Expression Of Adenosine Deaminase Research Articles

Neurochemical Research of LOXBlock-1 and ZnSO4 against Neurodegenerative Damage Induced by Amyloid Beta(1-42).

Synaptosomes offer an intriguing ex vivo model system for investigating the molecular mechanisms of neurodegenerative processes. Lipoxygenases significantly affect the course of neurodegenerative diseases. Homeostasis of trace elements such as zinc is necessary for the continuity of brain functions. In this study, we purpose to determine whether LOXBlock-1, a 12/15 lipoxygenase inhibitor, and zinc sulfate (ZnSO4) provide any biochemical protection during neurodegenerative damage in synaptosomes induced by amyloid beta 1-42 (Aβ1-42). In this study, animals (30 Wistar Albino male rats 30) were divided into 5 groups (6 animals in each group): Control, 10µM Aβ1-42, 10µM Aβ1-42+25mM LOXBlock-1, 10µM Aβ1-42+10µM ZnSO4, and 10µM Aβ1-42+25mM LOXBlock-1+10µM ZnSO4. Synaptosomes were isolated from the rat cerebral cortex. Following, 8-hydroxy-2-deoxyguanosine (8-OHdG) levels, malondialdehyde (MDA) levels, adenosine deaminase (ADA) levels, reduced-glutathione (GSH) levels, neuronal nitric oxide synthase (nNOS) levels, acetylcholinesterase (AChE) activity, catalase (CAT) activity, and 8-OHdG levels in synaptosomes were detected according to the ELISA method. ADA and AChE expression and protein levels were analyzed. MDA, nNOS, AChE, and 8-OHdG levels in synaptosomes treated with Aβ1-42 resulted in an increase, while there was a decrease in ADA, GSH, and CAT levels (p<0.001 vs. control). Conversely, LOXBlock-1 and ZnSO4 treatments in synaptosomes treated with Aβ1-42 decreased MDA, nNOS, AChE, and 8-OHdG levels, while ADA, GSH, and CAT levels increased. Moreover, the most effective improvement was seen in the co-treatment group of LOXBlock-1 and ZnSO4. Our data showed that LOXBlock-1 and ZnSO4 co-treatment may protect against Aβ1-42 exposure in rat brain synaptosomes.

Risperidone impedes glutamate excitotoxicity in a valproic acid rat model of autism: Role of ADAR2 in AMPA GluA2 RNA editing

Several reports indicate a plausible role of calcium (Ca2+) permeable AMPA glutamate receptors (with RNA hypo-editing at the GluA2 Q/R site) and the subsequent excitotoxicity-mediated neuronal death in the pathogenesis of a wide array of neurological disorders including autism spectrum disorder (ASD). This study was designed to examine the effects of chronic risperidone treatment on the expression of adenosine deaminase acting on RNA 2 (Adar2), the status of AMPA glutamate receptor GluA2 editing, and its effects on oxidative/nitrosative stress and excitotoxicity-mediated neuronal death in the prenatal valproic acid (VPA) rat model of ASD. Prenatal VPA exposure was associated with autistic-like behaviors accompanied by an increase in the apoptotic marker “caspase-3” and a decrease in the antiapoptotic marker “BCL2” alongside a reduction in the Adar2 relative gene expression and an increase in GluA2 Q:R ratio in the hippocampus and the prefrontal cortex. Risperidone, at doses of 1 and 3 mg, improved the VPA-induced behavioral deficits and enhanced the Adar2 relative gene expression and the subsequent GluA2 subunit editing. This was reflected on the cellular level where risperidone impeded VPA-induced oxidative/nitrosative stress and neurodegenerative changes. In conclusion, the present study confirms a possible role for Adar2 downregulation and the subsequent hypo-editing of the GluA2 subunit in the pathophysiology of the prenatal VPA rat model of autism and highlights the favorable effect of risperidone on reversing the RNA editing machinery deficits, giving insights into a new possible mechanism of risperidone in autism.

Increased RNA editing sites revealed as potential novel biomarkers for diagnosis in primary Sjögren's syndrome

BackgroundTranscriptome-wide aberrant RNA editing has been shown to contribute to autoimmune diseases, but its extent and significance in primary Sjögren's syndrome (pSS) are currently poorly understood. MethodsWe systematically characterized the global pattern and clinical relevance of RNA editing in pSS by performing large-scale RNA sequencing of minor salivary gland tissues obtained from 439 pSS patients and 130 non-pSS or healthy controls. FindingsCompared with controls, pSS patients displayed increased global RNA-editing levels, which were significantly correlated and clinically relevant to various immune features in pSS. The elevated editing levels were likely explained by significantly increased expression of adenosine deaminase acting on RNA 1 (ADAR1) p150 in pSS, which was associated with disease features. In addition, genome-wide differential RNA editing (DRE) analysis between pSS and non-pSS showed that most (249/284) DRE sites were hyper-edited in pSS, especially the top 10 DRE sites dominated by hyper-edited sites and assigned to nine unique genes involved in the inflammatory response or immune system. Interestingly, among all DRE sites, six RNA editing sites were only detected in pSS and resided in three unique genes (NLRC5, IKZF3 and JAK3). Furthermore, these six specific DRE sites with significant clinical relevance in pSS showed a strong capacity to distinguish between pSS and non-pSS, reflecting powerful diagnostic efficacy and accuracy. ConclusionThese findings reveal the potential role of RNA editing in contributing to the risk of pSS and further highlight the important prognostic value and diagnostic potential of RNA editing in pSS.

