Lubricin/proteoglycan 4 (PRG4) is a mucinous glycoprotein secreted by synovial fibroblasts and superficial zone chondrocytes. Recently, we showed that recombinant human PRG4 (rhPRG4) is a putative ligand for CD44 receptor. rhPRG4-CD44 interaction inhibits cytokine-induced rheumatoid arthritis synoviocyte proliferation. The objective of this study is to decipher the autocrine function of PRG4 in regulating osteoarthritic synoviocyte proliferation and expression of catabolic and pro-inflammatory mediators under basal and interleukin-1 beta (IL-1β)-stimulated conditions.Prg4+/+ and Prg4-/- synoviocytes were studied using western blot. Nuclear translocation of p50 and p65 proteins in osteoarthritis (OA) fibroblast-like synoviocytes (FLS) in response to IL-1β stimulation in the absence or presence of rhPRG4 was studied using DNA binding assays. OA synoviocyte (5000 cells per well) proliferation following IL-1β (20 ng/ml) treatment in the absence or presence of rhPRG4 (50–200 μg/ml) over 48 hours was determined using a colorimetric assay. Gene expression of matrix metalloproteinases (MMPs), tissue inhibitor of metallproteinases-1 (TIMP-1), TIMP-2, IL-1β, IL-6, IL-8, TNF-α, cycloxygenae-2 (COX2) and PRG4 in unstimulated and IL-1β (1 ng/ml)-stimulated OA synoviocytes, in the presence or absence of rhPRG4 (100 and 200 μg/ml), was studied following incubation for 24 hours.Cytosolic and nuclear levels of nuclear factor kappa B (NFκB) p50 and p65 subunits in Prg4-/- synoviocytes contained higher nuclear p50 and p65 levels compared to Prg4+/+ synoviocytes (p < 0.05). rhPRG4 (100 μg/ml) reduced p50 and p65 nuclear levels in Prg4+/+ and Prg4-/- synoviocytes (p < 0.001). Similarly, rhPRG4 (200 μg/ml) inhibited NFκB translocation and cell proliferation in OA synoviocytes in a CD44-dependent manner (p < 0.001) via inhibition of IκBα phosphorylation. IL-1β reduced PRG4 expression in OA synoviocytes and rhPRG4 (100 μg/ml) treatment reversed this effect (p < 0.001). rhPRG4 (200 μg/ml) reduced basal gene expression of MMP-1, MMP-3, MMP-13, IL-6, IL-8, and PRG4 in OA synoviocytes, while increasing TIMP-2 and cycloxygenase-2 (COX2) expression (p < 0.001). rhPRG4 (200 μg/ml) reduced IL-1β induction of MMP-1, MMP-3, MMP-9, MMP-13, IL-6, IL-8, and COX2 expression in a CD44-dependent manner (p < 0.001).PRG4 plays an important anti-inflammatory role in regulating OA synoviocyte proliferation and reduces basal and IL-1β-stimulated expression of catabolic mediators. Exogenous rhPRG4 autoregulates native PRG4 expression in OA synoviocytes. Prg4 gene in the autosomal recessive disease, camptodactylyl-arthropathy-coxa vara pericarditis (CACP) syndrome, characterized by juvenile-onset arthropathy [Prg4 knockout (Prg4-/-) mice display progressive synovial hyperplasia, cartilage surface fibrillations, and chondrocyte apoptosis, which may not be completely reversed by PRG4 re-expression [Lubricin/proteoglycan-4 (PRG4) is a glycoprotein secreted by synovial fibroblasts and superficial zone chondrocytes –3. The ihropathy . The joipression –8. Therapression –14.Prg4-/- synovial fibroblasts and human synovial fibroblasts derived from patients with RA [A biological role for recombinant human PRG4 (rhPRG4) was recently reported. rhPRG4 was shown to compete with hyaluronan for binding to the CD44 receptor resulting in downstream inhibition of nuclear factor kappa B (NFκB) nuclear translocation in synoviocytes from patients with rheumatoid arthritis (RA) . Further with RA . rhPRG4  with RA –17.PRG4 gene expression in OA fibroblast-like synoviocytes (FLS) in response to interleukin-1 beta (IL-1β) and rhPRG4 treatments. We hypothesized that PRG4 is an important autocrine modulator of synovial cells, mediated by its interaction with CD44.CD44 is a major cell surface receptor, with various isoforms generated by alternative splicing and glycosylations, which possesses the ability to bind different ligands and exert different biological functions in inflammation and cancer , 19. IncPrg4+/+ and Prg4-/- male mice 8–10 weeks old and synoviocytes were isolated as described [Prg4+/+ and Prg4-/- synoviocytes were cultured in T-25 flasks until confluence. Prg4+/+ and Prg4-/- synoviocytes were treated with rhPRG4 (100 μg/ml) [Synovial tissues were harvested from escribed . Prg4+/0 μg/ml) , CD44 moOA FLS  were harvested from patients undergoing total knee replacement. Cells were used between the third and sixth passages in all experiments. OA FLS  were stimulated with IL-1β  for 6 hours at 37 °C in the presence or absence of rhPRG4 (100 μg/ml), rhPRG4 (200 μg/ml), rhPRG4 (200 μg/ml) + CD44 Ab  or CD44 Ab (2.5 μg/ml). Cells were harvested and nuclear extraction was performed using a commercially available kit (Thermo Fisher Scientific). Total protein was quantified, and 3 μg of nuclear extract from each experimental group was used. The p50 and p65 proteins were detected in the nuclear extract using NFκB DNA binding assay kits (Abcam). Data are presented as p50 or p65 nuclear levels normalized to untreated controls. Data are presented as the average ± the standard deviation of four independent experiments, each with duplicate wells per group.OA FLS were seeded in T-25 flasks at 1,000,000 cells in DMEM + 10% FBS for 48 hours. The total volume was 4 ml. After 48 hours, medium was removed and replaced with DMEM + 1% FBS. Cells were incubated for 6 hours in the absence or presence of rhPRG4 (100 and 200 μg/ml) and/or CD44 Ab  followed by treatment with IL-1β (1 ng/ml) for 30 minutes.Subsequently, medium was removed and cells were rinsed twice with ice cold PBS (1 ml). Protein extraction reagent  supplemented with protease and phosphatase cocktail inhibitor  and EDTA  was added (300 μl per flask) and cells were collected. Protein samples  were loaded in 10% PAGE pre-cast gels . Following gel electrophoresis and transfer, membranes were blocked using 5% bovine serum albumin (BSA) for 1 hour at room temperature. Membranes were probed for phosphorylated inhibitor kappa B alpha (p-IκBα) or total IκBα using commercially available rabbit antibodies (Abcam). Antibodies were diluted 1:1000 in 5% BSA and incubated with membranes overnight at 4 °C. After washing with Tris-buffered saline Tween 20 (TBS-T), membranes were incubated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit (1:5000) for 1 hour at room temperature. Membranes were also probed for β-Actin using a commercially available β-Actin antibody  (Abcam). Protein bands were developed using SuperSignal West Pico PLUS chemiluminescent substrate (Thermo Fisher Scientific) and visualized using C-Digit Target .OA FLS in 96-well plates (5000 cells per well) in DMEM supplemented with 1% FBS and 1 mM pyruvate were stimulated with IL-1β (20 ng/ml) for 48 hours at 37 °C in the absence or presence of rhPRG4 at a final concentration of 50, 100, or 200 μg/ml. The total volume in each well was 200 μl. Cell proliferation was determined using the Cell Titer 96 AQueous one solution cell proliferation assay  and absorbance at 490 nm was measured. In a separate set of experiments, rhPRG4 (200 μg/ml) treatment was performed in the absence or presence of a CD44 Ab  and cell proliferation was determined as described previously. Data are presented as fold of OA FLS proliferation normalized to untreated controls. Data are presented as the average ± standard deviation of four independent experiments, each with triplicate wells per group.OA FLS and RA FLS (Cell Applications) were grown in DMEM + 10% FBS and used between the third and sixth passages: 20,000 cells per well were plated in sterile 96-well plates and incubated at 37 °C for 48 hours to allow cell attachment. The total volume per well was 200 μl. The medium was changed to DMEM + 1% FBS and cells were treated with IL-1β (1 ng/ml), tumor necrosis factor alpha (TNF-α) , or transforming growth factor beta (TGF-β)  for 72 hours. Subsequently, medium supernatants were collected and PRG4 concentrations were determined using an inhibition ELISA as previously described . PRG4 co-ΔΔCt method [OA FLS  were treated with IL-1β (1 ng/ml) in the absence or presence of rhPRG4 (100 or 200 μg/ml) for 24 hours followed by RNA extraction using Triazol reagent (Thermo Fisher Scientific), and RNA concentrations were determined using a NanoDrop ND-2000 spectrophotometer . cDNA was synthesized using Transcriptor First Strand cDNA Synthesis Kit . Quantitative PCR (qPCR) was performed on Applied Biosystems StepOnePlus Real-Time PCR System (Thermo Fisher Scientific) using TaqMan Fast Advanced Master Mix . The cycle threshold (Ct) value of PRG4  was normalized to the Ct value of GAPDH  in the same sample, and the relative expression was calculated using the 2n = 5)  following knee replacement surgery [Synovial fluid (SF) samples were collected from patients with OA ( surgery . Four of surgery . PRG4 de surgery .IL-1β-induced OA FLS proliferation was conducted as described previously in the absence or presence of pooled OA SF or PRG4-immunoprecipitated OA SF at 20 μl or 40 μl per well (corresponding to 10% or 20% dilution). Cell proliferation was determined using the Cell Titer 96 AQueous one solution cell proliferation assay  and the 490 nm absorbance was measured. Data are presented as fold OA FLS proliferation compared to untreated controls. Data are presented as the average ± standard deviation of four independent experiments, each with duplicate wells per treatment.PRG4 gene expression was determined in PRG4 siRNA and NC siRNA-treated OA FLS as described previously and compared to PRG4 gene expression in untreated control OA FLS. IL-1β stimulation of OA FLS proliferation in control, NC siRNA, and PRG4 siRNA-treated cells was performed as described previously. IL-1β stimulation was performed for 24 hours and cell proliferation was measured as described previously. OA FLS proliferation of the different experimental groups was normalized to untreated control cells. Data are presented as the average ± standard deviation of three independent experiments with duplicate wells per treatment.OA FLS  were treated with a pre-validated PRG4 small interfering RNA (siRNA) (Thermo Fisher Scientific) (25 pmol per well) or a non-targeted negative control (NC) siRNA (25pmoles) (Thermo Fisher Scientific) for 48 hours. Transfection was performed using Lipofectamine RNAiMAX (Thermo Fisher Scientific) per manufacturer’s recommendations. To confirm PRG4 knockdown, MMP-1 (Hs00899658_m1), MMP-2 (Hs00234422_m1), MMP-3 (Hs00968305_m1), MMP-9 (Hs00234579_m1), MMP-13 (Hs00233992_m1), TIMP-1 (Hs00171558_m1), TIMP-2 (Hs00234278_m1), ADAMTS4 (Hs00192708_m1), ADAMTS5 (Hs00199841_m1), IL-1β (Hs00174097_m1), IL-6 (Hs00985639_m1), IL-8 (Hs00174103_m1), TNF-α (Hs00174128_m1), and cycloxygenase-2 (COX2) (HS00153133_m1). All primers and probes are commercially available (Thermo Fisher Scientific). The Ct values of target genes were calculated and normalized to the Ct value of GAPDH in the same sample and relative gene expression was calculated as described previously. Data are presented as fold expression of target genes in rhPRG4-treated OA FLS compared to expression in untreated control OA FLS. Data are presented as the average ± standard error of the mean (SEM) of four independent experiments.OA FLS  were treated with rhPRG4 (100 and 200 μg/ml) for 24 hours followed by RNA isolation, cDNA synthesis and qPCR was performed as described previously. Target genes included MMP-1, MMP-2, MMP-3, MMP-9, MMP-13, TIMP-1, TIMP-2, ADAMTS4, ADAMTS5, IL-1β, IL-6, IL-8, TNF-α and COX2. The Ct values of target genes were calculated and normalized to the Ct value of GAPDH in the same sample, and relative gene expression was calculated as described previously. Data are presented as fold expression of target genes in the IL-1β group, IL-1β + rhPRG4 (100 μg/ml) or IL-1β + rhPRG4 (200 μg/ml)-treated OA FLS groups, compared to expression in the control group. Data are presented as the average ± SEM of four independent experiments.OA FLS  were treated with IL-1β (1 ng/ml) in the absence or presence of rhPRG4 (100 and 200 μg/ml) for 24 hours followed by RNA isolation, cDNA synthesis, and qPCR as described previously. Target genes included In another set of experiments, CD44 neutralization was achieved by pre-incubating OA FLS  with CD44 Ab  for 2 hours followed by treating OA FLS with IL-1β + rhPRG4 (200 μg/ml) for 24 hours followed by RNA isolation, cDNA, and qPCR as described previously. Data are presented as the average ± SEM of four independent experiments.t target gene-CtGAPDH) for each gene of interest in each experimental group and data were graphically