloromercuribenzoate (PCMB) treatment to apoenzymes, only GOT retained 47% of the original activity. Reconstitution of the apoenzymes with PLP also followed different course; activities of GOT and TD were completely restored while GOT remained partially inactivated. Treatment of apoenzymes with CP resulted in impairment of their reconstitution except GPT, activity of which could be completely restored. When PLP was pre-treated with CP before reconstitution, however, even GPT was only partially restored. The data indicated that CP affect activities of these enzymes at different levels, holoenzymes, PLP and probably apoenzymes. Under a concentration of PLP, activity of GOT would be most enhanced, followed by TD then GPT. In the presence of CP, this effect would be eliminated.

Adenosine deaminase-deficient severe combined immunodeficiency was the first disease investigated for gene therapy because of a postulated production or survival advantage for gene-corrected T lymphocytes, which may overcome inefficient gene transfer. Four years after three newborns with this disease were given infusions of transduced autologous umbilical cord blood CD34+ cells, the frequency of gene-containing T lymphocytes has risen to 1–10%, whereas the frequencies of other hematopoietic and lymphoid cells containing the gene remain at 0.01–0.1%. Cessation of polyethylene glycol-conjugated adenosine deaminase enzyme replacement in one subject led to a decline in immune function, despite the persistence of gene-containing T lymphocytes. Thus, despite the long-term engraftment of transduced stem cells and selective accumulation of gene-containing T lymphocytes, improved gene transfer and expression will be needed to attain a therapeutic effect.

The most important purine nucleotides (NAD, AMP, IMP, GMP, XMP, ADP, ATP, GDP, GTP) were analyzed by HPLC in the lymphocytes of healthy subjects and HIV-1 seropositive patients at different stages of the disease (ARC-AIDS). Several differences, which focus attention on the behaviour of purine nucleotide metabolism in the lymphocytes of these patients, were observed.

Anion-exchange, high performance liquid chromatography was used to determine purine nucleotides in lymphocytes of healthy males and females of various ages. We observed a considerable dispersion of values which were unrelated to age or sex, possibly linked to various states of activation of circulating lymphocytes and to other unknown factors. purine nucleotide / normal subject / HPLC

We compared the susceptibility of liver grafts from lean and obese Zucker rats to preservation injury, using two organ-preservation techniques: conventional static preservation (SP) and machine perfusion (MP) preservation. SP: livers preserved by UW solution at 4, 8 or 20 degrees C for 6-h. MP: livers perfused for 6-h with an improved oxygenated Krebs-Henseleit solution (KH) at 4, 8 or 20 degrees C. Reperfusion with KH (2-h) was performed either with the SP or MP preserved livers. Fatty livers tolerate SP poorly at 4, 8 and 20 degrees C as compared with MP at the same temperatures. SP induced a decrease in the ATP/ADP ratio both at 8 and 20 degrees C in obese rats while an increase in energy status was found with MP at 8 and 20 degrees C. Nitrate/nitrite (NOx) concentration was higher and bile flow lower in livers preserved with SP than MP. In lean rats, no differences were observed between MP and SP as regards enzyme release, bile production and NOx levels except for SP at 20 degrees C in which high enzyme release and low bile flow were observed. In lean rats ATP/ADP was higher and NOx was lower with MP at 20 degrees C than with SP at 20 degrees C. To optimize steatotic liver preservation SP should be avoided because it is particularly detrimental as compared with MP.

B-CLL is the most frequent type of leukemia in the Western countries. The disease, common among the elderly, follows a variable course in terms of survival time and symptoms. There is evidence that the accumulation of lymphocytes in peripheral blood and bone marrow is due to a cell resistance to apoptosis rather than to highly proliferative cells. Genetic mechanisms that lead to the development and progression of disease are mainly unknown, although a number of prognostically and diagnostically important genetic markers have been identified. The aim of this study is to investigate the gene expression profile, by a specific chip for microarray analysis, in B-CLL lymphocytes with regard to factors involved in apoptosis cascade, signal transduction, purine metabolism enzymes, interleukin expression, enzymes involved in the responses to oxidative stress. We found relevant results in a set of 19 of the 57 genes considered. IMP dehydrogenase, adenine phosphoribosyltransferase, adenylosuccinate lyase, adenylate kinase, ADORA1, G-protein-coupled receptor kinase 6, Bcl-2-like 1 isoform 2, caspase 6, and 8 were found underexpressed; while ADORA3, Gars-Airs-Gart, adenylate kinase 3, adenylate deaminase, NMN adenylyltransferase, CD26, CD38, interleukins 18 and 4 were found overexpressed. The microarray technique is a powerful method for identification of potential important diagnostic and prognostic markers, besides giving prominence to genes candidate for further studies.

