Metabolism of FAD, FMN and riboflavin (vitamin B2) in the human parasitic blood fluke Schistosoma mansoni.

BACKGROUND: Schistosomiasis is a parasitic disease caused by trematodes of the genus Schistosoma. The intravascular worms acquire the nutrients necessary for their survival from host blood. Since all animals are auxotrophic for riboflavin (vitamin B2), schistosomes too must import it to survive. Riboflavin is an essential component of the coenzymes flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD); these support key functions of dozens of flavoenzymes. METHODS: Here, using a combination of metabolomics, enzyme kinetics and in silico molecular analysis, we focus on the biochemistry of riboflavin and its metabolites in Schistosoma mansoni (Sm). RESULTS: We show that when schistosomes are incubated in murine plasma, levels of FAD decrease over time while levels of FMN increase. We show that live schistosomes cleave exogenous FAD to generate FMN and this ability is significantly blocked when expression of the surface nucleotide pyrophosphatase/phosphodiesterase ectoenzyme SmNPP5 is suppressed using RNAi. Recombinant SmNPP5 cleaves FAD with a Km of 178 ± 5.9 µM and Kcat/Km of 324,734 ± 36,347 M- 1.S- 1. The FAD-dependent enzyme IL-4I1 drives the oxidative deamination of phenylalanine to produce phenylpyruvate and H2O2. Since schistosomes are damaged by H2O2, we determined if SmNPP5 could impede H2O2 production by blocking IL-4I1 action in vitro. We found that this was not the case; covalently bound FAD on IL-4I1 appears inaccessible to SmNPP5. We also report that live schistosomes can cleave exogenous FMN to generate riboflavin and this ability is significantly impeded when expression of a second surface ectoenzyme (alkaline phosphatase, SmAP) is suppressed. Recombinant SmAP cleaves FMN with a Km of 3.82 ± 0.58 mM and Kcat/Km of 1393 ± 347 M- 1.S- 1. CONCLUSIONS: The sequential hydrolysis of FAD by tegumental ecto-enzymes SmNPP5 and SmAP can generate free vitamin B2 around the worms from where it can be conveniently imported by the recently described schistosome riboflavin transporter SmaRT. Finally, we identified in silico schistosome homologs of enzymes that are involved in intracellular vitamin B2 metabolism. These are riboflavin kinase (SmRFK) as well as FAD synthase (SmFADS); cDNAs encoding these two enzymes were cloned and sequenced. SmRFK is predicted to convert riboflavin to FMN while SmFADS could further act on FMN to regenerate FAD in order to facilitate robust vitamin B2-dependent metabolism in schistosomes.

Zinc transport via ZNT5-6 and ZNT7 is critical for cell surface glycosylphosphatidylinositol-anchored protein expression.

Glycosylphosphatidylinositol (GPI)-anchored proteins play crucial roles in various enzyme activities, cell signaling and adhesion, and immune responses. While the molecular mechanism underlying GPI-anchored protein biosynthesis has been well studied, the role of zinc transport in this process has not yet been elucidated. Zn transporter (ZNT) proteins mobilize cytosolic zinc to the extracellular space and to intracellular compartments. Here, we report that the early secretory pathway ZNTs (ZNT5-ZNT6 heterodimers [ZNT5-6] and ZNT7-ZNT7 homodimers [ZNT7]), which supply zinc to the lumen of the early secretory pathway compartments are essential for GPI-anchored protein expression on the cell surface. We show, using overexpression and gene disruption/re-expression strategies in cultured human cells, that loss of ZNT5-6 and ZNT7 zinc transport functions results in significant reduction in GPI-anchored protein levels similar to that in mutant cells lacking phosphatidylinositol glycan anchor biosynthesis (PIG) genes. Furthermore, medaka fish with disrupted Znt5 and Znt7 genes show touch-insensitive phenotypes similar to zebrafish Pig mutants. These findings provide a previously unappreciated insight into the regulation of GPI-anchored protein expression and protein quality control in the early secretory pathway.

Marine Invertebrates: A Promissory Still Unexplored Source of Inhibitors of Biomedically Relevant Metallo Aminopeptidases Belonging to the M1 and M17 Families.

