Prostaglandin E receptor 2 (EP2) is an immune modulatory molecule that regulates the balance of immunity. Here we investigated the role of EP2 in immune dysregulation in patients with acute-on-chronic liver failure (ACLF).Plasma Progstaglandin E2 (PGE2) levels and EP2 expression on immune cells were determined in blood samples collected from patients with chronic hepatitis B related ACLF(HB-ACLF), patients with chronic hepatitis B (CHB), acute decompensated cirrhosis without ACLF (AD) and healthy controls (HC). Cytokine production, bacterial phagocytosis and reactive oxygen species (ROS) production were detected to explore the role of EP2 in regulating immune cell functions.+ T cells was decreased in HB-ACLF compared with those in controls. The levels of PGE2 and EP2 were associated with systemic inflammation and disease severity. Small molecular chemicals against EP2 increased both cytokine secretion in PBMCs and ROS production in neutrophils and monocytes, but decreased monocytic phagocytosis. By contrast, an EP2-selective agonist reduced the production of a series of cytokines in PBMCs, but increased G-CSF.The plasma PGE2 levels were increased and EP2 expression on CD8E. coli in HB-ACLF. EP2 might be a new potential target for HB-ACLF treatment.Altered PGE2-EP2 augmented the excessive inflammation of innate and adaptive immune cells in response to LPS or The online version of this article (10.1186/s12967-019-1844-0) contains supplementary material, which is available to authorized users. Hepatitis B virus (HBV) infection, the most common chronic viral infection worldwide, could lead to chronic liver inflammation, cirrhosis and hepatocellular carcinoma . One of ACLF exhibits remarkable features of exacerbated systemic inflammation that are closely associated with poor outcomes . This syInhibitory check points, expressed on immune effector cells, maintain immune balance and prevent an excessive inflammatory response. We have already demonstrated that decreased Tim-3 is associated with the monocytic over response to LPS in decompensated cirrhosis . ProstanThis study was designed to investigate whether and how the PGE2-EP2 axis alter innate and adaptive immune function and the response to LPS in patients with hepatitis B- related ACLF (HB-ACLF).In total, 323 subjects were recruited from outpatients or inpatients of the First Affiliated Hospital of Zhejiang University between October 2016 and December 2017. These subjects were divided into four groups: (i) HB-ACLF: 135 CHB patients with ACLF diagnosed by the criteria of the Asian-Pacific Association for the Study of the Liver (APASL)—i.e., “acute hepatic insult manifesting as jaundice (≥ 5 mg/dL) and coagulopathy, complicated within 4 weeks by ascites and/or encephalopathy in a patient with previously diagnosed or undiagnosed chronic liver disease.” ; (ii) acCD3-percp-cy5.5 , CD8-APC , CD8-FITC , CD14-APC , CD56-FITC , CD16-PerCP-eFluor™710 , CD11b-PerCP-eFluor™710 , IFN-γ-PE , TLR2-FITC  and Mouse IgG1-PE  were all bought from eBioscience. HLA-DR-PE , IL-10-APC , CXCR3-APC  and IFN-γ-FITC , Rat IgG2a-APC  and mouse IgG-APC  were purchased from BD Biosciences. CD16-FITC , HLA-DR-APC , CD3-FITC  and TLR4-APC  were purchased from Biolegend. EP2-PE  and Rabbit IgG-PE  were purchased from Cayman Chemical.Whole-blood samples were collected within 24 h after admission. After centrifugation, the plasma was collected and stored immediately at − 80 °C. Peripheral blood mononuclear cells (PBMCs) were isolated using Ficoll-Hypaque density gradient centrifugation .The plasma cytokine levels of ACLF patients were measured using a multiplex panel , according to the manufacturer’s instructions. PGE2 was measured using the Prostaglandin E Metabolites ELISA kit . Both measurements were performed according to the manufacturer’s instructions. The detection limits were as follows:IL-1β, 17.3 pg/ml; IL-1rα, 15 pg/ml; IL-2, 16.5 pg/ml; IL-4, 11 pg/ml; IL-5, 27 pg/ml; IL-6, 24.8 pg/ml; IL-7, 11 pg/ml; IL-8, 14.5 pg/ml; IL-9: 21 pg/ml; IL-10: 33.3 pg/ml; IL-12: 15.5 pg/ml; IL-13: 13.5 pg/ml; IL-5; 57.3 pg/ml; IL-17: 24.8 pg/ml; eotaxin, 17 pg/ml; FGF basic, 14 pg/ml; G-CSF, 36.3 pg/ml; GM-CSF, 16 pg/ml; IFN-γ, 15.5 pg/ml; IP-10, 14.5 pg/ml; MCP-1, 18.5 pg/ml; MIP-1α, 8.8 pg/ml; PDGF-bb, 32.5 pg/ml; MIP-1β, 31.5 pg/ml; RANTES, 15.8 pg/ml; TNF-α, 12.5 pg/ml; VEGF, 109.3 pg/ml; PGE2, 2 pg/ml.Isolated PBMCs or 100 μl of heparin-treated peripheral whole blood was incubated with antibodies for 15 min at room temperature. After incubation, whole-blood samples were lysed with FACS lysing solution and were washed with phosphate-buffered saline before analysis using an Accuri C6 cytometer . The isotype IgG was used as the control.To explore factors driving EP2 expression, PBMC from healthy controls were incubated for 7 days with a range of IP-10 and MIP-1β and assessed for EP2 expression.+T cells (CD3+/CD8+), CD4+T cells (CD3+/CD8−), NK (CD3−/CD56+), NKT (CD3+/CD56+), monocytes (CD14+), neutrophils (CD16+).Gating strategies of flow cytometry for immune subsets are introduced in Figure S1  were stimulated with lipopolysaccharide (LPS)  for 72 h in the presence or absence of AH6809  or DMSO (0.1%) in 1640 RPMI medium  supplemented with 10% fetal bovine serum , 100 U/ml of penicillin, and 100 U/ml of streptomycin (Invitrogen) at 37 °C in 5% CO2. For the last 6 h, monensin  was added to the medium to inhibit cytokine secretion, and the cells were re-stimulated with LPS (100 ng/ml). Next, the cells were incubated with fixation/permeabilization solution (BD biosciences) at 4 °C for 20 min. After incubation, the cells were washed, stained with IFN-γ-PE and IL-10-APC for 15 min at room temperature and analyzed by flow cytometry.In vitro, PBMCs (2 × 105/well) were stimulated with LPS  for 72 h in the presence or absence of AH6809 (150 μM) or butaprost  or DMSO in 10% FBS RPMI medium. Next, the supernatants were collected and stored at − 80 °C immediately. Cytokines in the supernatants were measured using a multiplex panel  according to the manufacturer’s instructions. The limitations of detection have been mentioned above.In vitro, PBMCs (2 × 10Escherichia coli (K-12 strain) bio-particles  in 96-well plates and was analyzed by FACS according to previously described protocols [2.One hundred microliters of whole blood were incubated with AF488-conjugated E. coli (8.04*107cfu/ml) or PMA (50 ng/ml) in 96-well plates for 30 min. Next, the cells were assessed for oxidative burst using an ROS assay kit  and were analyzed by FACS according to the manufacturer’s protocol. For blocking experiments, blood was pre-incubated with AH6809 or DMSO for 1 h at 37 °C in 5% CO2.One hundred microliters of whole blood samples were incubated with or without heat-inactivated The data are shown as the mean ± standard deviation (SD), mean ± standard error of the mean (SEM), median (range) or number (percentage). For comparisons between two independent groups, the Mann–Whitney U test was used. Comparisons between paired groups were performed by the Wilcoxon signed-rank test. Correlations between variables were calculated by the Spearman’s rank correlation test. P < 0.05 at two sides was considered statistically significant. All statistical analyses were performed using GraphPad Prism 6 (GraphPad Software).We confirmed an elevation of various cytokines levels in HB-ACLF , MFI  and count . In contrast, no difference of EP2 expression on CD8+ T