SET/I2PP2A is a multifunctional protein that is up-regulated in head and neck squamous cell carcinoma (HNSCC). The action of SET in HNSCC tumorigenicity is unknown.Stable SET knockdown by shRNA (shSET) was established in three HNSCC cell lines: HN12, HN13, and Cal27. Protein expression and phosphorylated protein levels were determined by Western blotting and immunofluorescence, cell migration and invasion were measured by functional analysis, and PP2A activity was determined using a serine/threonine phosphatase assay. A real-time PCR array was used to quantify 84 genes associated with cell motility. Metalloproteinase (MMP) activity was assessed by zymographic and fluorometric assays. HN12shSET xenograft tumors (flank and tongue models) were established in Balb/c nude mice; the xenograft characteristics and cisplatin sensitivity were demonstrated by macroscopic, immunohistochemical, and histological analyses, as well as lymph node metastasis by histology.The HN12shSET cells displayed reduced ERK1/2 and p53 phosphorylation compared with control. ShSET reduced HN12 cell proliferation and increased the sub-G1 population of HN12 and Cal27 cells. Increased PP2A activity was also associated with shSET. The PCR array indicated up-regulation of three mRNAs in HN12 cells: vimentin, matrix metalloproteinase-9 (MMP9) and non-muscle myosin heavy chain IIB. Reduced E-cadherin and pan-cytokeratin, as well as increased vimentin, were also demonstrated as the result of SET knockdown. These changes were accompanied by an increase in MMP-9 and MMP-2 activities, migration and invasion. The HN12shSET subcutaneous xenograft tumors presented a poorly differentiated phenotype, reduced cell proliferation, and cisplatin sensitivity. An orthotopic xenograft tumor model using the HN12shSET cells displayed increased metastatic potential.in vivo. As a metastasis suppressor, SET regulates invasion, the epithelial mesenchymal transition, and metastasis.SET accumulation has important actions in HNSCC. As an oncogene, SET promotes cell proliferation, survival, and resistance to cell death by cisplatin  SET gene. SET was originally identified as a component of the SET-CAN fusion gene produced by somatic translocation in acute, undifferentiated leukemia [SET I2PP2A) is a 39 kDa phosphoprotein encoded by the leukemia . SET is PP2A is aleukemia . Recentlleukemia .The mitogen-activated protein kinases (MAPKs) transduce signals from the cell membrane to the nucleus in response to a wide range of stimuli. MAPKs include three family members: ERKs (ERK1 and ERK2), c-Jun NH2-terminal kinase (JNK), and p38MAPK. ERKs are activated by phosphorylation and translocation to the nucleus where they phosphorylate multiple substrates . It has The p53 protein is a tumor suppressor that protects the genome by preventing cell transformation and inducing cell cycle arrest, DNA repair, and apoptosis. p53 phosphorylation is required for signal transduction in response to DNA damage and p21 protein activation ,9. Indeein vitro and the HN12 cell line in vivo (xenograft tumor models in nude mice). Our studies focused on cell invasion, proliferation, and EMT characteristics, as well as the in vivo xenograft tumor models, response to cisplatin, and lymphnode metastasis.In the present study, we determined the effects of stable SET knockdown on tumorigenicity using three HNSCC cell lines, HN12, HN13 and Cal27, HN12 cells stably expressing shRNA against SET (shSET) and control shRNA (shControl) were selected using puromycin. SET protein knockdown in HN12shSET cells , and matrix metalloproteinase 9 (MMP-9) mRNAs were up-regulated in the HN12shSET cells. In contrast, the protooncogene c-Src (SRC), Wiskott-Aldrich syndrome-like (WASL) and LIM kinase (LIMK1) mRNAs were down-regulated , and increased MMP expression. These findings suggest that SET is involved in motility, actin dynamics (accumulation of F-actin and cofilin aggregates in cytoplasm), reduced cell adhesion, and increased MMP-9 and MMP-2 expression. Altogether, these effects confer a more aggressive phenotype to HN12shSET cells.Cell motility is a complex and dynamic process. The cofilin protein, a regulator of actin polymerization that defines the direction of cell motility , is phosin vitro by the BrdU assay  Figures A-C. In aAccordingly, histological analysis Figure  demonstrSer-15 and ERK1/2 phosphorylation levels  analysis confirmed SET protein knockdown in the HN12shSET xenograft tumor cells Figure A. The HNThe potential of the SET protein as a new target in cancer therapy has been explored using a peptide  and sphiAn orthotopic tongue xenograft tumor model in nude mice (n = 3) was adopted to evaluate the metastatic potential of the HN12shSET cells compared with the HN12shControl cells. Two out of three animals were lymph node positive in the HN12shSET orthotopic tumor model, and no mice were positive in the HN12shcontrol model Figure .in vitro and in vivo models, and these findings reinforce SET’s potential as a therapeutic target in HNSCC. Furthermore, the development of an orthotopic HN12shSET xenograft tumor model confirmed the gain of a mesenchymal-like behavior.SET is associated with many cellular processes, such as cell cycle control , apoptosin vivo. Importantly, an inflammatory infiltrate and necrosis were evident in the HN12shSET xenograft tumors, suggesting that SET accumulation in HNSCC plays an important role in cell survival in vivo.Previous reports have demonstrated the effects of a sphingolipid pharmacological inhibitor (FTY720) in lung cancer  and chroin vitro and in vivo.In numerous solid tumors, metastasis is preceded by EMT, which allows cells to repress epithelial characteristics and to acquire a mesenchymal-like phenotype that is associated with increased migration and invasion . An assoin vivo; as a metastasis suppressor, SET regulates invasion, the epithelial mesenchymal transition, and metastasis. (2) This is the first time a functional response initiated exclusively by shRNA-mediated stable knockdown of SET is achieved in cancer. (3) The stable SET knockdown standardized in this study may serve as a model to evaluate the effects of SET reduction and the mechanisms related to aggressive cancer behaviors.(1) SET accumulation has important actions in HNSCC: as an oncogene, SET promotes cell proliferation, survival, and resistance to cell death by cisplatin The Animal Care and Use Committee of the University of São Paulo (Ribeirão Preto Campus) approved the procedures used in this study (protocol numbers 11.1.168.53.2 and 11.1.193.53.7).2 at 37°C. The MISSION short-hairpin RNA (shRNA) plasmid TCR1 containing DNA against human SET  or the shRNA control  were transfected into HN12 cells using the Turbofect reagent . Stable transfectants were selected using puromycin (1 μg/mL). Lentivectors containing the shSET and shControl constructs were added to the HN13 and Cal27 cells for transduction. The transduced cells were selected using puromycin (1 μg/mL). For siRNA expression in the HNSCC cell lines, duplex RNA (siRNA) against SET was purchased from Qiagen; the protocol used for this experiment was previously reported [The HNSCC cell lines HN12 (tumorigenic and metastatic), HN13  and Cal2reported . The effSer15; #9286; Cell Signaling), p21WAF1/Cip1 , β-actin and tubulin were used. The reactions were developed using the chemiluminescent ECL Western blotting system . Densitometric analysis was performed using the ImageJ 6.4 software [Cell protein extracts were obtained using the CelLytic Mammalian Cell Lysis/Extraction Reagent (Sigma-Aldrich) with protease and phosphatase inhibitor cocktails (Sigma-Aldrich). Protein concentration was determined using the Bradford protein assay . Thirty micrograms of protein were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a PVDF membrane . The membranes were blocked in 5% non-fat dry milk in Tris-buffered saline containing 10% Tween 20. Antibodies against SET , ERK1/2 , phospho-p44/42 MAPK ERK1/2 , PP2Ac , p53 , phospho-p53  was used to determine cell viability according to the manufacturer’s instructions. The assays were performed in quintuplicate, and three independent biological experiments were considered. The cells were plated on 96-well plates 24 h before the addition of the MTS solution (tetrazolium compound and phenazine methosulfate). Next, the cells were incubated for 2 h, and the absorbance at 490 nm was recorded using a microplate reader (Bio-Rad).DAKO LSAB + System-HRP kit . The BrdU-labeling index, reported as the percentage of cells labeled with BrdU, was determined by counting 10,000 cells from two independent reactions using the Zeiss Axiovert 40 inverted microscope and the AxioVision Rel. 4.8.2 software .Cell proliferation was estimated using the bromodeoxyuridine  incorporation index as previously reported , with mo® easyCyte™ 8HT flow cytometry system, and the InCyte 2.2 software  was used for acquisition and analysis.The cell cycle was analyzed by flow cytometry using propidium iodide . Twenty-four hours after release from synchrony, the cells were maintained for an additional 24 h in DMEM 10% FBS. Next, the cells were collected and fixed in 70% ethanol. After washing in PBS and centrifugation, the cells were suspended in the PI (50 μg/mL) solution containing DNase-free RNAse (Invitrogen) and maintained for 30 min at 37°C. The assay was performed in a Guavain vitro cell migration and invasion assays were performed in 24-well plates  using modified Boyden chamber inserts with a polycarbonate filter membrane containing 8-μm pores. For the invasion assay, matrigel (BD Biosciences) was diluted 1:1 with serum-free medium and used to coat the filter membranes. The cells (1×105) were suspended in 250 μl of serum-free DMEM and seeded onto the upper compartment of the transwell chamber; DMEM containing 10% FBS was used in the lower chamber for stimulation. After a 24 h or 72 h incubation for the migration or invasion analysis, respectively, the medium in the upper chamber was removed, and the filters were fixed in 10% formalin for 15 min. The cells on the lower surface were stained with 4′, 6-diamidino-2-phenylindole . Five fields were photographed at ×200 original magnification using a Zeiss Axiovert 40 inverted microscope and processed using the AxioVision Rel. 4.8.2 software. The cells were counted using the ImageJ program [The  program . The mig2 First Strand kits , respectively. Total cDNA was used to quantify the mRNAs of 84 human genes related to cell motility in a 96-well plate array  according to the manufacturer’s instructions for the Eppendorf Mastercycler EP Realplex instrument . Up-and down-regulated genes were defined as genes with expression levels in HN12shSET cells that are >2.0 or < −2.0, respectively, [2 Profiler PCR Array Data Analysis, version 3.5.RNA isolation and cDNA synthesis were performed using the RNeasy and RTctively,  in relatImmunofluorescence was performed with primary antibodies against E-cadherin , pan-cytokeratin , cofilin , F-actin , β-actin , and vimentin . Cells were incubated with either an anti-rabbit antibody conjugated with Alexa 488 or an anti-mouse antibody conjugated with Alexa 594 (Invitrogen). The nuclei were stained with DAPI. The