presented as fold expression relative to untreated controls using the 2-ΔΔCt method. Continuous variables were initially tested for normality and equal variances. Variables that satisfied both assumptions were tested for statistical significance using Student’s t test or analysis of variance (ANOVA) with Tukey’s post-hoc test for comparisons of two groups and more than two groups, respectively. Variables that did not satisfy the normality assumption were tested using the Mann-Whitney U test or ANOVA on the ranks. The level of statistical significance was set at α = 0.05.The nuclear NFκB p50 and p65 protein levels in OA FLS treatments were normalized to untreated control levels. The average of the absorbance values in untreated control cells across the four independent experiments was used to normalize absorbance values in the different experimental groups across the four independent experiments. A similar approach was used to present OA FLS proliferation. Statistical analyses of gene expression data was performed using ΔCt values . There was no significant difference in cytosolic and nuclear levels of p50 between untreated control and CD44 Ab-treated Prg4+/+ synoviocytes or Prg4-/- synoviocytes. rhPRG4 treatment significantly reduced cytosolic and nuclear p50 levels compared to untreated control Prg4+/+ and Prg4-/- synoviocytes (p < 0.001). A trend towards an increase in cytosolic and nuclear p50 levels in the CD44 Ab + rhPRG4 group compared to the rhPRG4 alone group was observed although it was not statistically significant (p > 0.05).Cytosolic and nuclear p50 in rhPRG4 and CD44 antibody (Ab)-treated and untreated synoviocytes is shown in Fig. Prg4+/+ and Prg4-/- synoviocytes . CD44 Ab-treated Prg4-/- synoviocytes had significantly higher nuclear p65 levels compared to untreated controls (p < 0.01). rhPRG4 treatment significantly reduced cytosolic and nuclear p65 levels compared to untreated control Prg4+/+ and Prg4-/- synoviocytes (p < 0.001). Similarly, a trend towards an increase in cytosolic and nuclear p65 levels in the CD44 Ab + rhPRG4 group compared to the rhPRG4 alone group was observed although it was not statistically significant (p > 0.05).Cytosolic and nuclear p65 protein in rhPRG4 and CD44 Ab-treated and untreated synoviocytes is shown in Fig. tes Fig. . rhPRG4 p < 0.001) . Similarly, rhPRG4 (200 μg/ml) treatment significantly reduced IL-1β induced p50 and p65 nuclear translocation (p < 0.001). There was no significant difference in the p50 and p65 nuclear level in the IL-1β + CD44 Ab group and the IL-1β group. NFκB p50 and p65 nuclear levels in the IL-1β + rhPRG4 (200 μg/ml) + CD44 Ab group were significantly higher than corresponding levels in the IL-1β + rhPRG4 (200 μg/ml) group (p < 0.001) and were not significantly different from nuclear levels in the IL-1β + CD44 Ab group. The ability of rhPRG4 to lower NFκB nuclear levels was inhibited by co-incubation with CD44 Ab, which showed no difference from the IL-1β group.IL-1β treatment significantly increased NFκB p50 and p65 nuclear levels in OA FLS compared to untreated OA FLS (01) Fig. . rhPRG4 A representative p-IκBα western blot in control and IL-1β-treated OA FLS in the presence or absence of rhPRG4 with or without CD44 Ab co-treatment is shown in Fig. p < 0.001) . CD44 Ab and rhPRG4 co-treatment resulted in significantly greater OA FLS proliferation compared to rhPRG4 treatment (p < 0.01) and was not significantly different from OA FLS proliferation in the IL1β alone group. Finally, there was no significant difference in OA FLS proliferation between the IL-1β + CD44 Ab group and the IL-1β group.IL-1β induced OA FLS proliferation over 48 hours (01) Fig. . rhPRG4 p < 0.001) . TGF-β enhanced PRG4 production by OA FLS compared to control untreated cells (p < 0.001). IL-1β, TNF-α, and TGF-β did not significantly alter PRG4 production by RA FLS compared to untreated cells.Basal PRG4 production in OA FLS was significantly higher than PRG4 production by RA FLS (01) Fig. . IL-1β aPRG4 gene expression in OA FLS . IL-1β reduced PRG4 gene expression in OA FLS (p < 0.001). PRG4 expression in the IL-1β + rhPRG4 (100 μg/ml) group was significantly higher than PRG4 expression in IL-1β alone (p < 0.001) and the IL-1β + rhPRG4 (200 μg/ml) group (p < 0.01). Alternatively, there was no significant difference in PRG4 gene expression between the IL1β group and the IL1β + rhPRG4 (200 μg/ml) group. Finally, PRG4 gene expression in the IL-1β + rhPRG4 (200 μg/ml) treatment was significantly lower than PRG4 expression in untreated control OA FLS (p < 0.001).rhPRG4 (100 μg/ml) treatment did not alter basal FLS Fig. . In contp < 0.001) or no SF treatment (p < 0.01). OA SF (20% dilution) significantly reduced IL-1β-induced OA FLS proliferation (p < 0.01) . NC siRNA treatment did not significantly alter PRG4 gene expression in OA FLS compared to untreated OA FLS  compared to untreated or NC siRNA-treated OA FLS. PRG4 siRNA-treated OA FLS displayed significantly higher basal cell proliferation compared to NC siRNA-treated OA FLS (p < 0.01) or untreated OA FLS (p < 0.001) over 24 hours .Mean PRG4 concentration in OA SF after PRG4 immunoprecipitation was 25.11 ± 3.18 μg/ml, compared to 280.43 ± 14.76 μg/ml in OA SF with no PRG4 immunoprecipitation. OA SF treatment at 10% dilution did not significantly alter IL-1β-induced OA FLS proliferation Fig. . PRG4-de01) Fig. . PRG4-deFLS Fig. . In conturs Fig. . Similarp < 0.001). The magnitude of reduction in gene expression was approximately 45% for MMP-1 compared with 58% for MMP-3 and 62% for MMP-13. rhPRG4 (200 μg/ml) significantly increased basal expression of TIMP-2 (p < 0.001) (~50% increase) and did not alter the expression of MMP-2, TIMP-1, ADAMTS4, or ADAMTS5.rhPRG4 (100 μg/ml) did not significantly alter basal expression of MMP-1, MMP-2, MMP-3, MMP-9, MMP-13, TIMP-1, TIMP-2, ADAMTS4, or ADAMTS5 Fig. . rhPRG4 MMP-1, MMP-3, MMP-9, MMP-13, ADAMTS4, and ADAMTS5 gene expression in OA FLS (p < 0.001)  (~35% reduction) . The magnitude of reduction in gene expression was approximately 51% for MMP-1 compared with 76% for MMP-3, 49% for MMP-9, 77% for MMP-13, and 60% for ADAMTS5. rhPRG4 (100 or 200 μg/ml) treatment did not alter ADAMTS4 expression. Expression of MMP-9, MMP-13 and ADAMTS5 genes was significantly higher in the IL-1β + rhPRG4 + CD44 Ab group compared to the IL-1β + rhPRG4 group (p < 0.001)  Fig. . rhPRG4 on) Fig. . rhPRG4 01) Fig. . In contIL-1β, IL-6, IL-8, TNF-α, and significantly increased basal COX2 gene expression (p < 0.001) (~96% increase)   and significantly increased basal expression of COX2 (~61% increase) (p < 0.001). rhPRG4 (100 or 200 μg/ml) did not alter basal expression of TNF-α.rhPRG4 (100 μg/ml) did not significantly alter basal expression of se) Fig. . rhPRG4 IL-1β, IL-6, IL-8, TNF-α, and COX2 gene expression (p < 0.001)  group and the IL-1β group . The magnitude of reduction in gene expression was approximately 67% for IL-6 compared with 55% for IL-8 and 73% for COX2. Expression of IL-6, IL-8 and COX-2 genes was significantly higher in the IL-1β + rhPRG4 + CD44 Ab group compared to the IL-1β + rhPRG4 group (p < 0.001)  Fig. . There woup Fig. . rhPRG4 01) Fig. . FinallyPrg4-/- synoviocytes were higher compared to Prg4+/+ synoviocytes. Additionally, Prg4+/+ and Prg4-/- synoviocytes had similar levels of p65 subunits in the cytosol while the latter had elevated cytosolic p50 subunit levels. Enhanced nuclear localization of NFκB in Prg4-/- synoviocytes may explain their pro-inflammatory and proliferative capacity under unstimulated conditions and in response to inflammatory stimuli compared to their wildtype counterparts [Prg4-/- synoviocytes and Prg4+/+ synoviocytes. Interestingly, rhPRG4 treatment reduced cytosolic levels of p50 and p65 proteins in Prg4+/+ and Prg4-/- synoviocytes. This is a unique biological effect of rhPRG4 and may indicate that rhPRG4 functions to reduce the total cellular pool of NFκB, accounting for its anti-proliferative and anti-inflammatory activities. The observed effect of rhPRG4 in relation to nuclear NFκB translocation in murine synoviocytes may not be entirely due to its binding to CD44 receptor as neutralization of CD44 did not completely abolish the effect of rhPRG4.In this work, we have shown that nuclear levels of NFκB p50 and p65 in terparts . rhPRG4 Prg4-/- mice and the partial regression of synovial hyperplasia upon Prg4 re-expression [OA synovitis is characterized by synovial hyperplasia with an increased number of synovial lining cells, neovascularization and inflammatory cell infiltration –31. Patipression , 8.PRG4 expression by articular chondrocytes and synovial fibroblasts is modulated by mechanical and biological stimuli. Shear upregulates PRG4 gene expression in articular cartilage [PRG4 expression is reduced by IL-1β and is increased by TGF-β and TGF-β-linked PRG4 accumulation is counterbalanced by IL-1β [artilage , 40 and artilage . Cartilaartilage –44. In sby IL-1β , 45. In by IL-1β .PRG4 expression in OA synoviocytes following IL-1β challenge, rhPRG4 at a higher concentration did not antagonize IL-1β downregulation of PRG4 in OA synoviocytes. Moreover, exogenous PRG4 appeared to directly regulate PRG4 production by OA synoviocytes. At a low concentration, rhPRG4 treatment did not change basal expression of PRG4 in OA synoviocytes. Alternatively, exogenous PRG4 exhibited negative feedback on PRG4 expression in OA synoviocytes as rhPRG4 treatment downregulated basal PRG4 expression in OA synoviocytes. Our observations extend the understanding of how endogenous PRG4 expression is modulated by exogenous PRG4 in articular cartilage and in in vivo models of posttraumatic arthritis [rhPRG4 exhibited a bimodal concentration-dependent response related to its ability to modulate PRG4 expression in IL-1β-stimulated OA synoviocytes. While rhPRG4 at a low concentration restored rthritis , 46.MMP-1, MMP-3, MMP-9, MMP-13, TIMP-1, aggrecanase-1, aggrecanase-2, IL-1β, TNF-α, IL-6, IL-8, and COX2. Our results are in agreement with previous studies that reported on the effect of IL-1β on OA synoviocytes [MMP-1, MMP-3, MMP-9, MMP-13, aggrecanase-2, IL-6, IL-8, and COX2 in IL-1β-stimulated OA synoviocytes. This anti-inflammatory effect of rhPRG4 was partially mediated by the CD44 receptor, as blocking this receptor abolished the effect of rhPRG4 on MMP-9, MMP-13, aggrecanase-2, IL-6, IL-8, and COX2 expression.Synoviocytes play an important role in driving OA pathogenesis. OA synoviocytes express a wide-range of matrix-degrading catabolic enzymes and cytokines. In our study, and in response to IL-1β stimulation, OA synoviocytes induced the expression of viocytes , 48. rhPMMP-1, MMP-3, MMP-9, and MMP-13, which are involved in synovial fibroblast proliferation and migration [COX2 in IL-1β-stimulated OA synoviocytes, whereas rhPRG4 marginally increased the expression of COX2 in unstimulated OA synoviocytes. The significance of this observation in the overall biological effect of rhPRG4 remains to be elucidated.rhPRG4 dose-dependently downregulated basal expression of OA-associated enzymes and cytokines. Specifically, rhPRG4 downregulated the expression of igration , 50 and Prg4-/- synoviocytes and Prg4+/+ synoviocytes, despite the fact that Prg4-/- synoviocytes display enhanced CD44 protein expression compared to Prg4+/+ synoviocytes [In summary, we herein report on the biological effect of PRG4 on fibroblast-like synoviocytes derived from patients with OA. To our knowledge, this is the first report that delineates the biological role of PRG4 in regulating OA synoviocyte function. Our study adds to existing literature that describes a non-mechanical cartilage-protective effect of PRG4 through upregulation of hypoxia-inducible factor (HIF3α) . An inteviocytes . The CD4Our findings may be limited by the small number of OA SF aspirates included in the study. Additionally, we have not studied the biological effect of rhPRG4 on FLS from patients with varying degrees of synovitis. A third limitation of our study is that we cannot exclude the possibility that other SF proteins were co-precipitated in the PRG4 immunoprecipitation experiments.PRG4 is a glycoprotein secreted by synovial fibroblasts that regulates basal and IL-1-induced expression of matrix metalloproteinases that are involved in synovial proliferation and cartilage destruction. The full-length recombinant form of PRG4 inhibits proliferation of synovial fibroblasts through a mechanism that involves the CD44 receptor. Additionally, depletion of native PRG4 in OA SF stimulates synoviocyte proliferation.