Background: We investigated the long-term outcome of gene therapy for severe combined immunodeficiency (SCID) due to the lack of adenosine deaminase (ADA), a fatal disorder of purine metabolism and immunodeficiency. Methods: We infused autologous CD34+ bone marrow cells transduced with a retroviral vector containing the ADA gene into 10 children with SCID due to ADA deficiency who lacked an HLA-identical sibling donor, after nonmyeloablative conditioning with busulfan. Enzyme-replacement therapy was not given after infusion of the cells. Results: All patients are alive after a median follow-up of 4.0 years (range, 1.8 to 8.0). Transduced hematopoietic stem cells have stably engrafted and differentiated into myeloid cells containing ADA (mean range at 1 year in bone marrow lineages, 3.5 to 8.9%) and lymphoid cells (mean range in peripheral blood, 52.4 to 88.0%). Eight patients do not require enzyme-replacement therapy, their blood cells continue to express ADA, and they have no signs of defective detoxification of purine metabolites. Nine patients had immune reconstitution with increases in T-cell counts (median count at 3 years, 1.07 x 10(sup 9) per liter) and normalization of T-cell function. In the five patients in whom intravenous immune globulin replacement was discontinued, antigen-specific antibody responses were elicited after exposure to vaccines or viral antigens. Effective protection against infections and improvement in physical development made a normal lifestyle possible. Serious adverse events included prolonged neutropenia (in two patients), hypertension (in one), central-venous-catheter-related infections (in two), Epstein-Barr virus reactivation (in one), and autoimmune hepatitis (in one). Conclusions: Gene therapy, combined with reduced-intensity conditioning, is a safe and effective treatment for SCID in patients with ADA deficiency. (ClinicalTrials.gov numbers, NCT00598481 and NCT00599781.) N Engl J Med 2009;360:447-58. RI rossi, paolo/D-6504-2012; galimberti, stefania/H-2594-2012 BACKGROUND:We investigated the long-term outcome of gene therapy for severe combined immunodeficiency (SCID) due to the lack of adenosine deaminase (ADA), a fatal disorder of purine metabolism and immunodeficiency.METHODS:We infused autologous CD34+ bone marrow cells transduced with a retroviral vector containing the ADA gene into 10 children with SCID due to ADA deficiency who lacked an HLA-identical sibling donor, after nonmyeloablative conditioning with busulfan. Enzyme-replacement therapy was not given after infusion of the cells.RESULTS:All patients are alive after a median follow-up of 4.0 years (range, 1.8 to 8.0). Transduced hematopoietic stem cells have stably engrafted and differentiated into myeloid cells containing ADA (mean range at 1 year in bone marrow lineages, 3.5 to 8.9%) and lymphoid cells (mean range in peripheral blood, 52.4 to 88.0%). Eight patients do not require enzyme-replacement therapy, their blood cells continue to express ADA, and they have no signs of defective detoxification of purine metabolites. Nine patients had immune reconstitution with increases in T-cell counts (median count at 3 years, 1.07x10(9) per liter) and normalization of T-cell function. In the five patients in whom intravenous immune globulin replacement was discontinued, antigen-specific antibody responses were elicited after exposure to vaccines or viral antigens. Effective protection against infections and improvement in physical development made a normal lifestyle possible. Serious adverse events included prolonged neutropenia (in two patients), hypertension (in one), central-venous-catheter-related infections (in two), Epstein-Barr virus reactivation (in one), and autoimmune hepatitis (in one).CONCLUSIONS:Gene therapy, combined with reduced-intensity conditioning, is a safe and effective treatment for SCID in patients with ADA deficiency. (ClinicalTrials.gov numbers, NCT00598481 and NCT00599781.) BackgroundWe investigated the long-term outcome of gene therapy for severe combined immunodeficiency(SCID) due to the lack of adenosine deaminase (ADA), a fatal disorderof purine metabolism and immunodeficiency.MethodsWe infused autologous CD34+ bone marrow cells transduced with a retroviral vectorcontaining the ADA gene into 10 children with SCID due to ADA deficiency wholacked an HLA-identical sibling donor, after nonmyeloablative conditioning withbusulfan. Enzyme-replacement therapy was not given after infusion of the cells.ResultsAll patients are alive after a median follow-up of 4.0 years (range, 1.8 to 8.0). Transducedhematopoietic stem cells have stably engrafted and differentiated into myeloidcells containing ADA (mean range at 1 year in bone marrow lineages, 3.5 to 8.9%)and lymphoid cells (mean range in peripheral blood, 52.4 to 88.0%). Eight patientsdo not require enzyme-replacement therapy, their blood cells continue to expressADA, and they have no signs of defective detoxification of purine metabolites. Ninepatients had immune reconstitution with increases in T-cell counts (median countat 3 years, 1.07×109 per liter) and normalization of T-cell function. In the five patientsin whom intravenous immune globulin replacement was discontinued, antigenspecificantibody responses were elicited after exposure to vaccines or viral antigens.Effective protection against infections and improvement in physical development madea normal lifestyle possible. Serious adverse events included prolonged neutropenia(in two patients), hypertension (in one), central-venous-catheter–related infections (intwo), Epstein–Barr virus reactivation (in one), and autoimmune hepatitis (in one).ConclusionsGene therapy, combined with reduced-intensity conditioning, is a safe and effectivetreatment for SCID in patients with ADA deficiency. (ClinicalTrials.gov numbers,NCT00598481 and NCT00599781.)