Improving combination therapies: targeting A2B-adenosine receptor to modulate metabolic tumor microenvironment and immunosuppression.

We investigated the role of A2B-adenosine receptor in regulating immunosuppressive metabolic stress in the tumor microenvironment. Novel A2B-adenosine receptor antagonist PBF-1129 was tested for antitumor activity in mice and evaluated for safety and immunologic efficacy in a phase I clinical trial of patients with non-small cell lung cancer. The antitumor efficacy of A2B-adenosine receptor antagonists and their impact on the metabolic and immune tumor microenvironment were evaluated in lung, melanoma, colon, breast, and epidermal growth factor receptor-inducible transgenic cancer models. Employing electron paramagnetic resonance, we assessed changes in tumor microenvironment metabolic parameters, including pO2, pH, and inorganic phosphate, during tumor growth and evaluated the immunologic effects of PBF-1129, including its pharmacokinetics, safety, and toxicity, in patients with non-small cell lung cancer. Levels of metabolic stress correlated with tumor growth, metastasis, and immunosuppression. Tumor interstitial inorganic phosphate emerged as a correlative and cumulative measure of tumor microenvironment stress and immunosuppression. A2B-adenosine receptor inhibition alleviated metabolic stress, downregulated expression of adenosine-generating ectonucleotidases, increased expression of adenosine deaminase, decreased tumor growth and metastasis, increased interferon γ production, and enhanced the efficacy of antitumor therapies following combination regimens in animal models (anti-programmed cell death 1 protein vs anti-programmed cell death 1 protein plus PBF-1129 treatment hazard ratio = 11.74 [95% confidence interval = 3.35 to 41.13], n = 10, P < .001, 2-sided F test). In patients with non-small cell lung cancer, PBF-1129 was well tolerated, with no dose-limiting toxicities; demonstrated pharmacologic efficacy; modulated the adenosine generation system; and improved antitumor immunity. Data identify A2B-adenosine receptor as a valuable therapeutic target to modify metabolic and immune tumor microenvironment to reduce immunosuppression, enhance the efficacy of immunotherapies, and support clinical application of PBF-1129 in combination therapies.

Transamniotic Fetal Administration of Genetically Modified Hematopoietic Stem Cells Carrying a Human Transgene in a Syngeneic Rat Model.

Hematopoietic stem cell (HSC)-based gene therapy has already reached clinical reality in a few applications. Fetal administration of genetically modified HSCs has only been feasible to date through invasive and morbid methods. It has been recently shown that native donor HSCs can reach the fetal circulation and bone marrow after simple delivery into the amniotic fluid, at least in a syngeneic healthy model. We sought to determine whether the transamniotic route could also be a practical alternative for the fetal administration of genetically modified HSCs in a comparable model. Pregnant Lewis rat dams underwent volume-matched intra-amniotic injections in all their fetuses (n = 47) on gestational day 17 (E17; term = E21-22) of donor HSCs genetically modified using a custom lentiviral vector designed to constitutively express both a firefly luciferase reporter gene and a human adenosine deaminase (ADA) transgene. Donor HSCs consisted of syngeneic cells isolated from the amniotic fluid and phenotyped by flow cytometry. Fetuses were euthanized at term, when seven select sites relevant to HSC-based therapies were screened for either luciferase activity by luminometry or for the presence of human ADA mRNA by digital droplet polymerase chain reaction (ddPCR). Among survivors (30/47; 64%), positive luminescence and positive human ADA expression were detected in the bone marrow (respectively, 33% and 76%), liver (respectively, 11% and 81%), spleen (respectively, 11% and 67%), thymus (respectively, 33% and 67%), lungs (respectively, 44% and 86%), and brain (respectively, 22% and 90%). Nucleated peripheral blood cells were analyzed only by ddPCR, showing positive human ADA expression at 54%. We conclude that genetically modified HSCs can reach the fetal circulation and fetal bone marrow after simple intra-amniotic administration in a syngeneic rat model. Gene therapy by transamniotic HSC delivery may become a practicable, minimally invasive strategy for the prenatal treatment of select hemoglobinopathies, immunodeficiencies, and inherited metabolic disorders.

