Journal: Frontiers in immunology

Article Title: Immunometabolic interference between cancer and COVID-19.

doi: 10.3389/fimmu.2023.1168455

Figure Lengend Snippet: FIGURE 9 Flow cytometry analysis of NAMPT and NAD-dependent enzymes in the different patient cohorts. Box-and-whisker plots representing FACS quantification of (A) NAMPT, (B) pSIRT1, (C) CD38, (D) CD39 and (E) PARP expression in the indicated immune cell populations (CD3+ T cells, CD19+

Article Snippet: Cells were incubated with the following antihuman antibodies for 20 min at 4°C: CD33 PerCP Cy5.5 (WM53) (0.6:100, Biolegend cat No. 303402), CD14 BV650 (M5E2) (1.25:100, Biolegend cat. No. 301835), CD16 BV711 (3G8) (0.3:100, Biolegend cat. No. 302044), HLA-DR BV421 (L243) (1.25:100, Biolegend Cat No. 307636), CD15 BV786 (HI98) (1.25:100, BD Biosciences cat. No. 563838), CD11c BUV661 (B-ly6) (0.6:100, BD Biosciences cat. No. 612968), CD123 BUV395 (7G3) (0.6:100, BD Biosciences cat. No. 564195), CD38 PE-Cy7 (HIT2) (1.25:100, Invitrogen cat. No. 25- 0389-42), CD39 PE (MZ18-23C8) (0.6:100, MACSMiltenyi Biotec Inc. cat. No.130-118-668), CD39 FITC (MZ18-23C8) (1.25:100, MACS Miltenyi Biotec Inc. cat. No. 130-125-113), CD3 BV650 (OKT3) (1.25:100, Biolegend cat. No.317324), CD4 BV570 (OKT4) (2.5:100, Frontiers in Immunology 03 Biolegend cat. No. 317445), CD8 BV786 (RPA-T8) (0.6:100, BD Biosciences cat. No. 557085), CD19 AF-700 (HIB19) (2.5:100, BD Biosciences cat. No. 561031), CD45RA APC-Cy7 (HI100) (1.25:100, BD Biosciences cat. No. 560674), CD45RO BUV395 (UCHL1) (1.25:100, BD Biosciences 564292), CD27 PE-Cy5 (O323) (1.25:100, Invitrogen cat. No. 15295964), CD16 BV605 (3G8) (0.6:100, Biolegend 302040), CD3 BUV496 (UCHT1) (1.25:100, BD Biosciences cat. No. 612940), CD66b PE-Cy7(G10F5) (0.6:100 Biolegend cat. No. 305116) and IgD FITC (IA6-2) (1.25:100, Invitrogen cat. No. 11-9868-42).

Techniques: Flow Cytometry, Whisker Assay, Expressing

Journal: International Journal of Nanomedicine

Article Title: T Cell-Derived Apoptotic Extracellular Vesicles Ameliorate Bone Loss via CD39 and CD73-Mediated ATP Hydrolysis

doi: 10.2147/IJN.S491222

Figure Lengend Snippet: ApoEVs hydrolyze ATP to adenosine via surface CD39 and CD73. ( A ) The activity of ApoEVs and apoptotic T cells to hydrolyze ATP was measured in vitro (N = 3). ( B ) Extracellular ATP concentration in bone marrow plasma of sham and OVX animals (N = 6). ( C ) Western blot analysis of CD39 and CD73 in bone marrow of sham and OVX animals. ( D ) Extracellular adenosine concentration in bone marrow plasma of sham and OVX animals. ( E ) Western blot analysis of CD39 and CD73 on the membrane of apoptotic T cells and ApoEVs. ( F ) Immunoelectron microscopy detection of CD39 and CD73 on ApoEVs (scale bar = 200 nm). Yellow arrows indicate CD39 and red arrows indicate CD73 adhered by gold particles. ( G ) The activity of ApoEVs to hydrolyze ATP with or without POM (a CD39 inhibitor) or PSB (a CD73 inhibitor) was determined in vitro (N = 3). ( H ) The activity of ApoEVs in hydrolyzing ATP to adenosine with or without POM or PSB was determined in vitro (N = 3). Data are presented as mean ± SD; ns, not significant; ***P< 0.001 by one-way ANOVA with Tukey’s post hoc test or unpaired Student’s t test.

Article Snippet: To prepare CD39- or CD73-inhibited ApoEVs, 30 μg/mL of ApoEVs were preincubated with 100 μM POM (dissolved in DMSO, MedChemExpress) or PSB (dissolved in water, MedChemExpress) for 40 minutes at room temperature.