H-Cµ1-Cµ2-Cγ3 and VH-Cµ1-Cµ2-Hinge-Cγ2-Cγ3 functionally bound to the antigen, VH-Cγ1-Hinge-Cγ2-Cγ3, VH-Cµ1-Hinge-Cγ2-Cγ3, and VH-Cµ1-Cµ2-Cγ2-Cγ3 did not. Gel filtration analyses revealed that the functional molecules tend to form multimers and the multimeric forms retained antigen binding activity. Furthermore, the mutation of amino acid residue p.309Q > C of mouse IgG and addition of IgM tailpiece to the C-terminus of the molecules induced multimer formation, dramatically enhanced antibody functionality and all non-functional molecules became strongly functional. The functional molecules could be bound by protein A/protein G and other IgG specific reagents and therefore should be useful for further characterization of the antigen. Our study revealed that multimerization of converted IgM is functionally important for antigen binding activity of engineered IgM/IgG chimeric antibodies.Monoclonal antibodies offer great tools for research. We encountered a potentially useful mouse IgM monoclonal antibody whose antigen is expressed in normal skin but lost in human skin cancer. Because IgM is difficult to work with and the antigen was unknown, we decided to convert the IgM (µ) to IgG (γ) version. After cDNA for the antibody was obtained by RACE PCR, we made a series of molecules with different combinations of IgM and IgG domains. Whereas V Although IgM binds to bacterial protein L3, the binding occurs through the light chain3 and, in our hands, it is inefficient for immunoprecipitation, particularly when the antigen is large and there is a probability of steric hindrance. Therefore, when an antibody is of IgM type and RNA from its hybridoma is available, it is often desirable to convert it into an IgG format by cloning its cDNA to explore its full potential for many purposes.IgG by far is the most common Ig used in research and many reagents are available for use with IgG. IgM, being a very large molecule, is difficult to work with in terms of reagent availability, purification and specificity. IgM does not bind to common bacterial protein A and protein G4. Immunocytochemical studies revealed that the antibody recognized an antigen associated with human keratinocyte plasma membrane and, in particular, with desmosomes5, which are cell adhering apparatus that maintain normal skin integrity6. Studies also showed that the antigen is expressed in normal human skin but defective in human squamous cell carcinoma (SCC)7, suggesting that the antibody recognizes a potentially important antigen in the skin. Biochemical analyses revealed that the antigen is different from known desmosomal proteins8 and therefore may react with an unidentified novel molecule.An IgM monoclonal antibody, which is named KM48 and recognizes a skin related antigen, was generated after immunization of mouse with normal human epidermal cell suspension11. However, conversion of IgM to IgG is problematic in some situations12.However, attempts to identify the antigen for KM48 have failed, and the failure was mainly attributed to the antibody being of IgM class, which has limited usability for applications, such as immunoprecipitation. To overcome the obstacle of limited usability of the IgM antibody, we decided to convert the IgM antibody into an IgG format to enable us to identify or confirm its antigen using immunoprecipitation. Conversion of one form of Ig to another is usually done by joining the variable regions of the antibody of interest to the constant regions of a desired isotypeIn this study, we constructed and produced functional IgM/IgG hybrid molecules of KM48 by combining different portions of the µ chain and different portions of the γ chain. Eventually, we identified Ig domains and structures that are functionally required for antigen binding in engineered IgM/IgG antibodies.13, which was excluded from further analysis. PCR was also used to successfully amplify the constant region of mouse IgG2a from normal mouse spleen cDNA  PCR, approximately 2 kb product was obtained for IgM heavy chain and a 0.7 kb product was obtained for the light chain from our hybidoma RACE cDNA pool  VH-Cµ1-Cµ2-Cγ3 did not bind to protein G, all other molecules bound to protein G, suggesting that Cγ2 and Cγ3 domains of IgG are required for protein G binding  VH-Cµ1-Cµ2-Cµ3-Cµ4  VH-Cµ1-Cµ2-Cγ3, moderately to (v) VH-Cµ1-Cµ2-Cγ2-Cγ3 and (vi) VH-Cµ1-Cµ2-Hinge-Cγ2-Cγ3, but did not bind to (ii) VH-Cγ1-Hinge-Cγ2-Cγ3 and (iii) VH-Cµ1-Hinge-Cγ2-Cγ3  VH-Cµ1-Cµ2-Cµ3-Cµ4 and (iv) VH-Cµ1-Cµ2-Cγ3 Cγ3 Fig.  were alsCγ3 Fig. . On the Cγ3 Fig. , (iii) VCγ3 Fig.  and (v) Cγ3 Fig.  all failWe also compared the binding activities of the functional molecules with that of the original hybridoma-derived IgM molecule. Immunofluorescence of normal human skin revealed that the reactivities of the recombinant IgM/IgG molecules were similar to that of the original IgM molecule Fig. .H-Cµ1-Cµ2-Cµ3-Cµ4, (iv) VH-Cµ1-Cµ2-Cγ3 and (vi) VH-Cµ1-Cµ1-Hinge-Cγ2-Cγ3 showed positive reactivity, while (ii) VH-Cγ1-Hinge-Cγ2-Cγ3, (iii) VH-Cµ1-Hinge-Cγ2-Cγ3 and (v) VH-Cµ1-Cµ2-Cγ2-Cγ3 were negative  VH-Cµ1-Cµ2-Cγ3 and (vi) VH-Cµ1-Cµ2-Hinge-Cγ2-Cγ3 are functional but (ii) VH-Cγ1-Hinge-Cγ2-Cγ3, (iii) VH-Cµ1-Hinge-Cγ2-Cγ3 and (v) VH-Cµ1-Cµ2-Cγ2-Cγ3 are not functional.We also performed Western blotting of normal human epidermal extract to examine the reactivities of the antibodies. Similar to the results of immunofluorescence, the Western blot analysis showed that (i) Vive Fig. . The com15, we performed gel filtration to examine the multimeric state of the recombinant antibodies. We chose the 3 molecules containing the Cµ2 domain i.e. (iv) VH-Cµ1-Cµ2-Cγ3, (v) VH-Cµ1-Cµ2-Cγ2-Cγ3 and (vi) VH-Cµ1-Cµ2-Hinge-Cγ2-Cγ3 because both functional IgM/IgG chimeric antibodies, (iv) VH-Cµ1-Cµ2-Cγ3 and (vi) VH-Cµ1-Cµ2-Hinge-Cγ2-Cγ3 have the Cµ2 domain. By gel filtration analysis, we found that the functional molecules, but not the non-functional molecule, tend to form multimers  VH-Cµ1-Cµ2-Hinge-Cγ2-Cγ3. The amount of monomer was very low in (iv) VH-Cµ1-Cµ2-Cγ3  VH-Cµ1-Cµ2-Cγ2-Cγ3  VH-Cµ1-Cµ2-Hinge-Cγ2-Cγ3 to a lesser extent and very few fractions in (v) VH-Cµ1-Cµ2-Cγ2-Cγ3  VH-Cµ1-Cµ2-Hinge-Cγ2-Cγ3 but not in (v) VH-Cµ1-Cµ2-Cγ2-Cγ3  VH-Cµ1-Cµ2-Hinge-Cγ2-Cγ3  VCγ3 Fig. . None ofkin Fig. . We confkin Fig. , suggest17. In mouse, the amino acid residue at this position was 309Q. Because multimeric forms of our molecules showed positive reactions  of human IgG and addition of IgM tail piece (µTP), consisting of the last 18 amino acids of IgM to the C-terminus can induce multimerization of human IgGons Figs  and 5, wH-Cµ1-Cµ2-Hinge-Cγ2-Cγ3 with double mutation  induced multimer formation of all molecules tested in various patterns Fig. . Inclusiion Fig. . The mulion Fig. . The larBy immunofluorescence analyses of normal human skin, we found that all molecules became functional to varying degrees upon addition of each and both mutation(s) Fig. . Interes19. In most cases, the converted IgM fail to function12. Our studies suggest that these failures can be attributed to the lack of the multimeric forms in recombinant IgM/IgG molecules.In this study, we have successfully converted a mouse IgM monoclonal antibody into functional IgG-like molecules. We demonstrated that multimerization of the molecules is important for antigen binding. Only a few studies have documented the successful conversion of IgM into an IgG formatH-Cµ1-Cµ2-Hinge-Cγ2-Cγ3 but not (v) VH-Cµ1-Cµ2-Cγ2-Cγ3 was functional. The difference between the 2 molecules is the presence or absence of the 16-amino acid hinge domain of mouse IgG2a. The gel filtration analysis clearly showed that (vi) VH-Cµ1-Cµ2-Hinge-Cγ2-Cγ3, but not (v) VH-Cµ1-Cµ2-Cγ2-Cγ3, forms multimers  VH-Cµ1-Cµ2-Cγ3 formed more multimers than (vi) VH-Cµ1-Cµ2-Hinge-Cγ2-Cγ3  VCγ3 Fig. . It has ons Fig. , suggest17, but it was not demonstrated whether multimerization of IgG was required for functional antigen binding. The mutations greatly enhanced both multimerization and functionality of the hybrid molecules. Surprisingly and interestingly, even non-functional molecules became strongly functional after introducing the 2 mutations simultaneously. These results suggest that murine IgM can be converted into functional IgG-like molecules by mutating amino acid 309Q to 309 C and attaching the µTP to the C-terminus of the resultant molecules.To further improve multimerization, we introduced 2 previously described mutations into our molecules Fig. . It was 18. The functional antibody was in the single-chain Fv format18, but it was not clear whether the antibody formed multimers. In another study, a functional IgG form of anti-CD19 IgM antibody was made and expressed in SF9 insect cells19. Although the antibody was functional, it failed to be secreted from the cells, and it was also not clear whether the antibody formed multimers19. Thus, in this study, we succeeded in making IgG-like molecules from IgM, whose functionality is based on multimer formation. To the best of our knowledge, this is the first study demonstrating that multimerization is required for binding to the antigen. It is not clear whether this phenomenon is unique to our specific antibody or whether it is universal, but it must be worthy trying constructing multimers when an engineered IgG converted from IgM has no function.In a previous study, functional IgG form of anti-CD20 IgM antibody was made, after spatial conformations were determined with computer-guided homology modeling methodsH-Cµ1-Cµ2-Cγ3 forms multimers readily  VH-Cµ1-Cµ2-Cγ3  Vily Fig.  and reacily Fig. . Howeverily Fig. , which iers Fig. , and is Cγ3 Fig. . Some ofIn conclusion, we have successfully converted an IgM molecule into functional IgG-like molecules, which reacted strongly with a skin-related antigen. We demonstrated that the multimerization is important for functional binding of the chimeric antibodies to the antigen. The results in our study should provide important insights for future studies that aim