Correlations of Myeloperoxidase (MPO), Adenosine deaminase (ADA), C–C motif chemokine 22 (CCL22), Tumour necrosis factor alpha (TNFα) and Interleukin-6 (IL-6) mRNA expression in the nasopharyngeal specimens with the diagnosis and severity of SARS-CoV-2 infections

ABSTRACT Cytokine dynamics in patients with coronavirus disease 2019 (COVID-19) have been studied in blood but seldomly in respiratory specimens. We studied different cell markers and cytokines in fresh nasopharyngeal swab specimens for the diagnosis and for stratifying the severity of COVID-19. This was a retrospective case-control study comparing Myeloperoxidase (MPO), Adenosine deaminase (ADA), C–C motif chemokine ligand 22 (CCL22), Tumour necrosis factor alpha (TNFα) and Interleukin-6 (IL-6) mRNA expression in 490 (327 patients and 163 control) nasopharyngeal specimens from 317 (154 COVID-19 and 163 control) hospitalized patients. Of the 154 COVID-19 cases, 46 died. Both total and normalized MPO, ADA, CCL22, TNFα, and IL-6 mRNA expression levels were significantly higher in the nasopharyngeal specimens of infected patients when compared with controls, with ADA showing better performance (OR 5.703, 95% CI 3.424–9.500, p < 0.001). Receiver operating characteristics (ROC) curve showed that the cut-off value of normalized ADA mRNA level at 2.37 × 10–3 had a sensitivity of 81.8% and specificity of 83.4%. While patients with severe COVID-19 had more respiratory symptoms, and elevated lactate dehydrogenase, multivariate analysis showed that severe COVID-19 patients had lower CCL22 mRNA (OR 0.211, 95% CI 0.060–0.746, p = 0.016) in nasopharyngeal specimens, while lymphocyte count, C-reactive protein, and viral load in nasopharyngeal specimens did not correlate with disease severity. In summary, ADA appears to be a better biomarker to differentiate between infected and uninfected patients, while CCL22 has the potential in stratifying the severity of COVID-19.

ADAR1 Inhibits HBV DNA Replication via Regulating miR-122-5p in Palmitic Acid Treated HepG2.2.15 Cells.

Changes in living standards and diet structure, non-alcoholic fatty liver disease (NAFLD) is prevalent globally, including in Asia, where chronic hepatitis B (CHB) is endemic. As such, cooccurrence of NAFLD with CHB is common in Asia. However, the pathogenesis underlying the onset of fatty liver in CHB prognosis has not been fully elucidated. Therefore, we aimed to investigate the effects and mechanisms of lipotoxicity on hepatitis B virus (HBV) DNA replication. The expression of adenosine deaminase acting on RNA-1 (ADAR1) and miR-122 was evaluated in liver tissues from patients with CHB concurrent NAFLD. Palmitic acid-treated HepG2.2.15 cells were used as the cell model. The effect of lipotoxicity on HBV DNA replication was evaluated in vitro by transfecting the ADAR1 overexpression or knockdown lentiviral vector into HepG2.2.15 cells, respectively. qRT-PCR, western blotting and immunofluorescence were performed to determine ADAR1 expression. The expression of ADAR1 in the liver tissues of CHB patients with concurrent NAFLD was significantly down-regulated compared with that in CHB patients. Enforced expression of ADAR1 inhibited the HBV DNA replication, whereas ADAR1 knockdown resulted in increased HBV DNA expression in palmitic acid - treated HepG2.2.15 cells. Additionally, ADAR1 inhibited the HBV DNA replication by upregulating miR-122, which is most abundant in the liver and mainly inhibits HBV DNA replication. ADAR1 may act as a suppressor of HBV replication in palmitic acid -treated HepG2.2.15 cells by increasing miR-122 levels. Thus, ADAR1 may serve as a potential biomarker and therapeutic target for CHB with concurrent NAFLD.

Expression of Adenosine Deaminase and NLRP3 Inflammasome in Tuberculous Peritonitis and Their Relationship with Clinical Efficacy.

Objective Tuberculous peritonitis (TP) can cause multiple infections of surrounding organs and tissues, leading to organ failure and endangering life safety. In this research, the relationship between adenosine deaminase (ADA), NLRP3 inflammasome, and TP and its clinical significance will be deeply explored, so as to provide new directions and reliable reference opinions for future clinical diagnosis and treatment. Methods Altogether, 59 TP patients (research group, RG) and 52 non-TP patients (control group, CG) who were admitted to our hospital from May 2014 to June 2018 were regarded as research objects. Ascites samples of RG before treatment (admission) and one month after treatment and CG before treatment were obtained, and the ADA and NLRP3 levels were tested to evaluate the clinical and prognostic significance of the two in TP. Results Before treatment, ADA and NLRP3 in RG were higher than CG (P < 0.05), and the sensitivity and specificity of combined detection of the two in predicting TP occurrence were 89.83% and 73.08% (P < 0.05). In addition, ADA and NLRP3 in RG patients were positively correlated with the disappearance time of abdominal pain and ascites (P < 0.05) and had excellent predictive effect on the adverse reactions during treatment (P < 0.05). After treatment, both in RG patients decreased, which was inversely proportional to the clinical efficacy (P < 0.05). Prognostic follow-up manifested that ADA and NLRP3 in relapse patients were higher than those without recurrence after treatment (P < 0.05). Conclusion The increase of ADA and NLRP3 in TP is relevant to the adverse reactions during treatment, clinical efficacy, and prognosis recurrence after treatment. It can be used as a disease marker to confirm, intervene, and evaluate TP progression promptly.