digital images were obtained using a Zeiss Axiovert 40 inverted microscope and processed using the AxioVision Rel. 4.8.2 software (Carl Zeiss).5) were plated in a 6-well plate in triplicate. After 24 h, the medium was changed to DMEM without fetal bovine serum (FBS), and the cells were maintained for an additional 24 h and 72 h. The supernatant was collected from three wells and concentrated in an Amicon Ultra Centrifugal Filter Device 10,000 MWCO . The cells were then counted. Ten microliters of concentrated supernatant was activated with 1 mM 4-aminophenylmercuric acetate  for 1 h at 37°C or not treated. The samples were resolved by 12% SDS-PAGE containing 1 mg/mL gelatin. The gel was washed with 2% Triton X-100 for 40 min and incubated in a reaction buffer containing 10 mM Tris–HCl, pH 8.0, and 5 mM CaCl2 for 16 h at 37°C. The gel was then stained with 0.25% Coomassie blue. After removing the stain, the negative bands representing the MMP activity were visualized. Semiquantitative analysis using densitometry was performed with the ImageJ 6.4 software [Cells  activity was measured using the Serine/Threonine Phosphatase Assay system  and the synthetic peptide RRA(pT)VA (Promega). For this assay, cells were lysed with Cellytic (Sigma) containing a protease inhibitor cocktail (Sigma), and the free phosphate was eliminated from the lysates using a Sephadex G-25 resin (Promega). For measurements of phosphatase activity, a standard phosphate curve was first constructed with 0, 100, 200, 500, 1,000 and 2,000 ρmol of phosphate. The samples  were incubated with or without 12 nM or 5 μM okadaic acid for 15 minutes at room temperature. The reaction was performed by adding the PP2ase-2A 5× reaction buffer  and the Thr phosphopeptide to the samples in a 96-well plate for 10 minutes at 30°C. The reaction was stopped by incubation with the molybdate dye for 15 minutes, and the absorbance was determined at 595 nm using a microplate reader (BioRad).6 HN12shControl and HN12shSET cells were injected s.c. into the left and right flanks, respectively, of ten 8-week-old Balb/C male nude mice. The tumor size was measured weekly using a caliper; the volume was reported as mm3 and calculated using the formula 0.5 × length × width2. At 4 weeks post-injection, the mice were euthanized. The tumors were collected, fixed in 10% formalin, and embedded in paraffin. Five-μm sections were stained with H&E for histological analysis. Three-μm sections were used for the immunohistochemical analysis of SET, Ki67, pan-CTKR, p62, pERK1/2, and p-p53Ser15 proteins as previously described [Ser15. For Ki67 analysis, five microscopic fields were analyzed for the determination of percentage of positive cells.To assess the xenograft tumor growth, 2×10in vivo, five Balb/C nude mice were injected with cells as described above. Cisplatin was administered intraperitoneally at 3.5 mg/kg/day  15 days after cell injection, and the treatment continued for 5 days. The post-treatment procedures are the same as described above.To evaluate cisplatin sensitivity 5) were injected into the tongues of Balb/c nude mice after anesthesia (n = 3) according to the ethical conduct outlined in the Care and Use of Animals for Experimentation of the University of São Paulo. The mice were assessed daily and weighed once a week. The mice were euthanized 15 days post-injection. The tongue and lymph nodes were collected, fixed in 10% formalin, and embedded in paraffin. Five-μm sections were stained with H&E for histological analysis.We used an orthotopic human xenograft tumor model to evaluate metastatic potential. The stable SET knockdown HN12 (HN12shSET) and HN12shControl cells  Western blotting using antibodies against SET, total ERK1/2, pERK1/2, E-cadherin, vimentin and tubulin (as a constitutively expressed protein). The densitometric results are presented as the shSET/shControl ratio. ERK phosphorylation is indicated as phospho-ERK/total ERK. (B) Immunofluorescence using an anti-pan-CTKR in HN12siSET cells; nuclei were stained with DAPI (blue). (C) HN12 cells with SET knockdown (HN12siSET) and negative siRNA control cells in the matrigel invasion assay. The results are either representative of three independent experiments or are reported as the means and standard deviations of experiments performed in triplicate (*p < 0.05).Click here for fileSer-15 in HN13 and Cal27 cell lines with stable SET knockdown (shRNA). Western blotting was performed using antibodies against total p53, p-p53Ser-15 and tubulin. The densitometric values represent the shSET/shControl ratio. Tubulin was used as a constitutively expressed protein.Reduction of p-p53Click here for fileSer-15 and pERK1/2. The images are representative of three experiments. The immunocomplexes were visualized with a chromogenic substrate  and counterstained with hematoxylin.Reduction of p-p53 and pERK in the HN12shSET xenograft tumors compared with HN12shControl tumors. Three-μm sections from the HN12shSET xenograft tumors were used for immunohistochemical analysis with antibodies against p-53Click here for file


Antrodia cinnamomea mycelia, was demonstrated to exhibit significant anti-inflammatory bioactivity in vitro. However, its role in hepatic injury in vivo still remains unclear. We hypothesized that antrodan may have beneficial hepatoprotective effects. To verify this, a lipopolysaccharide (LPS)-Sprague-Dawley rat model was used. Antrodan protected against liver damage by suppressing LPS-stimulated serum glutamine-oxaloacetic transaminase (GOT), glutamic-pyruvic transaminase (GPT), interleukin (IL)-6, hepatic thiobarbituric acid reactive substances (TBARS), nitric oxide (NO), inducible NO synthase (iNOS) and nuclear factor (NF)-κB, and by effectively alleviating the downregulated hepatic superoxide dismutase (SOD), catalase, and glutathione peroxidase (GSH-Px). Hematoxylin-eosin staining revealed that antrodan at a dosage of 40 mg/kg was able to alleviate LPS-induced liver damage to a normal status. In addition, we identified the partial main architectural backbone of antrodan to have a 1→3 linear β-glycosidic backbone of mannan linked by β-1→3 glucosidic branches. Conclusively, antrodan can potentially ameliorate liver damage in vivo by suppressing oxidative stress induced by LPS.Antrodan, a protein-bound polysaccharide isolated from  Antrodia cinnamomea (syn. A. camphorata) (AC) fruiting bodies, in which 78 compounds have been identified Beta-glucans are commonly found at high levels in fungi, yeast, oats, barley, and bacteria β-Glucans can act on several immune receptors including Dectin-1, complement receptor (CR)3 and Toll-like receptor (TLR)-2/6, and can also trigger groups of immune cells including macrophages, neutrophils, monocytes, natural killer (NK) cells, and dendritic cells and enhance phagocytosis Decoking is thought to produce an extract comprising both triterpenes and a diversity of soluble polysaccharides, and we hypothesized that AC polysaccharides could contribute comparable, if not more-prominent, immunobioactivity that ultimately may be associated with certain promising hepatoprotective effects. To verify this, we carried out the present experiments.2), 3--2,5-diphenyl tetrazolium bromide (MTT), sodium bicarbonate, lipopolysaccharide , 2′,7-dichlorofluoresein, and N-(1-naphthyl)-ethylenediamine dihydrochloride were procured from Sigma . Fetal bovine serum (FBS), an L-glutamine solution (100 mM), penicillin-streptomycin solution  were purchased from Biological Industries . Dulbecco's modified Eagle medium (DMEM) and a trypsin-EDTA solution were provided by Hyclone . A protein assay kit was a product of Bio-Rad . Methanolic HCl (0.5 N), a Sylon HTP kit, and N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) were products of Supelco . The rat interleukin (IL)-6 enzyme-linked immunosorbent assay (ELISA) kit was supplied by R&D Systems .Trypan blue, sodium nitrite  at 80°C for 1 h. This extraction was repeated three times. After being cooled, the extracts were combined and filtered with the aid of aspiration. To the filtrate was added 1 N HCl to pH 4.0. A 2-fold volume of ethanol (95%) was then added. The precipitated polysaccharides were collected and re-purified in 4 L of hot water (90°C). In the final step, the soluble polysaccharides were re-precipitated by the addition of a 3-fold volume of ethanol (95%). The solution was left to stand for 4 h. The precipitate was collected, lyophilized, and pulverized . AC-II was further subjected to gel permeation chromatographic separation according to Carbonero et al. g for 5 min to eliminate any undissolved residue. The supernatant was subjected to a Sepharose CL-6B column (ℓ×id  = 82×3 cm) and eluted with DDW (adjusted to pH 11.0 with NaOH) at a flow rate 0.5 mL/min. The eluent was collected with a fractionator  at a velocity 1 tube/10 min. In total, 100 tubes were collected. The total sugar content of the eluent was determined, and the optical density (OD) at 280 nm was monitored. By referring to the OD at 280 nm, the main fractions (16th to 40th) were selected and combined. The combined solution was lyophilized to yield a pure glucan, called “antrodan” in the text hereafter. To analyze the glycosidic linkage of the polysaccharide moiety (2 mg), partially methylated alditol acetates were prepared from permethyl derivatives by hydrolysis (with 2 M trifluoroacetic acid at 121°C for 2 h), reduction (with 10 mg NaBD4/mL for 2 h at room temperature), and acetylation (with acetic anhydride at 100°C for 1 h). A gas chromatographic-mass spectrometric (GC-MS) analysis was performed on an Agilent Gas Chromatograph 6890  connected to an Agilent 5973 Mass Selective Detector. Samples were dissolved in hexane and injected into the HP-5MS fused silica capillary column  at an injector temperature of 250°C. The helium carrier gas was operated at a flow rate of 1 ml/min. The oven, initially held at 100°C for 1 min, was programmed at a rate of 10°C/min to 200°C and held for 1 min at 200°C, and finally increased to 300°C at a rate of 15°C/min Antrodan, a kind of glycoprotein, was prepared from mycelia of 2, and blood was collected from the hepatic portal vein and stored at 4°C. The heart, kidneys, and liver were excised, rinsed twice with phosphate-buffered saline (PBS), dewatered of soft skin tissues, weighed, and immediately immersed in liquid nitrogen for storage at −80°C for further use. The collected blood was centrifuged at 3000×g for 15 min to separate the serum, which was used in the following biochemical analyses.The animal experiment was approved by the Institutional Animal Care and Ethics Committee of Hungkuang University . Thirty male Sprague-Dawley rats, aged 6 weeks and weighing 265∼287 g, were purchased from the BioLasCo Animal Center . These rats were housed in stainless cages in an animal room maintained at 20±2°C, and a relative humidity of 65%±6%, with a 12/12-h light/dark cycle. These rats were fed ad libitum on basic granular chow  and water and acclimated in the animal room for the first week. The rats were randomly divided into five groups, with six in each group. The grouping involved the control, LPS control, antrodan control, LPS + low-dose antrodan (40 mg/kg) (antrodan L+LPS), and