cells was found between CHB and HC. There was a decrease of the count of EP2 positive CD4+ T cell and NK cells in ACLF than HC (p = 0.012) and CHB (p = 0.023) respectively  and HC , but no significant differences of the frequency of EP2+ monocyte were found between ACLF than HC (p = 0.16)  In the density plots of forward and lateral angular scatter, the human whole-blood gates were Lym (lymphocyte), Mo (monocyte) and Ne (neutrophil). (b) CD8+ T cells were located at the Lym gate and were double positive for CD3 and CD8, whereas CD3+CD8− cells were considered as CD4+ T cells in this experiment. (c) NK and NKT cells were also located in the Lym gate and were further confirmed by CD56 and CD3 markers. (d) Monocytes were present in the Mo-gate and were positive for CD14. (e) Neutrophils were present in the Ne-gate and were positive for CD16. Figure S2. Hyper inflammatory status in ACLF. (a) Plasma cytokines of ACLF (n = 24), CHB (n = 25) and HC (n = 24) were determined using a multi-plex cytokine assay. (b) Representative plots for spontaneous ROS production in monocytes and neutrophils from three groups. (c) Spontaneous ROS production in monocytes and neutrophils from HC (n = 19), CHB (n = 19) and ACLF (n = 15) was assessed. The error bar represented the SEM and the horizontal line represented the median. Statistical analyses were performed using the Mann–Whitney test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; ns, not statistically significant. Figure S3. Phenotype characterization of blood immune cells. Peripheral whole blood (at least 5 subjects each group) was stained with anti-CD14, anti-CD16, anti-CD3, anti-CD8, anti-TLR2, anti-HLA-DR, anti-CD11b or anti-TLR4. The percentages of different phenotypes of blood immune cells were determined by flow cytometry. The horizontal line represented the median. Statistical analyses were analyzed using the Mann–Whitney test. Four groups were compared with each other. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Figure S4. Plasma from ACLF promotes macrophage M1 transformation. (a-b) Peripheral monocytes from HC were differentiated into macrophage in vitro by the addition of M-CSF for 7 days. Differentiated macrophages from HC were incubated in 20% ACLF plasma or HC plasma for 24 h in the presence of (a) LPS (100 ng/ml) or (b) IL-4 (15 ng/ml). M1 phenotypes were assessed by the expression of CD80, CD86 and HLA-DR, and M2 phenotypes were assessed by the expression of CD206 and CD163. (c-d) PMA-differentiated THP-1 was incubated in 20% ACLF plasma or HC plasma for 24 h in the presence of (c) LPS (100 ng/ml) or (d) IL-4 (15 ng/ml). M1 phenotypes were assessed by the expression of CD86, and M2 phenotypes were assessed by PD-1. All experiments were analyzed by flow cytometry. The horizontal line represented the mean. Statistical analyses were performed using the Mann–Whitney test. *p < 0.05, **p < 0.01, ***p < 0.001. ns, not statistically significant. Figure S5. EP2 expression on other immune cells. The horizontal line represented the median, and statistical analyses were performed using the Mann–Whitney test. *p < 0.05, **p < 0.01. ns, not statistically significant. Figure S6. Frequency of CXCR3+ CD8+ T cells and CXCR3+ monocytes. The horizontal line represented the median. Statistical analyses were analyzed using the Mann–Whitney test. *p < 0.05, **p < 0.01. ns, not statistically significant. Figure S7. Association of PGE2-EP2 expression with disease severity in ACLF. (a) EP2 expression on CD8+ T cells in survivors or death or transplantation group during the 90-day follow-up. (b) Plasma PGE2 levels were measured in survivors or death or transplantation group during the 90-day follow-up. (c) Plasma PGE2 levels were measured in ACLF or ACLF-MOF during the 90-day follow-up. (d) Correlation analysis between the plasma PGE2 levels and prognostic score. The horizontal line represented the median. Statistical analyses were analyzed using the Mann–Whitney test or spearman correlation. ns, not statistically significant.


A dispiro compound bearing two cyclohexyl moieties and a central sulfur-rich seven-membered ring was re-synthesized and newly crystallized. The modified synthetic procedure, the compound’s purification and characterization and the crystal structure – which is only the second of its kind – including non-classical hydrogen-bonding interactions are reported and discussed. 12H20S5, crystallizes in the monoclinic space group P21/c with four molecules in the unit cell. In the crystal, the asymmetric unit comprises the entire molecule with the three cyclic moieties arranged in a line. The molecules in the unit cell pack in a parallel fashion, with their longitudinal axes arranged along a uniform direction. The packing is stabilized by the one-dimensional propagation of non-classical hydrogen-bonding contacts between the central sulfur atom of the S3 fragment and the C—H of a cyclohexyl group from a glide-related molecule [C⋯S = 3.787 (2) Å].The title compound, C This is similar in MOSYOI, with the four-membered rings being nearly perpendicular to the central seven-membered ring. The sulfur–sulfur bond distances range from 2.026 (1) to 2.035 (1) Å, which is a little bit shorter than the sum of the covalent radii of 2.06 Å  and 2.035 (1) Å]. The shorter S—S bond in the S3 moiety is the one that points towards a non-classical hydrogen-bonding contact (vide infra), implying that this interaction might influence the relative distances in the S3 fragment. The angles involving central S atoms range from 105.16 (5)° (around S1) to 106.89 (5)° (around S3) while the S—C—S angles are slightly wider with 111.58 (7)° (around C1) and 114.31 (7)° (around C7), i.e. they are more and less acute, respectively, than the ideal tetrahedral angle.The title compound 7,8,15,16,17-pentathiadispiro[5.2.5nt Fig. 1. The twoac planes. The crystallographic direction of these vectors approximates [403 fragment as acceptor and a C—H of one cyclohexyl moiety (C6—H6B) as donor, pointing roughly into opposite directions and protruding along the c-axis direction  x, y, z, (ii): x, y, −z)  Fig. 3.3 (1×100 ml) and water (1×100 ml), before being dried over Na2SO4. The solvent was reduced to 5 ml in vacuo and adsorbed onto isolute® HM-N, prior to purification by flash chromatography  in n-hexane, detection by thin layer chromatography and fluorescence quenching at 254 nm). Recrystallization from 0.1 ml mg−1 methanol yielded colorless block-like crystals, the identity of which was confirmed by melting point determination (356.5 K). As a result of the lipophilic nature of the analyte, the purity and stability of the colorless product was accessible to supercritical fluid chromatography  and methanol containing 20 mM ammonium formate (B), isocratic mode (5% B), oven temperature: 313 K). Yield: 5.0 g (14%).The title compound was synthesized based on a modified literature procedure  δ 1.45 ppm , 1.6 ppm , 1.9 ppm . 13C{1H} NMR  δ 25.4 ppm, 37.8 ppm. IR (FT–IR): (υ cm−1) = 2926 (s), 1439 (s). Elemental analysis calculated for C12H20S5: C 44.40; H 6.21; S 49.39. Found: C 44.72; H 6.03; S 49.25.Uiso(H) = 1.2Ueq(C).Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S2056989019007138/fy2136sup1.cifCrystal structure: contains datablock(s) I. DOI: 10.1107/S2056989019007138/fy2136Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989019007138/fy2136Isup3.cmlSupporting information file. DOI: 1916548CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