Inhibition of adenosine deaminase activity reverses resistance to the cytotoxic effect of high adenosine levels in cervical cancer cells

Adenosine (ADO) generation in the tumor microenvironment (TME) plays important roles in the promotion of tumor growth, invasion, and metastasis and in suppression of the antitumor immune response. Recently, adenosine deaminase (ADA) activity in the TME has been proposed to be a compensatory mechanism against toxic accumulation of ADO in cancerous tissues. In the present study, the expression and functional activity of ADA in cervical cancer (CeCa) tumor cells were analyzed: C33A (HPV-), CaSki (HPV + ), and HeLa (HPV + ) cells. CeCa tumor cells, as well as activated T lymphocytes (ATLs), which were used as a positive control, showed different ADA contents in the membrane and intracellularly and a strong ability to convert ADO into inosine (INO). Treatment of tumor cells with EHNA, a specific ADA inhibitor, decreased the viability of CeCa tumor cells in a dose-dependent manner. In C33A (EHNA half maximal inhibitory concentration (IC50) = 374 μM), CaSki (EHNA IC50 = 273.6 μM), and HeLa (EHNA IC50 = 252.2 μM) cells, EHNA strongly reversed the resistance of tumor cells to the cytotoxic effect of high concentrations of ADO; 38.82 ± 3.1%, 47.18 ± 4.7%, and 71.63 ± 6.9% of the cells were apoptotic, and 40 ± 4.8%, 52 ± 5.3% and 70 ± 6.8% of the cells had mitochondrial membrane damage, respectively. In ATLs (EHNA IC50 = 391.8 μM) treated with EHNA, 32.4 ± 4.4% were apoptotic, and 32 ± 4.3% had mitochondrial membrane damage. These results suggest that the presence and activity of ADA in CeCa tumor cells can provide protection against the cytotoxic effect of high ADO contents in the TME. Therefore, the inhibition of ADA could be a strategy for the treatment of CeCa.

Interleukin 32 as a Potential Marker for Diagnosis of Tuberculous Pleural Effusion.

ABSTRACTAccurate differential diagnosis is the key to choosing the correct treatment for pleural effusion. The present study aimed to assess whether interleukin 32 (IL-32) could be a new biomarker of tuberculous pleural effusion (TPE) and to explore the biological role of IL-32 in TPE. IL-32 levels were evaluated in the pleural effusions of 131 patients with undetermined pleural effusion from Wuhan and Beijing cohorts using an enzyme-linked immunosorbent assay method. Macrophages from TPE patients were transfected with IL-32-specific small interfering RNA (siRNA), and adenosine deaminase (ADA) expression was determined by real-time PCR and colorimetric methods. With a cutoff value of 247.9 ng/mL, the area under the curve of the receiver operating characteristic (ROC) curve for IL-32 was 0.933 for TPE, and the sensitivity and specificity were 88.4% and 93.4%, respectively. A multivariate logistic regression model with relatively good diagnostic performance was established. IL-32-specific siRNA downregulated ADA expression in macrophages, and IL-32γ treatment significantly induced ADA expression. Our results indicate that IL-32 in pleural effusion may be a novel biomarker for identifying patients with TPE. In addition, our multivariate model is acceptable to rule in or rule out TPE across diverse prevalence settings. Furthermore, IL-32 may modulate ADA expression in the tuberculosis microenvironment. (This study has been registered at ChiCTR under registration number ChiCTR2100051112 [https://www.chictr.org.cn/index.aspx].)IMPORTANCE Tuberculous pleural effusion (TPE) is a common form of extrapulmonary tuberculosis, with manifestations ranging from benign effusion with spontaneous absorption to effusion with pleural thickening, empyema, and even fibrosis, which can lead to a lasting impairment of lung function. Therefore, it is of great significance to find a rapid method to establish early diagnosis and apply antituberculosis therapy in the early stage. This study indicates that interleukin 32 (IL-32) in pleural effusion is a new high-potency marker to distinguish TPE from pleural effusions with other etiologies. A multivariate model combining age, adenosine deaminase (ADA), lactic dehydrogenase, and IL-32 may reliably rule in TPE in intermediate- or high-prevalence areas. Additionally, we observed that IL-32 might regulate ADA expression in macrophages in the tuberculosis microenvironment. Therefore, this study provides new insights into the role of IL-32 in the tuberculosis microenvironment.

Treatment With Methotrexate Associated With Lipid Core Nanoparticles Prevents Aortic Dilation in a Murine Model of Marfan Syndrome.