LPS + high-dose antrodan (80 mg/kg) (antrodan H+LPS) groups. Animals were separated and caged, with two or three rats in each cage. Antrodan was administered by gavage. The placebo group was fed basic chow only. Normal saline was used instead of antrodan in the LPS control. Access to chow and water was ad libitum. The entire experiment lasted 7 days. The initial and final body weights (BWs) were recorded. One day before being euthanized, LPS (5 mg/kg)  was intraperitoneally (i.p.) injected to induce acute hepatic injury. Then animals were anesthetized with COSerum levels of GOT and GPT were assayed with Colorimetrics Slides  according to instructions given by the manufacturer.3, rapidly followed by the addition of the Griess reagents . The absorbance was immediately measured at 540 nm after the mixture being reacted for 15 min in dark at RT. Nitrite concentration was calculated using a NaNO2 standard curve and expressed as micromoles per liter.Levels of serum IL-6 were determined by using a ELISA kit purchased from R&D Systems  according to the protocol provided by the manufacture. Briefly to a 96-well plate, 100 μL of capture antibody was added. The plate was tightly sealed and stored at room temperature (RT) overnight. After washes, the plates were blocked with Block Buffer at room temperature for 1 h. Authentic IL-6 or serum (100 μL) was added and left to react for 2 h at RT. Dilute detection antibody was loaded to each well, and the plate was incubated for an additional 2 h at RT. Avidin peroxidase (100 μL) was added and allowed to react for 30 min at ambient temperature. A substrate solution (ABTS liquid substrate solution) (100 μL) was added, and the mixture was allowed to react for 10 min while avoiding direct sunlight. The absorbance was read at 405 nm, with wavelength correction set at 650 nm using the ELISA reader . A calibration curve was similarly established using authentic IL-6. Serum nitrite (including nitrate) concentration was measured by a modified method of the Griess assay, described by Miranda et al. g for 15 min. The supernatant (1 mL) was transferred to a microcentrifuge tube and centrifuged at 4°C and 12,000×g for 10 min. The protein content of the supernatant was determined .The Bradford protein binding assay g for 5 min. The supernatant was decanted and transferred to a 5-mL reaction vessel. Tris-HCl  was added and agitated for 5 min. To 10 μL of the mixture, methanolic pyrogallol (50 mM) was added, immediately mixed, and read at 325 nm. Readings were successively made every 15 s for a total period of 3 min. Water was used as the blank, and a similar protocol was conducted. The activity is expressed in U/mg protein. The following equation was used to calculate SOD activity:S is the change in the OD/min after adding the sample solution, Vt is the total volume of the reaction mixture (mL), 4.8 is the dilution extent, Vs is the total sample volume (mL), P is the total protein content (mg), A1 is the OD at 0 s after adding water, A2 is the OD at 120 s after adding water, and t is the time (min).The method of Marklund and Marklund g for 5 min. The supernatant (5 μL) was decanted and transferred to a 5-mL reaction vessel and diluted with phosphate-EDTA buffer . To the diluted solution (2 mL), H2O2  was added and immediately mixed, and the OD was read at 240 nm. Readings were successively taken every 15 s for a total reading period of 3 min. CAT activity is expressed as U/mg protein.The method of Aebi 2 is the OD at 180 s, A1 is the OD at 0 s, t is the time (min), Vt is the total volume of the reaction mixture (mL), 4.8 is the dilution extent, ℓ is the light path (cm), ε240 is the extinction coefficient of 0.0395 mM−1cm−1, Vs is the total sample volume (mL), and P is the total protein content (mg).The following equation was used to calculate CAT activity:3, 0.2 mM NADPH, 1 U/mL GSH reductase, and 1 mM GSH) was added and mixed well. The mixture was left at ambient temperature for 5 min to facilitate the reaction. To this reaction mixture, 100 μL of 2.5 mM H2O2 was added and mixed well. The OD was read at 340 nm. Readings were successively taken every 5 s for a period 3 min. The GSH-Px activity is expressed as U/mg protein.The method of Lawerence and Burk 2 is the OD at 180 s, A1 is the OD at 0 s, t is the time (min), Vt is the total volume of the reaction mixture (mL), 4.8 is the extent of dilution, ℓ is the light path (cm), ε340 is the extinction coefficient of NADPH of 6.22 mM−1cm−1, Vs is the total sample volume (mL), and P is the total protein content (mg).The following equation was used to calculate GSH-Px activity:g for 10 min. The supernatant was separated and read at 535 nm. A calibration curve was established using trimethylolpropane as the reference compound and treated similarly. The hepatic tissue TBARS level was calculated using this curve.The method of Buege and Aust Liver tissue samples were collected at 24 h after LPS administration. After excision, the liver was cut into slices at a thickness of 0.5∼1.0 cm and immersed in a 10% neutral formalin solution for 3 days. Specimens were embedded in paraffin and frozen at 2∼8°C. The frozen slices were sliced with a rotary microtome to yield slices with a thickness of 4 μM. The microtomed slices were mounted onto microscopic glass slides, and treated with 50∼60°C water. After being tempered overnight in an oven held at 37°C, the slices were stained with H&E, sealed with fat-soluble gel, and examined microscopically .2, 10 mM KCl, 1 mM dithiothreitol, and 1 mM phenylmethylsulfonylfluoride) and incubated on ice for 10 min. The samples were then centrifuged at 850×g for 10 min at 4°C. The supernatants were discarded and the pellets were resuspended in 1 μL of buffer A with 0.1% Triton X-100 per μg of tissue, incubated for 10 min on ice and centrifuged as described above. The supernatant was removed and saved as the cytoplasmic fraction. The cytoplasmic protein was used for detection of inducible nitric oxide synthase (iNOS) and NF-κB p65 in Western blot analysis. Sample (30 μg of protein) was subjected to SDS-polyacrylamide gel electrophoresis (8% gel for iNOS and 12% for NF-κB p65). Proteins in the gel were transferred to a polyvinylidene difluoride membrane by electroblotting, and the membrane was incubated sequentially with primary antibodies that recognize iNOS , NF-κB p65  and β-actin  for 2 h, and horseradish peroxidase-conjugated secondary antibody for 1 h at room temperature. After washing with phosphate-buffered saline (PBST), goat anti-rabbit IgG horseradish peroxidase (HRP)-conjugated secondary antibodies (Santa Cruz) (1∶1000 dilution in PBST) were incubated for1 h at roomtemperature. Immunoreactive proteins were detected using an enhanced chemiluminescence kit , and the relative expression of the protein bands was quantified by densitometry with ImageQuant TL software .Liver samples were homogenized and cytoplasmic proteins were isolated as following. One gram liver tissue was homogenized in 10 mL of ice-cold buffer A . Tukey's test or a least significant different (LSD) test was used to analyze differences in significance. 3-Manp was found to be the most abundant among all monosaccharide derivatives. The second most abundant was 2,4,6-Me3-Glcp ). Interestingly, as to inhibition of IL-6 production, antrodan alone showed a rather prominent suppressive effect. However, in treated groups, the higher dose of antrodan was shown to be much inferior in a dose-dependent manner (p<0.05).LPS highly stimulated serum GOT, GPT, and IL-6 levels to 168±45 U/L, 160±76 U/L, and 68.3±20.2 pg/mL, respectively . Antrodat manner  (p<0.05)p<0.05) and antrodan by 52% (p<0.05) , LPS reduced SOD by about 77% ((p<0.05) . The low(p<0.05) .p<0.05). It is worth noting that antrodan L+LPS and antrodan H+LPS showed no better effect, and respective CAT activities were still 61.55±4.68 and 46.23±7.18 U/mg protein . Antrodan alone suppressed the CAT level to 68.54±15.74 U/mg protein, a 57% decrease ( protein . GSH-Px  protein .p<0.05) (p<0.05). In contrast, in the antrodan L+LPS and antrodan H+LPS groups, TBARS levels were significantly ameliorated to 107.48±14.37 and 107.95±19.84 nmole/mg protein, respectively. It seemed that a low dose of antrodan (40 mg/kg) had a full-strength alleviating effect  . To our g effect .p<0.05).LPS highly stimulated serum NO to 55.5±6.0 μM . AntrodaThe histopathological examination revealed no morphological alterations in either the control  or antroLPS highly upregulated cytosolic NF-κB and substantially downregulated iNOS activity . AntrodaHericium erinaceus was been found to have immunomodulatory activity Recently, we showed antrodan characteristically to be a protein-bound polysaccharide in which β-glucans occupied a higher percentage (14.20%) than α-glucans (1.45%). In addition, a large amount of uronic acid (152.6 mg/g) coexists in the complex Ganoderma atrum also revealed multiple strong antioxidant activities in vivoNonetheless, the β-glucan content of antrodan is slightly lower compared to yeast which has a β-(1→3) glucan content of 45.47% in vivo are stimulated monocytes/macrophages, fibroblasts, and vascular endothelial cells IL-6 has been found both in local and systemic acute inflammatory responses. However, the main sources of IL-6 LPS exerts oxidative stress and damages rat livers β-Glucan stimulates lymphocyte formation in the injured body, producing the cytokine, IL-1, and an immunoglobulin M (IgM) antibody and adjusting the immune function -) production and protein nitration in the mitochondrial matrix, resulting in mitochondrial dysfunction and contractile failure One current hypothesis for the molecular mechanisms of septic shock is that enhanced NO production by mitochondrial (mt) NOS leads to excessive peroxynitrite  TBARS, 13,14-dihydro-15-keto-prostaglandin F(2α) and NO in situ in the liver and skeletal muscles. Liver chemiluminescence in inflammatory processes and phagocyte chemiluminescence were found to spectrally differ from spontaneous liver chemiluminescence with increased emission at 440∼600 nm − and ONOO− could participate in reactions leading to the formation of excited species Antrodan showed a promising  content , Table 1While evidence for ‘baseline’ iNOS expression has been elusive, IRF1 and NF-κB-dependent activation of the iNOS promoter supports inflammation-mediated stimulation of this transcript. iNOS produces large quantities of NO upon stimulation, such as by proinflammatory cytokines  The phenomena that antrodan alone actively raised the hepatic TBARS to a high level but was only moderately effective in counteracting LPS-induced TBARS elevation  imply duAgaricus blazei exhibited main components of FI0, a β-(1→6)-; (1→3)-β-D-glucan; FA-1, an-α acidic (1→6)-; (1→4)-α-D-glucan; FA-1, an acidic β-(1→6)-; α-(1→3)-D-glucan; and FA-2, a β-acidic RNA-protein complex It is worth noting that the active polysaccharide fractionated from the fruiting body of In conclusion, a low dose of antrodan (40 mg/kg) can act as a promising hepatoprotective agent. In addition, we identified the partial main architectural backbone of antrodan to have a 1→3 linear β-glycosidic backbone of mannan linked by β-1→3 glucosan branches. The findings of certain adverse effects that might be caused by the higher dose of antrodan inspired us to conduct further studies on i) reducing the molecular weight (or molecular size); ii) eliminating most of the Core2 O-glycans, if any; and iii) eliminating the N-glycan content, if any. However, according to previous experiences