to convert IgM to IgG-like antibody for research and therapeutic applications.The studies were approved by the internal review board of Kurume University School of medicine. All described experiments were performed following guidelines of ethical committee of Kurume University School of Medicine and in accordance with Declaration of Helsinki Principles. Informed consent was obtained from skin donors. Adjacent normal skin obtained during operation for other skin problems was used in this study. Unwanted human foreskin was also used.4. The cells were cultured in RPMI-1640 medium supplemented with 20% fetal calf serum (FCS). Total RNA was isolated with RNeasy Kit  following the manufacturer’s instructions. cDNA synthesis and PCR was performed using the RNA-ligase mediated GeneRacer RACE Kit  according to the manufacturer’s instructions. cDNA also was synthesized from total mouse spleen RNA  with Superscript III reverse transcriptase (Invitrogen) and oligo (dT) primer. For PCR, forward primer included in the kit was used along with 5′-GGTTGAGCGCTAGCATGG-3′ and 5′-GGCGTCTCAGGACCTTTGTC-3′ reverse primers to amplify full-length mouse IgM heavy and light chains, respectively. PCR was performed with high fidelity PrimeStar Max DNA polymerase . The amplified products were cloned into PCR-blunt vector (Invitrogen) and sequenced to determine the variable regions of heavy and light chains.Hybridoma secreting an IgM antibody against a human skin antigen was established as described previouslyatcACCATGGGATGGAGCTGG-3′ was used along with the listed IgM reverse primers to amplify IgM portions. IgG2a reverse primer, 5′-tacaagatctttgatatcGGTGTCTCTTTGGACCTGAGAGT-3′ was used along with the listed IgG forward primers to amplify IgG portions. For full-length IgM, 5′-tacaagatctttgatatcGGTTGAGCGCTAGCATGG reverse primer was used. For the kappa light chain, 5′-cggagtatacggatccAAAATGATGAGTCCTGCCCAG-3′ (forward) and 5′-taggcgtacgggatccGGCGTCTCAGGACCTTTGTC-3′ (reverse) primers were used. The primers were designed with additional 15 bases to the 5′-end (non-underlined lower case) to facilitate cloning by sequence- and ligation-independent cloning technology. Additional bases (underlined) were added to recreate BamHI or EcoRV restriction enzyme site. PCR was performed with PrimeStar Max DNA polymerase (Takara Bio). Amplified fragments were cloned simultaneously into pVITRO2-MCS dual expression vector  that was digested with Fastdigest BamHI/EcoRV , using Gibson assembly  or In-fusion HD  cloning kits. The heavy chain was inserted into BamHI site under control of mouse EF1α promoter and the light chain was inserted into EcoRV site under control of rat EF1α promoter. The hygromycin resistance gene in the original vector was replaced with puromycin resistance gene in some cases. The assembled fragments were transformed into NEBstable competent cells (New England BioLab) and transformants were selected on low salt LB agar plates supplemented with 25 µg/ml puromycin or 50 µg/ml hygromycin (InvivoGen) and 50 mM potassium phosphate buffer, pH 8.0. The sequences were verified by direct DNA sequencing.Various primer pairs (Supplementary Table Primers 5′-CCCATCtgtCACCAGGACTGGATGAGT-3′ (forward) and 5′-CTGGTGacaGATGGGGAGGGCACTGAC-3′ (reverse) were used to mutate the molecules to introduce 309 C mutation (lower case in primer). Whole circular plasmid was amplified as above. For introduction of µTP, primers 5′-atgtctgacacaggcggcacctgctatTGAGCTCAGCACCCACAAAAC-3′ (forward) and 5′-gatcagggagacattgtacagtgtgggTTTACCCGGAGTCCGGGA-3′ (reverse) were used. Half each of the 54-bp µTP (lower case) was attached to the forward and reverse primers. Primers were phosphorylated and PCR was performed as above. Whole linear plasmid PCR product was self-ligated using Ligation High kit (Toyobo). Plasmid was obtained after transformation and selection as described above. Sequences were confirmed by direct DNA sequencing.Lenti-X 293 T cells (Clontech), a form of HEK293T cells, were cultured in DMEM supplemented with 10% FCS. For transfections, cells were seeded into 12-well plates and transfected with 2.5 µg of plasmid DNA using 0.75 µl of Xfect transfection reagent (Clontech). Stable cells were generated by selection with 0.4–0.8 µg/ml puromycin or 200 µg/ml hygromycin and resistant cells were expanded and cultured until 80% confluent. After rinsing with PBS, medium was switched to MAB serum-free medium supplemented with 1% KOSR  and cultured for 6 days. Antibodies were purified from culture supernatant using protein A , Protein G  or protein L (GeneScript) after buffer was exchanged to 20 mM sodium phosphate buffer, pH 7.2, containing 150 mM NaCl using Amicon centrifugation units. Antibodies were eluted with 100 mM glycine, pH 2.7, and immediately neutralized with 22 µl of 2 M Tris solution, per 1 ml of eluate.Samples were mixed with 5X SDS sample buffer with or without 5% β-mercaptoethanol, heated for 5 minutes at 80 °C (non-reducing) or 95 °C (reducing) and, separated by 5–20% gradient PAGE gel . Proteins were transferred onto nitrocellulose membrane using iBlot 2 transfer device (Invitrogen). Membranes were blocked with 4% skim milk in TBS and incubated with HRP- or ALP- conjugated anti-mouse IgG (H + L or L) or anti-mouse IgM. After washing, proteins bands were visualized with TMB for HRP (ATTO) or 1-Step NBT/BCIP for ALP .Serum-free cell culture supernatant was diluted 5X in 20 mM sodium phosphate buffer, pH 7.2, containing 150 mM NaCl and added to washed protein G beads (Merck Millipore). The suspension was rotated at room temperature (RT) for 30 minutes. After washing extensively, bound material was eluted by heating at 80 °C in non-reducing SDS sample buffer, separated by SDS-PAGE and detected by immunoblotting as described above.Normal human skin sections were blocked with 1% BSA/PBS for 30 min and incubated with antibodies at RT for 1 hour. After washing with PBS, sections were incubated with Alexa Fluor 488-conjugated anti-mouse IgG (H + L) (Invitrogen) diluted 1:1000 with 1% BSA/PBS. The sections were observed with BX51 fluorescence microscope .27. Epidermis was homogenized in 20 mM Tris, pH 7.2, containing 150 mM NaCl and 1% Triton X-100. After centrifugation to remove insoluble materials, 20 μg total protein in the supernatant was used per lane in Western blotting. Blotted membranes were incubated with recombinant antibodies at RT for 2 hours. After washing, membranes were incubated with ALP-conjugated anti-mouse IgG (H + L), washed and detected with 1-Step NBT/BCIP.Human skin, obtained at circumcision, was processed as described previouslyWe developed an ELISA system to monitor the amount of recombinant antibodies in various samples. Maxisorp plates were coated with 100 μl of 0.5 μg/ml anti-mouse kappa light chain . Plates were blocked at RT for 3 hours with TBS containing 10% adult bovine serum (Sigma), 5% sucrose and 0.05% sodium azide, dried at RT for 2 hours, stored at 4 °C and used with 1 month. Antibody samples were diluted in TBS containing 2% adult bovine serum and 0.01% Tween 20. Diluted samples were incubated with the plates at RT for 1 hour and washed 4 times with TBS containing 0.05% Tween 20. The plates were incubated with 1:4,000 dilution of HRP-conjugated anti-mouse IgM  or 1: 20,000 anti-mouse IgG  at RT for 1 hour. After washing, color was developed with TMB. Reactions were stopped with 0.5 M HCl and the optical density was read with plate reader at 450 nm. Antibody amounts were estimated with the built-in software of the plate reader using appropriate Ig as standard ranging from 3.125 to 200 ng/ml.Gel filtration analysis was carried out using Superdex 200 Increase 10/300 GL column  in PBS (pH 7.4) at 0.5 ml/min. 100 μl of purified antibodies was injected and optical density at 280 nm was continuously monitored by an AKTApurifier system . Samples were fractionated manually to measure the concentrations by ELISA and the antigen binding activity by immunofluorescence.All data generated or analyzed during this study are included in this published article and its Supplementary Information files.Supplementary Information


Diffusion-Tensor cardiovascular magnetic resonance (DT-CMR) is capable of mapping myocardial fiber orientation . It has been demonstrated in myocardial infarction (MI) murine models that DT-CMR can identify the effects of stem cell therapy on myocardial fiber orientation . HoweverMI was induced in 7 Yucatan mini pigs by balloon occlusion of the mid-LAD for 2.5 hours. The MI was allowed to heal for 4 weeks for all pigs defining baseline. Group 1 (N = 4) were treated with exosome proteins derived from CDCs. Group 2 was given a placebo.2, 8 avg, 2 avg/BH), and WH viability CMR . Viability and function CMR yielded scar size (SS) and ejection fraction (EF) [CMR was performed at baseline and 4 weeks after treatment on a 3T Siemens Verio with the following: whole-heart 2D multi-slice (WH) morphological CMR, WH function CINE CMR, 3 short axis slice DT-CMR  before and after treatment (p < 0.017 significance). Change (Δ) in MD, FA, and HATS were correlated (R^2) with ΔSS and ΔEF.2/ms, 0.01 ± 0.03, and 0.05 ± 0.15°/%depth, respectively), while SS was significantly reduced . In contrast, Group 2 (placebo) exhibited significant (p < 0.01) adverse changes with decreased EF (Δ: -4 ± 2%), increased SS (Δ: 3 ± 1%), decreased FA (Δ: -0.03 ± 0.02), and less helical HATS (-1.2 ± 0.1 vs -0.9 ± 0.2°/%depth). ΔMD and ΔFA weakly correlated with ΔEF  and ΔSS . However, ΔHATS significantly (p < 0.01) correlated highly with ΔEF (R^2: 0.85) and ΔSS (R^2: 0.67).For Group 1 (treated), EF, MD, FA, and HATS did not significantly change (Δ: -1 ± 2%, -0.1 ± 0.2 umIn a MI porcine model, in vivo DT-CMR revealed that myocardial fiber orientation was preserved with CDC-derived exosome treatment and adversely changed with placebo treatment consistent with observed viability and function changes. Furthermore, changes in helix transmurality highly correlated with changes in viability and function.