In the crystal, the molecules are linked  23H23NO, the two ring systems are twisted by 51.40 (11)° relative to each other. In the crystal, the molecules are connected by weak C—H⋯π interactions, generating a three-dimensional supramolecular structure. Hirshfeld surface analysis and two-dimensional fingerprint plots indicate that the most important contributions to the crystal packing are from H⋯H (67.2%), C⋯H/H⋯C (26.7%) and C⋯C (2.5%) interactions.In the title Schiff base compound, C We report herein the synthesis, crystal structure and Hirshfeld structural analysis of the title compound.Schiff bases have found wide use as a ligands in coordination chemistry , contains one independent molecule Fig. 1. the twoIn the crystal, the molecules are connected by C—H⋯π interactions, generating a three-dimensional supramolecular structure Table 1.et al., 2016E)-N-benzylidene-5,6,7,8-tetrahydronaphthalen-1-amine and (E)-N-[methylene]aniline including 2-(4-isopropylphenyl)-1,3-diphenyl-2,3-dihydro-1H-naphthooxazine -1,2-dihydro-3′H-spiro-1-amine -4-benzoyl-7-methyl4,5,5a,6,6a,7,8,9,10,10adecahydroindoloquinoline-9-carboxylate dihydrate CrystalExplorer  shows the two-dimensional fingerprint of the  points associated with hydrogen atoms. It is characterized by an end point that points to the origin and corresponds to di = de = 1.08 Å, which indicates the presence of the H⋯H contacts in this study (67.2%). The graph shown in Fig. 5b (C⋯H/H⋯C) shows the contacts between the carbon atoms inside the surface and the hydrogen atoms outside the surface and vice versa. The plot shows two symmetrical wings on the left and right sides (26.7%). Further, there are C⋯C (2.5%), C⋯O/O⋯C (2%), N⋯H/H⋯N (1.4%) and O⋯H/H⋯O (0.2%) contacts.Fig. 4A view of the three-dimensional Hirshfeld surface of the title compound plotted over electrostatic potential energy in the range −0.048 to 0.033 a.u. using the STO-3G basis set at the Hartree–Fock level of theory is shown in Fig. 6The title compound was prepared Fig. 7 by refluUiso(H) = 1.2Ueq(C).Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S2056989018013117/xu5940sup1.cifCrystal structure: contains datablock(s) I. DOI: 10.1107/S2056989018013117/xu5940Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989018013117/xu5940Isup3.cmlSupporting information file. DOI: 1843572CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Scientific Reports 10.1038/s41598-019-47964-1, published online 09 August 2019Correction to: This Article contains errors in Table 5. The ‘No. of KI offspring (%)’ value is missing and the ‘No. of offspring with partial insertion (%)’ value should read “1 (5.3)” and not “9 (47.4)”. The correct Table 5 appears below as Table


In all three molecules, the seven-membered thiazepan ring exhibits a chair conformation. Except for the nitro groups, the three molecules have similar conformations when overlayed in pairs.The crystal of 6-(4-nitrophenyl)-7-phenyl-5-thia-7-azaspiro[2.6]nonan-8-one ( 1), C19H18N2O3S, has monoclinic (P21/n) symmetry while that of its isomer 6-(4-nitrophenyl)-7-phenyl-5-thia-7-azaspiro[2.6]nonan-8-one (2), has orthorhombic (Pca21) symmetry: compound 1 has two molecules, A and B, in the asymmetric unit while 2 has one. In all three molecules, the seven-membered thiazepan ring exhibits a chair conformation with Q2 and Q3 values (Å) of 0.521 (3), 0.735 (3) and 0.485 (3), 0.749 (3) in 1 and 0.517 (5), 0.699 (5) in 2. In each structure, the phenyl rings attached to adjacent atoms of the thiazepan ring have interplanar angles ranging between 41 and 47°. Except for the nitro groups, the three molecules have similar conformations when overlayed in pairs. Both crystal structures are consolidated by C—H⋯O hydrogen bonds.The crystal of 6-(3-nitrophenyl)-7-phenyl-5-thia-7-azaspiro[2.6]nonan-8-one ( For example, the Bristol-Myers Squibb ACE/NEP inhibitor omapatrilat advanced to Phase II clinical trials (Graul 1) and 6-(3-nitrophenyl)-7-phenyl-5-thia-7-azaspiro[2.6]nonan-8-one (2), in which a nitro group substitutes at the para and meta positions, respectively, of the C-2 aromatic ring.Previously we reported the synthesis and crystal structure of 6,7-diphenyl-5-thia-7-azaspiro[2.6]nonan-8-one  and B (containing S2), in the asymmetric unit  at C1 and (R) at C20 for 1 and (S) at C1 for 2; in both structures, crystal symmetry generates a racemic mixture. These molecules adopt similar conformations and overlay closely ] as calculated by PLATON , 0.735 (3) and 0.485 (3), 0.749 (3), respectively, with equivalent values of 0.517 (5), 0.699 (5) for 2. The dihedral angles between the aromatic rings attached to the 2 and 3 positions of the thiazepan rings are 46.93 (15) (molecule 1A), 42.50 (15) (1B) and 42.0 (3)° (2).Compound it Fig. 1 and 2 crle Fig. 2. The conly Fig. 3 apart fr1 has more extensive hydrogen bonding compared to that of 2 , aniline , and [1-(sulfanylmethyl)cyclopropyl] acetic acid  were added. Pyridine  was added. Finally, 2,4,6-tripropyl-1,3,5,2,4,6-trioxatriphosphorinane-2,4,6-trioxide (T3P) in 2-methyltetrahydrofuran  was added. The reaction was stirred at room temperature and followed by TLC. The mixture was poured into a separatory funnel with dichloromethane and distilled water. The layers were separated and the aqueous layer was then extracted twice with dichloromethane. The organic fractions were combined and washed with saturated sodium bicarbonate and saturated sodium chloride and then dried over sodium sulfate and concentrated under vacuum. Further purification was carried out as indicated below for each compound.A two-necked 25 ml round-bottom flask was oven-dried, cooled under N1): Chromatography on 30 g flash silica gel with mixtures of ethyl acetate and hexanes gave a solid. Recrystallization from 2-propanol solution gave crystals , m.p. 457–458 K. X-ray-quality crystals were grown by slow evaporation from a 2-propanol solution.6-(3-Nitrophenyl)-7-phenyl-5-thia-7-azaspiro[2.6]nonan-8-one (2): Chromatography on 30 g flash silica gel with mixtures of ethyl acetate and hexanes gave a solid. Recrystallization from ethyl acetate solution gave colorless crystals , m.p. 480–482 K (decomposition). X-ray-quality crystals were grown by slow evaporation from an ethyl acetate solution.6-(4-Nitrophenyl)-7-phenyl-5-thia-7-azaspiro[2.6]nonan-8-one (Uiso(H) = 1.2Ueq(C).Crystal data, data collection and structure refinement details are summarized in Table 310.1107/S2056989019010429/hb7839sup1.cifCrystal structure: contains datablock(s) 1, 2. DOI: 10.1107/S2056989019010429/hb78391sup2.hklStructure factors: contains datablock(s) 1. DOI: Click here for additional data file.10.1107/S2056989019010429/hb78391sup5.molSupporting information file. DOI: 10.1107/S2056989019010429/hb78392sup4.hklStructure factors: contains datablock(s) 2. DOI: Click here for additional data file.10.1107/S2056989019010429/hb78392sup6.molSupporting information file. DOI: Click here for additional data file.10.1107/S2056989019010429/hb78391sup6.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S2056989019010429/hb78392sup7.cmlSupporting information file. DOI: 1942358, 1942357CCDC references:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Scientific Reports 10.1038/s41598-018-30786-y, published online 17 August 2018Correction to: In Figure 5b, “EV Spike glucuronidase (UV)” should read “EV spike glucuronidase (90° LS)”. The correct Figure 5b appears below as Figure.