Proteolytic enzymes, also known as peptidases, are critical in all living organisms. Peptidases control the cleavage, activation, turnover, and synthesis of proteins and regulate many biochemical and physiological processes. They are also involved in several pathophysiological processes. Among peptidases, aminopeptidases catalyze the cleavage of the N-terminal amino acids of proteins or peptide substrates. They are distributed in many phyla and play critical roles in physiology and pathophysiology. Many of them are metallopeptidases belonging to the M1 and M17 families, among others. Some, such as M1 aminopeptidases N and A, thyrotropin-releasing hormone-degrading ectoenzyme, and M17 leucyl aminopeptidase, are targets for the development of therapeutic agents for human diseases, including cancer, hypertension, central nervous system disorders, inflammation, immune system disorders, skin pathologies, and infectious diseases, such as malaria. The relevance of aminopeptidases has driven the search and identification of potent and selective inhibitors as major tools to control proteolysis with an impact in biochemistry, biotechnology, and biomedicine. The present contribution focuses on marine invertebrate biodiversity as an important and promising source of inhibitors of metalloaminopeptidases from M1 and M17 families, with foreseen biomedical applications in human diseases. The results reviewed in the present contribution support and encourage further studies with inhibitors isolated from marine invertebrates in different biomedical models associated with the activity of these families of exopeptidases.

Inhibition of ATP hydrolysis as a key regulator of temozolomide resistance and migratory phenotype of glioblastoma cells.

Glioblastoma (GBM) is the most lethal among malignant gliomas. The tumor invasiveness and therapy-resistance are important clinical hallmarks. Growing evidence emphasizes the purinergic signaling contributing to tumor growth. Here we exposed a potential role of extracellular ATPase activity as a key regulator of temozolomide cytotoxicity and the migration process in GBM cells. The inhibition of ATP hydrolysis was able to improve the impact of temozolomide, causing arrest mainly in S and G2 phases of the cell cycle, leading M059J and U251 cells to apoptosis. In addition to eradicating GBM cells, ATP hydrolysis exhibited a potential to modulate the invasive phenotype and the expression of proteins involved in cell migration and epithelial-to-mesenchymal-like transition in a 3D culture model. Finally, we suggest the ATPase activity as a key target to decline temozolomide resistance and the migratory phenotype in GBM cells.

Detailed analyses of the crucial functions of Zn transporter proteins in alkaline phosphatase activation.

Numerous zinc ectoenzymes are metalated by zinc and activated in the compartments of the early secretory pathway before reaching their destination. Zn transporter (ZNT) proteins located in these compartments are essential for ectoenzyme activation. We have previously reported that ZNT proteins, specifically ZNT5-ZNT6 heterodimers and ZNT7 homodimers, play critical roles in the activation of zinc ectoenzymes, such as alkaline phosphatases (ALPs), by mobilizing cytosolic zinc into these compartments. However, this process remains incompletely understood. Here, using genetically-engineered chicken DT40 cells, we first determined that Zrt/Irt-like protein (ZIP) transporters that are localized to the compartments of the early secretory pathway play only a minor role in the ALP activation process. These transporters included ZIP7, ZIP9, and ZIP13, performing pivotal functions in maintaining cellular homeostasis by effluxing zinc out of the compartments. Next, using purified ALP proteins, we showed that zinc metalation on ALP produced in DT40 cells lacking ZNT5-ZNT6 heterodimers and ZNT7 homodimers is impaired. Finally, by genetically disrupting both ZNT5 and ZNT7 in human HAP1 cells, we directly demonstrated that the tissue-nonspecific ALP-activating functions of both ZNT complexes are conserved in human cells. Furthermore, using mutant HAP1 cells, we uncovered a previously-unrecognized and unique spatial regulation of ZNT5-ZNT6 heterodimer formation, wherein ZNT5 recruits ZNT6 to the Golgi apparatus to form the heterodimeric complex. These findings fill in major gaps in our understanding of the molecular mechanisms underlying zinc ectoenzyme activation in the compartments of the early secretory pathway.

Altered aryl-hydrocarbon-receptor signalling affects regulatory and effector cell immunity in autoimmune hepatitis.