Mutations in the adenosine deaminase (ADA) gene are responsible for a form of severe combined immunodeficiency (SCID) caused by the lymphotoxic accumulation of ADA substrates, adenosine and 2'-deoxy-adenosine. The molecular mechanisms underlying T-cell dysfunction in humans remain to be elucidated. Here, we show that CD4(+) T cells from ADA-SCID patients have severely compromised TCR/CD28-driven proliferation and cytokine production, both at the transcriptional and protein levels. Such an impairment is associated with an intrinsically reduced ZAP-70 phosphorylation, Ca(2+) flux, and ERK1/2 signaling and to defective transcriptional events linked to CREB and NF-kappaB. Moreover, exposure to 2'-deoxy-adenosine results in a stronger inhibition of T-cell activation, mediated by the aberrant A(2A) adenosine receptor signaling engagement and PKA hyperactivation, or in a direct apoptotic effect at higher doses. Conversely, in T cells isolated from patients after gene therapy with retrovirally transduced hematopoietic stem/progenitor cells, the biochemical events after TCR triggering occur properly, leading to restored effector functions and normal sensitivity to apoptosis. Overall, our findings provide a better understanding of the pathogenesis of the immune defects associated with an altered purine metabolism and confirm that ADA gene transfer is an efficacious treatment for ADA-SCID. The trials in this study are enrolled at www.ClinicalTrials.gov as #NCT00598481 and #NCT0059978 Mutations in the adenosine deaminase (ADA) gene are responsible for a form of severe combined immunodeficiency (SCID) caused by the lymphotoxic accumulation of ADA substrates, adenosine and 2-deoxy-adenosine. The molecular mechanisms underlying T-cell dysfunction in humans remain to be elucidated. Here, we show that CD4 T cells from ADA-SCID patients have severely compromised TCR/CD28-driven proliferation and cytokine production, both at the transcriptional and protein levels. Such an impairment is associated with an intrinsically reduced ZAP-70 phosphorylation, Ca2 flux, and ERK1/2 signaling and to defective transcriptional events linked to CREB and NF-B. Moreover, exposure to 2- deoxy-adenosine results in a stronger inhibition of T-cell activation, mediated by the aberrant A2A adenosine receptor signaling engagement and PKA hyperactivation, or in a direct apoptotic effect at higher doses. Conversely, in T cells isolated from patients after gene therapy with retrovirally transduced hematopoietic stem/progenitor cells, the biochemical events after TCR triggering occur properly, leading to restored effector functions and normal sensitivity to apoptosis. Overall, our findings provide a better understanding of the pathogenesis of the immune defects associated with an altered purine metabolism and confirm that ADA gene transfer is an efficacious treatment for ADA-SCID. The trials in this study are enrolled at www.ClinicalTrials. gov as #NCT00598481 and #NCT0059978.