Within a single coordination polymer strand, the axially chiral 2,2′-dithiobis(pyridine N-oxide) ligands are homochiral, but the enantiomeric conformation is present in adjacent strands. Within a coordination polymer strand, the iodido ligands point towards the centroids of the aromatic rings of the pyridine N-oxide moieties in the coordination sphere of HgII. Moreover, intra-strand C—H⋯O and C—H⋯I interactions, and inter-strand short S⋯I and S⋯O contacts are observed.The title compound, [HgI Recently, we reported one-dimensional coordination polymers from 2,2′-dithiobis(pyridine N-oxide), and ZnII and CdII halides  as a spacer ligand (i.e. involving both pyridine N-oxide moieties as coordinating groups). As a continuation of this work, we herein report the crystal structure of a one-dimensional coordination polymer formed from 2,2′-dithiobis(pyridine N-oxide) and HgI2.Research into one-dimensional coordination polymers has been an active field of research, not only due to the usually easy and straightforward synthesis, but also due to interesting structural features and introduction of these compounds as new materials such as coordination polymeric gels, fibres and nanostructures  as bridging ligands in a μ-κ2O:O′ coordination mode. Fig. 1II ion. The HgII ion is tetracoordinated by two iodide ligands and two O atoms of the bridging ligands, with a coordination sphere that is best described as a severely distorted tetrahedron or a seesaw form °.The title compound, (I)1-screw axis parallel to the b axis  exhibits exclusively either the right-handed P or the left-handed M conformation, i.e. the bridging ligands in each coordination polymer chain are homochiral. The centrosymmetric crystal structure (space group P21/n) features, however, both enantiomeric conformations in adjacent strands, as shown in Fig. 2The strand of the coordination polymer propagates along a 2is Fig. 2. Within N-oxide moieties in the coordination sphere of HgII, but I⋯π interactions are not observed. The I⋯Cg distances are long [I1⋯Cg1 = 3.940 (2) Å and I2⋯Cg2ii = 4.205 (2) Å] and the Hg—I⋯Cg angles are acute [Hg1—I1⋯Cg1 = 77.94 (3)° and Hg1—I2⋯Cg2ii = 68.79 (3)°] , connect adjacent chains into a layer structure parallel to  in the Cambridge Structural Database  ]n  ]n diiodidomercury(II) °, which is similar to that of 155.113 (16)° observed in (I)The isomorphous series of one-dimensional Zn2 in 2 ml of methanol was mixed with a solution of 12 mg (0.048 mmol) 2,2′-dithiobis(pyridine N-oxide) (Acros Organics) in 8 ml of methanol. The reaction mixture was left at room temperature and the solvent was allowed to evaporate slowly. Colourless crystals of (I)ca four weeks.A solution of 20 mg (0.044 mmol) HgI2I2(C5H4NOS)2] (C5H4NOS− is pyrithionate) in a preliminary X-ray analysis.After prolonged standing, colourless crystals of a second product appeared in the crystallization vessel, which were identified as [HgUiso(H) = 1.2Ueq(C). The C—H bond lengths were set at 0.95 Å.Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S2056989018003055/is5492sup1.cifCrystal structure: contains datablock(s) global, I. DOI: 10.1107/S2056989018003055/is5492Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989018003055/is5492Isup3.molSupporting information file. DOI: 1825194CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Scientific Reports 10.1038/s41598-017-07033-x, published online 31 July 2017Correction to: B/f.u.)’. The correct Figure 3a appears below as Figure In Figure 3a, the y-axis ‘M (arbitrary units)’ is incorrectly given as ‘M (µ


In all three structures, the nitramide is mainly  H-tetrazol-5-yl)methyl]nitramide, (I), C4H6N10O2, there are two intramolecular N—H⋯O hydrogen bonds. In the crystal, N—H⋯N hydrogen bonds link molecules, forming a two-dimensional network parallel to (-201) and weak C—H⋯O, C—H⋯N hydrogen bonds, and intermolecular π–π stacking completes the three-dimensional network. The anion in the molecular salt, triaminoguanidinium 5-({[(1H-tetrazol-5-yl)methyl](nitro)amino}methyl)tetrazol-1-ide, (II), CH9N6+·C4H5N10O2−, displays intramolecular π–π stacking and in the crystal, N—H⋯N and N—H⋯O hydrogen bonds link the components of the structure, forming a three-dimensional network. In the crystal of diammonium bis[(tetrazol-1-id-5-yl)methyl]nitramide monohydrate, (III), 2NH4+·C4H4N10O22−·H2O, O—H⋯N, N—H⋯N, and N—H⋯O hydrogen bonds link the components of the structure into a three-dimensional network. In addition, there is intermolecular π–π stacking. In all three structures, the central N atom of the nitramide is mainly sp2-hybridized. Bond lengths indicate delocalization of charges on the tetrazole rings for all three compounds. Compound (II) was found to be a non-merohedral twin and was solved and refined in the major component.In the molecule of neutral bis[(1 Interest is high in improving energetics to reduce environmental impact and to improve safety and performance cis to one another er Fig. 3.This unusual conformation is likely due to the intramolecular π–π stacking interactions observed between the tetrazole rings [centroid–centroid distance = 3.4978 (10) Å]. Both tetrazole rings are nearly planar with an r.m.s. deviation of 0.0007 for the protonated ring and 0.00004 Å for the deprotonated ring.et al., 1987For all three compounds, the C—N (ranging from 1.321 to 1.338 Å) and N—N (ranging from 1.301 to 1.362 Å) bond lengths for the tetrazole rings were found to match more closely with bonds of multiple character than of discrete single and double bonds, signifying a delocalized aromatic system (Allen In structure (II)n, the angle between the Namine—Nnitro vector and the Cmethylene1/Namine/Cmethylene2 plane, described by Allen et al. (1995n of 13.1 (5)° for vector N2–N1 and plane C11/C5/N1, structure (II)n of 26.11 (18)° for vector N14–N7 and plane C6/N7/C8, and structure (III)n of 6.21 (11)° for vector N7A–N7 and plane C6/N7/C8. This indicated the hybridization of the central nitrogen atom is mainly 2sp hybridized for all three structures .The pyramidality of the amine functionality for the central tertiary amine was examined for all three structures by examining χ al. 1995. Structuvia N—H⋯N bonds, forming a two-dimensional network parallel to is Fig. 4. Additioc axis is Fig. 5.b axis is Fig. 6.Although compounds (I)−3 (173 K). This is similar to the density, determined by X-ray crystallography, of the well known energetics RDX  and HMX  at 1.794 g cm−3 (298 K) and 1.948 g cm−3 (120 K) respectively , and the density of compound (III)−3 (296 K).The neutral complex, compound (I)et al., 2016H-tetrazole N,N-bis(cyanomethyl)nitramide , zinc bromide , 30 ml water, and a magnetic stirbar. The reaction was heated to 323 K with stirring. Sodium azide  was dissolved in 30 ml water and added to the heated reaction. A reflux condenser was fitted to the flask and the reaction was heated to 363 K for 1 h causing a gradual color change to light brown and the formation of a precipitate. The reaction was allowed to cool to room temperature, then 37% HCl (5 ml) was added and the mixture was allowed to stir for 30 min. The product was collected by vacuum filtration using a Buchner funnel and recrystallized from hot water. Yield 95%, 4 g. Melting point 475–477 K (dec.). CHN: Expected: C, 21.24; H, 2.67; N, 61.93. Found: C, 21.82(0.08); H, 2.96(0.08); N, 62.20(0.30). 1H NMR (DMSO-d6): 4.15 , 5.49  ppm. 13C NMR (DMSO-d6): 40.33, 152.74 ppm. IR: 637, 685, 765, 875, 933, 1042, 1088, 1111, 1246, 1284, 1408, 1481, 1524, 1557, 2864, 3022 cm−1.A 100 ml round-bottom flask was charged with Compound (II)1H NMR (DMSO-d6): 4.65 , 5.20 , 8.6  ppm. 13C NMR (DMSO-d6): 46.95, 157.60, 159.64 ppm. IR: 637, 685, 765, 875, 933, 1042, 1088, 1111, 1246, 1284, 1408, 1481, 1524, 1557, 2864, 3022 cm−1.A 50 ml round-bottom flask was charged with a stir bar, barium hydroxide octahydrate  and 20 mmol water. The base was stirred until fully dissolved. Compound (I)Compound (III)1H NMR (DMSO-d6): 5.13 , 3.70 (broad) ppm. 13C NMR (DMSO-d6): 40.05; 155.80 ppm. IR: 2908; 2149; 1869; 1844; 1717; 1700; 1684; 1676; 1653; 1636; 1617; 1540; 1521; 1456; 1419; 1364; 1270; 1209; 1159; 1140; 1113; 1076; 920; 877; 809; 706; 612 cm−1.A 50 ml round-bottom flask was charged with (I)Uiso(H) = 1.2Ueq(C). All other H atoms were located in a difference Fourier map using. Compound (II)Crystal data, data collection and structure refinement details are summarized in Table 410.1107/S2056989017008817/lh5843sup1.cifCrystal structure: contains datablock(s) I, II, III. DOI: 10.1107/S2056989017008817/lh5843Isup2.hklStructure factors: contains datablock(s) I. DOI: 10.1107/S2056989017008817/lh5843IIsup3.hklStructure factors: contains datablock(s) II. DOI: 10.1107/S2056989017008817/lh5843IIIsup4.hklStructure factors: contains datablock(s) III. DOI: Click here for additional data file.10.1107/S2056989017008817/lh5843Isup5.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S2056989017008817/lh5843IIsup6.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S2056989017008817/lh5843IIIsup7.cmlSupporting information file. DOI: 1555912, 1555911, 1555910CCDC references:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Their planar structures were elucidated by a comprehensive analysis of NMR and MS spectroscopic spectra. The absolute configurations were determined by Marfey’s method and single-crystal X-ray diffraction (Cu Kα) analysis. Xylapeptide A (1) is the first example of cyclopentapeptide with L-Pip of terrestrial origin and showed strong antibacterial activity against Bacillus subtilis and B. cereus with MIC value of 12.5 μg/mL.Two new cyclopentapeptides, xylapeptide A ( Endophytes are increasingly attracting attention as sources of potentially valuable compounds3. The secondary metabolites produced by endophytes have received enduring development for chemical diversity and antimicrobial activities4. Peptides are investigated intensively because they are target specific with higher degree of interactions5. In recent years, a quite number of peptides isolated from endophytes have been reported. It is noteworthy that peptides with good antimicrobial activities were isolated mainly from Streptomyces genus, such as kakadumycin A6 showed potent antibacterial activity against Staphylococcus aureus, S. simulans, S. pneumonia, and Shigella dysenteriae; munumbicin C7 displayed strong activity against vancomycin-sensitive S. aureus; actinomycin D8 exhibited strong activity against methicillin-resistant S. aureus and multidrug-resistant Mycobacterium tuberculosis, and munumbicin E-48 demonstrated strong activity against S. aureus. However, few peptides isolated from fungi exhibited good antimicrobial activities, the example was the cyclo-(Pro-Thr) and cyclo-(Pro-Tyr)9 (produced by a Penicillium sp. isolated the mangrove Acrostichum aureurm) showed a strong antibacterial activity against S. aureus and antifungal activity against Candida albicans.New antibiotics are in need of development against the resistant microorganisms in controlling bacterial infectionsSophora tonkinensis is an important traditional Chinese medicinal plant used widely to treat acute pharyngolaryngeal infections and sore throats11. In the course of our studies to investigate antimicrobial constituents from associated fungi, the fungus Xylaria sp. (GDG-102) was isolated from S. tonkinensis. The crude extract of the fermentation broth exhibited antibacterial activity. Preliminary UPLC-MS and 1H NMR analysis showed that this fungus produced relatively polar peptide compounds  and B (2)  was obtained as a colorless crystal. Its HRESIMS gave a [M + H]+ at m/z 556.3505, indicating a molecular formula of C30H45N5O5 and requiring 11 degrees of unsaturation. The 1H NMR spectrum  and 170.2, six nitrogenated sp3 carbon resonance at δC 64.2, 59.0, 57.0, 48.1, 47.5 and 47.4. These above NMR features accounted for 9 of the 11 unsaturations. Further analysis of 2D NMR spectrum allowed five subunits to be established, an N-Me-phenylalanine (N-Me-Phe), a valine , an alanine (Ala), a leucine (Leu), and a pipecolinic acid (Pip) . Furthermore, the ESI MS2 -L-alalinamide , and UPLC-MS analysis of the derivatives with comparison to the standards  was also obtained as a colorless crystal. Its molecular formula C29H43N5O5 (eleven degrees of unsaturation) was determined on the basis of HRESIMS data. The 1H and 13C NMR spectrum ) in 1 were absent. Examination of the 2D NMR spectra revealed that 2 shared the same N-Me-Phe, Val, Ala, and Leu units with 1, the only difference is that the Pip in 1 was replaced by a proline (Pro) subunit in 2. The sequence of the amino acids of 2 was also assigned by HMBC correlations  of the standard L-Pro and D-Pro were 6.262 and 6.689 min, respectively. The retention times for the acid hydrolysate of 2 were 6.285, 6.818, 7.707, 9.004, and 9.318 min, respectively, which correspond to L-Pro, L-Leu, L-Val, N-Me-L-Phe, and D-Ala , apratoxin H (cyanobacterium Moorea producens17), petrosifungins A and B (fungus Penicillium brevicompactum18), cyclohexapeptide similanamide (fungus Aspergillus similanensis19), cyclohexadepsipeptides somamides A and B (cyanobacteria Lyngbya majuscule and Schizothrix sp20.), symplostatins (cyanobacterium Symploca