BACKGROUND & AIMS: In autoimmune hepatitis (AIH), the imbalance between regulatory T cells (Tregs) and T-helper type 17 (Th17) cells has been linked to low levels of CD39, an ectoenzyme that hydrolyses ATP, ultimately generating immunosuppressive adenosine. Upregulation of CD39 results from activation of aryl hydrocarbon receptor (AHR), which mediates toxin responses to modulate T-cell immunity. In this study, we investigated whether altered AHR signalling underlies defective CD39 expression and function in AIH Tregs and Th17 cells, therefore contributing to regulatory/effector cell imbalance. METHODS: Tregs and Th17 cells, obtained from the peripheral blood of 49 patients with AIH and 21 healthy individuals (HI), were tested for response to endogenous and exogenous AHR ligands. RESULTS: When compared to those of HI, AIH-derived Tregs and Th17 cells displayed impaired responses to AHR activation, reflected by impaired upregulation of CD39, delayed increase in ectoenzymatic activity, and defective Treg suppressive function. These impairments resulted, at least in part, from heightened levels of AHRR and Erα in Tregs and high HIF-1α in Th17 cells, and were reverted upon molecular blockade. Importantly, in AIH-derived Tregs, the binding affinity of AHR was higher for Erα than ARNT. CONCLUSIONS: In AIH, high levels of AHRR and HIF-1α inhibit AHR signalling in Tregs and Th17 cells. AHR non-canonical binding to Erα further amplifies the lack of effective CD39 upregulation. Blockade of these inhibitory and/or non-canonical activation pathways represents a potential therapeutic approach to restore CD39 and immunohomeostasis in AIH. LAY SUMMARY: In patients with autoimmune hepatitis, the imbalance between regulatory T cells and T helper type-17 cells is linked to dysfunction of the aryl hydrocarbon receptor pathway, resulting from aberrant inhibition or non-canonical activation. These alterations impair Treg- and Th17 cell-induced upregulation of CD39, an ectoenzyme key to immunoregulation. Blockade of excessive inhibition or non-canonical activation of the aryl hydrocarbon receptor pathway might represent a novel therapeutic strategy to control inflammation while restoring immune balance in autoimmune hepatitis.

HIF-1α-induced xenobiotic transporters promote Th17 responses in Crohn's disease.

In Crohn's disease, pathogenic Th17-cells express low levels of CD39 ectonucleotidase and are refractory to the immunosuppressive effects of unconjugated bilirubin (UCB), an endogenous ligand for aryl-hydrocarbon-receptor (AhR). This resistance to AhR ligation might be associated with alterations in responses to hypoxia. Limited exposure to hypoxia appears beneficial in acute tissue injury. However, in protracted inflammation, hypoxemia may paradoxically result in Th17-cell activation. We report here that in vitro exposure of Th17-cells from Crohn's disease patients to hypoxia limits responsiveness to AhR stimulation by UCB, as reflected by lower CD39 levels. Blockade of hypoxia-inducible-factor-1alpha (HIF-1α) upregulates CD39 and favors Th17-cell regulatory responses. Resistance of Th17-cells to AhR signaling results, in part, from HIF-1α-dependent induction of ATP-binding cassette (ABC) transporters: multidrug-resistance-protein-1 (MDR1) and multidrug-resistance-associated-protein-4 (MRP4). Increased ABC transporters promote efflux of suppressive AhR ligands, such as UCB, from Th17-cells. Inhibition of MDR1, MRP4 and/or HIF-1α with ritonavir (RTV) reconstitutes AhR function in Th17-cells, enhancing therapeutic effects of UCB in dextran-sulfate-sodium-induced experimental colitis. Deleterious effects of hypoxia on Th17-cells in Crohn's disease can be ameliorated either by inhibiting HIF-1α or by suppressing ABC transporters to increase UCB availability as an AhR substrate. Targeting HIF-1α-ABC transporters could provide innovative therapeutic pathways for IBD.

NPP1 is responsible for potent extracellular ATP hydrolysis as NTPDase1 in primary cultured murine microglia.