Methylmalonyl-coenzyme A mutase (MCM) is a 5'-deoxyadenosylcobalamin-linked mitochondrial enzyme that catalyzes the isomerization of L-methylmalonyl-coenzyme A to succinyl-coenzyme A. We described a method for methylmalonyl-CoA and succinyl-CoA separation by CE, suitable for the evaluation of MCM activity. The working conditions for optimal separation were obtained in order to achieve the best resolution in the shortest analysis time. The optimization of buffer composition together with other variables, such as injection time, separation voltage, migration time, and capillary temperature, resulted in a solution of 30 mM NaH 2 PΟ 4 containing 15 mM SDS, pH 3.2. Separations were carried out in an uncoated fused-silica capillary (55 cm, 50 μm id) at -25 kV, reading at 254 nm. The method performance was evaluated by measuring total and holo-MCM activity in biological matrices such as rat liver and human peripheral blood lymphocytes (PBL). The mean MCM activity was expressed in nmol/h/mg protein of tissue/cell extract and was calculated from the amount of reaction product formed. The rapidity of analysis and utmost precision (repeatability and within-laboratory reproducibility) point out the potentialities of the proposed method for the differential diagnosis of methylmalonic acidemia, in relation to protein or coenzyme defects.

N-terminal pro-B-type natriuretic peptide (NT-proBNP) is a sensitive functional marker in heart disease, including left ventricular hypertrophy (LVH) secondary to valvular aortic stenosis (AS). We evaluated the association between NT-proBNP changes, oxidative stress, energy status and severity of LVH in patients with AS. Ten patients undergoing aortic valve replacement for AS were studied. Plasma NT-proBNP concentrations were performed by electroluminescence immunoassay 15min after the induction of anesthesia (t0), before aortic cross-clamping (t1), before clamp removal (t2), 15min after myocardial reperfusion (t3), and 24h after surgery (t4). Heart biopsies were obtained and high energy phosphates (ATP, ADP, AMP) were analyzed by capillary electrophoresis (CE). In plasma samples from the coronary sinus, nitrate plus nitrite (NOx) concentrations were also analyzed by CE. Echocardiographic measurements were acquired and correlations between biochemical markers and severity of AS were assessed. NT-proBNP peaked significantly at t4 (p

Mycophenolate mofetil (MMF) is a widely used drug for the maintenance of immunosuppressive therapy in renal-transplant recipients. MMF is rapidly metabolized in vivo to mycophenolic acid (MPA), a reversible, noncompetitive inhibitor of inosine monophosphate dehydrogenase, which represents a limiting enzyme in lymphocyte proliferation. MPA shows large interindividual pharmacokinetic variability: its monitoring is therefore of primary importance to achieve adequate immunosuppression with minimized risk of graft rejection or toxicity. We developed a CE method for the determination of total MPA (tMPA) in plasma, based on easy sample preparation; CE evaluation of tMPA was performed in 30 mmol/L sodium-borate with 10 mmol/L SDS (pH 10.00) at 25 degrees C using a 60 cm (54.5 to window) uncoated capillary with UV detection at 254 nm wavelength. MPA was readily detectable in plasma; the CE method was linear in the range of 0.7-120 microg/mL (r0.992). Intra- and interassay imprecision was7% except for the lowest spiked MPA concentration, which had an intra-assay RSD% of 14.7 compared to 18.3 interassay. Data by CE were compared with results obtained by a validated HPLC method. CE assay of tMPA exhibited a very good correlation (r(2)0.988) with respect to HPLC; Bland-Altman difference versus average showed a mean of -0.18 microg/mL +/- 1.14 SD. CE determination of tMPA is a robust, sensitive and reproducible method with the advantage over HPLC of being fast, simple and unexpensive, also enabling quick assessment of MPA for pharmacokinetic studies.

Organ dysfunction secondary to ischemia-reperfusion (I/R) injury still represents a major problem in liver transplantation. Apoptosis has been observed in hepatocytes and sinusoidal endothelial cell, following I/R injury and it has been postulated as a contributing factor in ischemia-reperfusion graft dysfunction, involving a complex series of events, as changes of protein tyrosine-kinase phosphorylation. We evaluated hepatic purine metabolites, protein tyrosine phosphatases (PTPs), nitrate plus nitrite levels (NOx), caspase-3 (C-3) activity and DNA fragmentation in the time course of twelve pig orthotopic liver transplantation. Biopsies were taken before explantation (t0), after cold ischemic storage (t1) and 30 min from reperfusion (t2). During the ischemic period we observed a reduction of high energy phosphates and an increase of purine bases; PTP activity was largely increased. At t2 high energy phosphates showed a tendency to increase with respect to t1, with a partial restoration of phosphorylation potential, measured as ATP/ADT ratio. PTP activity was significantly reduced, with a concomitant increase of NOx production and C-3 activity; in a considerable number of cases we observed a sustained DNA fragmentation. We speculate that NOx production could be related to nitrosative stress, which in turn leads to dynamic alteration in PTP balance and cell signalling, regulating the activity of a number of proteins implicated in apoptotic cell death. These findings could be of interest in new potential strategy to prevent and treat I/R injury.