RGS14-SLC34A1-PFN3-F12 on 5q35.3  = 1.19), INMT-FAM188B-AQP1 on 7p14.3 , and DGKH on 13q14.1 . Subsequent analyses in 21,842 Japanese subjects revealed the association of SNP rs11746443 with the reduction of estimated glomerular filtration rate (eGFR) (P = 6.54×10−8), suggesting a crucial role for this variation in renal function. Our findings elucidated the significance of genetic variations for the pathogenesis of nephrolithiasis.Nephrolithiasis is a common nephrologic disorder with complex etiology. To identify the genetic factor(s) for nephrolithiasis, we conducted a three-stage genome-wide association study (GWAS) using a total of 5,892 nephrolithiasis cases and 17,809 controls of Japanese origin. Here we found three novel loci for nephrolithiasis:  P<1×10−8). The top two significant SNPs, rs11746443 and rs1000597, are located upstream of the SLC34A1 and the AQP1 genes that play important roles in kidney function and the urine-concentration process, respectively. We also found that SNP rs11746443 is associated with the reduction of estimated glomerular filtration rate (eGFR), indicating the role of this variation in kidney function. Although nephrolithiasis is considered as one of the lifestyle-related diseases, the results of dietary intervention studies to reduce the recurrence incidence have been unsuccessful. Our findings could contribute to a better understanding of the pathogenesis of nephrolithiasis and lead to the development of new therapeutics.Although nephrolithiasis is one of the most common nephro-urological disorders with high prevalence (4%–9%) and extremely high recurrence rate (60% within ten years), little is known about the role of common variations in its pathogenesis. Through a GWAS using a total of 5,892 cases and 17,809 controls, we identified three novel nephrolithiasis loci: rs11746443, rs1000597, and rs4142110 ( CLDN14 gene with nephrolithiasis and bone mineral density Nephrolithiasis, also called kidney stone, is a common disorder which causes severe acute back pain and sometimes leads to severe complications such as pyelonephritis or acute renal failure. Lifetime prevalence of nephrolithiasis is estimated to be 4–9% in Japan P≥1×10−6 in controls), we conducted principal component analysis and found that all subjects were of Asian ancestry (P<5×10−8), we selected top 100 SNPs (P≤7.85×10−5) showing the smallest P-value for further association analysis   . TherefoP<5×10−8) without heterogeneity between three studies (−7) , these two SNPs did not indicate significant association in stage 3  and exhibited heterogeneity between three studies  = 1.19), INMT-FAM188B-AQP1 on 7p14.3 , and DGKH on 13q14.1 .Subsequently, we genotyped 11 SNPs in the additional cohort consisting of 2,109 nephrolithiasis cases and 4,622 controls. We excluded SNP rs10866504 from further analysis due to departure from Hardy-Weinberg equilibrium (HWE) in control samples. Five SNPs among ten cleared the significant threshold (P<0.005) in stage 3 . A meta- studies . SNP rs3ies (−7) . Althoug studies . TherefoD' = 0.992 and 2r = 0.600), while SNP rs1000597 in 7q14 was also in high linkage disequilibrium with rs12669187 (D' = 0.87 and 2r = 0.64). To further evaluate the effects of these variations, we conducted the conditional analyses. As a result, all four SNPs remained to be significantly associated with nephrolithiasis  using data from HAPMAP phase II (JPT). The regional association plots revealed that all modestly-associated SNPs are confined within an ectively . The res2 region . SLC34A1n kidney . MutatioFAM188B gene and at 14.2 kb upstream of the AQP1 gene which encodes aquaporin-1  but revealed some trend of the association with P-value of 3.29×10−3 and OR of 1.10 , metanalysis of three studied indicated the suggestive association (P = 5.72×10−4 and OR of 1.10). Taken together, CLDN14 would be a common genetic locus for nephrolithiasis in both Caucasian and Japanese populations.The  of 1.10 . To furtThen we conducted the multiple logistic regression analysis of three significant variations  including age, gender, and BMI as covariates. As a result, these variations remained as factors that were strongly associated with nephrolithiasis with OR of 1.18  of 1.12–1.25), 1.21 (95% C.I. of 1.14–1.27), and 1.14 (95% C.I. of 1.09–1.19), respectively , indicatThrough the GWAS and two sets of replication studies using a total of 5,892 cases and 17,809 controls, we identified three significantly-associated loci for nephrolithiasis. Although the molecular mechanisms how these variations could increase the risk of nephrolithiasis should be further investigated, our results elucidated crucial roles of genetic factors related with renal function as well as pathogenesis of nephrolithiasis. Nephrolithiasis is considered as one of lifestyle-related diseases; low fluid intake, low dietary calcium, and high dietary salt have been shown to increase the disease risk. However the results of dietary intervention studies to reduce the recurrence incidence of nephrolithiasis has been unsuccessful All subjects provided written informed consent. This project was approved by the ethical committees at the Institute of Medical Science, University of Tokyo, and Center for Genomic Medicine, Institutes of Physical and Chemical Research (RIKEN).Characteristics of each cohort are shown in P-value of Hardy-Weinberg equilibrium test of <1.0×10−6 in controls, and minor allele frequency (MAF) of <0.01 in each stage. Finally, we analyzed 556,249 SNPs in GWAS. In stage2, we genotyped 2,783 cases and 5,251 non-nephrolithiasis controls  by using multiplex PCR-based Invader assay (Third Wave Technologies) or Human Omni Express, respectively. In replication/stage 3, we genotyped 2,109 cases and 4,622 non-nephrolithiasis controls  using multiplex PCR-based Invader assay (Third Wave Technologies). For the QTL analyses, we genotyped 27,323 samples using Illumina HumanHap610-Quad Genotyping BeadChip as described previously Genomic DNA was extracted from peripheral blood leukocytes using a standard method. In the GWAS, a total of 1000 cases and 7,936 non-nephrolithiasis controls  were genotyped at 712,726 SNPs using Human Omni Express . We perf−8 in meta-analysis of all stages −5 (TOP 100 SNPs) in GWAS, 8.47×10−4 (0.05/59) in stage2, and 5.00×10−3 (0.05/10) in stage 3, respectively. We selected SNPs which satisfied all these four criteria. Heterogeneity across three stages was examined by using the Breslow-Day test −5), 96.38% in stage2 (P = 0.05/59), 95.54% in stage3 (P = 0.05/10), and 99.37% in all stage (P = 5.00×10−8) at minor allele frequency of 0.3 and Odd ratio of 1.2. For multiple logistic regression analysis at rs11746443, rs1000597 and rs4142110, we considered age at recruitment, gender, and BMI as covariates using the R program.The association of SNPs with nephrolithiasis in each stage was tested by a 1-degree-of-freedom Cochran-Armitage trend test using PLINK We used a Hidden Markov model programmed in MACH 2) = 194×serum creatinine (mg per 100 ml)−1.094×age−0.287 . For eGFR, a linear regression model was used to analyze the associations of quantitative traits with SNP genotypes by incorporating alcohol, smoking status, as covariates.We assessed the effect of genetic variations on serum calcium, phosphorus, urate, BMI, and eGFR, as described previously mRNA or total RNA from normal tissues were purchased from Calbiochem and BioChain. cDNAs were synthesized with the SuperScript Preamplification System (Invitrogen). Quantitative real-time PCR was conducted using the SYBR Green I Master on a LightCycler 480 (Roche). The primer sequences are indicated in i is the weight for SNPi, and Xi is the number of risk alleles .The wGRS was calculated by multiplying the number of risk alleles for each SNP by weight for that SNP  , and thehttp://frodo.wi.mit.edu) web tool was used to design primers. We employed LocusZoom (http://csg.sph.umich.edu/locuszoom/) for plotting regional association plots. We used SNP Functional Prediction http://snpinfo.niehs.nih.gov/snpfunc.htm). We used MACTH http://www.gene-regulation.com/index.htm).For general statistical analysis, we employed R statistical environment version 2.9.1 (cran.r-project.org) or plink-1.06 (pngu.mgh.harvard.edu/∼purcell/plink/). The Haploview software version 4.2 Figure S1Summary of study design and results.(TIF)Click here for additional data file.Figure S2Principal Component Analysis (PCA) plots showing the distribution of case and control used in the GWAS. CEU; Utah residents with Northern and Western European ancestry from the CEPH collection. YRI; Yoruban in Ibadan, Nigeria. CHB; Han Chinese in Beijing, China. JPT; Japanese in Tokyo, Japan. Case and control samples belonged to Hondo cluster according to PCA were used in this study.(TIF)Click here for additional data file.Figure S3Association analysis using all samples included in Hondo and Ryukyu clusters. Quantile-quantile plots for test statistics (Cochran-Armitage trend test) for 556,249 SNPs passing the quality control. Black dots are the uncorrected test statistics (λ = 1.123). Under the null hypothesis of no association at any loci, the points would be expected to follow the red line (y = x).(TIF)Click here for additional data file.Figure S4P values of association. The P values were calculated by Cochran-Armitage trend test. The y axis represents the −log10 P values of 556,249 SNPs, and x axis shows their chromosomal positions. The horizontal blue line shows the threshold of P≤7.85×10−5 for selecting top 100 SNPs for replication.a) Association analysis using all samples. Quantile-quantile plots for test statistics  for 556,249 SNPs passing the quality control. Black dots are the uncorrected test statistics (λ = 1.054). Under the null hypothesis of no association at any loci, the points would be expected to follow the red line (y = x). b, Association analysis using samples included in a Hondo cluster. Quantile-quantile plots for test statistics (Cochran-Armitage trend test) for 556,249 SNPs passing the quality control. Black dots are the uncorrected test statistics (λ = 1.042). Under the null hypothesis of no association at any loci, the points would be expected to follow the red line (y = x). c, Association analysis using samples included in a Hondo cluster. Manhattan plot showing the genome-wide (PDF)Click here for additional data file.Figure S5P values of heterogeneities (Phet) across three stages examined by using the Breslow-Day test.Forest plots for stratified association analysis at rs11746443 (a), rs1000597 (b) and rs4142110 (c). Case and control samples from GWAS, replication 1, and replication 2 were stratified by age , gender , and BMI . Odds ratios and confidence intervals are calculated from fixed effect model implemented in R. The (PDF)Click here for additional data file.Figure S6P values of genotyped SNPs (circle) and imputed SNPs (cross) are