TCR Vβ21 T cell clone which was specific to CML patients, and demonstrated that TCR Vα13/β21 gene-modified CD3+ T cells had specific cytotoxicity for HLA-A11+ K562 cells. However, it remains unclear which antigen is specifically recognized by the TCR Vβ21 T cell clone. In this study, CD3+ T cells from healthy donor peripheral blood were stimulated with the WT1 peptide or mixed BCR-ABL peptides in the presence or absence of IL-2 and IL-7. The distribution of the TCR Vβ repertoire was analyzed after different stimulations. We found that the mixed BCR-ABL peptides induced clonally expanded Vβ7–9-Dβ2-Jβ2–7 T cells while the Wilms Tumor 1 peptide induced clonally expanded Vβ11–2-Dβ1-Jβ1–1 T cells by high-throughput TCRβ sequencing and GeneScan. Interestingly, the sequence and CDR3 motif of Vβ11–2 T cell clone are similar to the TCR Vβ21 (a different TCR V region naming system) T cell clone that we previously found in CML patients. Thus, our findings suggest that the TCR Vβ21 T cell clone found in CML patients might be a T cell clone that specifically recognizes WT1.We previously identified a The online version of this article (10.1186/s40364-019-0163-1) contains supplementary material, which is available to authorized users. Chronic myelogenous leukemia (CML) is a common hematological malignancy in adults and has the molecular characteristic of BCR-ABL fusion proteins, which exhibit abnormal kinase activity . AlthougMechanistically, infusing donor-derived cytotoxic T lymphocytes (CTLs) induces CTL-mediated leukemia cell death through the recognition of leukemia-associated antigens. However, DLI also causes graft-versus-host disease (GVHD), mainly because CTLs are multi-clonal T cells that also recognize allo-antigens expressed in host-normal tissues . TherefoTCR Vβ + T cell clone both in blood and bone marrow [TCR Vβ T cells after injection with a WT1 vaccine.Wilms Tumor 1 (WT1) is a tumor suppressor gene involved in the etiology of Wilms’ tumor. It is overexpressed mainly in myeloid leukemias, such as acute myeloid leukemia (AML) and CML, myelodysplastic syndrome (MDS), and several solid tumors –14. There marrow –23. HoweVβ21 monoclone in blood from patients with CML and demonstrated that TCR Vα13/β21 gene-modified T cells could induce cell death in HLA-A11+ K562 cells [TCR Vβ21 T cell clones specifically recognize BCR-ABL or other CML-associated antigens. Therefore, in this study, we analyzed the distribution of the TCR Vβ repertoire in CD3+ T cells from healthy donor peripheral blood after different stimulations with a WT1 peptide or mixed BCR-ABL peptides in the presence or absence of interleukin (IL)-2 and IL-7.We previously identified a TCR 62 cells , 25. How+ donor with informed consent by Ficoll-Hypaque gradient centrifugation. CD3+ T cells were then sorted by immunomagnetic beads from the PBMCs. The immunnomagnetic beads were purchased from MACS, and the sorting operation was performed according to the manufacturer’s instructions . This study was approved in writing by the Ethics Committee of the first affiliated hospital of Jinan University.Peripheral blood mononuclear cells (PBMCs) were isolated from the peripheral blood of a healthy HLA-0201+ T cells (2.5 X 106 cells/mL) were cultured in RPMI 1640 without fetal bovine serum overnight. Fresh media containing 100 UI/mL IL-7, 100 UI/mL IL-2, and antigen peptides was then added to the cells. Untreated cells served as the control group, and the cytokines group comprised cells treated only with IL-2 and IL-7. The cells in the WT1 group were treated with a WT1-specific antigen peptide (RMFPNAPYL HLA A0201), while the cells in the BCR-ABL (B3A2) group were treated with six mixed antigen BCR-ABL peptides . After amplification, the concentration and integrity of the fragments were determined by Qubit, Agilent, and Q-PCR. Qualified libraries were sequenced by HiSeq or MiSeq. The mixcr (v1.8.2) program was used to identify the sequences in each sample. Sequences containing the complementarity-determining region 3 (CDR3) that had greater than four amino acids and a nucleic acid length that was a multiple of three without stop codon were retained as qualified clones. Bioinformatics analysis was performed after obtaining qualified clones. The amplification and sequencing of TCR Vβ and primary analysis were performed by the Huayin Health Company.Total RNA was extracted from samples with TRIzol  according to the manufacturer’s instructions. The RNA was dissolved by ddHTCR Vβ primers and a TCR Cβ primer were used in unlabeled PCR to amplify the TCR Vβ subfamily members. PCR was performed as described in our previous study. A portion of the PCR product was used for direct sequencing, which was performed by Invitrogen Biotechnology Company. The sequences of the different samples were analyzed with BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi). The remaining PCR product was used to perform runoff PCR with the addition of fluorescent primers labeled at the 5′ end with a FAM (5-Carboxyfluorescein) fluorophore (Cβ-FAM) . Then, the labeled runoff PCR products (2.0 μL) were mixed with 9.5 μL formamide  and 0.5 μL Size Standards  and heat-denatured at 94 °C for 4 min. The samples were resolved by electrophoresis using a 310 DNA sequenator  in a 310 POP-4™ gel . The size and fluorescence intensity were determined by GeneScan software. The PCR protocol was performed as described in our previous study [Twenty-four us study , 27.+ T cells from the same healthy donor’s PBMCs under different conditions and then analyzed the distribution of the TCR Vβ repertoire by TCRβ gene sequencing. Approximately 16.1 million effective reads were generated from the CD3-positive T cell populations (Table Vβ gene and 14 types of the Jβ gene detected in each group. The number of unique VDJ rearrangements was approximately 1.4 thousand in each group. The number of unique CDR3 amino acid sequences (corresponding to an in-frame effective rearrangement of the CDR3 nucleotide sequence) in the control group was 17,789, while it was 13,828 in the cytokines group. For the WT1 and B3A2 groups, the numbers were 14,472 and 11,747, respectively. From these data, we could also determine that there was no significant difference in the numbers of Vβ genes, Jβ genes, VDJ gene rearrangements, and unique CDR3 amino acid sequences in the four groups.We treated four groups of CD3Vβ genes and Jβ segments among the four groups , Vβ5–1 (11.1%), and Vβ29–1 (9.2%), which was similar to that found in the cytokines-treated group , Vβ12–3 (13.5%), and Vβ20–1 (10.8%), while in the B3A2 group, Vβ7–9 (30.3%), Vβ20–1 (24.8%), and Vβ5–1 (15.4%) were the most frequent. These data demonstrate that stimulation with cytokines did not change the type of Vβ subfamilies used in normal T cells as expected, while stimulation with different peptides could induce the selective proliferation of different Vβ subfamily T cell clones.However, there were some differences in the usage of the ups Fig. a, b. To Vβ20-1-Dβ2-Jβ2-1 (3.1%), Vβ9-Jβ1-6 (2.6%), and Vβ20-1-Dβ2-Jβ2-7 (1.7%), while in the cytokines group, it was TCR Vβ29-1-Jβ2-4 (5.9%), Vβ20-1-Dβ1-Jβ2-4 (4.9%), and Vβ24-1-Dβ2-Jβ2-1 (4.5%). The VDJ rearrangement usage pattern changed in the WT1 and B3A2 groups. The top three VDJ rearrangements in the WT1 group were Vβ29-1-Dβ1-Jβ1-2 (12.2%), Vβ11-2-Dβ1-Jβ1-1 (4.7%), and V12-3-Dβ2-Jβ2-1 (4.2%). In the B3A2 group, they were Vβ7-9-Dβ2-Jβ2-7 (29.0%), Vβ20-1-Dβ2-Jβ2-3 (12.4%), and Vβ20-1-Dβ1-Jβ2-1 (6.7%). Similarly, the expression types and frequencies of the CDR3 amino acid sequences were different in the four groups  (4.7%) in the WT1 group was identified as a TCR Vβ11-2-Dβ1-Jβ1-1 rearrangement  in T cells from CML patients that we identified previously.It is well known that VDJ recombination is an important event during the proliferation of the T cells, and CDR3 is an antigen recognition region that could be used as a specific marker for each T cell clone. Thus, we analyzed the usage frequency of VDJ recombination and the expression frequency of the predicted CDR3 amino acid sequences in the four groups. As shown in Fig. Vβ21 clone in the WT1 group was biclonal (bi-peaks) and the size of the fragment was approximately 150 bp. In the B3A2 group . The sequencing results  and CASSLAEREYPLEQYF (BCR-ABL related). Therefore, both novel TCR clones might be responsible for BCR-ABL or WT1 epitopes, and whether they could be used to produce TCR-modified T cells requires further investigation. Interestingly, we found that the TCR Vβ11-2-Dβ1-Jβ1-1 sequence is similar to the sequence in the Vβ21 T cell clone (a different TCR V region naming system) in CML patients that