The use of cyclosporine in the 1980s has led to the acceptance of cardiac transplantation as a therapeutic option for end-stage cardiomyopathy; the number of cardiac transplantation has hugely increased in the last fifteen years with a patient survival higher than 80%1. However the primary cause of death in the first 30 days after transplantation is from cardiac origin rather than from rejection. Despite many recent researches 2-3 , myocardial protection from ischemia-reperfusion injury has not been optimized and the mortality rates correlate with increased ischemic intervals4 . These findings suggest that myocardial preservation remains suboptimal and that these results may be improved allowing longer ischemic periods. Up today the cold static myocardial preservation cannot be prolonged beyond 5h The lack of oxygen and the reperfusion after different periods of hypoxia during transplantation lead to ATP breakdown and depletion of the naturally occurring defences against free radical injury. Nucleotides degrade rapidly during cold storage, forming end-products which flow freely through the plasma membrane.

Ecto-5’ -nucleotidase is an extracellular enzyme which is anchored to the plasma membrane of various cells, including human lymphocytes through a glycosylphosphatidylinositol (GPI) linkage. The enzyme dephosphorylates purine and pyrimidine nucleoside monophosphates to the corresponding nucleosides and generates adenosine from extracellular AMP. In addition to its function as an enzyme, the ecto-5’ -nucleotidase mediates costimulatory signals in T cells, lymphocyte adhesion to endhotelium, and has a role in controlling B cell-follicular dendritic cell interactions. Ecto-5’ -nucleotidase is recognized as a maturation marker for both T and B lymphocytes. Abnormally low ecto-5’-nucleotidase activity has been found on lymphocytes of pat ients suffering with a variety of immunodeficiency diseases characterized by a block in lymphocyte maturation 1. In previous studies we found a strong reduction of ecto-5’-nucleotidase activity in lymphocytes from patients affected by B-cell chronic lymphocytic

The liver is a key-organ in the regulation of energetic metabolism; it supplies lipids, nucleotides and vitamins to the whole organism. Almost all these synthesis are highly dependent from oxygen availability which may lead to decreased liver functions, cellular suffering, death and inflammation. A better understanding of tissue energy metabolism and antioxidant status is of great interest in the field of liver preservation for transplantation. The lack of oxygen and the reperfusion after periods of hypoxia during transplantation, leads to ATP breakdown and depletion of the naturally occurring defences. Nucleotides degrade rapidly during cold storage, forming end-products which flow freely through the plasma membrane. Depletion of mitochondrial superoxide-dismutase (SOD) and glutathione makes the liver vulnerable to oxygen free radicals1. It is well known that reactive oxygen species resulting from the ischemia-reperfusion sequence are mediators of cellular damage2. A burst of free radical production is thought to occur when the organ undergoes global ischemia, overwhelming the scavenging capacity of the hepathocytes and preventing restoration of the normal energy balance. Nucleotide and glutathione determinations are widely accepted tools in the evaluation of cellular energy and antioxidant status3.

The aim of this work is to analyse the activities of the enzymes metabolising adenosine in fragments of neoplastic and normal‐appearing mucosa, surrounding the tumour in 20 patients affected by colorectal cancer. The results show that the activities of the enzymes are markedly higher in tumour in comparison to normal mucosa to coope with the accelerated purine metabolism in cancerous tissues.

A capillary electrophoresis (CE) method was developed for ADA/SCID diagnosis and monitoring of enzyme replacement therapy, as well as for exploring the transfection efficiency for different retroviral vectors in gene therapy.

In order to investigate the behaviour of biochemical parameters in children from Mozambique, we have determined the serum levels of folic acid and vitamin B12, two well known markers of nutritional anemia. We have correlated their values with other blood parameters and have evidenced potential interesting relationship between folate content and platelets count.