plotted  against their physical position on chromosome 5 (a), 7 (b), and 13(c) (NCBI Build 36). SNPs rs11746443 on 5q35 (a), rs1000597 on 7p14 (b), and rs4142110 on 13q14 (c) are represented by purple diamonds. The genetic recombination rates estimated from 1000 Genomes samples (JPT+CHB) are shown with a blue line. SNP's color indicates LD with rs11746443 (a), rs1000597 (b), and rs4142110 (c) according to a scale from r2 = 0 to r2 = 1 based on pair-wise r2 values from HapMap JPT. Middle Panel; Gene annotations from the University of California Santa Cruz genome browser. Lower Panel; We drew the LD map based on D' values using the genotype data of the cases and controls in the GWAS samples.Enlarged regional plots of three significant susceptibility loci at 5q35, 7p14, and 13q14. (a–c) Upper panel; (PDF)Click here for additional data file.Figure S7SLC34A1, AQP1 and DGKH genes in 27 normal tissues. Quantitative PCR analysis of SLC34A1 (a), AQP1 (b), and DGKH (c) in normal tissues. ACTB was used for normalization of expression levels.Expression of (TIF)Click here for additional data file.Figure S8ENST00000434909. (a) Gene Structure of FAM188B, AQP1 and ENST00000434909. Locations of rs12669187 and rs1000597 are intronic 4 and intronic 6 of ENST00000434909. (b) Protein structure of AQP1, FAM188B, and ENST00000434909. (c) Quantitative PCR analysis of ENST00000434909 in normal tissues. ACTB was used for normalization of expression levels.Expression of (TIF)Click here for additional data file.Figure S9P-values of genotyped SNPs (circle) and imputed SNPs (cross) are plotted  against their physical position on chromosome 21 (NCBI Build 36). SNPs rs2835349 on 21q22 are represented by purple diamonds. The genetic recombination rates estimated from 1000 Genomes samples (JPT+CHB) are shown with a blue line. SNP's color indicates LD with rs2835349 according to a scale from r2 = 0 to r2 = 1 based on pair-wise r2 values from HapMap JPT. Middle Panel; Gene annotations from the University of California Santa Cruz genome browser. Lower Panel; We drew the LD map based on D' values using the genotype data of the cases and controls in the GWAS samples.Regional association plots at rs2835349 loci. Upper panel; (TIF)Click here for additional data file.Table S1Characteristics of samples and methods used in this study.(DOCX)Click here for additional data file.Table S2The Result of GWAS for nephrolithiasis (Top 100 SNPs).(DOCX)Click here for additional data file.Table S3Results of association analysis for nephrolithiasis in stage2 (59 SNPs).(DOCX)Click here for additional data file.Table S4Results of meta-analysis between GWAS and Stage 2 for nephrolithiasis (59 SNPs).(DOCX)Click here for additional data file.Table S5a) Results of association analysis for nephrolithiasis in stage3/replication (11 SNPs). b) Results of meta analysis of 3 stage (11SNPs).(DOCX)Click here for additional data file.Table S6The results of conditional analysis for significantly SNPs in 5q35.3 or 7p14.3 associated with nephrolithiasis in all stages.(DOCX)Click here for additional data file.Table S7Result of imputation analysis at 5q35.3.(DOCX)Click here for additional data file.Table S8Result of imputation analysis and snp location at 7p14.3 (a) and 13q14.1 (b).(DOCX)Click here for additional data file.Table S9Association of rs2835349 with nephrolithiasis in the all stage and combined analysis.(DOCX)Click here for additional data file.Table S10Multiple logistic regression analysis for risk of nephrolithiasis.(DOCX)Click here for additional data file.Table S11a) SNPs that compose the weighted genetic risk score and weights assigned to each marker. b) wGRS scores and odd ratios of nephrolithiasis by wGRS groups.(DOCX)Click here for additional data file.Table S12Sequences of primers.(DOCX)Click here for additional data file.


We aimed to examine associations among serum 25-hydroxyvitamin D (25OHD) levels, 1,25-dihyroxyvitamin D  levels, vitamin D receptor (VDR) polymorphisms, and renal function based on estimated glomerular filtration rate (eGFR) in patients with type 2 diabetes.I (rs10735810) polymorphisms by an ordered logistic regression model adjusted for the following confounders: disease duration, calendar month, use of angiotensin converting enzyme inhibitors/angiotensin receptor blockers or statins, and serum calcium, phosphate, and intact parathyroid hormone levels.In a cross-sectional study of 410 patients, chronic kidney disease (CKD) stage assessed by eGFR was compared with 25OHD, 1,25OHD, and VDR FokP<0.0001), but it had stronger linearity with 1,25OHD (r = 0.73) than 25OHD (r = 0.22) levels. Using multivariate analysis, 1,25OHD levels (P<0.001), but not 25OHD levels, were negatively associated with CKD stage. Although FokI polymorphisms by themselves showed no significant associations with CKD stage, a significant interaction between 1,25OHD and FokITT was observed (P = 0.008). The positive association between 1,25OHD and eGFR was steeper in FokICT and CC polymorphisms (r = 0.74) than FokITT polymorphisms (r = 0.65).1,25OHD levels, rather than 25OHD levels, showed seasonal oscillations; peak levels were seen from May to October and the lowest levels were seen from December to February. These findings were evident in patients with CKD stage 3∼5 but not stage 1∼2. eGFR was in direct proportion to both 25OHD and 1,25OHD levels (I polymorphisms.These results suggest that higher 1,25OHD levels may be associated with better CKD stages in patients with type 2 diabetes and that this association was modified by Fok Worldwide, the number of people with diabetes is projected to rise from 171 million in 2000 to 366 million in 2030 25-hydroxyvitamin D (25OHD) is the primary circulating form of vitamin D and is converted into 1,25-dihyroxyvitamin D  through 1alpha-hydroxyvitamin D (1aOHase); this conversion takes place primarily in the kidneys. As a result, 1,25OHD levels can be decreased in patients with renal dysfunction. Low levels of 1,25OHD increase renal renin production, thus activating the renin-angiotensin-aldosterone system (RAAS), reduce renal expression of klotho, increase fibroblast growth factor-23 levels, and consequently suppress 1aOHase, further lowering 1,25OHD levels, all of which are associated with progression of renal damage I polymorphisms in the vitamin D receptor (VDR) gene differ between patients with diabetic nephropathy and healthy subjects I TT genotype, FokI CC had 1.7-fold greater function of vitamin D-dependent transcriptional activation of a reporter construct under the control of a vitamin D response element in transfected HeLa cells I T) to ACG (FokI C) polymorphism, the first potential start site moves to the 3′ direction, resulting in proteins that are three amino acids shorter and more functional Furthermore, FokTaken together, this evidence suggests that 25OHD, 1,25OHD, and VDR may play a role in exacerbation of diabetic nephropathy, at least in part. However, there are no reports studying these three factors together. Therefore, we conducted a cross-sectional study of patients with type 2 diabetes to elucidate 1) which is a stronger factor in the association with eGFR levels and CKD stages, 25OHD or 1,25OHD, and 2) if there is any interaction between 25OHD/1,25OHD and VDR polymorphisms, in association with CKD stages, after adjusting for other confounders.This cross-sectional study was carried out as collaboration among the Division of Kidney, Hypertension; Division of Diabetes, Metabolism and Endocrinology; and Division of Molecular Epidemiology, Jikei University School of Medicine. The study protocol was reviewed and approved by the ethics committee of the Jikei Institutional Review Board, Jikei University School of Medicine, as well as the clinical study committee of the Jikei University hospital and Shin-Kashiwa clinic.Patients aged 20 to 80 years old with type 2 diabetes, diagnosed by doctors based on Japanese diagnostic criteria −1.094× age−0.287, if female, ×0.739 2; Stage 2 CKD, eGFR ≥60 to <90 ml/min/1.73 m2; Stage 3 CKD, eGFR ≥30 to <60 ml/min/1.73 m2; Stage 4 CKD, eGFR ≥15 to <30 ml/min/1.73 m2; and Stage 5 CKD, eGFR <15 ml/min/1.73 m2.Disease duration (years) was defined as the period of time between diagnosis of diabetes and clinical evaluation for entry into the study. Age, gender, height, weight, and blood pressure as well as laboratory data of peripheral blood calcium (Ca) , phosphate (P) , and intact parathyroid hormone  levels were recorded. eGFR was calculated according to the following Japanese standard formula based on insulin clearance: 194× creatinineSerum levels of 25OHD (ng/mL) and 1,25OHD (pg/mL) were measured at SRL Inc.  as described previously I single nucleotide polymorphisms as described previously We used polymerase chain reaction and direct sequencing to analyze VDR polymorphisms according to the FokI polymorphisms, we made a new variable by multiplying the 25OHD/1,25OHD levels and the FokICC/FokICT/FokITT polymorphisms. Two-sided P values <0.05 were considered statistically significant. All statistical analyses were performed using STATA 12.0 .Associations between CKD stage and patients' characteristics were evaluated using single-ordered logistic regression models and chi-square test. Associations between CKD stage and vitamin D-related factors as well as other possible confounders were evaluated using a multiple-ordered logistic regression model. Hardy-Weinberg equilibrium was assessed by the chi-square test. To identify any interaction between 25OHD/1,25OHD and FokA total of 410 patients agreed to participate in this study. Baseline characteristics of the study population divided by CKD stage are shown in Histograms of circulating 25OHD  and 1,25OHD levels  are shown in P = 0.001∼0.027)  . In cont1∼0.027)  but not 1∼0.027) . 25OHD lP<0.0001) and 1,25OHD levels (P<0.0001), but had a stronger linearity for 1,25OHD (r = 0.73) than 25OHD (r = 0.22). 