we previously identified, which could mediate specific cytotoxicity against CML with the TCR-modified T cell technique [Vβ21 T cell clones from CML patients specifically recognize the WT1 peptide or cross respond based on different individuals requires more investigation. Previous studies have been showed that WT1 specific T cell clones were induced in AML patients by the same WT1126–134 peptide vaccines which was used in this study, such T cell clone also expressed TCR Vβ11, however, the CDR3 sequence (ASSDYNEQF) is different from the TCR Vβ11-2-Dβ1-Jβ1-1 (CASSSSGTGPNTEAFF) which we found in the study and the CML patients [TCR Vβ5-1-Dβ2-Jβ2-5) inducted by WT1 (CMTWNQMNL) peptides [TCR Vβ11-2-Dβ1-Jβ1-1 clone in this study may provide new data of WT1 specific TCR clone bank. Moreover, on the other hand, all of this identified TCR clone may be thought as one of the immune biomarker of WT1 specific T cell clone in WT1 + malignancies.In this study, we first compared the frequent usage of the angement . Signifiechnique . Whetherpatients , 41–43. peptides . OverallTCRVβ repertoire in T cells after WT1 and BCR-ABL peptide stimulation and identified two novel TCR clones (Vβ11-2-Dβ1-Jβ1-1 and Vβ7-9-Dβ2-Jβ2-7) related to both antigens. Functional studies will be performed to confirm their anti-CML cytotoxicity by producing TCR gene-modified T cells, it may be possible to provide a new TCR-T cell clone for WT1 + leukemia and maybe for WT1 + solid tumors immunotherapy.In summary, we characterized the different usage patterns of the Additional file 1:Table S1. Sequences of the BCR-ABL antigen peptides. (DOCX 17 kb)


N-acetylglucosamine was used to maximize the chemical diversity of sponge-derived actinomycete Actinokineospora spheciospongiae sp. nov. HR–ESI–MS was employed for dereplication study and orthogonal partial least square-discriminant analysis was applied to evaluate the HR–ESI–MS data of the different fractions. As a result, two new fridamycins H (1) and I (2), along with three known compounds actinosporin C (3), D (4), and G (5) were isolated from the solid culture of sponge-associated actinomycete Actinokineospora spheciospongiae sp. nov., elicited with N-acetylglucosamine. Characterization of the isolated compounds was pursued using mass spectrometry and NMR spectral data. Fridamycin H (1) exhibited significant growth inhibitory activity towards Trypanosoma brucei strain TC221. These results highlight the potential of elicitation in sponge-associated actinomycetes as an effective strategy for the discovery of new anti-infective natural products.Several approaches have been dedicated to activate the cryptic gene clusters in the genomes of actinomycetes for the targeted discovery of new fascinating biomedical lead structures. In the current study, The online version of this article (10.1186/s13568-018-0730-0) contains supplementary material, which is available to authorized users. Actinokineospora spheciospongiae was first isolated by Abdelmohsen et al. in 2010 from the Red Sea sponge Spheciospongia vagabunda that was collected from Ras Mohamed, Egypt  and the metabolic profile was investigated using 1H NMR fingerprint methodology which resulted in identification of four new actinosporins (E–H) , along with three known compounds actinosporin C (3), D (4), and G (5). Moreover, the antitrypanosomal activity of all isolated pure compounds was also investigated.Marine sponge-derived actinomycetes have been recognized as potential producers of fascinating chemical scaffolds such as polyketides, alkaloids, peptides, sterols, terpenes and fatty acids  was isolated from the Red Sea sponge Spheciospongia vagabunda that was collected by SCUBA diving from offshore Ras Mohamed, Egypt  in August 2006. Two hundred ISP2 agar plates (square 120 × 120 mm), each inoculated with 100 µL of a 5 day old liquid culture of Actinokineospora spheciospongiae and incubated at 30 °C for 6 h. The agar plates were then elicited with GlcNAc . The plates were then incubated at 30 °C for 7 days. The agar medium with bacterial biomass was cut into small pieces and transferred to 1 L Erlenmeyer flasks. One liter of EtOAc/flask was added to submerge the agar pieces and macerate the medium culture under shaking at 150 rpm for overnight. The macerated medium was subsequently filtered using filter paper . The filtrates were combined and evaporated in a rotary evaporator under vacuum  to yield 850 mg of dried crude EtOAc extract, which was fractionated on a Sephadex LH20  column eluting with H2O/MeOH (90:10%) to 100% methanol, to yield 12 fractions (50 mL each). The highly active fractions (3 and 5) were further purified by semi-preparative HPLC  using H2O/acetonitrile (95:5%) initially for 5 min, followed by a linear gradient to 100% acetonitrile within 40 min and maintained isocratically for further 5 min. Separation was achieved on a preparative C18 column , with a flow rate of 3.0 mL/min yielding compounds 1 (1.6 mg) and 2 (1.8 mg) from fraction 3 and compounds 3–5  from fraction 5.One milligram of dried microbial extracts and/or fractions was dissolved in HPLC-grade MeOH to a final concentration of 1 mg/mL and subjected to high resolution LC–ESI–MS using Thermo Scientific Exact mass analysers  coupled to a Dionex UltiMate 3000 HPLC system. The samples were eluted through a C-18 column  with a length of 75 mm, internal diameter of 3.0 mm and particle size 5 μm. The mobile phase consisted of 0.1% formic acid in HPLC-grade water (solvent A) and acetonitrile (solvent B). The flow rate was set at 300 µL/min. Gradient elution was initiated with 10% B for 5 min, which was increased to 100% B over 30 min, which was maintained for another 5 min before decreasing to 10% B in the next min. The column was then equilibrated with 10% B for 4 min until the end of the run. MS/MS was measured using LTQ Orbitrap Thermo scientific mass analysers .Trypanosoma brucei strain TC 221 . 96-well plate chambers were used for testing Trypanosomes versus various concentrations of examined compounds at 0.25–40 μM in DMSO (1%) to a final volume of 200 μL. For controls, DMSO (1%) plus parasites without any examined compound were used concurrently in each plate. After that, the plates were incubated in an atmosphere of CO2 5% at 37 °C for 24 h. Then, 20 μL of Alamar Blue was added in each plate and the activity was measured by light absorption at ʎ 550 nm with a reference wavelength of 650 nm using an MR 700 Microplate Reader  after 48 and 72 h. The quantification of the IC50 values of the examined compounds were processed by linear interpolation in triplicate.Antitrypanosomal action had been investigated following the method reported by Abdelmohsen et al. . In brie5 cells/mL at 37 °C, 5% CO2, and 95% humidity for 24 h, were used for investigation of their cytotoxic properties against Macrophages (J774.1). After adding of Alamar Blue (20 μL), the plates were incubated and the then, determination of the optical densities at ʎ 540 nm and a reference wavelength of 630 nm were done after 48 and 72 h, in the same way as described for the anti-trypansomal activity   were cultivated in Complete Baltz Medium free from phenol red. Different concentrations of tested compounds (0.25–200 μM) at a cell density of 1 × 101): Orange powder; [α]D25 + 12° ; UV (MeOH) λ max (log ε) 231 (4.45), 253 (4.51), 293 (4.37) nm; IR (KBr)νmax 3420, 2962, 1710, 1436, 1340, 1275, 1065 cm−1; 1H NMR  and 13C NMR  data, see Table m/z 503.1524 [M+H]+ .Fridamycin H (2): Yellow powder; [α]D25 + 25° ; UV (MeOH) λ max (log ε) 232 (4.54), 253 (4.53), 294 (4.17) nm; IR (KBr)νmax 3419, 3030, 1700, 1650, 1432, 1345, 1275, 1065 cm−1; 1H NMR  and 13C NMR  data, see Table m/z 614.2036 [M+Na]+ .Fridamycin I (m/z (retention time in minutes) 387.0865 (6.65) [M−H]− and 401.1025 (10.44) [M−H]−, were dereplicated as G-2N (C23H16O6), an antibiotic formerly reported from actinomycete Frankia sp.