The movement of microglia is regulated mainly by P1 and P2 purinergic receptors, which are activated by various nucleotides and their metabolites. Recently, such purinergic signalling has been spotlighted because of potential roles in the pathophysiologies of neurodegenerative and neuropsychiatric disorders. To understand the characteristics of microglia in relation of P1 and P2 signalling, we investigated the ectoenzymes expressed in microglia. At first, we profiled the expression of all known ectoenzymes in cultured microglia. We found that, like NTPDase1 (ectonucleoside triphosphate diphosphohydrolase 1, CD39), NPP1 (ectonucleotide pyrophosphatase/phosphodiesterase 1, PC-1) is also highly expressed in primary cultured murine microglia. Knockdown of NPP1 significantly reduced ATP hydrolysis and Pi production in cultured microglia. In addition, the knockdown of NPP1 enhanced basal nucleotide-stimulating responses of cultured microglia, such as phagocytosis and cell migration, and these results were very similar to NTPDase1 knockdown results. Moreover, inhibition of the adenosine receptors by caffeine treatment reduced phagocytosis of NPP1 knock downed-cultured microglia. In conclusion, we suggest that these potent ectoenzymes of primary cultured murine microglia, NPP1 together with CD73 (ecto-5'-nucleotidase) maintain the adenosine levels for triggering nucleotide-stimulating responses.

Advances in TRH signaling.

The activity of the hypothalamus-pituitary-thyroid axis (HPT) is coordinated by hypophysiotropic thyrotropin releasing hormone (TRH) neurons present in the paraventricular nucleus of the hypothalamus. Hypophysiotropic TRH neurons act as energy sensors. TRH controls the synthesis and release of thyrotropin, which activates the synthesis and secretion of thyroid hormones; in target tissues, transporters and deiodinases control their local availability. Thyroid hormones regulate many functions, including energy homeostasis. This review discusses recent evidence that covers several aspects of TRH role in HPT axis regulation. Knowledge about the mechanisms of TRH signaling has steadily increased. New transcription factors engaged in TRH gene expression have been identified, and advances made on how they interact with signaling pathways and define the dynamics of TRH neurons response to acute and/or long-term influences. Albeit yet incomplete, the relationship of TRH neurons activity with positive energy balance has emerged. The importance of tanycytes as a central relay for the feedback control of the axis, as well as for HPT responses to alterations in energy balance, and other stimuli has been reinforced. Finally, some studies have started to shed light on the interference of prenatal and postnatal stress and nutrition on HPT axis programing, which have confirmed the axis susceptibility to early insults.

Impact of ENPP1 K121Q on change of insulin resistance after web-based intervention in Korean men with diabetes and impaired fasting glucose.

Ectoenzyme nucleotide pyrophosphate phosphodiesterase 1 (ENPP1) gene has been studied in relation to type 2 diabetes mellitus (T2DM) and insulin resistance (IR). We hypothesized that the difference in genotype may be one of the factors that affect the outcome of intervention. We genotyped 448 men with fasting glucose≥5.6 mM/L, including 371 in subjects with K allele (KK) (69 control group [CG]; and 302 intervention group [IG]) and 77 in subjects with Q allele (KQ+QQ) (13 CG and 64 IG). The web-based intervention based on a lifestyle modification was delivered by e-mail once a month for 10 months. In the KK, IG demonstrated significantly decreased levels of fasting serum insulin (FSI) as compared to CG and homeostasis model of assessment of insulin resistance (HOMA-IR). In the KQ+QQ IG group, hemoglobin A1c (HbA1c), FSI and HOMA-IR were significantly decreased, and showed further reduction in the HOMA-IR than KQ+QQ CG. After analysis of covariance, K121Q did significantly influence the change of HbA1c in CG after appropriate adjustment. In a multivariate model, BMI change predicted HOMA-IR change (adjusted ß=0.801; P=0.022) in KK IG subjects with T2DM. ENPP1 K121Q did not influence the change in IR. However, individuals with T2DM carrying the K121 variant are very responsive to the effect of BMI reduction on HOMA-IR.

Decoupling between the genetic potential and the metabolic regulation and expression in microbial organic matter cleavage across microbiomes.