In Marfan syndrome (MFS), dilation, dissection, and rupture of the aorta occur. Inflammation can be involved in the pathogenicity of aortic defects and can thus be a therapeutic target for MFS. Previously, we showed that the formulation of methotrexate (MTX) associated with lipid nanoparticles (LDE) has potent anti-inflammatory effects without toxicity. To investigate whether LDEMTX treatment can prevent the development of aortic lesions in the MFS murine model. MgΔloxPneo MFS (n = 40) and wild-type (WT, n = 60) mice were allocated to 6 groups weekly injected with IP solutions of: (1) only LDE; (2) commercial MTX; (3) LDEMTX (dose = 1mg/kg) between 3rd and 6th months of life. After 12 weeks of treatments, animals were examined by echocardiography and euthanatized for morphometric and molecular studies. MFS mice treated with LDEMTX showed narrower lumens in the aortic arch, as well as in the ascending and descending aorta. LDEMTX reduced fibrosis and the number of dissections in MFS but not the number of elastic fiber disruptions. In MFS mice, LDEMTX treatment lowered protein expression of pro-inflammatory factors macrophages (CD68), T-lymphocytes (CD3), tumor necrosis factor-α (TNF-α), apoptotic factor cleaved-caspase 3, and type 1 collagen and lowered the protein expression of the transforming growth factor-β (TGF-β), extracellular signal-regulated kinases ½ (ERK1/2), and SMAD3. Protein expression of CD68 and CD3 had a positive correlation with an area of aortic lumen (r2 = 0.36; p < 0.001), suggesting the importance of inflammation in the causative mechanisms of aortic dilation. Enhanced adenosine availability by LDEMTX was suggested by higher aortic expression of an anti-adenosine A2a receptor (A2a) and lower adenosine deaminase expression. Commercial MTX had negligible effects. LDEMTX prevented the development of MFS-associated aortic defects and can thus be a candidate for testing in clinical studies.

Adenosine deaminase modulates metabolic remodeling and orchestrates joint destruction in rheumatoid arthritis

Rheumatoid Arthritis (RA) is a chronic autoimmune disease associated with inflammation and joint remodeling. Adenosine deaminase (ADA), a risk factor in RA, degrades adenosine, an anti-inflammatory molecule, resulting in an inflammatory bias. We present an integrative analysis of clinical data, cytokines, serum metabolomics in RA patients and mechanistic studies on ADA-mediated effects on in vitro cell culture models. ADA activity differentiated patients into low and high ADA sets. The levels of the cytokines TNFα, IFNγ, IL-10, TGFβ and sRANKL were elevated in RA and more pronounced in high ADA sets. Serum metabolomic analysis shows altered metabolic pathways in RA which were distinct between low and high ADA sets. Comparative analysis with previous studies shows similar pathways are modulated by DMARDs and biologics. Random forest analysis distinguished RA from control by methyl-histidine and hydroxyisocaproic acid, while hexose-phosphate and fructose-6-phosphate distinguished high ADA from low ADA. The deregulated metabolic pathways of High ADA datasets significantly overlapped with high ADA expressing PBMCs GEO transcriptomics dataset. ADA induced the death of chondrocytes, synoviocyte proliferation, both inflammation in macrophages and their differentiation into osteoclasts and impaired differentiation of mesenchymal stem cells to osteoblasts and mineralization. PBMCs expressing elevated ADA had increased expression of cytokines and P2 receptors compared to synovial macrophages which has low expression of ADA. Our data demonstrates increased cytokine levels and distinct metabolic signatures of RA based on the ADA activity, suggests an important role for ADA in the pathophysiology of RA joints and as a potential marker and therapeutic target in RA patients.

Autologous Ex Vivo Lentiviral Gene Therapy for Adenosine Deaminase Deficiency

BackgroundSevere combined immunodeficiency due to adenosine deaminase (ADA) deficiency (ADA-SCID) is a rare and life-threatening primary immunodeficiency.MethodsWe treated 50 patients with ADA-SCID (30 in the United States and 20 in the United Kingdom) with an investigational gene therapy composed of autologous CD34+ hematopoietic stem and progenitor cells (HSPCs) transduced ex vivo with a self-inactivating lentiviral vector encoding human ADA. Data from the two U.S. studies (in which fresh and cryopreserved formulations were used) at 24 months of follow-up were analyzed alongside data from the U.K. study (in which a fresh formulation was used) at 36 months of follow-up.ResultsOverall survival was 100% in all studies up to 24 and 36 months. Event-free survival (in the absence of reinitiation of enzyme-replacement therapy or rescue allogeneic hematopoietic stem-cell transplantation) was 97% (U.S. studies) and 100% (U.K. study) at 12 months; 97% and 95%, respectively, at 24 months; and 95% (U.K. study) at 36 months. Engraftment of genetically modified HSPCs persisted in 29 of 30 patients in the U.S. studies and in 19 of 20 patients in the U.K. study. Patients had sustained metabolic detoxification and normalization of ADA activity levels. Immune reconstitution was robust, with 90% of the patients in the U.S. studies and 100% of those in the U.K. study discontinuing immunoglobulin-replacement therapy by 24 months and 36 months, respectively. No evidence of monoclonal expansion, leukoproliferative complications, or emergence of replication-competent lentivirus was noted, and no events of autoimmunity or graft-versus-host disease occurred. Most adverse events were of low grade.ConclusionsTreatment of ADA-SCID with ex vivo lentiviral HSPC gene therapy resulted in high overall and event-free survival with sustained ADA expression, metabolic correction, and functional immune reconstitution. (Funded by the National Institutes of Health and others; ClinicalTrials.gov numbers, NCT01852071, NCT02999984, and NCT01380990.)