Background: The diagnosis and monitoring of severe combined immunodeficiency disease (SCID) attributable to adenosine deaminase (ADA) deficiency requires measurements of ADA, purine nucleoside phosphorylase (PNP), and S-adenosyl-l-homocysteine-hydrolase (SAHH) activity and of deoxyadenosine metabolites. We developed capillary electrophoresis (CE) methods for the detection of key diagnostic metabolites and evaluation of enzyme activities. Methods: Deoxyadenosine metabolites were separated in 30 mmol/L sodium borate–10 mmol/L sodium dodecyl sulfate (pH 9.80) at 25 °C on a 60-cm uncoated capillary. For determination of enzyme activities, substrate–product separation and measurements were carried out in 20 mmol/L sodium borate (pH 10.00) at 25 °C on a 42-cm uncoated capillary. Results: Deoxynucleotides and deoxyadenosine were readily detectable in erythrocytes and urine, respectively. Both methods were linear in the range 2–500 μmol/L (r >0.99). Intra- and interassay CV were 0.99 for both). In erythrocytes from healthy individuals, mean (SD) ADA activity was 5619 (2584) nmol/s per liter of packed cells. In erythrocytes of SCID patients at diagnosis, ADA activity was 56.9 (48.3) nmol/s per liter of packed cells; SAHH activity was also much reduced. PNP activity was similar in patients and controls. Conclusions: CE can be used to test ADA deficiency and enables rapid assessment of ADA expression in hematopoietic cells of SCID patients during therapy.

Compared to murine and human hemoglobin, bovine hemoglobin has a less exothermic oxygen binding and delivers oxygen even at low temperatures. This property could improve oxygen availability for myocytes during hypothermic arrest of hearts. The aim of this study was to evaluate the advantage of using cardioplegic solutions enriched with bovine hemoglobin when storing rat hearts. Hearts excised from rats after perfusion with different cardioplegic solutions (Celsior, Celsior plus 4% human hemoglobin, Celsior plus 4% and 8% bovine hemoglobin) were compared. Biopsies were obtained from the beating hearts before cardioplegic infusion and during a 48 h period of cold storage. Adenosine triphosphate, its catabolites and markers of oxidative stress were measured as indices of preservation. The results show that bovine hemoglobin-enriched solutions highly improve adenosine triphosphate content, decreasing its catabolites; no significant changes in antioxidant status were evident. The statistically significant difference was evident up to 6 h of storage. Doubling the concentration of bovine hemoglobin produces only slight improvement. Alternative hemoglobins with different properties may improve and prolong heart storage. As bovine hemoglobin delivers oxygen even at low temperatures, it improves energy content and anabolic reactions, without decreasing oxidative stress.

Hematopoietic stem cell (HSC) gene therapy for adenosine deaminase (ADA)–deficient severe combined immunodeficiency (SCID) has shown limited clinical efficacy because of the small proportion of engrafted genetically corrected HSCs. We describe an improved protocol for gene transfer into HSCs associated with nonmyeloablative conditioning. This protocol was used in two patients for whom enzyme replacement therapy was not available, which allowed the effect of gene therapy alone to be evaluated. Sustained engraftment of engineered HSCs with differentiation into multiple lineages resulted in increased lymphocyte counts, improved immune functions (including antigen-specific responses), and lower toxic metabolites. Both patients are currently at home and clinically well, with normal growth and development. These results indicate the safety and efficacy of HSC gene therapy combined with nonmyeloablative conditioning for the treatment of SCID.

Adenylosuccinate lyase deficiency, which is associated with severe mental retardation and autistic features, was discovered in 1984. Since then this enzyme has been analyzed in many human tissues and it is now generally agreed that screening for this enzyme defect should be performed in all unexplained neurological diseases. The aim of the present study was to analyze adenylosuccinate lyase activity in blood cells by a fast simple method adaptable to screening purposes. The activity was also analyzed in B-lymphocytes from patients with B-cell chronic lymphocytic leukemia. The biological role of adenylosuccinate lyase and its importance in regulating cellular levels of AMP is discussed.

An understanding of tissue energy metabolism and antioxidant status is of major interest in the field of organ preservation for transplantation. Nucleotide and glutathione are indicators of cell damage occurring during ischemia and reperfusion. A high performance capillary electrophoresis (HPCE) method with UV detection (185 nm) for the simultaneous analysis of intracellular free ribonucleotides, nucleosides, bases and glutathione (oxidized and reduced form) in myocardial tissues is described. The method does not involve thiol derivatization. The separations were carried out in an uncoated fused-silica capillary, 60 cm long, 52.5 cm to detector, 75 microm ID, with 20 mM Na-borate buffer, pH 10.00, at 20 kV voltage and reading at 185 nm. Injection was hydrostatic for 12 s and total analysis time was 20 min. The technique enables optimum separation of all the compounds examined and has a resolution similar to that of HPLC analysis, with the advantage of fast simultaneous measurement of cell nucleotide metabolism and redox state, not possible with HPLC.