Metagenomics, metatranscriptomics, and metaproteomics are used to explore the microbial capability of enzyme secretion, but the links between protein-encoding genes and corresponding transcripts/proteins across ecosystems are underexplored. By conducting a multi-omics comparison focusing on key enzymes (carbohydrate-active enzymes [CAZymes] and peptidases) cleaving the main biomolecules across distinct microbiomes living in the ocean, soil, and human gut, we show that the community structure, functional diversity, and secretion mechanisms of microbial secretory CAZymes and peptidases vary drastically between microbiomes at metagenomic, metatranscriptomic, and metaproteomic levels. Such variations lead to decoupled relationships between CAZymes and peptidases from genetic potentials to protein expressions due to the different responses of key players toward organic matter sources and concentrations. Our results highlight the need for systematic analysis of the factors shaping patterns of microbial cleavage on organic matter to better link omics data to ecosystem processes. IMPORTANCE: Omics tools are used to explore adaptive mechanism of microbes in diverse systems, but the advantages and limitations of different omics tools remain skeptical. Here, we reported distinct profiles in microbial secretory enzyme composition revealed by different omics methods. In general, the predicted function from metagenomic analysis decoupled from the expression of corresponding transcripts/proteins. Linking omics results to taxonomic origin, functional capability, substrate specificity, secretion preference, and enzymatic activity measurement suggested the substrate's source, concentration and stoichiometry impose strong filtering on the expression of extracellular enzymes, which may overwrite the genetic potentials. Our results present an integrated perspective on the need for multi-dimensional characterization of microbial adaptation in a changing environment.

Immune interactions and regulation with CD39+ extracellular vesicles from platelet concentrates.

Introduction: CD39 plays an important role in the immunoregulation and inhibition of effector cells. It is expressed on immune cells, including Tregs, and on extracellular vesicles (EVs) budding from the plasma membrane. Platelet transfusion may induce alloimmunization against HLA-I antigens, leading to refractoriness to platelet transfusion with severe consequences for patients. Tregs may play a key role in determining whether alloimmunization occurs in patients with hematologic disorders. We hypothesized that CD39+ EVs might play an immunoregulatory role, particularly in the context of platelet transfusions in patients with hematologic disorders. Such alloimmunization leads to the production of alloantibodies and is sensitive to the regulatory action of CD39. Methods: We characterized CD39+ EVs in platelet concentrates by flow cytometry. The absolute numbers and cellular origins of CD39+ EVs were evaluated. We also performed functional tests to evaluate interactions with immune cells and their functions. Results: We found that CD39+ EVs from platelet concentrates had an inhibitory phenotype that could be transferred to the immune cells with which they interacted: CD4+ and CD8+ T lymphocytes (TLs), dendritic cells, monocytes, and B lymphocytes (BLs). Moreover, the concentration of CD39+ EVs in platelet concentrates varied and was very high in 10% of concentrates. The number of these EVs present was determinant for EV-cell interactions. Finally, functional interactions were observed with BLs, CD4+ TLs and CD39+ EVs for immunoglobulin production and lymphoproliferation, with potential implications for the immunological management of patients.

Soil extracellular enzyme stoichiometry reveals the nutrient limitations in soil microbial metabolism under different carbon input manipulations.

Changes in the quality and quantity of litter and root inputs due to climate change and human activities can influence below-ground biogeochemical processes in forest ecosystems. However, it is unclear whether and how much aboveground litter and root inputs affect soil microbial metabolism and nutrient limitation mechanisms. In this study, according to a 4-years field manipulation experiment, litter and root manipulations (control (CK), double litter input (DL), no litter (NL), no root (NR), and no inputs (NI)) were set up to analyze the extracellular enzyme activities and stoichiometric ratios characteristics of 0-10 cm and 10-20 cm soils, explore the metabolic limitations of microorganisms, and clarify the main driving factors restricting nutrient limitation. The results showed that the enzyme activities associated with the C cycling (ß-1,4-glucosidase (BG), cellulose disaccharide hydrolase (CBH)) and N cycling (ß-1,4-N-acetylglucosaminidase (NAG), leucine aminopeptidase (LAP)) in DL treatment were significantly higher than those in NR treatment. Moreover, enzyme activities related to P cycling are significantly higher in comparison to other treatments. The acid phosphatase (AP), which is related to the P cycle, showed the highest activity under NR treatment. In addition, there was no significant difference in soil microbial metabolic limitation by the different carbon inputs, which did not change the original nutrient limitation pattern. The main drivers of microbial nutrient metabolic limitation included soil physicochemical properties, soil total nutrients, and available nutrients, among which soil SWC and pH presented the greatest influence on microbial C limitation and soil total nutrients showed the greatest influence on microbial N limitation. Changes in soil carbon input altered soil extracellular enzyme activities and their stoichiometric ratios by affecting soil physicochemical properties, total nutrients. This study provides data for the understanding of material cycling in forest ecosystems under environmental change.