Gene delivery using AAV8 in vivo for disease stabilization in a bimodal gene therapy approach for the treatment of ADA-deficient SCID

Adenosine deaminase (ADA) deficiency is an inborn error of metabolism affecting multiple systems and causing severe combined immunodeficiency. We tested intravenous administration of recombinant adeno-associated virus (AAV) 2/8-ADA vector in ADA-deficient neonate and adult mice or as part of a bimodal approach comprised of rAAV treatment at birth followed by infusion of lentiviral vector (LV)-modified lineage-depleted bone marrow cells at 8 weeks. ADA−/− mice treated with rAAV and enzyme replacement therapy (ERT) for 30 days were rescued from the lethal pulmonary insufficiency, surviving out to 180 days without further treatment. rAAV vector copy number (VCN) was highest in liver, lung, and heart and was associated with near-normal ADA activity and thymocyte development. In the bimodal approach, rAAV-mediated ADA expression supported survival during the 4 weeks before infusion of the LV-modified bone marrow cells and during the engraftment period. Conditioning prior to infusion may have resulted in the replacement of rAAV marked cells in marrow and liver, with LV VCN 100- to 1,000-fold higher in hematopoietic tissue compared with rAAV VCN, and was associated with immune cell reconstitution. In conclusion, a bimodal approach may be an alternative for patients without reliable access to ERT before receiving a stem cell transplant or gene therapy.

In ovo feeding of vitamin C regulates splenic development through purine nucleotide metabolism and induction of apoptosis in broiler chickens.

Nutrition in early life has a long-term influence on later health. In order to the explore effects of in ovo feeding (IOF) of vitamin C on splenic development, splenic metabolism and apoptosis were detected in embryo, adult chickens and in vitro. A total of 360 fertile eggs were selected and randomly assigned to control (CON) and vitamin C (VC) groups which were injected with saline and vitamin C on embryonic day 11, respectively. Functional enrichment of differentially expressed genes by transcriptome on embryonic day 19 suggested that purine nucleotide metabolism might be a potential pathway for the IOF of vitamin C to regulate spleen development. Additionally, the IOF of vitamin C significantly increased splenic vitamin C content on post-hatch day 21. Meanwhile, the splenic expression of adenosine deaminase, serine/threonine kinase 1 and proliferating cell nuclear antigen was down-regulated, whereas the expression of cysteinyl aspartate specific proteinase 9 was up-regulated in the VC group. On post-hatch day 42, the IOF of vitamin C significantly down-regulated the splenic expression of B-cell lymphoma 2 and increased the mRNA level of cysteinyl aspartate specific proteinase 9. The IOF of vitamin C could regulate the expression of genes related to adenylate metabolism and increased the apoptosis rate in vitro, which is consistent with the result in vivo. In conclusion, the IOF of vitamin C regulated splenic development and maturation by affecting purine nucleotide metabolism pathway and promoting apoptosis.

Methotrexate associated with a lipid core nanoparticle prevented the dilation and dissection of the aortic arch in mice with Marfan syndrome

Abstract Introduction Mutations in the fibrillin-1 gene result in dilation, dissection and rupture of the aorta, the main cause of mortality in patients with Marfan syndrome (MFS). Inflammation is associated with greater susceptibility to dissection and rupture of the aorta. Previously, we showed that methotrexate (MTX) carried in lipid core nanoparticles (LDE) had powerful anti-inflammatory and anti-proliferative effects on rheumatoid arthritis and atherosclerotic lesions induced in rabbits and improved myocardial infarction morphological and functional aspects in rats with ligation of left coronary artery. When MTX is associated to LDE, the uptake of the drug by the cells is increased several-fold, which conceivably endows MTX with enhanced action mechanisms and the drug toxicity is diminished. Purpose To investigate whether treatment with LDE-MTX can prevent the development of aortic arch lesions in a murine model of MFS. Methods mgΔloxPneo MFS murine model and wild-type mice were allocated in 3 groups of treatment: LDE only; MTX in commercial formulation; LDE-MTX. The treatment occurred weekly at a dose of 1mg/kg ip, between the 3rd and 6th month of life, period in which both dilation and dissection of the aortic arch are observed. After 12 weeks, the animals were submitted to echocardiography, morphometry and protein expression of the aortic arch. Results Compared to LDE and MTX groups, LDE-MTX treatment showed smaller lumen area of ascending and descending aorta and aortic arch segments in MFS mice. LDE-MTX also decreased the collagen volume fraction and the amount of aortic dissections, but did not affect the thickness of the vessel wall and the number of elastic fiber ruptures. The protein expression of the inflammatory markers CD68 (macrophages), CD3 (lymphocytes) and tumor necrosis factor alpha, of the apoptotic marker caspase 3 and of type 1 collagen were lower in MFS LDE-MTX group when compared to MFS mice treated with LDE and MTX. Moreover, treatment with LDE-MTX reduced TGF-β, ERK and the SMAD3 protein expression. Of note, CD68 and CD3 protein expression was positively correlated with the lumen area of the aortic arch (r2=0.36; p&amp;lt;0.001), indicating the importance of inflammation for the aortic dilation. The increase in bioavailability of intracellular adenosine in MFS animals treated with LDE-MTX was suggested by the higher expression of A2a adenosine receptor and the lower expression of adenosine deaminase in the aortic arch. Conclusion Possibly by increasing the bioavailability of intracellular adenosine, LDE-MTX treatment had the ability to reduce the processes of inflammation, apoptosis and fibrosis that are consequent to fibrillin-1 mutation. By these means, LDE-MTX may conceivably prevent the development of the dilation and dissection in the aortic arch, the hallmarks of MFS, in this animal model. Funding Acknowledgement Type of funding source: Public grant(s) – National budget only. Main funding source(s): FAPESP (Fundação de Amparo à Pesquisa do Estado de São Paulo)