(Nancy and Mary 24H18O6), an antibiotic previously isolated from Streptomyces sp. was performed for the MS dataset of active versus inactive fractions to pinpoint the putatively active metabolites in the antitrypanosomally-active fractions prior to any further purification attempts.2 = 0.99) and high predictive ability of Q2 = 0.96. The current OPLS-DA model produced the highest predictive power (Q2) when assessed by cross validation. Moreover, the statistical significance of estimated predictive power of the respective model was tested by permutation test which exhibited Q2Y intercept at − 0.81 that indicated a valid model. A particularly useful tool that compares the variable magnitude against its reliability is the S-loadings plot obtained from the OPLS-DA model and represented in Fig. To achieve the best discrimination between the active and inactive fractions, a supervised orthogonal partial least square discriminant analysis (OPLS-DA) was carried out. The OPLS-DA scores plot Fig. a showed m/z 501.159 (Rt = 9.86 min) [M−H]−, equivalent to C25H26O11 did not match any compounds reported in the DNP database while the m/z ion at 357.097 (Rt = 9.88 min) [M+H]+ for the predicted molecular formula C19H16O7 was dereplicated for fridamycin E, the simplest member of the angucycline family of antibiotics that was previously isolated from a mutant of Streptomyces parvulus (strain Tu 1989)  requiring thirteen degrees of unsaturation  in DMSO-d6 was used for processing the NMR spectral analyses. Initial investigation of NMR spectral data of 1 (Table R)-fridamycin E moiety  indicating similar chromophores. A molecular formula of C1H NMR spectrum  and 7.70 (J = 8.4 Hz) assignable to H-1 and H-3, respectively, in addition to a one proton doublet of doublet at δH 7.86 (J = 7.8 and 8.4 Hz), of H-2. Another spin system consists of two one-proton doublets at δH 7.78 (J = 7.8 Hz) and 7.61 (J = 7.8 Hz) assignable to protons H-6 and H-7, respectively, of the tetra-substituted aromatic system. Moreover, the 1H NMR spectrum showed two one-proton doublets at δH 2.88 (J = 13.2 Hz) and 3.03 (J = 13.2 Hz), attributed to the two geminally coupled protons, characteristic for H-11a and H-11b, respectively. Whereas, the two-protons singlet at δH 2.40 was assignable for H-13, belonging to the protons of a methylene group directly linked to the carboxyl group. One singlet at δH 1.19 together with its corresponding carbon signal at δC 26.8 was for the methyl group (CH3-15) which was considerably downfield shifted due to its direct attachment to oxygenated carbon at δC 71.5 (C-12) , one methyl doublet at δH 1.09 (J = 6 Hz), as well as four one-proton multiplets at δH (3.33–3.99) indicated the presence of the rhamnose moiety in the structure , while the others resonated at δC 181.2 and 188.9 were assigned to quinone carbonyl carbons C-5 and C-10, respectively. Additionally two methylene carbons C-11 and C-13 were observed; the latter was deshielded to 46.7 ppm in agreement with direct attachment to carboxylic group, while the former one was detected at 40.1 ppm due to its direct attachment to the aromatic ring that could be confirmed by HMBC correlations from H-11a and H-11b (δH 2.88 and 3.03) to C-7 (δC 141.0) and C-9 (δC 160.3). Two oxygenated aromatic carbons were observed at δC 156.8 and 160.3, assignable to C-4 and C-9, respectively. The former (C-4) displayed HMBC correlation to the anomeric proton of l-rhamnose , confirming the attachment of l-rhamnose to C-4 of the anthraquinone motif  configuration (α-OH and β-CH3) at C-12. Whereas the (S) isomers, (α-CH3 and β-OH) at C-12, have been only obtained chemosynthetically as the less bioactive isomers of fridamycin E analogues  [M+Na]+) requiring seventeen degrees of unsaturation Additional file 2 (Table 1 (fridamycin H) except for the substitution of the carboxyl group of 1 with N-benzyl amide moiety that was represented by multiplet signals at δH 7.25, 7.30 and 7.24 assignable to ,  and H-4″, respectively, in accordance with mono-substituted benzene ring. Two-protons singlet at δH 3.54 along with its corresponding carbon signal at δC 40.9 were for the methylene group (CH2-16) directly attached to the benzene ring and one singlet at δH 8.17 corresponding to an amine proton  to C-1″ and C-2″, 6″ at δC 135.45 and 129.7, respectively and to amide-type carbonyl carbon at (δC 173.2) were observed confirming direct attachment of the methylene group (CH2-16) to the benzene ring from one side and to the amide moiety from the other side , D (4) and G (5).Separation of fraction 5 delivered three actinosporins with similar UV spectra . By comparing their spectra . While the rest displayed no significant antitypanosomal or cytotoxic activity at the concentration tested.All the isolated compounds were tested against Actinokineospora spheciospongiae sp. nov. showed unexplored chemical potential. Actinosporins A and B had been isolated using broth culture of Actinokineospora sp. strain EG49  and I (2), along with three known actinosporins C (3), D (4) and G (5).Bioinformatic analyses have revealed the presence of large fractions of silent gene clusters in the genome sequences of actinomycetes. Innovative strategies had been used to induce production of weakly expressed secondary metabolites which are not expressed under standard fermentation conditions and several approaches including biological, chemical, and molecular, have been applied to activate those silent natural products in the laboratory culture of actinomycetes  and I (2), along with three known actinosporins C (3), D (4) and G (5) were considered for the examination of the antitrypanosomal property versus Trypanosoma brucei TC221 in addition to cytototoxic properties towards Macrophages (J774.1). The results obtained displayed that fridamycin H showed significant antitrypanosomal activity after 48 and 72 h with IC50 values of 3.38 and 5.26 μM, respectively, with no cytotoxicity against J774.1 macrophages (IC50 > 200 μM). These results demonstrate the potential of elicitation via compound induction as a tool for discovery of new antitrypanosomal chemical scaffolds.On the other side, trypanosomiasis, commonly known as sleeping sickness, still has been considered one of the most serious challenges to human health, particularly in underdeveloped regions of Africa (Jacobs et al. Additional file 1. Additional figures.


H-chromene derivative molecules are linked by N—H⋯O and N—H⋯N hydrogen bonds, forming double layers or ribbons.In the structures of two title compounds, the 4 16H9Cl2N2O2·C4H8O2 and C16H9Cl2N2O2, the bicyclic 4H-chromene cores are nearly planar with maximum deviations of 0.081 (2) and 0.087 (2) Å. In both structures, the chromene derivative molecules are linked into centrosymmetric dimers by pairs of N—H⋯O hydrogen bonds, forming 22R(16) motifs. These dimers are further linked in the 3,4-dichlorophenyl derivative by N—H⋯N hydrogen bonds into double layers parallel to (100) and in the 2,6-dichlorophenyl derivative by O—H⋯N hydrogen bonds into ribbons along the [1via O—H⋯O hydrogen bonds.In the title compounds, C The pyran ring is a core unit found in benzopyrans, chromones, coumarins and flavanoids. Numerous naturally occurring compounds containing a pyran ring show therapeutic activities such as antiviral  act as synthetic building blocks for the design of various pyran-containing bio-active molecules -7-hydroxy-4H-benzopyran 1,4-dioxane mono solvate (I)H-benzopyran (II)Against this background we carried out the crystallographic studies of the title 4H-chromene derivatives differ only in the positions of the chlorine atoms attached to the phenyl ring, and the key bond dimensions in (I)sp3-hybridized C7 atom in both cases  or 2,4-dichlorobenzaldehyde  [for (II)], malononitrile  and resorcinol  in 150 ml of water were refluxed for about 10-20 minutes in 250 ml round-bottom flasks. The progress of the reaction was monitored by thin layer chromatography using silica gel-G plates. After the product had formed, the reaction mixtures were kept in the refrigerator overnight. The solid mass that settled was filtered using a suction pump, washed well with a mixture of methanol and water and dried in air. The crude products were recrystallized from methanol giving white powders. Single crystals were grown by slow evaporation of solutions in 1,4-dioxane (I)Uiso(H) = 1.2Ueq and C—H = 0.98 Å for methine, 0.97 Å for methylene and 0.93 Å for aromatic C atoms, and N—H = 0.86 Å. In (I)Uiso value was allowed to refine freely. In (II)Crystal data, diffraction data and structure refinement details for (I)10.1107/S2056989019013197/yk2126sup1.cifCrystal structure: contains datablock(s) I, II, global. DOI: 10.1107/S2056989019013197/yk2126Isup2.hklStructure factors: contains datablock(s) I. DOI: 10.1107/S2056989019013197/yk2126IIsup3.hklStructure factors: contains datablock(s) II. DOI: Click here for additional data file.10.1107/S2056989019013197/yk2126Isup4.