The synthesis of purine nucleotides has been studied in human peripheral blood lymphocytes from healthy subjects and patients affected by B-cell chronic lymphocytic leukemia (B-CLL). The rate of the synthesis was measured by following the incorporation of 14C-formate into the nucleotides of lymphocyte suspensions. The whole sequence AMP→ADP→ATP was found reduced in B-CLL lymphocytes; in the case of guanylates only the last step of the sequence GMP→GDP→GTP was significantly lower in the same cells. From the analysis of these results, combined with previous data, we conclude that purine metabolism undergoes an imbalancement during CLL, which is partially compensated, and point out the importance of studying concomitantly purine metabolism and nucleic acid synthesis in leukemia cells.

The behaviour of 5'-nucleotidase isoenzymes (ecto-5'-nucleotidase, e-Ns and c-N-II soluble 5'-nucleotidases) was studied in lymphocytes from patients with B-cell chronic lymphocytic leukemia. A strong reduction in ecto- and soluble activities was observed, although the pattern of the three 5'-nucleotidases did not always strictly overlap. A significant decrease (p0.05) in ecto-5'-nucleotidase, e-Ns and c-N-II was found in B and T populations (B lymphocytes: 1.13, 0.88 and 1.26 nmol/h/10(6) cells versus 95.96, 9.64 and 13.73 nmol/h/10(6) cells in controls; T lymphocytes: 1.31, 0.23 and 0.06 nmol/h/10(6) cells versus 9.25, 1.31 and 2.10 nmol/h/10(6) cells in healthy subjects). The percentage of ecto-5'-nucleotidase-positive cells (CD73+) was reduced in leukemia patients, indicating a lower number of active molecules on the cell surface. The results of RT-PCR analysis showed that the ecto-5'-nucleotidase mRNA of leukemia patients was not defective.

Objectives: The activity of membrane-bound ecto-5′-nucleotidase and soluble e-Ns and c-N-II 5′-nucleotidases was evaluated on lymphocytes from patients affected by B-cell chronic lymphocytic leukemia (B-CLL). A statistically significative decrease in ecto-5′-nucleotidase, e-Ns, and c-N-II activities was observed in peripheral blood lymphocytes and in B and T populations from affected individuals. Design and Methods: For the assay of ecto-5′-nucleotidase, e-Ns, and c-N-II activity we used a radioactive procedure coupled to HPLC. Since the ecto-5′-nucleotidase is identified as CD73 antigen, we performed immunofluorescence analysis using a specific monoclonal antibody. We analyzed ecto-5′-nucleotidase mRNA by RT-PCR to ascertain the possibility of an alteration in the transcription of its gene. Results: A decrease in ecto-5′-nucleotidase activity was correlated to reduction in ecto-5′-nucleotidase positive cells (CD73 + ) in leukemia patients. RT-PCR produced a fragment of the expected size and the specific mRNA was found expressed in both healthy subjects and leukemia patients. Conclusions: The decrease in ecto-5′-nucleotidase activity in patients with B-CLL is not due to loss of transcription of the specific mRNA. The presence of point mutations, splicing alteration, or posttranslational modifications must be investigated. If a defect at DNA or RNA level will be detected, the molecular analysis will be considered for diagnosis of B-cell chronic lymphocytic leukemia.

Objectives: The HER2 gene has been found amplified in a number of human adenocarcinoma leading to elevated levels of expression of its encoded product, p185 protein. Because little information is available on the tissue and tumor specificity of this gene product, we studied the expression of p185 protein in preneoplastic colon lesions. Adenylosuccinate lyase (ASL, EC 4.3.2.2) is known to increase in malignancies such as colorectal, breast, and prostate cancer. In order to evaluate the potential of ASL as a tumor marker, its activity was determined and compared with the expression of p185. Design and methods: p185 was determined by an immunohistochemical procedure in patients with the preneoplastic lesions. ASL activity was evaluated in intestinal mucosa adjacent to colorectal cancers (patient group A) and in preneoplastic colorectal lesions (group B). The enzyme activity was evaluated in dialyzed supernatants, following the disappearance of substrate (adenylosuccinate AMP-S) and the formation of product (adenosine 5′-monophosphate -AMP), separated by high performance liquid chromatography. Results and conclusions: Increased expression of p185 and elevated ASL activity were observed in tubular and tubulo-villous adenoma and may, therefore, be associated with the early stages of colorectal cancer.