Fibroblast growth factor 2 upregulates ecto-5'-nucleotidase and adenosine deaminase via MAPK pathways in cultured rat spinal cord astrocytes.

Adenosine triphosphate (ATP) and adenosine are neurotransmitters and neuromodulators in the central nervous system. Astrocytes regulate extracellular concentration of purines via ATP release and its metabolisms via ecto-enzymes. The expression and activity of purine metabolic enzymes in astrocytes are increased under pathological conditions. We previously showed that fibroblast growth factor 2 (FGF2) upregulates the expression and activity of the enzymes ecto-5'-nucleotidase (CD73) and adenosine deaminase (ADA). Here, we further demonstrate that this occurs in concentration- and time-dependent manners in cultured rat spinal cord astrocytes and is suppressed by inhibitors of the FGF receptor as well as the mitogen-activated protein kinases (MAPKs). We also found that FGF2 increased the phosphorylation of MAPKs, including extracellular signal-regulated kinase, c-Jun N-terminal kinase, and p38 MAPK, leading to the increased expression and activity of CD73 and ADA. Our findings reveal the involvement of FGF2/MAPK pathways in the regulation of purine metabolic enzymes in astrocytes. These pathways may contribute to the control of extracellular purine concentrations under physiological and pathological conditions.

Abstract A095: Metabolic rewiring in African-American prostate cancer: A role for adenosine-inosine axis in tumor progression

Abstract Introduction: Prostate cancer (PCa) prevalence and mortality are remarkably higher in African-American (AA)men compared to European-American (EA) men. In addition to known differences in socioeconomic status and access to health care, there is increasing evidence to suggest biological differences between AA and EA PCa. Our lab has pioneered the understanding of reprogrammed metabolism, a hallmark of tumor progression, in AA and EA PCa. Analysis of a compendium of 190 metabolites in ancestry verified, pathologically confirmed AA and EA PCa tissues followed by validation of key pathway components in plasma and urine revealed high inosine to adenosine ratio in AA PCa compared to EA PCa. Consistent with this, the enzyme adenosine deaminase (ADA), which converts adenosine to inosine was also found to be elevated in AA PCa. The current study will address the role of adenosine-inosine axis in AA PCa progression and attempt to delineate its significance in the clinical setting. Methods: Analysis of metabolites in tissues, plasma and urine was done using LC/MS. Tissue microarray analysis (TMA) was done to check the expression of ADA in tissues and correlate it to clinical outcomes. To determine the function of ADA in PCa progression, the gene was overexpressed in MDA-PCa-2A (AA) and LNCaP (EA) cells using lentiviral transduction (termed ADA OE). In vitro invasion was assessed using Boyden chamber assay and in vivo tumorigenic properties were examined in nude mice. Molecular studies were performed using qPCR, ELISA and western blotting. Results: Overexpression of the enzyme ADA in PCa cells confers an anchorage-independent growth phenotype (a.k.a. Anoikis Resistance) associated with a reduction in Integrin β1 (ITGB1) levels. Anoikis resistance is a key prerequisite for metastasis and a hallmark of circulating tumor cells (CTCs). ADA OE AA cells exhibited higher invasive potential compared to the EA cells. Molecular analysis revealed downregulation E-cadherin (epithelial marker) and Bim (pro-apoptotic marker), and upregulation of XIAP (anti-apoptotic marker), all of which support anoikis resistance phenotype in these cells. ADA OE cells had reduced cAMP levels, and addition of external cAMP resulted in reversal of anoikis resistance, increased cell adhesion and rescued ITGB1 levels. Mechanistically, it is postulated that high inosine upon ADA OE activates adenosine receptors A1 and A3, which causes a decrease in cAMP levels. Decreased cAMP affects the Epac-Rap1 signaling, a key regulator of integrin-mediated adhesion. Concomitantly, ADA OE cells also showed a decrease in Rap1 levels. Conclusion: Elevated inosine to adenosine ratio associated with increased expression of ADA is a metabolic hallmark in AA PCa, associated with anoikis resistance and possibly increased CTCs. Our findings also strongly support the potential of using inosine to adenosine ratio in plasma or urine as a predictive marker for PCa incidence or progression in AA men. In addition, ADA could serve as a potential therapeutic target for AA PCa. Citation Format: Christy Charles, Jie Gohlke, Stacy Lloyd, James Henderson, Balasubramaniam Karanam, Nora Navone, Rick Kittles, Stefan Ambs, George Michailidis, Nagireddy Putluri, Arun Sreekumar. Metabolic rewiring in African-American prostate cancer: A role for adenosine-inosine axis in tumor progression [abstract]. In: Proceedings of the Twelfth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2019 Sep 20-23; San Francisco, CA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2020;29(6 Suppl_2):Abstract nr A095.