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S2056989019013197/yk2126IIsup5.cmlSupporting information file. DOI: 1865757, 1865762CCDC references:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Context:Cussonia arborea Hochst. ex A. Rich  is a folk medicine used to treat various diseases. However, there is no report of the root phytochemistry.Objective: This study isolates and identifies the immunomodulatory compounds from root-bark of C. arborea.Materials and methods: The methanol extract (18 g) was subjected to repeated column chromatography resulting in isolation of five compounds (1–5). Structure determination was achieved by analysis of their 1 D and 2 D NMR, and mass spectroscopy. The compounds (100–1.0 μg/mL) were examined immunomodulatory for effect on production of reactive oxygen species (ROS) from whole blood phagocytes and on proliferation of T-cells. The compounds cytotoxicity (100–1.0 μg/mL) was evaluated on NIH-3T3 normal fibroblast cells.Results: Three pentacyclic triterpenoids , two phytosterols: [stigmasterol (3)] and [3-O-β-d-glucopyranosyl stigmasterol (4)] were all isolated from the methanol soluble extract. All the tested compounds (1–4) were found to be nontoxic on NIH-3T3 cells. Compound 1 and 2 moderately inhibited the production of ROS  whereas compound 2 exhibited the highest inhibitory effect (IC50 = 12.6 ± 0.4 μg/mL) on proliferation of phytoheamagglutinin (PHA) activated T-cells.Conclusions: The isolated compounds (1–5) are reported for the first time from this species. In addition, compound 2 with suppressive potential on production of intracellular ROS and proliferation of T-cells could be of immense value in control of autoimmune diseases as well as in immune compromised patients. Cussonia  is well known in folk medicines for the treatment of malaria, mental illness, eye problems, sexually transmitted diseases, skin problems, cancer, wounds, etc. -β-d-glucopyranosyl-(1 → 6)-β-d-glucopyranoside of 23-hydroxyursolic acid only, were reported to have been isolated from the stem bark of this species  3β-hydroxylolean-12-en-28-oic, (2) stigmasterol, (3) 3-O-β-d-glucopyranosyl stigmasterol, (4) and 23-hydroxy-3-oxo-urs-12-en-28-oic acid and (5) from the root-bark of this plant. In present study, we examined the immunomodulatory activities of the isolated compounds (1–4) using two different parameters of innate and adaptive immune responses, that is, effect on production of intracellular reactive oxygen species (ROS) from zymosan activated whole blood phagocytes and on proliferation of phytoheamagglutinin (PHA) activated T-lymphocytes to evaluate their potential for the control of harmful immune responses. The cytotoxicity of compounds was evaluated on NIH-3T3 fibroblast by MTT -2,5-diphenyltetrazolium bromide) assay. This is the first report of immunomodulatory study of C. arborea.The genus The nuclear magnetic resonance (NMR) spectra were recorded in deuterated chloroform or methanol or pyridine on a Bruker Avance 500 and 400 MHz NMR spectrometers. IR spectra were recorded using JASCO 302-A spectrometer, respectively. High resolution electron ionized mass spectrometry (HR EIMS) and fast atomic bombardment mass spectrometry (FABMS) were carried out using MAT 95XP and JMS HX-110 mass spectrometers, respectively. All reagents used were of analytical grade.C. arborea was collected in May, 2012, from a farm Land at Eruwa in Ibarapa East Local Government area of Oyo state, Nigeria. The root-barks were air-dried at room temperature and pulverized. The voucher specimen (UIH-22340) was deposited at the Herbarium unit of Botany and Microbiology Department, University of Ibadan, Nigeria after identification and authentication by Mr. D. O. Esimekhuai.The whole plant of C. arborea root-bark (1 kg) was extracted with methanol (3 × 2.5 L) for two weeks at room temperature (20–25 °C). The combined extracts were concentrated under pressure using rotary evaporator, preset at 37 °C yielding a light greenish powder (18 g). The methanol extract (18 g) was pre-adsorbed on silica gel and introduced to column  packed with silica gel as stationary phase. The solvent system used in eluting the column are hexane , hexane:EtOAc , EtOAc , EtOAc:MeOH , successively. Collection volume of eluent employed was 1000 mL/vial. A total of 80 fractions were collected (F1–F80). Fraction F10 and F12 eluted with hexane:EtOAc 17:3 and 7:3 afforded compounds 3 (8.0 mg) and 2 (22.0 mg), respectively, as white solids. Fraction F25 eluted with hexane:EtOAc (1:1) was concentrated under reduced pressure to yield a white precipitate amorphous powder (250 mg). The precipitate was then collected by filtration and purified by recrystallisation from MeOH by the slow evaporation of the solvent at room temperature to give the compound 1 (150.0 mg) as pure colourless crystal. Compound 4, a white amorphous compound, was also obtained from fraction F37, which was eluted with 80% EtOAc in hexane. Fraction F37 was re-subjected to silica gel Column Chromatography eluted with mixture of dichloromethane and methanol (97:3) to afford compound 5 (5.0 mg).The air dried 1–5 : 472 , 454 (7), 396 (7), 248 (100), 223 (23), 203 (86), 175 (69), 161 (9), (45), 119 (25); HREIMS: 472.3547 . 1H-NMR: : δ 0.69 , 0.81 , 0.90 , 0.93 , 0.97 , 1.12 , 2.83 , 3.50 , 3.57 , 5.21 . 13 C-NMR : See Melting point: 292 °C; Colourless crystalline solid; IR(KBr): 3450 (OH), 2943, 1699 (C = O), 1463, 1038 cmm/z (rel. int.): 456 , 410 (2), 300 (2), 248 (100), 233 (10), 203 (77), 189 (12), 133 (12), 189 (100), 133 (13), 119 (9); HREIMS: 456.3566 . 1H-NMR : δ 0.75 , 0.88 , 0.88 , 1.11 , 0.96 , 0.73 , 0.89 , 1.57 , 2.78 , 3.18 , 5.26 . 13 C-NMR : See Melting point: 271–273 °C; White amorphous solid; EI-MS −1; EI-MS: m/z (rel. int): 412 , 396 (47), 381 (34), 369 (17), 329 (40), 303 (46), 255 (66), 233 (42), 213 (48), 159 (43), 107 (52), 83 (44); HREIMS: 412.3700 . 1H NMR: : δ 0.66 , 0.78 , 0.82 , 1.01 , 0.99 , 4.96 , 5.10 , 5.32 . 13 C-NMR : See Melting point: 168–169 °C; White needle solid; IR (KBr): 1727, 1239, 3060, 1637, 865 cm−1; EI-MS: m/z (rel. int): 412 , 394 (100), 381 (23), 369 (9), 351 (32), 255 (80), 213 (36), 159 (43), 147 (50), 83 (72); FABMS (−ve) = 573.3674[M–H]− . 1H NMR: : δ 0.64 , 0.88 , 0.86 , 1.06 , 0.93 , 5.02 , 5.18 , 5.34 , 5.05  13 C-NMR: : See Melting point: 290–292 °C; White amorphous solid; IR (KBr): 3450, 1727, 1637, 1239, 865 cm−1; EI-MS: m/z (rel. int): 470 , 426 (9), 424 (5), 248 (100), 203 (73), 189 (32), 175 (24), 133 (85), 119 (34), 91 (25), 81 (35), 71 (40), 57 (66), 44 (57). HREIMS: 470.3421 . 1H NMR: : δ 0.77 , 0.83 , 0.87 , 0.92 , 1.06 , 1.23 , 2.15 , 3.40 , 3.70 , 5.23 .Melting point: 170–172 °C; Colourless solid; IR (KBr): 3453 (OH), 2944, 1699 (C = O), 1463, 1384, 1038 cmAll studies on human blood samples were carried out after an approval from independent ethics committee, International Center for Chemical and Biological Sciences, University of Karachi , No: ICCBS/IEC-008-BC-2015/Protocol/1.0, and with written informed consent from the volunteers. In this study, the blood used for both oxidative burst and lymphocyte proliferation test was from same volunteer.g for 5 min at room temperature. Supernatant was discarded and pellet was resuspended in 10 mL of Trisbase NaCl. Final concentration of 0.075% SOZ was achieved in each experimental well.Exact 10 mL of 0.3% SOZ was prepared by adding 30 mg of zymosan powder  in 2 mL of pooled human serum, then volume was made by adding 8 mL of Tris base NaCl. The mixture was vortexed and incubated in shaking water bath at 37 °C for 30 min then centrifuged at 2000 −5 M (10 mL) was prepared by dissolving 1.8 mg of luminol  in 1 mL of borate buffer. 9.0 mg of gelatin was then dissolved in 9.0 mL of HBSS++   then the mixture of luminol + borate buffer was transferred in it. Final concentration of 1.7 × 10−5 M was achieved in each experimental well. The assay was performed on heparinized human whole blood collected from healthy volunteer. The blood was diluted in HBSS++ with a dilution of 1:50. SOZ was used as an activator. As phagocytes have receptors for SOZ on their surface so they are the major producer of ROS in activated cells. Luminol-enhanced chemiluminescence assay was performed, as described by Helfand et al. . Results were monitored as relative light units (RLU) reading, with peak and total integral values set with repeated scans at 50 s intervals, and 1 s point of measuring time. Results were collected as separate graph for each well showing peak value, peak time of cell activity and total integral. The drug Ibuprofen was used as standard.Luminol solution 7 × 10d et al.  with somg for 20 min at room temperature. The buffy layer appeared at the interface of LSM and plasma was carefully collected and washed at 4 °C for 10 min at 300 g. The supernatant was discarded and pellet containing peripheral blood mononuclear cells (PBMNCs) was collected. Cell viability and counting was performed using trypan blue dye.Lymphocytes were isolated from human heparinized blood. Briefly 10 mL of blood was aseptically collected in heparin containing tube from healthy volunteer. Blood was