Techniques: Activity Assay, In Vitro, Concentration Assay, Clinical Proteomics, Western Blot, Membrane, Immuno-Electron Microscopy

Journal: International Journal of Nanomedicine

Article Title: T Cell-Derived Apoptotic Extracellular Vesicles Ameliorate Bone Loss via CD39 and CD73-Mediated ATP Hydrolysis

doi: 10.2147/IJN.S491222

Figure Lengend Snippet: ApoEVs promote bone regeneration via surface CD39 and CD73. OVX mice were divided into four groups and injected with PBS, ApoEVs, and POM or PSB pretreated ApoEVs, respectively (N = 5–6). Extracellular ATP ( A ) and adenosine ( B ) concentration in bone marrow plasma in different groups of mice were detected. ( C ) Micro-CT analyses of trabecular bone mass in the femurs. ( D-G ) Quantitative analyses of BMD ( D ), BV/TV ( E ), Tb. N ( F ) and Tb. Sp ( G ). ( H-J ) ELISA assays of IFN-γ ( H ), IL-17 ( I) and TNF-α ( J ) concentrations in the serum of peripheral blood from indicated groups. Data are presented as mean ± SD; ns, not significant; *P< 0.05; **P< 0.01; ***P< 0.001 by one-way ANOVA with Tukey’s post hoc test.

Article Snippet: To prepare CD39- or CD73-inhibited ApoEVs, 30 μg/mL of ApoEVs were preincubated with 100 μM POM (dissolved in DMSO, MedChemExpress) or PSB (dissolved in water, MedChemExpress) for 40 minutes at room temperature.

Techniques: Injection, Concentration Assay, Clinical Proteomics, Micro-CT, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Immunology

Article Title: Unveiling spatial complexity in solid tumor immune microenvironments through multiplexed imaging

doi: 10.3389/fimmu.2024.1383932

Figure Lengend Snippet: Immunophenotyping panel for multiplexed tissue imaging of cancer.

Article Snippet: CD39 , REA739 , 50 , 130-110-650 , PE , Miltenyi Biotec.

Techniques: Imaging

Journal: Frontiers in Immunology

Article Title: Unveiling spatial complexity in solid tumor immune microenvironments through multiplexed imaging

doi: 10.3389/fimmu.2024.1383932

Figure Lengend Snippet: Cellular neighborhood analysis of PD1 high/low T cells in the tumor margin and core. (A–D) Topology of PD1 high (left) and PD1 low (right) T cells and their cellular neighborhood within a 5 µm range. (A, B) represent tumor margin and (C, D) show tumor core areas. Cell types showing different distribution patterns around PD1 high and PD1 low T cells (mDCs, M1-like M, M2-like M, MDSCs, Fibroblasts, vessels, tumor cells) are highlighted by arrowheads. (E, F) Quantification of cells in a 5 µm range around of PD1 high/low T cells for tumor margin and tumor core, (E) represents immune cells and (F) stroma/tumor cells. (G) Violin plots for expression levels of eight immune-modulating markers (CD112, CD155, CD276, CD39, CD73, IDO, PD-L1, and VISTA) for the most important immune and tumor cells around PD1 high/low T cells in the tumor core area. Violin plots for tumor margin are shown in Supplementary Figure S5E . Depicted markers and annotated cell types as indicated by the color code. ROI sizes: Tumor margin (ROI15) and tumor core (ROI16): 975 x 769 µm.

Article Snippet: CD39 , REA739 , 50 , 130-110-650 , PE , Miltenyi Biotec.

Techniques: Expressing

Journal: Cardiovascular Diabetology

Article Title: Elevated glucose levels increase vascular calcification risk by disrupting extracellular pyrophosphate metabolism

doi: 10.1186/s12933-024-02502-w

Figure Lengend Snippet: Impact of high glucose on extracellular pyrophosphate metabolism. Aortic smooth muscle cells were cultured for one month in media containing either low (1 g/L) or high (4.5 g/L) glucose. A Measurement of extracellular pyrophosphate levels. B Extracellular pyrophosphate-to-ATP ratio. C , D Analysis of the gene expression of key enzymes involved in extracellular pyrophosphate metabolism, including eNTPD1, eNPP1, and TNAP, from isolated total RNA. (E) Immunoblot analysis of proteins associated with extracellular pyrophosphate metabolism. F , G Quantification of protein levels via ELISA, highlighting significant differences. The data are shown as the mean ± SEM, with data derived from 4 independent experiments, each containing 4 replicate plates. Statistical significance was determined via Student’s t test, with asterisks denoting significance levels: * P < 0.05; ** P < 0.01; *** P < 0.001

Article Snippet: The recombinant enzymes eNPP1 (catalog number 6136-EN) and eNTPD1 (catalog number 4397-EN) were obtained from R&D Systems (Minneapolis, MN, USA).