Effects of elevated carbon dioxide (CO2)and ozone (O3)concentrations on ectoenzyme activities in rice rhizospheric soil.

Rising atmospheric carbon dioxide (CO2) and ozone (O3) concentrations are the main global change drivers. Soil ectoenzymes play an important role in maintaining soil ecosystem services. Exploring the responses of soil ectoenzymes to elevated CO2 and O3 concentrations is important for combating global climate change. In this study, we simulated elevated CO2 concentrations (+200 µmol·mol-1, eCO2), elevated O3 concentrations (0.04 µmol·mol-1, eO3), and their combination (eCO2+eO3) in open-top chambers (OTCs), and investigated the responses of rhizospheric soil ectoenzyme activities. The results showed that eCO2 significantly increased the ß-D-Glucosidase (ßG) activity by 73.0%, and decreased that of polyphenol oxidase (PHO), peroxidase (PEO), and acid phosphatase (AP) by 48.9%, 46.6% and 72.9% respectively, but did not affect that of cellulose hydrolase (CBH) and ß-N-Acetylglucosaminidase (NAG). eO3 significantly reduced the activities of CBH and AP by 34.2% and 30.4%, respectively. The activities of PHO and AP were reduced by 87.3% and 32.3% under the eCO2+eO3 compared with the control, respectively. Results of the principal coordinate analysis, permutation multivariate analysis of variance and redundancy analysis showed that both elevated CO2 and O3 significantly affected soil ectoenzyme activities, with stronger effects of elevated CO2 than elevated O3. Root nitrogen content, root carbon to nitrogen ratio, soil microbial biomass carbon and nitrate nitrogen were the main drivers of soil ectoenzyme activities under elevated CO2 and O3. Elevated O3 could partially neutralize the effects of elevated CO2 on soil ectoenzyme activities. In conclusion, elevated CO2 and O3 restrained the activities of most soil ectoenzyme, suggesting that climate change would threat soil ecosystem services and functions in the agroecosystem.

The Impact of CD37 Ectoenzyme Expression in Benign and Malignant Colorectal Tumors.

The depth of invasion of colorectal cancer through the bowel wall provided the basis for pathological staging. The establishment of prognostic markers for CRC is important in the search for potential targets for therapeutic intervention. The cell surface nucleotidase (CD73) is an enzyme involved in tumor progression and metastasis. Its main function is to convert adenosine monophosphate (AMP) to adenosine. Preclinical studies suggest that CD73 can be targeted for cancer treatment. This study was designed to investigate the prognostic value of CD73 in malignant and benign colorectal tumors among Iraqi patients and its correlation with clinicopathological features using the immunohistochemistry (IHC) technique. In the current study, 60 cases of formalin-fixed paraffin-embedded (FFPE) tissues of colorectal tumors were collected from the Teaching Laboratories of Baghdad Medical City Teaching Hospital, Baghdad - Iraq & private labs in Baghdad. Of them, 52 were malignant, and 8 of benign cases. In addition, 10 cases of the non-pathological significance of colorectal tissues were used as a control group. This study's results revealed significant differences (P=0.001) in the expression of CD73 between malignant tumors and benign and non-significant pathology groups. The highest expression of CD73 was detected in 76.9% of malignant cases, while in the benign group, only 12.5% of cases showed weak positive expression for this marker. Furthermore, significant differences had been reported in CD73 expression with patients' age groups for malignant (P=0.0335), tumor side (P=0.0409), and tumor grade & stage (P=≤0.01). No significant differences had been seen between CD73 expression with tumor diameter and patients' gender.

Additive Effect of CD73 Inhibitor in Colorectal Cancer Treatment With CDK4/6 Inhibitor Through Regulation of PD-L1.