1,25OHD levels were positively associated with in Ca levels (P<0.0001), whereas they were negatively associated with P levels (P<0.0001) and iPTH levels (P<0.0001).eGFR levels were compared with 25OHD  and 1,25I (P = 0.23): Genotype frequencies: CC: 175; CT: 177; TT: 58; Allele frequencies: C: 527; T: 293. Associations between VDR polymorphisms, serum 25OHD/1,25OHD levels, their interactions, and CKD stages were computed with a multiple ordered logistic regression model after adjustment for possible confounders including disease duration, calendar month, use of ACEI/ARB, use of statins, and levels of Ca, P, and iPTH (P<0.001), but not 25OHD levels, were negatively associated with CKD stage. Although FokI polymorphisms by themselves showed no significant associations with CKD stages, a significant interaction between 1,25OHD and FokITT was observed (P = 0.008). The positive association between 1,25OHD and eGFR was steeper in patients with FokICC + CT polymorphisms (r = 0.74) than those with FokITT polymorphisms (r = 0.65)  . There wIn this study, we found that 1,25OHD levels, rather than 25OHD levels, showed seasonal oscillations in our study population. Peak values were seen from May to October and the lowest values were seen from December to February. Of interest, this oscillation of 1,25OHD levels was observed only in diabetic patients with advanced CKD (stage 3∼5), but not in patients with early CKD (stage 1∼2). Seasonal oscillations have been well documented in 25OHD levels I polymorphisms by themselves showed no significant associations with CKD stage. A recent article showing the significance of FokI polymorphisms compared patients with ESRD with healthy controls ITT polymorphisms demonstrated impaired renal function with increased serum cystatin C levels, glomerular dysfunction with proteinuria, as well as significant tubulopathy with hypercalciuria and increased levels of urinary β2-microglobulin I and TaqI was protective against nephropathy in patients with type 1 diabetes, although FokI polymorphisms had no significant association with the nephropathy I T alleles were independently associated with lower plasma renin activity IT) to ACG (FokIC), they were considered to be more transcriptionally potent In our study population of diabetic patients, FokITT: The negative association between 1,25OHD and CKD stages was more repressed in FokITT polymorphisms than FokICC polymorphisms. Moreover, the positive association between 1,25OHD and eGFR was steeper in patients with FokICC + CT polymorphisms than those with FokITT polymorphisms. Some studies have reported interactions between VDR polymorphisms and 25OHD in diseases such as colorectal adenoma Our study showed a significant interaction between 1,25OHD and FokThere are several limitations to this study. First, the study design was cross-sectional. Thus, we cannot determine whether decreased 25OHD and 1,25OHD levels are an aggravate cause of CKD or a consequence of exacerbation of CKD in diabetic patients. There was a seasonal oscillation in 25OHD levels among different patients . Thus information related to seasonal variations of 25OHD levels should be interpreted with caution. Second, interaction tests should be used cautiously in data analyses, as most studies are not powered to detect such interaction effects, and results of such tests are always exploratory in nature I polymorphisms.In conclusion, these results suggest that higher 1,25OHD levels may be associated with better CKD stages in patients with type 2 diabetes and that this association was modified by Fok


Rivaroxaban reduces stroke in patients with atrial fibrillation (AF). Left atrium (LA) plays a critical role in the pathophysiology of AF. However, the electromechanical effects of rivaroxaban on LA are not clear.n = 7) AP durations at 90% repolarization (APD90) from 76 ± 2 to 79 ± 3, 67 ± 4 , 59 ± 5, , and 56 ± 4 ms , respectively. Rivaroxaban  concentration-dependently increased the LA (n = 7) diastolic tension by 351 ± 69 , 563 ± 136 , 582 ± 119 , and 603 ± 108 mg , respectively, but did not change LA contractility. In the presence of L-NAME (100 μM) and indomethacin (10 μM), additional rivaroxaban (300 nM) treatment did not significantly further increase the LA (n = 7) diastolic tension, but shortened the APD90 from 73 ± 2 to 60 ± 6 ms . Rivaroxaban (100 nM) increased the L-type calcium current and ultra-rapid delayed rectifier potassium current, but did not change the transient outward potassium current in isolated LA cardiomyocytes.Conventional microelectrodes and a whole-cell patch-clamp were used to record the action potentials (APs) and ionic currents in rabbit LA preparations and isolated single LA cardiomyocytes before and after the administration of rivaroxaban. Rivaroxaban  concentration-dependently reduced LA (Rivaroxaban modulates LA electrical and mechanical characteristics with direct ionic current effects. LA plse (NOS) . In varise (NOS) . Accordise (NOS) ,6.Rivaroxaban selectively binds to the FXa and reduces thrombin formation, which prevents stroke in AF patients . HoweverGuide for the Care and Use of Laboratory Animals. Male rabbits (weighing 1 ~ 2 kg) were anesthetized with an intraperitoneal injection of sodium pentobarbital (100 mg/kg). A midline thoracotomy was then performed, and the heart and lungs were removed as described previously [3, 0.5 NaH2PO4, 0.5 MgCl2, 2.7 CaCl2, and 11 dextrose. Preparations were connected to a WPI model FD223 electrometer under a tension of 150 mg as described previously [2-3% CO2 gas mixture. The temperature was maintained at 37°C, and the preparations were allowed to equilibrate for 1 h before the electrophysiological assessment.Experiments in this study conformed to the institutional eviously ,9. To dibarbital 0 mg/kg. 90, APD50, and APD20. The RMP, APA, APD90, APD50, APD20, and contractile forces were measured under 2-Hz pacing of the LA before and after the sequential administration of rivaroxaban . In order to study the mechanical effects of rivaroxaban, an NG-nitro-L-arginine methyl ester (L-NAME) solution  and indomethacin  solution and rivaroxaban solution (300 nM) were sequentially added to LA preparations.Transmembrane action potentials (APs) were recorded by machine-pulled glass capillary microelectrodes filled with 3 mol/L of KCl which were connected to a WPI Duo 773 electrometer under a tension of 150 mg. Electrical and mechanical events (contractile force and diastolic tension) were simultaneously displayed on a Gould 4072 oscilloscope and a Gould TA11 recorder. Using a data acquisition system, signals were recorded with DC coupling and a 10-kHz low-pass filter cutoff frequency. Signals were recorded digitally with a 16-bit accuracy at a rate of 125 kHz. Electrical stimulation was provided using a Grass S88 stimulator through a Grass SIU5B stimulus isolation unit. The AP amplitude (APA) was obtained by measuring the difference between the resting membrane potential (RMP) or maximum diastolic potential and the peak of AP depolarization. AP durations (APDs) at repolarization of 90%, 50%, and 20% of the APA were respectively measured as APDICa-L) or ruptured (for others) patch clamp was performed in single isolated cardiomyocyte before and after the administration of rivaroxaban (100 nM) using an Axopatch 1D amplifier  at 35 ± 1°C[2 0.5, CaCl2 1.8, and glucose 10. The solution was titrated to a pH of 7.4 with NaOH. Micropipettes were filled with a solution containing (in mM) CsCl 130, MgCl2 1, Mg2ATP 5, HEPES 10, EGTA 10, NaGTP 0.1, and Na2 phosphocreatine 5, (adjusted to a pH of 7.2 with CsOH) for the ICa-L and containing (in mM) KCl 20, K aspartate 110, MgCl2 1, Mg2ATP 5, HEPES 10, EGTA 0.5, LiGTP 0.1, and Na2 phosphocreatine 5, (adjusted to a pH of 7.2 with KOH) for the AP and potassium currents. The ICa-L was measured as an inward current during depolarization from a holding potential of −50 mV to testing potentials ranging from −40 to +60 mV in 10-mV steps for 300 ms at a frequency of 0.1 Hz. The NaCl and KCl in the external solution were respectively replaced by TEACl and CsCl. The transient outward potassium current (Ito) was studied with a double-pulse protocol. A 30-ms pre-pulse from −80 to −40 mV was used to inactivate the sodium channels, followed by a 300-ms test pulse to +60 mV in 10-mV steps at a frequency of 0.1 Hz. CdCl2 (200 μM) was added to the bath solution to inhibit the ICa-L. The Ito was measured as the difference between the peak outward current and steady-state current [IKur) was studied with a double-pulse protocol, consisting of a 100-ms depolarizing pre-pulse to +40 mV from a holding potential of −50 mV, followed by 150-ms voltage steps from −40 to +60 mV in 10-mV increments at room temperature to provide an adequate temporal resolution. The IKur was measured as 4-aminopyridine (1 mM)-sensitive currents [Single LA myocytes were enzymatically dissociated through the same procedure as described previously . A whole 35 ± 1°C. Borosilcurrents ,15.t-test depending on the outcome of the normality test. A P value of less than 0.05 was considered statistically significant.All continuous variables are expressed as the mean ± standard error of the mean (SEM). A one-way repeated-measures analysis of variance (ANOVA) was used to compare the difference before and after drug administration on LA. The electrophysiological and mechanical characteristics between different groups were compared by a Wilcoxon rank-sum test or unpaired 20, APD50, and APD90 in LA. However, rivaroxaban did not significantly affect the RMP, APA, or contractile force of LA. Moreover, rivaroxaban  concentration-dependently increased LA diastolic tension, which could not be reversed after washout for 30 min . Superfusion with rivaroxaban significantly reduced APD50 and APD20 in LA myocytes. However, rivaroxaban (100 nM) did not affect the APA or RMP in LA myocytes.Rivaroxaban had rapid effects on the electrical activity of LA myocytes and reached a steady state in 3 ~ 5 min after its superfusion. Figure ICa-L before and after rivaroxaban (100 nM) administration in LA myocytes, and it was found that rivaroxaban increased the peak ICa-L by 11.8% . However, as shown in Figure Ito. Moreover, Figure IKur by 29.6% .Figure max), of around 125 μg/L or 300 nM, of rivaroxaban was reached within 2 ~ 4 h after oral administration after a single oral dose in healthy volunteers [To reduce thromboembolic complications with AF, several new antithrombotic agents were developed with non-inferior antithrombotic effects and fewer hemorrhagic complications than the traditional vitamin K antagonist ,17. It’slunteers ,23. Ther90 compared to the baseline  did not significantly increase the wall tension, but the total increment in the wall tension was comparable to the increase by rivaroxaban (300 nM) as shown in Figure e Figure . It is n20 and APD50, but did not affect the APD90, RMP, or APA. The less shortening of the APD in isolated single cardiomyocyte by rivaroxaban suggested that the greater shortening of APD in LA tissue preparations may have been caused by mechanoelectrical feedback due to the increased wall tension with stretched myocytes in tissue preparation experiments. The ionic current experiments showed that rivaroxaban did not change the Ito but significantly increased the IKur, which may have contributed to its effect in shortening the APD. Rivaroxaban also increased the ICa-L, which may have been a result of repolarization homeostasis and implies that the increasing LA wall tension was due to intracellular calcium loading. These findings suggest that rivaroxaban at clinically relevant concentrations directly affects ionic currents in LA myocytes. In order to correlate the clinical settings, we use 100 nM of rivaroxaban to measure its ionic current effects, which are closely related to the known plasma concentrations in humans [In experiments on isolated single myocytes, we found that rivaroxaban (100 nM) shortened the APDn humans . HoweverRivaroxaban modulates LA electrical characteristics with direct effects on ionic currents, and regulates mechanical characteristics, which can be attenuated by the inhibitions on NOS and COX.The authors declare that they have no competing interests.CJC and YKL interpreted the data and drafted the manuscript. YCC and JHH performed the experiments and revised it for scientific content. SAC and YJC conceived of this study, and participated in its design and coordination. All authors read and approved the final manuscript.