Objectives: High-energy phosphates and their catabolic products were determined in myocardium during coronary artery bypass surgery with blood cardioplegic reperfusion in order to evaluate the effects of aortic cross-clamping and reoxygenation on myocardial purine metabolism. Design and Methods: Transmural left ventricular biopsy specimens were taken with Tru-Cut biopsy needles, before aortic cross-clamping, before cross-clamp removal and after 30′ of reperfusion; perchloric extracts of the material were analyzed for nucleotide content by capillary zone electrophoresis (CZE). The CZE procedure used separates the complete spectrum of purine metabolites in myocardial extracts obtained from 0.6–8.6 mg biopsy material. Results: The basal values of ATP/ADP ratio and energy charge were low, IMP content was high. After the ischemic period, ATP levels further decreased and IMP, nucleosides and bases accumulated. After reperfusion, nucleoside and base basal levels, but not energy charge, were restored to some extent. Conclusions: The study arises the problem of myocardial preservation during heart surgery. In this investigation, capillary electrophoresis was an extremely adaptable technique for the evaluation of ischemic injury and could be useful in studying the effects of cardioplegic solutions.

The enzymes that phosphorylate nucleosides fall into two groups on the basis of the phosphate donors and the reaction mechanism. One group, the nucleoside kinases, utilizes nucleoside triphosphates as donors in direct displacement reactions; the other group, the nucleosides phosphotransferases, catalyzes the phosphorylation reaction with nucleoside monophosphates as donors, converting the acceptor nucleoside to its monophosphate and the nucleoside monophosphate to its unphosphorylated nucleoside.

Ecto-5′-nucleotidase (ecto-5′-NT, CD73) is a 69 KDa purine catabolic-pathway enzyme attached via a GPI-linkage to the external plasma membrane of various cell types, including subsets of human lymphocytes.1 In addition to purine catabolism, ecto-5′-NT serves as a maturation marker.2,3

Prolonged in vivo and ex vivo myocardial protection is one of the primary factors for the success of the procedure in the therapy of patients with end-stage cardiac disfunction. Advances in myocardial preservation for routine heart surgery and transplantation have been closely linked. Future advances in the understanding of myocardial energy metabolism and the limitation of oxygen-derived free radical induced injury will have wide application in both fields.

Adenosine deaminase (ADA) deficiency is a severe combined immunodeficiency (SCID) characterized by systemic toxicity and profoundly impaired lymphoid cell development and functions due to the accumulation of purine metabolites. ADA-deficient (ADA KO) mice die within 3 weeks of age due to metabolic toxicity and can be rescued by enzyme replacement therapy with PEG-ADA. The aim of our study was to investigate the efficacy of gene therapy (GT) in comparison to bone marrow transplantation (BMT) in the ADA KO mouse model. A SIN HIV-1-based lentiviral vector encoding human ADA cDNA under the control of PGK promoter was constructed. ADA KO mice were maintained short-term on PEG-ADA and then transplanted, after non-myeloablative conditioning either with WT BM cells or with ADA KO BM cells transduced ex vivo. We first showed that BMT with donor WT cells rescued mice from lethality, restored T-cell and B-cell counts in the peripheral blood and led to normal proliferation after T or B cells specific stimulation. Similarly, GT with transduced BM cells rescued ADA KO mice from lethality and restored their growth to normal levels. Quantitative PCR for donor cells (Y chromosome in sex-mismatched transplant) and vector-positive cells showed the presence of ADA transduced cells in the peripheral circulation of transplanted ADA KO mice (30|[ndash]|40% 5 months post-GT). Two months after GT, ADA activity in red blood cells reached normal levels and dAXP toxic metabolites decreased to undetectable levels. GT resulted in normal lymphoid differentiation in primary and secondary lymphoid organs, as well as restoration of T-cell and B-cell in vitro proliferative responses after stimulation with anti-CD3/anti-CD28 and anti-IgM antibody, respectively. Finally, serum levels of IgM and IgG were comparable between GT and BMT mice. These studies demonstrate that lentiviral-mediated ADA gene transfer associated with non-myeloablative conditioning corrects both the metabolic and the immunological defects in the ADA KO animal model. We are currently evaluating the long-term safety related to the use of hematopoietic stem/progenitor cells transduced with lentiviral vectors.