hydnoides21), tasipeptins A and B (cyanobacterium Symploca sp22.), kurahamide (cyanobacterium Moorea sp23.), and dolastatins (sea hare Dolabella auricularia24), cycloheptadepsipeptides neamphamides B−D (sponge Neamphius huxleyi25), cyclooctadepsipeptide homophymine A (sponge Homophymia sp26.), and pipecolidepsins A and B (sponge Homophymia lamellose27). Cyclic peptides with Pip from terrestrial origins are rare including cyclohexapeptides PF1171 A−G (unknown ascomycete OK-12813), and cyclodecadepsipeptides, clavariopsins A and B (aquatic hyphomycetes Clavariopsis aquatic28). Up to now, the Pip in all the peptides is L-configuration. Xylapeptide A (1) is the first cyclopentapeptide from a terrestrial origin with L-Pip.Naturally occurring cyclic peptides with nonproteinogenic amino acids mainly contained pipecolinic acid (Pip) and anthranilic acid (Ant)14C-L-Lys was transported rapidly in to the shoots and then degraded to Pip in many pathways30. In the Rhizoctonia leguminicola fungus, the L-[U-14C]-Lys is used to form L-Pip32. Although Gatto and co-workers reported the first in vitro characterization of a lysine cyclodeaminase (RapL) which utilized the NAD+ as a cofactor in a catalytic manner in Streptomyces hygroscopicus to catalyst lysine into Pip33, the genes controlling the biosynthesis of Lys to Pip have not been described yet. It is suggested that the Pip in xylapeptide A (1) may also be converted from lysine, and a radioactive labeling experiment is needed.The transformation of Lys to Pip in plants and fungus has been studied by radioactive labeling experiment. In the Ca-deficient wheat plants, the 1) and B (2) were evaluated for their antibacterial activity. Compound 1 showed strong and selective antibacterial activity against Bacillus subtilis and B. cereus with MIC value of 12.5 μg/mL. Compound 2 exhibited strong and broad antibacterial spectrum against B. subtilis, B. cereus, B. megaterium, Micrococcus luteus, Staphylococcus aureus, and Shigella castellani with MIC values of 12.5, 6.25, 6.25, 12.5, 12.5, and 12.5 μg/mL, respectively. Compound 2 also showed a strong antifungal activity against Canidia albicans with MIC value of 12.5 μg/mL. This suggested that the L-Pip residue and L-Pro residue play different roles in the antimicrobial activity. The cytotoxic, α-glucosidase inhibitory, and antiviral activities of 1 and 2 were also evaluated. However, they were all inactive.Xylapeptides A (1) and B (2), were isolated from a Xylaria sp. fungus cultured from the Chinese medicinal plant S. tonkinensis. Xylapeptide A (1) contains a non-proteinogenic amino acid, L-pipecolinic acid (L-Pip), which is the first example of cyclopentapeptide from a terrestrial origin with L-Pip. Compound 1 displayed a strong antibacterial activity against Bacillus subtilis and B. cereus. Xylapeptide B (2) has a broader antibacterial spectrum as well as antifungal activity.In summary, two new cyclopentapeptides, xylapeptides A  column coupled with a Waters 2996 photodiode array detector. UPLC MS was performed on Waters UPLC® system using a C18 column  and ACQUITY QDa ESIMS scan from 150 to 1000 Da. Silica gel , and octadecylsilyl silica gel , were used for column chromatography (CC). Precoated silica gel plates  were used for thin-layer chromatography.Optical rotations were measured on a JASCO P-1020 digital polarimeter. UV spectra were determined on a Shimadzu double-beam 210 A spectrometer. IR spectra were taken on a Bruker EQUINOX 55 spectrometer using KBr pellets. 1D and 2D NMR spectra were obtained on an Agilent DD2 400 MHz NMR spectrometer or a DRX-500 spectrometer, respectively, with TMS as the internal standard. ESIMS and HRESIMS spectra were obtained from a Micromass Q-TOF spectrometer and a Thermo Scientific LTQ Orbitrap XL spectrometer. Semi-preparative HPLC was performed on a Waters 1525 system using a C18  was isolated from the Chinese medicinal plant S. tonkinensis collected in Hechi, Guangxi Province, in October, 2014. The fungus Xylaria sp. (strain # GDG-102) was isolated from fresh healthy leaves of Chinese medicinal plant S. tonkinensis collected in Hechi, Guangxi Province, in October, 2014. The leaves were designed to undergo a process described as surface sterilization34. The surface-sterilized leaves were aseptically sectioned into small pieces and plated onto the PDA plates containing an antibiotic to suppress bacterial growth . After incubation at 27 °C, the fungal strain under investigation was isolated from the growing cultures by repeated reinoculation on PDA plates. The fungus was identified as Xylaria sp. (GenBank accession number KU645984) using a molecular biological protocol by DNA amplification and sequencing of the ITS region as described in ref. The fungus 2Cl2-CH3OH  for three times, respectively. The mycelia extraction were concentrated to about 0.3 L and extracted with EtOAc. The EtOAc layer was evaporated to dryness under reduced pressure to give broth extract (4.5 g) and mycelia extract (4.8 g), respectively. The broth extract was subjected to silica gel CC (petroleum ether (PE)−EtOAc, v-v, gradient) to afford five fractions (Fr.1–Fr.5). Fr.4 was repeated purified by ODS CC (CH3OH−H2O) and then recrystallized in MeOH to obtain 2 (20 mg). In search for similar compounds, the mycelia extraction was subjected to silica gel CC  to afford six fractions (Fr.1–Fr.6). Under the guidance of 1H NMR, Fr.4 was chromatographed on ODS with CH3OH−H2O, and then purified by semi-preparative HPLC  to yield 1 (10 mg).The fungal strain was cultivated in 20 L of liquid medium  at room temperature without shaking for 7 weeks. Then the culture was filtered to separate the culture broth from the mycelia. The culture broth was extracted with an equal volume of ethyl acetate (EtOAc) and the fungal mycelia were extracted with CHXylapeptide A (1). colorless crystals; c 0.1, CH3OH); UV (CH3OH) λmax (log ε) 205 (4.44) nm; IR (KBr) νmax 3446, 1683, 1521 cm−1; 1H and 13C NMR data, see Table 2 (fragmentation of m/z 556.4 [M + H]+) m/z 445.2 [M − Pip + H]+, 332.2 [M − Pip − Leu + H]+, 261.1 [M − Pip − Leu − Ala + H]+, 162.1 [M − Pip − Leu − Ala − Val + H]+; HRESIMS m/z 556.3505 [M + H]+ .ptide A . colorlXylapeptide B (2). colorless crystals; c 0.2, CH3OH); UV (CH3OH) λmax (log ε) 206 (4.47) nm; IR (KBr) νmax 3434, 2959, 1648, 1523, 1454 cm−1; 1H and 13C NMR data, see Table 2 (fragmentation of m/z 542.3 [M + H]+) m/z 443.2 [M − Val + H]+, 372.2 [M − Val − Ala + H]+, 259.1 [M − Val − Ala − Leu + H]+, 162.0 [M − Val − Ala − Leu − Pro + H]+; HRESIMS m/z 542.3345 [M + H]+ .ptide B . colorl3OH-H2O, the regular needle crystals of 1 and 2 were obtained. The single-crystal X-ray diffraction data were collected at 293(2) K for 1 and 296.15 K for 2 on a Bruker APEX-II CCD diffractometer with Cu Kα radiation (λ = 1.54178 Å). Crystallographic data for 1 and 2 have been deposited with the Cambridge Crystallographic Data Centre. Copies of the data can be obtained, free of charge, on application to the Director, CCDC, 12 Union Road, Cambridge CB2 1EZ, UK (fax: + 44-(0)1223-336033, or e-mail: deposit@ccdc.cam.ac.uk).Upon crystallization from CHCrystal data for1: C30H45N5O5∙2(H2O) Mr = 591.74, monoclinic, space group P2(1) with a = 13.3173 (2) Å, b = 6.90755 (8) Å, c = 17.8276 (3) Å, α = 90.00°, β = 93.7299 (14)°, γ = 90.00°, V = 1636.50 (4) Å3, Z = 2, Dx = 1.201 mg/m3, μ (Cu Kα) = 1.54178 mm−1, and F(000) = 640. Crystal dimensions: 0.40 × 0.28 × 0.10 mm3. Independent reflections: 20526, the final R1 value was 0.0337, wR2 = 0.0824 (I > 2σ(I)), Flack parameter = 0.04(15). CCDC number: 1501650.Crystal data for2: C29H43N5O5∙H2O∙CH3OH, Mr = 591.74, monoclinic, space group P2(1) with a = 9.9670 (5) Å, b = 12.5923(7) Å, c = 26.2574 (14) Å, α = 90.00°, β = 90.00°, γ = 90.00°, V = 3295.5(3) Å3, Z = 4, Dx = 1.193 mg/m3, μ (Cu Kα) = 1.54184 mm−1, and F(000) = 1280. Crystal dimensions: 0.30 × 0.15 × 0.08 mm3. Independent reflections: 20526, The final R1 value was 0.0756, wR2 = 0.1010 (I > 2σ(I)), Flack parameter = −0.1(2). CCDC number: 1502010.L- and D/L-configurations) relevant to 1 and 2 were obtained commercially and 0.5 mg was dissolved in 20 μL H2O. Each standard was then derivatized for Marfey’s analysis by adding 1 M NaHCO3 (10 μL) and L-FDAA . The mixture was heated at 45 °C for 1 h with continuous stirring, then neutralized with 1 M HCl after cooling at room temperature. The derivatives were then dried and diluted with CH3OH and analyzed by UPLC MS  CH3CN and (B) H2O with 0.1% HCOOH 5–50% A (0–13 min), 50–100% A (13–15 min), 100% A (15–17 min), 100–5% A (17–18 min), 5% A (18–20 min). Retention times (min) of the standard amino acid derivatives of L-Ala, D-Ala, L-Pro, D-Pro, L-Val D-Val, L-Leu, D-Leu, N-Me-L-Phe and N-Me-D-Phe were 5.817, 6.811, 6.262, 6.689, 7.692, 8.977, 8.996, 10.212, 9.281, and 9.736 min, respectively.The amino acid standards , 7.676 , 8.975 (L-Leu), and 9.263 (N-Me-L-Phe) min. The retention times of that in 2 were: 6.285 (L-Pro), 6.818 (D-Ala), 7.707 , 9.004 (L-Leu), and 9.318 (L-N-Me-Phe) min.Xylapeptide A  was hydrolyzed with 6 M HCl (2.0 mL) at 110 °C for 15 h. The solution was evaporated to dryness, and derivatized for Marfey’s analysis in a similar manner to the derivatized standard amino acids. The derivatives of the acid hydrolysate of B. subtilis, B. cereus, B. megaterium, Micrococcus luteus, Staphylococcus aureus, and Shigella castellani, and antifungal activity against C. albicans were evaluated by using 96-well microtitre plates35, with ampicillin as positive control. The cytotoxic activity against human colon carcinoma (HCT-116), human cervical cancer (HeLa), non-small cell lung carcinoma (A549), human breast cancer (MCF-7), human pancreatic carcinoma (BXPC-3), and chronic myelocytic leukemia (K562) cell lines was evaluated by the SRB method36 and MTT method37, with adriamycin as a positive control. The antiviral activity against human cytomegalovirus (HCMV), and herpes simplex virus type 1 (HSV-1) virus was evaluated by the cytopathic effect inhibition assay38, with cidofovir and acyclovir as positive control. The α-glucosidase inhibitory activity was evaluated by Yilmazer-Musa’s method39, with acarbose as a positive control.The antibacterial activity against Supplementary Information


Graptopetalum paraguayense E. Walther is a popular traditional Chinese herb and possesses several health benefits. In earlier studies, we demonstrated that G. paraguayense showed no genotoxicity and showed several biological activities. However, the constituents of G. paraguayense have not been studied yet. In this present study, we isolated and identified the constituents of the leaves of G. paraguayense E. Walther.G. paraguayense. The four major compounds isolated were flavonoid glucoside derivatives of quercetin  and kampferol , each presenting a 3-hydroxyl-3-methylglutaroyl (HMG) substituent; compounds 3 and 4—the 2´´-acetyl derivatives of 1 and 2, respectively—are novel compounds isolated from nature for the first time. High-performance liquid chromatography for the quantitative analyses of the four major HMG-substituted flavonoid glycosides in G. paraguayense E. Walther were accomplished to acquire the high yields of 1–4 in the methanolic extract . Furthermore, the antioxidant activities, including radical-scavenging, reducing power and lipid peroxidation inhibitory effects of these isolated flavonoids were also evaluated. All seven of the isolated flavonoid compounds possessed antioxdative activity.A total of seven flavonoid compounds were isolated from the methanolic extract of G. paraguayense E. Walther, we isolated four major components from its methanolic extract and determined their structures to be (acetylated) HMG-substituted flavonol glycosides, which are rare in nature. All seven of the isolated compounds possessed antioxdative activity, and those flavonoid compounds may be responsible for the functional ingredients in G. paraguayense. Further investigation of their bioactivities or pharmacological activities will be continued.In this study of the constituents of the leaves of The online version of this article (doi:10.1186/s40529-015-0088-4) contains supplementary material, which is available to authorized users. Graptopetalum paraguayense E. Walther is a popular traditional Chinese herb and possesses several health benefits. According to an archaic Chinese prescription, G. paraguayense, belonging to the Crassulaceae family, is used for alleviating hepatic disorders, lowering blood pressure, relieving pains and infections. We had shown that G. paraguayense showed no genotoxicity by using Ames test, in vitro chromosome aberration assay and in vivo erythrocyte micronucleus assay. Additionally, the acute oral toxicity and 28-day repeated feeding toxicity tests showed that the aqueous extract of G. paraguayense is generally considered to be safe, with an no observed adverse effect level (NOAEL) value of 1.0 g/kg BW in rats  acts as a mixed-type inhibitor of angiotensin-converting enzyme (ACE)  for 4 weeks reduces blood pressure in spontaneously hypertensive rats -induced liver damage  completely recovered from hepatic steatosis, thus, G. paraguayense is effective in patients with fatty liver.u et al. ; Chung eu et