Abstract A84: Clinical correlates and rationale for PEGylated adenosine deaminase 2 as a novel cancer immunotherapy

Abstract Extracellular adenosine accumulates in solid tumors due to direct release or due to dephosphorylation of ATP, ADP, and AMP by extracellular enzymes such as CD39 and CD73. Adenosine signaling promotes metastasis and suppress antitumor immune responses. CD73 expression is associated with poor survival and chemotherapy resistance in triple-negative breast tumors. Adenosine deaminase 2 is a serum protein that catalyzes the deamination of adenosine to inosine. Expression of Adenosine deaminase 2 increases in pathologic conditions such as inflammation and cancer. Here we show that expression of the gene coding for ADA2 (CECR1) in lung and breast tumors is associated with increased expression of gene signatures associated with immune checkpoint blockade responsiveness. Our analyses have also indicated that increased ADA2 expression is associated with favorable outcomes among Her2+ and triple-negative breast cancer patients. Further stratification based on the expression of adenosine-generating enzymes, CD39/CD73, revealed that increased ADA2 expression is particularly associated with increased survival among patients with elevated CD39 and CD73 expression. ADA2 expression is found to be higher in stroma areas of the tumor as compared with tumor cells. Stromal expression but not tumor expression of ADA2 is associated with increased T-cell costimulation and infiltration scores. To support clinical evidence in preclinical models and to show the potential of PEGylated Adenosine Deaminase 2 (PEGADA2) as a novel therapy, we have shown that an engineered high-catalytic activity PEGADA2 variant (PEGADA2HCA) can slow growth of EMT6 syngeneic breast tumors. PEGADA2HCA also completely reversed adenosine inhibition of human T-cell activation. PEGADA2HCA treatment in humanized mice caused a linear dose-exposure relationship after repeat dosing. These results provide a strong rationale for testing PEGylated Adenosine Deaminase 2 alone or in combination in a clinical setting. Citation Format: Ozge Saatci, Luz Marina Londoño, Renee Clift, Curt Thompson, Mike LaBarre, Ozgur Sahin, Caglar Cekic. Clinical correlates and rationale for PEGylated adenosine deaminase 2 as a novel cancer immunotherapy [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr A84.

Ammonium induced dysfunction of 5-HT2B receptor in astrocytes

Previously we reported that gene expression of astrocytic 5-HT2B receptors was decreased in brains of depressed animals exposed to chronic mild stress (CMS) (Li et al., 2012) and of Parkinson's disease (Song et al., 2018). Depression is also one of the psychiatric symptoms in hyperammonemia, and astrocyte is a primary target of ammonium in brain in vivo. In the present study, we have used preparations of the brains of urease-treated mice and ammonium-treated astrocytes in culture to study gene expression and function of 5-HT2B receptors. The urease-treated mice showed depressive behaviour. Both mRNA and protein of 5-HT2B receptors were increased in the brains of urease-treated mice and in ammonium-treated cultured astrocytes. Further study revealed that mRNA and protein expression of adenosine deaminase acting on RNA 2 (ADAR2), an enzyme catalyze RNA deamination of adenosine to inosine was increased in the brains of urease-treated mice and in ammonium-treated cultured astrocytes. This increase in ADAR2 induced RNA editing of 5-HT2B receptors. Cultured astrocytes treated with ammonium lost 5-HT induced Ca2+ signalling and ERK1/2 phosphorylation, indicating dysfunction of 5-HT2B receptors. This is in agreement with our previous observation that edited 5-HT2B receptors no longer respond to 5-HT (Hertz et al., 2014). Ammonium effects are inhibited by ADAR2 siRNA in cultured astrocytes, suggesting that increased gene expression and editing and loss of function of 5-HT2B receptors are results of increased activity of ADAR2. In summary, we have demonstrated that functional malfunction of astrocytic 5-HT2B receptors occurs in animal models of major depression, Parkinson depression and hepatic encephalopathy albeit via different mechanisms. Understanding the role of astrocytic 5-HT2B receptors in different pathological contexts may instigate development of novel therapeutic strategies for treating disease-specific depressive behaviour.