BACKGROUND & AIMS: Although cancer immunotherapies are effective for advanced-stage cancers, there are no clinically approved immunotherapies for colon cancers (CRCs). Therefore, there is a high demand for the development of novel therapies. Extracellular adenosine-mediated signaling is considered a promising target for advanced-stage cancers that are nonresponsive to programmed death 1 (PD-1)-/programmed death-ligand 1 (PD-L1)-targeted immunotherapies. In this study, we aimed to elucidate novel tumorigenic mechanisms of extracellular adenosine. METHODS: To investigate the effects of extracellular adenosine on tumor-associated macrophages, peripheral blood-derived human macrophages were treated with adenosine and analyzed using flow cytometry and Western blot. Changes in adenosine-treated macrophages were further assessed using multi-omics analysis, including total RNA sequencing and proteomics. Colon cancer mouse models were used to measure the therapeutic efficacy of AB680 and palbociclib. We also used tissue microarrays of patients with CRC, to evaluate their clinical relevance. RESULTS: Extracellular adenosine-mediated reduction of cyclin D1 (CCND1) was found to be critical for the regulation of immune checkpoint molecules and PD-L1 levels in human macrophages, indicating that post-translational modification of PD-L1 is affected by adenosine. A potent CD73 selective inhibitor, AB680, reversed the effects of adenosine on CCND1 and PD-L1. This result strongly suggests that AB680 is a combinatory therapeutic option to overcome the undesired side effects of the cyclin-dependent kinase 4/6 inhibitor, palbociclib, which increases PD-L1 expression in tumors. Because palbociclib is undergoing clinical trials for metastatic CRC in combination with cetuximab (clinical trial number: NCT03446157), we validated that the combination of AB680 and palbociclib significantly improved anti-tumor efficacy in CRC animal models, thereby highlighting it as a novel immunotherapeutic strategy. We further assessed whether the level of CCND1 in tumor-associated macrophages was indeed reduced in tumor sections obtained from patients with CRC, for evaluating the clinical relevance of this strategy. CONCLUSIONS: In this study, we demonstrated that a novel combination therapy of AB680 and palbociclib may be advantageous for the treatment of CRC.

CD26 expression on donor harvest as a risk predictive biomarker for developing graft-versus-host disease post-allogeneic hematopoietic stem cell transplantation: A ten-year follow-up study.

BACKGROUND: Allogeneic hematopoietic stem cell transplantation (ASCT) is the preferred treatment option for patients with several hematologic disorders and immunodeficiency syndromes. Graft-versus-host disease (GVHD) is an immune mediated post-transplant complication which has a major impact on long-term transplant outcomes. OBJECTIVE: Current efforts are focused on identification of new markers that serve as potential predictors of GVHD and other post-transplant clinical outcomes. METHODS: This study includes donor harvests collected from twenty-three allogeneic donors during period 2008-2009 and respective transplant recipients followed for clinical outcomes till March 2019. Percent CD26+ and CD34+ cells in donor harvest were analyzed using flow cytometry. Percent expression and infused dose of CD26+ and CD34+ cells were evaluated for association with various clinical outcomes. RESULTS: Total 23 healthy donors with median age of 28 years (13 males), and transplant recipients with median age of 24 years (17 males) formed the study cohort. The diagnosis included malignant (n= 13) and non-malignant (n= 10) hematological disorders. Median CD34brCD45lo HSC expression was 0.57% (IQR 0.24-1.03) while median CD26 expression was 19.64% (IQR 8.96-33.56) of all nucleated cells. CD26 expression was associated with donor age (P= 0.037). CD26 percent expression correlated with WBC engraftment (P= 0.015) and with acute GVHD (P= 0.023) whereas infused CD26 cell dose correlated with WBC engraftment (P= 0.004) and risk of CMV reactivation (P= 0.020). There was no statistically significant correlation of either CD26 expression or cell dose with chronic GVHD, EFS or OS. CONCLUSIONS: Our findings suggest a role of CD26 expression on human donor harvest as a potential predictor of acute GVHD. This association warrants further exploration.

Leukocyte Membrane Enzymes Play the Cell Adhesion Game.