al. ). In earg et al. ), radicag et al. ), and ang et al. ). Furthen et al. ), and oug et al. ) and prou et al. ). In an n et al. ). Duh eth et al. ). A reposis (Hsu ). FurtheG. paraguayense have not been studied yet. In this present study, we isolated and identified several components of the herb and by using high-performance liquid chromatography (HPLC), we performed quantitative analysis for the four major compounds 1–4  are expressed herein in units of ppm, with coupling constants (J) in Hz. HR-FAB/MS spectra were recorded using a Finnigan Thermo Quest MAT 95XL mass spectrometer. Column chromatography (CC) was performed using silica gel 60 F254 , an ODS column [Lichroperp RP-18 ], and Sephadex LH-20 . Thin layer chromatography (TLC) and preparative TLCs were performed using precoated silica gel plates; spots were visualized at 254 and 365 nm. Preparative HPLC was performed using an Agilent 1100 series system coupled with a UV detector.Optical rotations were measured using a Perkin–Elmer 241 polarimeter. IR spectra were recorded using a Perkin–Elmer Spectrum 100 FT-IR spectrometer. UV spectra were recorded using a Cintra UV 101 spectrometer.G. paraguayense, was identified by an herb expert, Mr. Chiu Nien-Yuan, Department of Pharmacology, China Medical University (Taiwan). The leaves of G. paraguayense were collected from a farm in Yunlin County in Taiwan. A few batches of leaves samples were bought and found consistent by their HPLC profiles.The plant, G. paraguayense  were extracted with n-hexane; the residue was extracted with methanol and then this extract was concentrated. The methanolic extract (89.6 g) of G. paraguayense was partitioned between EtOAc and water, the aqueous layer discarded, and the organic layer dried over Na2SO4. After evaporation of the solvent under reduced pressure, we obtained 10.0 g of EtOAc extract . The GP-EtOAc extract was coated with silica gel and subjected to CC , eluting with a gradient of hexane, EtOAc, and MeOH  to yield seven fractions. Fraction 6 was subjected to ODS CC , eluting with a gradient of MeOH and H2O  to yield three fractions. Fraction 3  was further purified six times through ODS CC (gradient from MeOH/H2O to MeOH) to obtain compounds 1 (30 mg), 2 (46 mg), 3 (51 mg), and 4 (35 mg).The freeze-dried leaves of O-[6´´-(3-hydroxyl-3-methylglutaroyl)]-[2´´-acetyl]-β-D-glucopyranoside (3): C29H30O17; [α]D24 = −46.6° ; UV (MeOH) λmax(log ε): 208 (3.17), 257 (2.81), 354 (2.76) nm; IR νmax (MeOH) cm−1: 1764; (+) ESI-MS m/z: 673 [M + Na]+, 651 [M + H]+ ; (+) ESI-MS2 m/z: 349 [M + H – quercetin]+, 303 [quercetin + H]+; HR(+)ESI/MS [M + H]+m/z, 651.1587 . 1H NMR  and 13C NMR  spectral data of 3 are provided in Table O-[6´´-(3-hydroxyl-3-methylglutaroyl)]-[2´´-acetyl]- β-D- glucopyranoside (4): C29H30O16; [α]D24 = −43.0° ; UV (MeOH) λmax (logε): 209 (3.18), 266 (3.00), 350 (2.94) nm; IR νmax (MeOH) cm−1: 1764; (+)ESI-MS m/z: 657 [M + Na]+, 635 [M + H]+; (+)ESI-MS2 m/z: 349 [M + H – kaempferol]+, 287 [kaempferol + H]+; HR(+)ESI/MS: [M + H]+ m/z 635.1632 . 1H NMR  and 13C NMR  spectral data of 4 are provided in Table Quercetin 3-1–4 were prepared at concentrations of 2–500 μg/mL. The analysis was performed using a reversed-phase Lichrospher 100 RP-18 column . The extracts were separated using aqueous solutions containing 0.1% phosphoric acid (A) and acetonitrile (B). The eluent flow rate was maintained at 1 mL/min; the injection volume was 20 μL; the detection wavelength was 270 nm; the column temperature was set at ambient temperature. The elution program with a linear gradient was optimized as follows: 0 min, 6% B; 30 min, 45% B; 33 min, 100% B; 40 min, 6% B, followed by a 5-min conditioning period. The identities of the analytes were confirmed by comparing their HPLC retention times with that of the analytical standard. Calibration was achieved through linear regression analysis performed at eight different concentrations . Quantitative analysis was performed after integration of the peaks. All experiments in this study were carried out in triplicate.Calibration solutions of standards G. paraguayens were measured using a method described previously .The DPPH radical-scavenging activities of the compounds isolated from n et al. ). An ali+ radical-scavenging activities of the isolated compounds were determined through ABTS decolorization assays  and 7 mM ABTS salt in 0.01 M phosphate-buffered saline (pH 7.4), was maintained for 16 h at room temperature in the dark. Each tested compound (0.01–600.0 μM) was diluted (100X) with the ABTS+ solution to a total volume of 1 mL and then left to react for 10 min. The control (without sample) was used as a blank; 990 μL of PBS was added to these control samples instead. All assays were conducted at least three times with three different sample preparations. All data are expressed as the mean ± standard deviation (SD).The ABTSn et al. ). The AB2+-ascorbate and aquatinted by the malondialdehyde-thiobarbituric acid (MDA-TBA) adduct, according to the method described by Liao and Yin but with slight modification  used for liposome preparation were removed and a lipid film was formed through rotary evaporation under a N2 flush. The liposomes were then suspended in 50 mM sodium phosphate buffer . A mixture of the liposome suspension (1 mL), sodium phosphate buffer (0.6 mL), 25 mM FeCl3 (0.05 mL), 25 mM ascorbic acid (0.05 mL), and the isolated compounds  was incubated for 1 h at 37°C. After incubation, the solution was mixed with TBA (0.4% in 0.2 M HCl) and BHT (0.2% in 95% EtOH) at a ratio of 1:2:0.3 and then heated at 100°C for 20 min. After cooling, an equal volume of n-BuOH was added to the mixture to extract the chromogen. The absorbance of the n-BuOH layer was measured spectrophotometrically at 532 nm. The ability to inhibit MDA formation was calculated using the equation: inhibition effect (%) = [1 – (absorbance of sample at 532 nm/absorbance of control at 532 nm)] × 100%. The MDA level for no compound added was 18.04 μM in the control. The standard reference compound for all antioxidative tests was quercetin. All assays were conducted at least three times with three different sample preparations. All data are expressed as the mean ± standard deviation (SD).Liposome peroxidation was induced by Fe and Yin ). LiposoG. paraguayense extracts and the isolated compounds were determined according to the method of Yen and Chen (-β-d- glucopyranoside (1), kampferol 3-O-[6´´-(3-hydroxyl-3-methylglutaroyl)]-β-d- glucopyranoside (2), quercetin 3-O-[6´´-(3-hydroxyl-3-methylglutaroyl)-2´´-acetyl]- β-d-glucopyranoside (3), and kampferol 3-O-[6´´-(3-hydroxyl-3-methylglutaroyl)- 2´´-acetyl]-β-d- glucopyranoside (4); the three minor components were isoquercetin (5), kaempferol 3-O-β-d-glucopyranoside (6), and kaempferol (7). Among these components, compounds 1, 2, and 5–7 were known; compounds 3 and 4–derivatives of 1 and 2, respectively—are novel compounds isolated from nature for the first time in this study. Compounds 3 and 4 are new acylated derivatives of 1 and 2, respectively. The characteristic feature of these major flavonoid glycosides is the 3-hydroxyl-3- methylglutaroyl (HMG) substituent on their sugar moieties. Compound 1 had been isolated previously as a UV shield from Himalayan Rheum species and from another Euphorbia species , compouw et al. ). We use1–4 and the presence of signals for 15 aromatic carbon atoms in the downfield region in their 13C NMR spectra indicated their flavonoid skeleton structures. We deduced that the A-ring of each of the flavonoids 1–4 featured a 5,7-disubsituted structure, due to the appearance of a small coupling constant (2 Hz) between H-6 and H-8 in the 1H NMR spectrum; the B-ring featured either the ABX  or A2X2  system of protons that is characteristic of quercetin or kaempferol, respectively. For each of these compounds, we confirmed that the sugar moiety was a glucose unit, as revealed by the relationships among the coupled protons in the 1H-2D COSY spectra and by the chemical shifts of the corresponding signals of six carbon atoms-for example, at 104.2 (C-1´´), 75.2 (C-2´´), 77.7 (C-3´´), 70.6 (C-4´´), 75.0 (C-5´´), and 64.0 (C-6´´) ppm for compound 1 - in 13C NMR spectra. The β-glycoside linkage was established on the large coupling constant (7.8 Hz) of the glucose proton H-1´´. We established the site of attachment of the glucose moiety to the flavonoid as being the C-3 position, due to the correlation found between proton H-1´´ and C-3 in the HMBC spectra.The characteristic UV absorptions at 256 and 357 nm of compounds 1H and 13C NMR spectra for the glucose moieties in these compounds, the remaining signals including an upfield (ca. 2.45-2.55 ppm) multiplet corresponding to four protons, a signal at ~1.2 ppm for methyl protons, and peaks for six carbon atoms-two C = O carbon atoms ; one oxygenated quaternary carbon atom (69.9 ppm), two upfield methylene carbon atoms , and one methyl carbon (27.5 ppm)-that characterized the HMG substituent on these flavonoid glycosides. We confirmed that the attachment of the HMG units to the sugar moieties occurred at the C-6 position of the glucose unit, through HMBC correlation of the H-6´´ protons of the glucose with the carbonyl carbon (C = O) of the HMG unit.In addition to the signals in the 3 and 4, spectroscopic analysis revealed one extra substituent, which corresponded to an acetyl group . From the correlations between the glucose H-2´´ protons and the acetyl C = O carbon atoms in the HMBC spectra, we deduced that the site of attachment was the C-2´´ hydroxyl group.For compounds G. paraguayense powder yielded the four major compounds 1–4. According to HPLC analysis, with UV detection at 270 nm, the retention times of compounds 1–4 were 17.0, 19.0, 21.1, and 23.0 min, respectively. Figure Figure 1–4 to be 0.6, 0.8, 0.5, and 0.4 μg/mL, respectively.Through quantitative analysis based on triplicate injections into the HPLC system, we found that the linearity of detection of the four compounds ranged from 2 to 500 ppm (μg/mL), with the squared correlation coefficient exceeding 0.999 in each case . Figureand Chen ). Lipid h et al. ; Yin andh et al. ; Tsuda eh et al. ). The an50) for the radical-scavenging capacities and anti-lipid peroxidation activities. In terms of their IC50 values, the radical-scavenging activities and reducing powers of the isolated compounds decreased in the order 1 > 3 > 5 > 7 > 2 > 4 > 6; in contrast, the anti-lipid peroxidation capacities followed the order 5 > 7 > 2 > 1 > 4 > 3 > 6. Based on these results, the radical-scavenging capacities of the glycosides are lower than that of their aglycones. In addition, the acetylated HMG-substituted flavonoid glycosides 3 and 4 possessed weaker antioxidative activities than those of 1 and 2  HMG-substituted flavonol glycosides, which are rare in nature. Quantitative analysis results indicated that the four major flavonol glycosides totaled 17.3 mg/g of the extract , a notably high abundance of these special flavonoids in the leaves of this plant. All seven of the isolated compounds possessed antioxdative activity, including radical scavenging, reducing capability and lipid peroxidation inhibitory effect. In future work, due to the similarities of the structures of the four main constituents isolated herein to those commercialized inhibitors as HMG-CoA reductase, further investigation of their bioactivities or pharmacological activities will be continued.In this study of the constituents of the leaves of