Techniques: Cell Culture, Gene Expression, Isolation, Western Blot, Enzyme-linked Immunosorbent Assay, Derivative Assay

Journal: Cardiovascular Diabetology

Article Title: Elevated glucose levels increase vascular calcification risk by disrupting extracellular pyrophosphate metabolism

doi: 10.1186/s12933-024-02502-w

Figure Lengend Snippet: High glucose levels impair the pyrophosphate-to-phosphate ratio. Aortic smooth muscle cells were incubated for one month in medium containing 1 g/L or 4.5 g/L glucose. A Autoradiograph displaying representative products from the hydrolysis of ATP (1 µmol/L ATP, 10 µCi/mL [γ 32 Pi]ATP) incubated with or without recombinant eNPP1 (ectonucleotide pyrophosphatase/phosphodiesterase 1) or eNTPD1 (ectonucleoside triphosphate diphosphohydrolase 1) enzymes. Enzymatic hydrolysis generated radiolabeled 32 PPi (32-pyrophosphate) and 32 Pi (32-phosphate), which, alongside unreacted [γ 32 Pi]ATP, were separated by thin-layer chromatography (TLC), as detailed in the section. B Representative time course of ATP hydrolysis showing the products released over time. C Synthesis of the pyrophosphate 32 PPi via hydrolysis of [γ 32 Pi]ATP (10 µCi/mL; 1 µmol/L ATP) in the absence or presence of 100 µmol/L SBI245 (a specific TNAP inhibitor) or inorganic pyrophosphatase (PPase). D The pyrophosphate-to-phosphate ( 32 PPi/ 32 Pi) ratio was quantified following hydrolysis of [γ 32 Pi]ATP (10 µCi/mL; 1 µmol/L ATP) under various conditions: in the absence of inhibitors (Control), in the presence of an ectonucleoside triphosphate diphosphohydrolase (eNTPD) inhibitor (INH, 200 µmol/L), or with the recombinant enzymes eNPP1 and eNTPD1 (100 ng/mL). Experiments were conducted in media containing either physiological (1 g/L) or elevated (4.5 g/L) glucose concentrations. D) Synthesis of 32 PPi by hydrolysis of [γ 32 Pi]ATP (10 µCi/mL and 1 µmol/L ATP). E Hydrolysis of 32 PPi (10 µCi/mL and 5 µmol/L PPi). The results are shown as the mean ± SEM (4 independent experiments with 4 plates per experiment). Student’s t test ( E, F ) or one-way ANOVA with Tukey’s post hoc test ( C, D ) was used for statistical analysis. Asterisks indicate a statistically significant difference compared with the control group: * P < 0.05; *** P < 0.001. ### Indicates a value of P < 0.001 compared with the control group (1 g/L)

Article Snippet: The recombinant enzymes eNPP1 (catalog number 6136-EN) and eNTPD1 (catalog number 4397-EN) were obtained from R&D Systems (Minneapolis, MN, USA).

Techniques: Incubation, Autoradiography, Recombinant, Generated, Thin Layer Chromatography, Control

Journal: Cardiovascular Diabetology

Article Title: Elevated glucose levels increase vascular calcification risk by disrupting extracellular pyrophosphate metabolism