The configurations with two LEDs and four LEDs are developed for measuring PPG signal and Blood Oxygen Saturation (SpO2). N-LEDs configuration is proposed for multichannel SpO2 measurements. The approach resulted in better spectral sensitivity, increased and adjustable resolution, reduced noise, small size, low cost and low power consumption.A fully digital photoplethysmographic (PPG) sensor and actuator has been developed. The sensing circuit uses one Light Emitting Diode (LED) for emitting light into human tissue and one LED for detecting the reflectance light from human tissue. A Field Programmable Gate Array (FPGA) is used to control the LEDs and determine the PPG and Blood Oxygen Saturation (S It is based on the determination of optical properties of vascular tissue using a probe, which consists of LED-photodiode configuration ,2. The LOn the other hand, it has been neglected that LED in reverse operation mode can also detect light and can be used in a wide range of applications as an inexpensive, readily available optical detector –6. TypicThe second challenge is in the fact that, today, numerous miniature, battery powered medical devices tend to be simple, robust and multifunctional . SolutiopO2, while N-LEDs system can be employed for multichannel SpO2 measurements.Inspired by the possibility to use LEDs as an optical detector as well as by the advantages of nowadays highly integrated chips like FPGA, a PPG sensor requiring only standard LEDs as sensors and FPGA as actuator has been developed. Thus, fully digital, single-chip, solution for multichannel PPG measurement is obtained. 2-LEDs configuration is used for PPG measurement, 4-LEDs configuration for measurement of SSection 2 continues with the explanation of proposed sensing methodology. The implementation of sensing algorithm in FPGA technology is elaborated in Section 3, while Section 4 presents the test results.2.d. The algorithmic steps and electrical models are given in cT (0.1 μs–0.5 μs), charging LED2 to voltage VDD (+5 V or +3.3 V), across very low output impedance pinoR. This charge is held by the sum of the inherent capacitances of the diode and I/O pin itself, rC = rLEDC + rpinC. Typically rLEDC = 10 pF–15 pF and rpinC = 3 pF–5 pF. Then, pin P2 is switched into high-impedance (Hi-Z) mode, approximately 1015 Ώ, and digital counter/timer starts.The basic sensing circuit is shown in rC discharged by a current source iR(Φ), which models the optically induced photocurrent for incoming light intensity Φ. The process of rC discharging, assuming that iR(Φ) remains constant during short time, can be expressed as:νP2(t) linearly decreases with time t to zero. During discharging νP2(t) is continually polled through its digital equivalent P2in. When P2in changes from logical “1” to “0”, νP2(t) falls below the threshold voltage TRV. For CMOS FPGA devices VTR is about Td) is read as a number of clock ticks N:tclkf presents frequency of timer/counter clock.Under reverse bias conditions and high impedance of pin P2, the LED2 acts as a capacitor νP2(t) curves, obtained through this project, follow 1Φ, 2Φ and 3Φ, 1Φ < 2Φ < 3Φ. It is apparent that dT decreases when the amount of received light increases and vice versa. For light intensities 1Φ, 2Φ and 3Φ, N corresponds to 1N, 2N and 3N. The resolution of dT can be adjusted by tclkf ¸ taking care about overflow, 2l − 1 < maxN, l is counter length. Usually, tclkf is obtained from main clock clkf by frequency divider with selectable prescale factor pN, tclkf = clk/Npf, pN = 1, 2, 4, 8, 16…. This gives a possibility to adjust resolution of dT, marked as ΔTd, with prescaler pN as:Experimentally recorded clkf = 200 MHz and pN =1, dΔT can reach value of 5 ns. The sensing cycle repeats with frequency slf = 1/slT, which is in fact the sampling frequency of PPG signal.As example, for −12A (1 pA) in total darkness to about 10−6A (1 μA) under maximal reflectance. The blood cells  play role of obstacle. Performed experiments with volunteers shown that for rC = 15 pF and TRV = DDV/2, dT varies from 0.1 ms to 1 ms that corresponds to the counter resolution of above 210 to 215.In the case of human finger, ICCH and ICCL are maximal currents of FPGA's O/I pins during their high and low states, Tsl ≫ Td, Td ≫ Tc, then power dissipation of 2-LEDs system is given as:The LED-LED sensing probe is of low energy consumption. If VDD = 3.3 V, ICCH ≈ ICCL = 25 mA, Td = 0.15 ms and Tsl = 10 ms (fsl = 100 Hz), the dissipation of LED1-LED2 system is about 1.2 mW, while for Tsl = 100 ms (fsl = 10 Hz) it decreases to 0.12 mW.As an example, for pO2 meter, by adding two more red LEDs (2) and lower optical absorption in the near infrared region (IR) [2, and then used to calculate the “normalised ratio”:pO2 according to empiric equation:The PPG configuration from red LEDs . Light fion (IR) . These dpO2 can assist in the diagnosis of various respiratory conditions and ischemic states. Normal value is from 95%–100%. A smokers could have values as low as 90% and someone with Chronic Obstructive Pulmonary Disease (COPD) <85% [Knowledge of SpO2 measurements is practically double algorithm from Td = Tdred and Td = Tdir are measured in parallel with separated timers/counters. The power consumption is also double, but does not exceed 2.5 mW.Sensing algorithm for S3.pO2 detection algorithms could be implemented by using general-purpose microcontrollers (MCs) or PLDs. Designers often choose MCs because they traditionally offer better on-chip peripherals, such as timers, ADCs, UARTs, pulse width modulation (PWM) and other integrated I/O for multiple, asynchronous tasks [etc.Previously described PPG and Sus tasks ,10,11. BUnlike processors, FPGAs use dedicated hardware for processing logic and do not have an operating system (OS). Since FPGA are there truly parallel in nature, different processing operations do not have to compete for the same resources. FPGAs and processors also differ in compilation. When an application for an FPGA device is compiled, the result is a highly optimized silicon implementation that provides parallel processing with the performance and reliability benefits of dedicated hardware circuitry. Because there is no OS on the FPGA, the code is implemented in a way that ensures maximum performance and reliability. In contrast to early families, nowadays FPGAs do not only offer a lot of logic base cells, but also huge register blocks and memory areas and powerful processor cores, which speed may achieve 500 MHz.pO2 and to perform on-line signal processing.Considering experience and results obtained during development and investigation of MC based PPG sensors , this prLED drivers and Td meters simultaneously excite emitter diodes LED1and LED3 and simultaneously measure the decay times dirT and rT (IR and RED) by tracking voltages on LED2 and LED4. Note that in case of classical pulse oxymeters the time multiplex is used to distinguish between the signals from different light sources. Such multiplex is not necessary here because the LED1, LED3 and LED2, LED4 are switched on and read in parallel. The digital equivalents of dirT and rT in form of counter/timer values, are sent to the UART directly or via a Digital filter section, depending on the MUX selection. CLK_DIV determines counter frequency tclkf for different prescaled factors. String fitter (SF) packs the samples in predefined order, IR-RED…-IR-RED, while UART sends the original or filtered data to the host computer in selectable speeds. All functional modules are realised in form of VHDL code functions with generic arguments .The FPGA architecture, proposed in this paper, consists of several functional units, Mean, FIR, and IIR. The filter function is implemented through the transposed structure which handles all types:n-1 is the filter order, an is the feedback filter order, and bn is the feedforward filter order. As example, for c = 1/N and b(1) = 0, b(2) = … = b(nb + 1) = 1, a(1) = a(2) = … = a(na + 1) = 0 it becomes mean filter and for c = 1, a(1) = a(2) = … = a(na + 1) = 0 symmetric FIR, etc. Coefficients a(i), b(i) and c are multiplied and rounded in order to be pre-scaled in integer arithmetic [In order to optimize the design and improve Signal to Noise Ratio (SNR) we investigated several types of digital filters like ithmetic .pO2 probes can be connected to a single FPGA chip and processed in parallel.Today, physiology and cardiology is facing the challenge of performing pulse oximetry measurements at other body locations where arterial blood perfusion is severely degraded. Low and heterogeneous perfusion conditions can be overcome by a multi-channel oximetry approach that simultaneously exploits spatial, frequencies and temporal diversities. The proposed LED-LED FPGA approach is very suitable for such applications, as is shown in 4.pO2 configurations against qualitative and quantitative criteria. In PPG configuration, the sensing probe comprises an IR(910 nm)-IR(910 nm) 5 mm LED combination and the signal is collected in reflectance mode from a finger. As a substrate, an FPGA chip from Altera, Cyclone III, series, EP3C10 was chosen (device EP3C10E144C8) [etc., all in real time.The proposed PPG sensor-actuator was tested in both PPG and SMean filter is shown in tclkf = 48 MHz, pN = 1 and fsl = 200 Hz.low pass FIR filter with cut-off frequency fc = 10 Hz and fsl = 200 Hz, producing an output given in Nonin Model 8000AA [(Adult Articulated Finger Clip), is presented in Qualitative comparison between proposed sensor (A) and its conventional FDA approved equivalent (B), l 8000AA   = 1,896.95, RMS(RED) = 4.96, MEAN(IR) = 6,165.14, RMS(IR) = 35.14. Using pO2 is calculated to 98.53%.pO2 measurement against clinical reference and maximal average dissipation for 2-LEDs and 4-LEDs configurations.Quantitative testing was performed considering following criteria: occupation of silicon resources for hardware implementation in different configurations, comparison of accuracy PPG and SpO2 instruments. It is very useful for multi-point blood saturation examination. As example in EP3C120 family about 30 SpO2 instruments in configuration 4 LEDs + UART + MUX + ST + FIR F. can be implanted.pO2 accuracy are performed on volunteers of different age and health conditions. About 120 different subjects were examined. The measured value of %SpO2 and %HR were compared with Nonin's system, 8000AA sensor + OEM III Eval Kit + Software [The testing experiments for PPG and SSoftware . The exppO2 instruments in a single FPGA, the consumption falls to 25 mW per instrument that is better than in Nonin's case.Additionally, two systems are compared in term of maximal average power consumption. The results are given in Obviously, the proposed system is a low cost taking in account prices of LEDs (less than 0.5$ per piece) and FPGA chip (about 15$). The cost of experimental system, presented here, was about 50$, six times less than Nonin's equivalent 8000AA sensor + OEM III Eval Kit.5.We have demonstrated the possibility to design an effective system for measurement of PPG signals and blood oxygen saturation by using a LED-LED based configuration in conjunction with a FPGA chip. The proposed sensing technique is fully digital and provides good spectral sensitivity, increased resolution, satisfactory signal to noise ratio, low power consumption, in system flexibility and reconfigurability and miniaturisation. By using the presented approach the challenges of multi-channel oximetry can be overcome.