Antibody-dependent cellular cytotoxicity (ADCC), which mainly mediated by natural killer (NK) cells, may play a critical role in slowing human immunodeficiency virus type-1 (HIV-1) disease progression and protecting from HIV-1 infection. Besides classic NK cells, CD56+ T cells also have some NK cell-like properties, such as the large granular lymphocyte morphology and the capacity to destroy NK-sensitive target cells. However, little is known about the potentials of antibody-dependent CD56+ T cell responses and the association between antibody-dependent CD56+ T cell responses and HIV-1 disease progression.Ex vivo study showed that FcγRIII (CD16)-mediated CD56+ T cell responses were distinctly induced by IgG antibody-bound P815 cells. Comparatively, CD56− T cells and invariant NKT (CD3+ 6B11+) failed to induce antibody-dependent activation. Antibody-dependent CD56+ T cell responses were mainly ascribed to CD4/CD8 double negative subset and were functionally impaired in long-term HIV-1-infected former plasma donors, regardless of hepatitis C virus (HCV) coinfection status. Also, CD56+ T cell-mediated HIV-1-specific antibody-dependent responses were declined in men who have sex with men with HIV-1 infection over 3 years. Finally, we showed that matrix metalloprotease (MMP) inhibitor GM6001 could partially restored antibody-dependent CD56+ T cell responses of chronic HIV-1-infected subjects.In the present study, we showed evidences that, in addition to NK cells, CD56+ T cells could generate degranulation upon CD16 cross-linking. Our results suggested that CD56+ T cells could mediate ADCC responses and the responses were impaired in chronic HIV-1 infection.The online version of this article (doi:10.1186/s12977-016-0313-6) contains supplementary material, which is available to authorized users. Antibody-dependent cellular cytotoxicity (ADCC) is a combined immune responses involving both innate and adaptive immunity . In humaCD56+ T cells, also termed CD3+ CD56+ NKT-like cells, comprise approximately 5–15% of peripheral circulating T cells in human and express cell-surface molecule-CD56, a typical marker for natural killer (NK) cells . CompareIn this study, ex vivo ADCC activities mediated by CD56+ T cells were investigated in healthy versus chronic HIV-1 infected subjects and were compared with NK-mediated ADCC responses. Potenial factors that may influence ADCC activities, such as FcγR expression, CD56+ T subset distribution, and MMPs inhibitor, were also evaluated.P < 0.001) but higher than on CD56− T cells . On the contrary, the percentage of CD32+ CD56+ T cells was found very low (less than 2%) on all three cell populations , a low-affinity receptor, could bind to the Fc region of IgG and trigger ADCC responses. Besides CD16, FcγRIIc (CD32) was also reported responsible for ADCC effect , 21. In 6, a low-P < 0.001 for all). In addition, CD56+ T cells expressed a significantly lower CD107a (P < 0.001) while similar level of IFNγ when compared with CD56+ NK cells, indicating a weaker degranulation capacity of CD56+ T cells versus NK cells  were stimulated with P815 cells coated with P815-specific antibodies (P815/Abs) and ADCC response was reflected by CD107a and interferon-γ (IFNγ) production of CD56+ T, CD56+ NK and CD56− T cells. As shown in Fig. lls Fig. b. In addP < 0.001) in CD56+ T cell-mediated ADCC responses , confirming that peripheral CD56+ T cells possessed the potential to mediate ADCC function. Moreover, to address antibody-dependent lytic capacity of purified CD56+ T cell, target P815 cells were pre-stained with PKH26 and CFSE, and a rapid fluorometric ADCC (RFADCC) assay was employed to detect the frequencies of CFSE−/low target cells . A similar trend of antibody-dependent lytic capacity was found in NK cells but not in CD56− T cells. Taken together, these results indicated that ADCC responses could be mediated by CD56+ T cells though the intensity was generally lower than the response mediated by classic CD56+ NK cells.To confirm the capacity of CD56+ T cells to mediate ADCC responses, purified CD56+ T cells were co-cultured with P815 cells or Ab-opsonized P815 cells, and supernatants were collected for IFNγ detection by ELISA. As shown in Fig. P = 0.006). No differences of CD161+ and CD69+ cells were presented between these two types of cells. Furthermore, the capacity of iNKT to mediate ADCC was evaluated by detecting the frequency of IFNγ+ cells using the strategy for ADCC detection above. Compared to CD56+ T cells, IFNγ-producing iNKT cells were extremely lower after stimulating with Ab-bound P815 cells , in contrast to CD56+ T cells which showed an dramatically increased IFNγ+ production after stimulating with Ab-bound P815 cells (P < 0.001)  and HIV-1/HCV coinfected individuals , which was similar to the trends of ADCC mediated by CD56+ NK cells     and healthy controls (n = 36). Compared to healthy controls, CD56+ T cell-mediated nonspecific ADCC responses were significantly lower in both HIV-1 monoinfected individuals (P = 0.007) and HIV-1/HCV-coinfected  subjects  and HIV-1/HCV coinfection   subjects with HIV-1-infection for 1–3 years (n = 22) and over 3 years (n = 13). HIV-specific ADCC responses were identified by the frequencies of CD107a+ or IFNγ+ CD56+ T cells in response to HIV-1 specific peptide pools , while the frequency of CD8+ subset was increased (P = 0.023) when compared to healthy controls , CD4− CD8+ (CD8+ subset), CD4− CD8−   and IFNγ production  in healthy controls, though CD16+ frequency was lower on DN subset compared with CD4+ subset , indicating a functional impairment of the subset  and healthy controls (P = 0.038)    and enhanced the secretion of IFNγ (P < 0.001) -mediated cleavage of cell surface CD16 –26. MMP 05) Fig. a and cou01) Fig. c, d. TheAccumulated evidence showed that antibody-dependent cellular cytotoxicity may play an important role in the control of HIV-1 infection –32. HoweiNKT cells was identified as CD3+ Vα24-Jα18(6B11)+ cells and responded to CD1d-restricted lipid ligands . DiffereIn this study, we found the capacity of CD56+ T cells to mediate nonspecific ADCC function in long-term HIV-infected FPDs was significantly weaker than in healthy controls. The decreased CD56+ T cell-mediated ADCC activities in HIV-1 infection might be ascribed to the functional impairment since the frequencies of CD56+ T cells were comparable among groups Fig. c. In addTheoretically, nonspecific ADCC activities detected by Ab-opsonized P815 cells merely evaluated the potentials of effector cells to mediate degranulation and cytokine secretion, while failed to consider other important participants involved in ADCC response, such as Ab factor. In this study, we employed a HIV-1-specific CD56+ T cell-mediated ADCC assay using whole blood samples incubating with HIV-1 peptide pools to identify HIV-1-specific ADCC activities in HIV-1-infected individuals. Our data indicated that HIV-1-specific ADCC activity was also declined in MSM with HIV-1 infection longer than 3 years. To rule out TCR-dependent CD56+ T responses, whole blood samples used in HIV-1-specific ADCC assay were replaced by PBMCs samples. The results indicated that TCR-dependent CD56+ T cell responses (indicated by intracellular IFNγ production) were significantly lower than TCR-dependent CD56− T cell responses  in liquid nitrogen till use. Serum and EDTA anti-coagulated plasma were stored at −80 °C until use.A total of 111 HIV-1-infected participants and 36 healthy controls were recruited in this study. Fasted venous blood samples were collected from each participant. Routine blood tests, clinical biomedical tests, and CD4 +/CD8+ T cell counts were performed by local CDC. Peripheral blood mononuclear cells (PBMCs) were prepared from fresh EDTA anti-coagulated peripheral blood using Histopaque-1077  as described elsewhere  and wereHIV-1 infection was screened by an ELISA assay  for HIV antibody, and positive tests were confirmed by HIV Blot 2.2 WB assay . The status of anti-HCV antibodies was determined using the ARCHITECT Anti-HCV System . Plasma level of HIV-1 RNA was measured with the Standard Amplicor HIV Monitor assay, version 2.0 , according to the manufacturer’s protocols. The lower limit of detection was 40 copies/ml. Plasma HCV RNA level was quantified with 2nd Cobas Ampliprep/CobasTaqman (CAP/CTM) HCV test system  with a detection limit of 15 IU/ml.The study involved 76 anti-HIV-positive former plasma donors (FPDs) Table , recruit35 HIV-1 positive men who have sex with men (MSM) were recruited from Beijing city. Subjects were divided into two groups according to the length of HIV-1 infection: 1–3 years n = 22) and >3 years (n = 13) , anti-CD56 PE-Cy7 (clone B159), anti-CD16 APC-Cy7 (clone 3G8), and anti-CD32 PerCP-eFluor 710 (clone 6C4). All antibodies were purchased from BD Biosciences  except CD32 from eBiosciences . Samples were analyzed on BD FACS Fortessa  and the frequencies of CD16+ or CD32+ cells in CD56+ T cells, CD56+ NK cells and CD56− T cells were calculated and compared.5 PBMC from three healthy controls were stained with anti-CD3 Pacific Blue (clone UCHT1), anti-CD56 PE-Cy7 (clone B159), anti-CD16 APC-Cy7 (clone 3G8) and anti-CD32 PerCP-eFluor 710 (clone 6C4) and were run on an ImageStreamχ MarkII system . 20,000 events were collected for each sample and single color control was used to create a compensation matrix to correct for spectral overlap. Collected data were analyzed using IDEAS 3.0 software .To identify the expression of CD16 and CD32 on CD56+ T cells, CD56+ NK cells and CD56− T cells at single-cell level, 1 × 10PBMCs were stained with anti-CD3 Pacific Blue , anti-CD56 PE-Cy7  for 30 min at room temperature. CD56+ T cells, CD56+ NK cells and CD56− T cells were sorted by BD FACS AriaIII  and only cells with purity >95% were used in subsequent experiments.5 purified CD56+ T cells from healthy controls were stimulated with 10 μg/ml of purified anti-CD16 antibody  or 10 μg/ml purified anti-CD32 antibody  or mouse IgG1(κ)  isotype control for 30 min on ice. Cells were washed to remove unbound antibody and incubated with 10 μg/ml of goat anti-mouse IgG1 F(ab′)2 for 5 h  at 37 °C. Cells were washed and stained with anti-CD3 Pacific Blue (clone UCHT1), anti-CD56 PE-Cy7 (clone B159) and anti-CD107a PE-Cy5 (clone H4A3) and fixed by 2% paraformaldehyde (PFA). All data were acquired on BD FACS Fortessa  and analyzed by FlowJo software .1 × 10The nonspecific ADCC assay was performed as previously described, in which the mouse mastocytoma cell line P815 was used as target cells . Briefly6 target cells (P815) were double-stained with 5 μM PKH-26  and 5 μM 5-(and-6)-carboxyfluorescein diacetate succinimidyl ester (CFSE) . Purified CD56+ T cells, NK cells and T cells were used as effector cells and incubated with P815/Abs complex or P815 alone in a microtiter plate with an E:T ratio of 1:1. After 10 h incubation, cells were washed and fixed in cold 2% paraformaldehyde. Samples were run on BD FACS Fortessa  and data were analyzed by FlowJo software .Based on methods adapted from a rapid fluorometric ADCC (RFADCC) assay , 1 × 106HIV-1 gag, pol, env solid-phase peptide pools containing 20-mers (overlapping by 10 amino acids) from HIV-1 strain CN54 were synthesized to 95% purity by Bio-Scientific Co., Shanghai, China. Peptides were dissolved to a concentration of 1 mg/ml in RPMI 1640 containing 10% dimethyl sulphoxide (DMSO) as stock solutions, and working solutions were obtained by further dilution to a final concentration of 1 μg/ml.Whole blood samples were incubated with HIV-1 gag, pol and env peptide pools respectively at 37 °C for 5 h. Brefeldin-A , GolgiStop  and anti-CD107a APC  were added immediately to the cell medium and incubated for 6 h. Samples incubated with phorbol-12-myristate-13-acetate (PMA) (1 mg/ml) plus ionomycin (1 mg/ml) were used as positive controls and samples incubated with DMSO  were used as negative controls. Samples were surface-stained with live/dead dye Amcyan , anti-CD3-PerCP (clone SK7) and anti-CD56-PE (clone B159), and then lysed, permeabilized and intracellularly stained with anti-IFNγ Alexa Fluor700  and anti-CD107a APC (clone H4A3). All data were acquired on BD FACS Fortessa  and analyzed by FlowJo software .5 sorted CD56+ T cells were co-cultured with 1 × 105 P815 cells alone, 1 × 105 P815 cells plus P815-specific Abs, or medium alone for 24 h at 37 °C. The level of secreted IFNγ in culture supernatants was detected by ELISA  according to the manufacturer’s instruction. The sensitivity of the ELISA kit was 4 pg/ml.1 × 10Human peripheral invariant NKT (iNKT) cells were characterized as CD3+ T cells expressing a specific TCR (Vα24-Jα18-Vβ11). To identify iNKT cells, PBMCs were surface-stained with anti-CD3 Pacific Blue  and anti-Vα24-Jα18 FITC . CD3+ Vα24-Jα18+ double positive cells were defined as iNKT cells. To detect CD16, CD161 and CD69 expression on iNKT and CD56+ T cells, PBMC were surface-stained with anti-CD3 Pacific Blue, anti-Vα24-Jα18 FITC (clone 6B11), anti-CD56 PE-Cy7 (clone B159), anti-CD16 , anti-CD161  and anti-CD69 . Samples were analyzed on BD FACS Fortessa  and the frequencies of CD16+ or CD32+ cells in CD56+ T cells, CD56+ NK cells and CD56− T cells were calculated and compared.5 purified DN CD56+ T cells were cultured in interleukin-2-supplemented medium in the presence or absence of broad-spectrum MMP inhibitor GM6001 (5 mM) or DMSO for 0, 3, 6 and 12 h. CD16 expression (MFI) was detected by flow cytometry at each point. Additionally, changes in P815/Ab-stimulated purified CD56+ T cell responses (at an E:T ratio of 1:1) following GM 6001 (50 μM) (Sigma) treatment was analyzed after 6 h of coculture. An equivalent volume of DMSO (Sigma) was used as control. Cells were washed/stained as described above and fixed in cold 2% PFA. All data were acquired on BD FACS Fortessa  and analyzed by FlowJo software .1 × 10To determine whether GM6001 could restore the capacity of dysfunctional CD56+ T cell-medicated ADCC in HIV-1 infected subjects, purified DN (CD4-CD8-) CD56+ T cells were cultured in interleukin-2 (IL2)-supplemented medium in the presence of 5 μM GM6001 or an equivalent amount of DMSO vehicle alone. The degranulating capacities of CD56+ T cells following stimulation with P815 cells or Ab-coated P815 cells were analyzed and compared. After stained with anti-CD107a PE-Cy5  and anti-IFNγ FITC , cells were washed by PBS and fixed by 2% paraformaldehyde (PFA). All data were acquired on BD FACS Fortessa and analyzed by FlowJo software.U test, nonparametric t test, or Wilcoxon matched-pairs signed rank test when necessary. The Spearman’s correlation test was used to evaluate correlations between groups. All P values were two-tailed and considered significant when lower than 0.05.All the statistical and graphic analyses were performed using GraphPad Prism 5.0  or Microsoft Excel 2007. Comparisons between groups were performed using Mann–Whitney