doi: 10.1186/s12933-024-02502-w

Figure Lengend Snippet: STZ-treated rats exhibit impaired extracellular pyrophosphate metabolism in the aortic wall. A A representative time course of ATP hydrolysis was conducted using a 1 µmol/L ATP solution containing 10 µCi/mL [γ- 32 P]ATP as a radiotracer. The products of hydrolysis, 32 PPi (32-pyrophosphate), 32 Pi (32-phosphate), and [γ- 32 P]ATP-, were separated and quantified via thin layer chromatography, as outlined in the section. B The synthesis of pyrophosphate (PPi) was analyzed by hydrolyzing 1 µmol/L ATP containing 10 µCi/mL [γ- 32 P]ATP as a radiotracer. The reactions were carried out in the absence or presence of either a specific TNAP inhibitor (SBI-425) or inorganic pyrophosphatase (PPase). C The ratio of 32 PPi to 32 Pi generated by ATP hydrolysis was calculated to assess the efficiency and specificity of pyrophosphate synthesis. D The synthesis of 32 PPi was evaluated by hydrolyzing 1 µmol/L ATP containing 10 µCi/mL [γ- 32 P]ATP. E The release of 32 Pi was measured following the hydrolysis of 5 µmol/L pyrophosphate, which contained 10 µCi/mL 32 PPi as a radiotracer. F Quantification of protein levels via ELISA. G , H Total RNA was isolated from rat aortas to evaluate the expression levels of key enzymes involved in extracellular pyrophosphate metabolism, including eNTPD1 (ectonucleoside triphosphate diphosphohydrolase 1), eNPP1 (ectonucleotide pyrophosphatase/phosphodiesterase 1), and tissue-nonspecific alkaline phosphatase (TNAP) (panel G . Additionally, the expression of calcification-related proteins, such as matrix Gla protein (MGP) and osteopontin (OPN), was assessed (panel H). The data are shown as the mean ± SEM and represent data from 12–16 independent aortas. Statistical analyses were performed via Student’s t test. Asterisks indicate a significant difference with *** P < 0.001

Article Snippet: The recombinant enzymes eNPP1 (catalog number 6136-EN) and eNTPD1 (catalog number 4397-EN) were obtained from R&D Systems (Minneapolis, MN, USA).

Techniques: Thin Layer Chromatography, Generated, Enzyme-linked Immunosorbent Assay, Isolation, Expressing

Journal: Nature Communications

Article Title: CD39 polymorphism enables lung thrombosis in sickle cell disease

doi: 10.1038/s41467-026-68396-2

Figure Lengend Snippet: a Experimental scheme for ( b – f , j ). EVs Extracellular vesicles, SEC Size exclusion chromatography, NTA Nanoparticle tracking analysis. Created in BioRender. Brzoska, T https://BioRender.com/t6bmaj7 . b NTA plot showing concentration vs size distribution of EVs isolated from a control and an SCD mouse plasma. c EV concentration in plasma of SCD (n = 7) and control (n = 7) mice. d Western blot micrograph and e the densitometry analysis (arbitrary units) of CD39 protein expression in control (n = 5) and SCD (n = 5) mice EVs. Ponceau-S, loading control. f ADPase activity in control (n = 5) and SCD (n = 5) mice EVs ± incubation with CD39 inhibitor (500 µM ARL67156 ). g Experimental scheme for ( h , i ). Platelet-rich plasma, PRP. Created in BioRender. Brzoska, T. (2025) https://BioRender.com/vq6jifk . h In vitro platelet aggregation kinetics in a control mouse PRP sample following the addition of ADP (black), ADP + control mouse EVs (red), ADP + SCD mouse EVs (blue), and ADP + SCD mouse EVs + POM-1 (green). i Area under the curve (AUC) in four groups shown in ( h ). N = 4 per group. j Imaging flow cytometry images of CD39 + /CD31 + /CD144 + (row #1) or CD39 + /CD31 + /CD106 + (row #2) EVs isolated from SCD mice plasma. Bottom row- isotype control Ab stained EVs. Scale bar, 5 µm. Data representative of 3 independent experiments. k Experimental scheme for ( l – n ). In vitro cultured human lung microvascular endothelial cells (HMVECs-L) ± incubation with 20 µM hemin and EVs isolated from cell culture supernatant. Created in BioRender. Brzoska, T https://BioRender.com/yfnjlra . l NTA plot showing concentration vs size distribution of HMVECs-L EVs. m EV concentration in the supernatant of HMVECs-L incubated with (n = 6 independent experiments) or without (n = 6 independent experiments) hemin. n ADPase activity in EVs isolated from the supernatant of HMVECs-L incubated with (n = 4 independent experiments) or without (n = 4 independent experiments) hemin ±500 µM ARL67156 . Means were compared using unpaired two-tailed Student’s t test. Data represent mean ± SEM. Exact P values shown in the graphs.

Article Snippet: For each treatment group, 20 μl of EVs suspension was diluted with 80 μl of sterile PBS and incubated in the dark for 15 min (4 °C) with Alexa Fluor 647 anti-human CD39 antibody (clone: 498403; R&D Systems cat# FAB4397R) and Phycoerythrin (PE) anti-human CD31 antibody (clone: WM59, BD Pharmingen cat# 560983) for in situ staining of CD39 and CD31, respectively.