then mixed with equal volume (1:1) of incomplete RPMI  media in 50 mL sterile falcon tube. Lymphocyte separation media (LSM) (5 mL) was added into two sterile 15 mL falcon tubes and 10 mL of the mixture containing blood and RPMI was gently layered on LSM in each tube. Tubes were then centrifuged at 400 3H-Thymidine incorporated T-cell proliferation assay was performed as described by Mesaik et al. (6 cell/mL). Cells were then stimulated by adding 50 μL of 7.5 μg/mL phytoheamagglutinin-L (PHA-L) which acts as mitogen only for T-cells among other PBMNCs. The final volume of 200 μL was achieved in each well. The wells received 50 μL cells and 150 μL of 5% FCS/RPMI served as negative control where as positive control contains 50 μL cells, 50 μL of PHA-L and 100 μL of 5% FCS/RPMI. The prednisolone was used as standard. Plates were then incubated for 72 h at 37 °C in 5% CO2 incubator. After 72 h, cultures were pulsed with 25 μL of 0.5 μCi/well 3H-thymidine , and plates were further incubated for 18 h. After incubation cells were harvested using a glass fibre filter and cell harvester . The level of the 3H-thymidine incorporated into the cells was measured by a LS65000 liquid scintillation counter . Results were expressed as mean count per minutes (CPM). The 50% inhibitory concentration (IC50) values were calculated for all tested compounds.k et al. . Briefly4 cells/mL in Dulbacco’s modified eagles medium (DMEM) supplemented with 10% foetal bovine serum (FBS) were plated into 96-wells flat bottom plate and incubated overnight at 37 °C in 5% CO2. Three different concentrations of test compound  were added to the plate in triplicates and incubated for 48 h. 0.5 mg/mL MTT (50 µL) was added to each well, the plate was then further incubated for 4 h. MTT was aspirated and 100 µL of dimethyl sulfoxide (DMSO) was then added to each well. The extent of MTT reduction to formazan within cells was calculated by measuring the absorbance at 540 nm, using spectrophotometer . The cytotoxic activity was recorded as concentration causing 50% growth inhibition (IC50) for 3T3 cells. Cycloheximide was used as standard drug.Cytotoxicity of test compounds on NIH-3T3 fibroblast cells  was evaluated by using the standard MTT colorimetric assay. Briefly 100 μL of 5 × 1050 values were calculated using Excel based program. One-way ANOVA: Post hoc Dunnett test was also used and p < 0.05 was considered to indicate a statistically significant difference.All data are reported as mean ± SD of the mean and the ICC. arborea using various chromatographic techniques yielded compounds 1–5 , corresponding to molecular formula C30H48O4 in HR EIMS. The base peak at m/z 248, produced through retro-Diels-Alder fragmentation, and further loss of the carboxylic group led to the peak at m/z 203. The IR spectrum of compound 1 showed absorption of hydroxyl (3453 cm−1) and carbonyl (1699 cm−1) groups. The 1H-NMR spectrum exhibited signals due to 6 methyl singlets , an olefinic proton , a hydroxyl-methine group , one proton doublet of doublet at δ 2.83 (H-18) and a methylene attached to oxygen . The rest of signals were the sp3 CH and CH2 unit found at upfield region. The broad-band decoupled 13 C-NMR  by comparison with existing literature  consistent with 7 degree of unsaturation. The base peak at m/z 248 and the fragment ion at m/z 203 showed a characteristic for a pentacyclic triterpene of β-amyrin series with a double bond between C-12 and C-13. The 1H-NMR spectrum showed 7 tertiary methyl groups at δ 0.96 (H-23), 0.75 (H-24), 0.88 (H-25), 0.73 (H-26), 1.11 (H-27), 0.88 (H-29) and 0.89 (H-30) on an oleanane skeleton. One proton doublet of doublet at δ 2.78 and a singlet olefinic proton at δ 5.26 were assigned to H-18 and H-12, indicating an olea-12-ene skeleton. The 1H-NMR spectrum also showed a deshielded signal for methine proton δ 3.18 , which was assigned for H-3 proton. The 13 C-NMR spectrum of compound 2 indicated the presence of 30 carbon atoms: 7 methyl, 10 methylene, 5 methine, and 8 quaternary carbons. The presence of oxygenated carbon at C-3 showed resonance at δ 78.9. The signal at δ 183.13 was due to carbon of carboxylic acid at C-28. On the basis of the spectra data  in the HR EIMS. In the 1H-NMR, 6 methyls appeared at δ 0.67 (H-18), 0.99 (H-19), 1.01 (H-21), 0.82 (H-26), 0.76 (H-27) and 0.78 (H-29). Three olefinic signals of 1 proton each were observed at δ 5.10 , δ 4.96  and 5.32  and their corresponding carbons resonated at δc 138.3, 129.3 and 121.7, respectively, which signified the presence of two double bonds in the compound. One proton signal at δ 3.46 ppm belongs to methine at H-3 (71.81 ppm) revealed that hydroxyl function was attached to it. This proton H-3 was coupled to methylene protons at H-2 (2.21 ppm), and this correlation between H-3 and H-2 was established by COSY analysis. The 13 C-NMR revealed 29 signals, which were resolved using DEPT experiments into 6 methyl, 9 methylene, 11 methine, and 3 quaternary carbons. The spectra data (3 were in good agreement with one reported for stigmasterol (Forgo and Kövér Compound tra data  of compo4 was isolated as a white amorphous compound from fraction F37 (80% EtOAc in Hex). The 1H NMR spectrum showed signals for 6 methyl groups: 2 tertiary, 3 secondary, and 1 primary at δ 0.64 (H-18), 0.93 (H-19), 1.06 (H-21), 0.86 × 2 (H-26 & 27) and 0.88 (H-29), as well as 3 olefinic protons at 5.34 (H-6), 5.18 (H-22) and 5.02 (H-23) and an anomeric proton at 5.05 , which were features of a triterpene glycoside. The 13 C NMR spectrum of compound 4 was similar to that of 3 except for the signals that appeared between δ 70 and 80 region associated with sugar moiety. The anomeric carbon signal appeared at 102.59 and its proton at 5.05 ppm appearing as a doublet with coupling constant of 7.8 Hz. The most downfield peak at δ 140.94 was assigned to olefinic bonded quaternary carbon C-5. In HMBC spectrum H-1 of glc at δ of 5.05 exhibited a long range correlation with C-2 of aglycone at δ 32.12 and C-2 of glc at δ 78.10. Also, HMBC correlation was observed between H-23 of the aglycone at δH 5.02 and C-22 at δ 138.85. The FABMS of 4 (negative-ion mode) gave peak at m/z 573[M-H]−, indicating a molecular weight of 574 . The EIMS of 4 showed ion peaks at m/z 412, 394, 381, 351 and 255, which indicated that the aglycone is a stigmasterol. Compound 4 was characterized to be 3-O-β-d-glucopyranosyl stigmasterol on the basis of the spectra data (Compound tra data  as well 5 was obtained as a colourless solid. Its IR spectrum exhibited absorptions at 3453 (OH) and 1699 cm−1 (C = O). The positive-ion HR EIMS of compound 5 showed a molecular ion peak at m/z 470.3421, corresponding to C30H46O4. The peaks at m/z 248 (100%) and 203 (73%) supported the characteristics retro-Diels-Alder cleavage of Δ12-pentacyclic triterpenoid compound. The 1H NMR spectrum displayed signals due to 4 tertiary methyl groups at δ 0.77, 0.87, 1.06 and 1.23  and 2 secondary methyl groups at δ 0.83  and 0.92 . These characteristics together with the singlet olefinic proton at δ 5.23 ruled out an oleanane skeleton and confirmed the ursane framework , compounds 1 and 2 inhibited the production of ROS with an IC50 =  24.4 ± 4.3 and 37.5 ± 0.1 µg/mL, respectively, whereas compounds 3 and 4 did not inhibit the production of ROS (50 = 11.2 ± 1.9 µg/mL). Compound 2 also exhibited marked inhibition of T-cell proliferation IC50 =  12.6 ± 0.4 µg/mL among others which either showed low level of activity (compound 3 with an IC50 =  86.8 ± 0.1 µg/mL) or no inhibition (compounds 1 and 4), indicating good immunomodulatory potential of compound 2  and they also proposed a mechanism by which pentacyclic triterpenoids could bring about this effect. They stated that the combination of E ring size as well as C-19, C-20 and C-28 positions could be responsible for the differences in biological effects in pentacyclic triterpenes analogues. However, in this study, hederagenin (1), a pentacyclic triterpenoid showed no inhibition. Thus, methylene oxide at position 4 could be a major factor, if not the only one, causing no antiproliferative activity observed in compound 1. Compound 5 was not tested because the quantity isolated was not sufficient for the assay.The data collected revealed that among all tested compounds  are being reported and results obtained suggests that compound 2 may be a potential therapeutic agent in treatment of various immune disorders, including organ rejection after transplant. Further studies are suggested on making new derivatives of 2 which could improve and enhance the immunomodulatory potential of compound 2.The present phytochemical investigation of root-bark of