For a long time, proteins with enzymatic activity have not been usually considered to carry out other functions different from catalyzing chemical reactions within or outside the cell. Nevertheless, in the last few years several reports have uncovered the participation of numerous enzymes in other processes, placing them in the category of moonlighting proteins. Some moonlighting enzymes have been shown to participate in complex processes such as cell adhesion. Cell adhesion plays a physiological role in multiple processes: it enables cells to establish close contact with one another, allowing communication; it is a key step during cell migration; it is also involved in tightly binding neighboring cells in tissues, etc. Importantly, cell adhesion is also of great importance in pathophysiological scenarios like migration and metastasis establishment of cancer cells. Cell adhesion is strictly regulated through numerous switches: proteins, glycoproteins and other components of the cell membrane. Recently, several cell membrane enzymes have been reported to participate in distinct steps of the cell adhesion process. Here, we review a variety of examples of membrane bound enzymes participating in adhesion of immune cells.

Realization of Osteolysis, Angiogenesis, Immunosuppression, and Drug Resistance by Extracellular Vesicles: Roles of RNAs and Proteins in Their Cargoes and of Ectonucleotidases of the Immunosuppressive Adenosinergic Noncanonical Pathway in the Bone Marrow Niche of Multiple Myeloma.

Angiogenesis and immunosuppression promote multiple myeloma (MM) development, and osteolysis is a primary feature of MM. Although immunomodulatory drugs and proteasome inhibitors (PIs) markedly improve the survival of patients with MM, this disease remains incurable. In the bone marrow niche, a chain of ectoenzymes, including CD38, produce immunosuppressive adenosine, inhibiting T cell proliferation as well as immunosuppressive cells. Therefore, anti-CD38 antibodies targeting myeloma cells have the potential to restore T cell responses to myeloma cells. Meanwhile extracellular vesicles (EVs) containing microRNAs, proteins such as cytokines and chemokines, long noncoding RNAs, and PIWI-interacting RNAs have been shown to act as communication tools in myeloma cell/microenvironment interactions. Via EVs, mesenchymal stem cells allow myeloma cell dissemination and confer PI resistance, whereas myeloma cells promote angiogenesis, myeloid-derived suppressor cell proliferation, and osteoclast differentiation and inhibit osteoblast differentiation. In this review, to understand key processes of MM development involving communication between myeloma cells and other cells in the tumor microenvironment, EV cargo and the non-canonical adenosinergic pathway are introduced, and ectoenzymes and EVs are discussed as potential druggable targets for the treatment of MM patients.

Vitamin B6 Acquisition and Metabolism in Schistosoma mansoni.

Schistosomes are parasitic platyhelminths that currently infect >200 million people globally. The adult worms can live within the vasculature of their hosts for many years where they acquire all nutrients necessary for their survival and growth. In this work we focus on how Schistosoma mansoni parasites acquire and metabolize vitamin B6, whose active form is pyridoxal phosphate (PLP). We show here that live intravascular stage parasites (schistosomula and adult males and females) can cleave exogenous PLP to liberate pyridoxal. Of the three characterized nucleotide-metabolizing ectoenzymes expressed at the schistosome surface (SmAP, SmNPP5, and SmATPDase1), only SmAP hydrolyzes PLP. Heat-inactivated recombinant SmAP can no longer cleave PLP. Further, parasites whose SmAP gene has been suppressed by RNAi are significantly impaired in their ability to cleave PLP compared to controls. When schistosomes are incubated in murine plasma, they alter its metabolomic profile-the levels of both pyridoxal and phosphate increase over time, a finding consistent with the action of host-exposed SmAP acting on PLP. We hypothesize that SmAP-mediated dephosphorylation of PLP generates a pool of pyridoxal around the worms that can be conveniently taken in by the parasites to participate in essential, vitamin B6-driven metabolism. In addition, since host PLP-dependent enzymes play active roles in inflammatory processes, parasite-mediated cleavage of this metabolite may serve to limit parasite-damaging inflammation. In this work we also identified schistosome homologs of enzymes that are involved in intracellular vitamin B6 metabolism. These are pyridoxal kinase (SmPK) as well as pyridoxal phosphate phosphatase (SmPLP-Ph) and pyridox(am)ine 5'-phosphate oxidase (SmPNPO) and cDNAs encoding these three enzymes were cloned and sequenced. The three genes encoding these enzymes all display high relative expression in schistosomula and adult worms suggestive of robust vitamin B6 metabolism in the intravascular life stages.