Techniques: Size-exclusion Chromatography, Concentration Assay, Isolation, Control, Clinical Proteomics, Western Blot, Expressing, Activity Assay, Incubation, In Vitro, Imaging, Flow Cytometry, Staining, Cell Culture, Two Tailed Test

Journal: Nature Communications

Article Title: CD39 polymorphism enables lung thrombosis in sickle cell disease

doi: 10.1038/s41467-026-68396-2

Figure Lengend Snippet: a Number of SCD patients without (-PT) vs with (+PT) medical history of pulmonary thrombosis in Walk-PHASST registry. Created in BioRender. Brzoska, T https://BioRender.com/aovqt1d . Frequency of b rs3176891 GG genotype and ( c ) rs3176891G allele among -PT vs + PT SCD patients in ( a ). Data compared using four-fold table analysis with two-tailed χ2 test. d Odds Ratio for association of rs3176891G allele with risk of pulmonary thrombosis in SCD (n = 437) and non-SCD (n = 1891) humans. Data represent point estimate of Odds Ratio ±95% CI. e Number of non-SCD humans of African ancestry without (-PT) vs with ( + PT) medical history of pulmonary thrombosis in TOPMed database. Created in BioRender. Brzoska, T. (2025) https://BioRender.com/aovqt1d . f Frequency of rs3176891G allele among non-SCD humans in ( e ). g Experimental scheme for ( h – n ): blood from SCD patients with AA or AG or GG genotype of rs3176891 processed to generate platelet-rich plasma (PRP) and platelet-free plasma (PFP). Created in BioRender. Brzoska, T https://BioRender.com/ d8skzoa. h ADPase activity in EVs of SCD patients with AA (n = 4) vs AG/GG (n = 10) genotype. ADPase activity in EVs of SCD patients with i AA (n = 4) and j AG/GG (n = 10) genotype ± incubation with 500 µM ARL67156 . k Imaging flow cytometry images showing CD39 + /CD31 + EVs in the plasma of an SCD patient with AA genotype. Scale bar 5 µm. l Concentration of CD39 + /CD31 + EVs in PFP of SCD patients with AA (n = 3) vs AG/GG (n = 4) genotype. In vitro platelet aggregation kinetics shown as the percent increase in light transmission in PRP of SCD patients with AA (black), AG (blue) and GG (red) genotypes, following the addition of m 1 µM ADP or n 3 µg/ml collagen. Data from more patients are shown as Supplementary Fig. . Data compared using unpaired two-tailed Student’s t test in ( h , l ) (mean ± SEM), paired two-tailed Student’s t test in ( i ), and two-tailed Wilcoxon matched-pairs signed rank test in ( j ). Exact P values shown in the graphs.

Article Snippet: For each treatment group, 20 μl of EVs suspension was diluted with 80 μl of sterile PBS and incubated in the dark for 15 min (4 °C) with Alexa Fluor 647 anti-human CD39 antibody (clone: 498403; R&D Systems cat# FAB4397R) and Phycoerythrin (PE) anti-human CD31 antibody (clone: WM59, BD Pharmingen cat# 560983) for in situ staining of CD39 and CD31, respectively.

Techniques: Two Tailed Test, Clinical Proteomics, Activity Assay, Incubation, Imaging, Flow Cytometry, Concentration Assay, In Vitro, Transmission Assay

Journal: Nature Communications

Article Title: CD39 polymorphism enables lung thrombosis in sickle cell disease

doi: 10.1038/s41467-026-68396-2

Figure Lengend Snippet: Although ADP released during acute intravascular hemolysis can trigger in situ pulmonary thrombosis by stimulating platelet-purinergic P2Y1 and P2Y12 receptors, the sterile inflammatory milieu in SCD also promotes the generation of endothelium-derived CD39 + EVs that phosphohydrolize ADP to prevent pulmonary thrombosis. However, ENTPD1 rs3176891G allele is associated with impaired generation of CD39 + EVs, thus increasing the risk of pulmonary thrombosis in some SCD patients. Created in BioRender. Brzoska, T. (2025) https://BioRender.com/x4rvhil .

Article Snippet: For each treatment group, 20 μl of EVs suspension was diluted with 80 μl of sterile PBS and incubated in the dark for 15 min (4 °C) with Alexa Fluor 647 anti-human CD39 antibody (clone: 498403; R&D Systems cat# FAB4397R) and Phycoerythrin (PE) anti-human CD31 antibody (clone: WM59, BD Pharmingen cat# 560983) for in situ staining of CD39 and CD31, respectively.

Techniques: In Situ, Sterility, Derivative Assay