Our in vitro results show that MSC-secreted factors increase NSC proliferation and induce higher expression of GFAP, indicating a tendency toward differentiation into astrocytes. In vivo experiments showed that MSC injection at an acute model of brain injury diminishes a broad profile of cytokines in the tissue, suggesting that MSC-secreted factors may modulate the inflammation at the injury site, which may be of interest to the development of a favorable microenvironment for endogenous NSC and consequently to repair the injured tissue.Therapy with mesenchymal stem cells (MSCs) has showed to be promising due to its immunomodulatory function. Traumatic brain injury (TBI) triggers immune response and release of inflammatory mediators, mainly cytokines, by glial cells creating a hostile microenvironment for endogenous neural stem cells (NSCs). We investigated the effects of factors secreted by MSCs on NSC  For many years, the central nervous system (CNS) was considered an immunologically privileged site, due to its particular and limited immune response, different from other systems . It is nα, IL-1β, IL-6, TNF-α, and TGF-β, chemokines, and prostaglandins, which increase the blood brain barrier permeability and facilitate the invasion of peripheral immune cells, inducing the secretion of toxic molecules [Inflammatory molecules are important in healthy nervous tissue and their expression is promptly upregulated when an injury occurs . Cytokinolecules , 7. In the adult brain, chemoattractants, including the chemokine CXCL12 produced at the injury site induce neuroblasts originated by neural stem cells (NSC) at neurogenic niches, such as the subventricular zone (SVZ), to migrate towards the injury in order to regenerate the nervous tissue –11. AlthCell-based therapy using adult-derived mesenchymal stem cells (MSC) have been tested in several disease models –24. It iin vitro. Our results show a decrease in the production of inflammatory cytokines 24 hours after MSC transplantation. We also show that soluble factors secreted by MSC increase proliferation and modulate gene expression of neural stem cells in vitro, increasing the expression of GFAP, a marker of glial cells. In order to better understand the paracrine effects of MSC transplanted into an experimental brain injury site on local cytokine production we have used a TBI model in mice, transplanted bone marrow-derived MSC into the lesion, and analyzed the production of cytokines. We also investigated the effects of soluble factors secreted by MSC on neural stem cells survival, proliferation, and differentiation ad libitum). All the experimental procedures were conducted according to international regulation and were approved by the Committee for Ethics in Research of Universidade Federal de São Paulo . Adult  C57BL/6 male mice were used in this study. The animals were maintained under standard conditions . Inside the laminar flow, the epiphyses were cut and bone marrow was flushed by DMEM into a culture dish using a syringe. The volume obtained from each femur was treated separately. Bone marrow was suspended and centrifuged for 5 minutes at 400 ×g. The supernatant was discarded and the cell pellet derived from each femur was suspended in DMEM low glucose containing 10% fetal bovine serum , 1% glutamine , and 1% penicillin/streptomycin (GibcoBRL) and cultured in six-well culture dishes  at a volume of 3.5 mL/well. Cultures were kept in a humidified 5% CO2 incubator at 37°C for 72 hours, when nonadherent cells were removed by changing the medium. Culture medium was changed every 3 days until adherent cells (MSC) reached 70–80% confluence, then cells were washed with 0.03% PBS/EDTA, detached with 0.1% trypsin (Cultilab), and replated in a 1 : 2 split ratio. Differentiation potential was checked by culturing the MSC under favorable conditions for adipogenic and osteogenic differentiation, as previously described [After euthanasia in COescribed . To obtaμg/mL heparin (Sigma). After mechanical dissociation, cells were plated on a polyHEMA (Sigma) precoated 75 cm2 flask at a density of 2.4 × 107 cells/flask or on a 24-well plate at a density of 2 × 106 cells/well. Neurosphere formation takes up to 14–21 days to occur, and during that time culture medium was changed every 4-5 days by centrifugation for 5 minutes at 400 ×g. Half volume of the medium was replaced by fresh medium. Neurospheres were cultured with or without MSC-CM for 2 days to evaluate NSC viability and for 4 days to evaluate NSC survival and differentiation. Adult NSC were obtained from adult C57BL/6 male mice SVZ. After euthanasia by cervical dislocation, brain was removed, the SVZ dissected under a microscope, and the cells maintained in DMEM high glucose (GibcoBRL). After sedimentation, supernatant was discarded, and cells were dissociated by incubation with 0.1% trypsin/EDTA during 5 minutes at 37°C. FBS was added to stop trypsin action, cells were centrifuged for 5 minutes at 400 ×g, and supernatant was removed. Isolated cells were then suspended in DMEM/F12 7 : 3 (v/v) (GibcoBRL), supplemented with 2% B27 (GibcoBRL), 20 ng/mL EGF (Sigma), 20 ng/mL FGF2 , 1% penicillin/streptomycin (GibcoBRL), and 5 μL of culture medium and 30 μL of MTT solution (5 mg/L) were added to each well and incubated for 3 hours at 37°C. After this period, MTT solution was aspirated and the formazan reaction products were dissolved by the addition of 180 μl of dimethyl sulfoxide  to each well. The plate was shaken for 15 minutes, the content was transferred to a 96-well plate, and the optical density was read at 540 nm on an ELISA plate reader . The MTT 2,5-diphenyltetrazolium bromide, Sigma) assay was performed to evaluate NSC viability cultured for 48 hours in MSC-CM. At the end of this period, neurospheres were centrifuged for 5 minutes at 400 ×g, supernatant was discarded, and 270 In vitro adult NSC proliferation was measured by flow cytometry , after BrdU incorporation. Adult NSC were cultured as neurospheres and maintained for 4 days in MSC-CM or in control medium  at 37°C. BrdU was added during the last 24 hours before analysis. Neurospheres were dissociated with 0.1% trypsin for 5 minutes at 37°C, fixed by 1% paraformaldehyde (PFA) in 0.1 M PBS, incubated in 2 M HCl for 1 hour at 37°C, washed with 0.1 M PBS, blocked, and permeabilized with 10% FBS and 0.1% TritonX-100 in 0.1 M PBS. Cells were incubated with anti-BrdU  for 1 hour and washed with 0.1M PBS. Cells were then incubated with secondary antibody  for 40 minutes. 5 cells in 3 μL) were injected at the injury site using a Hamilton syringe under the same stereotaxic coordinates and the same anesthesia, immediately after the injury. Control consisted of animals without injury and animals that were submitted to the injury but did not receive MSC . Brain tissue was collected 24 hours (acute injury) or 30 days (chronic injury) after MSC injection. Mice were anesthetized by i.p. injection of sodium thiopental , brain was removed from skull, the motor cortex was dissected and total RNA was extracted by Trizol Reagent . Animal's blood was collected by cardiac puncture, centrifuged to separate the serum, which was immediately frozen in dry ice and kept at −80°C until use. Adult C57BL/6 male mice were submitted to surgery under anesthesia with intraperitoneal administration of xilasine (32 mg/kg)/ketamine chloridrate (66 mg/kg) . Traumatic injury to mice motor cortex was performed according to previously described protocol , 29. Briμg of total RNA. Total RNA was isolated from dissected motor cortex or neurospheres using Trizol Reagent (Life Technologies), and RNA concentration was determined using NanoDrop ND-1000 . Reverse transcriptase reactions were performed with ImProm-II Reverse Transcription System  using 2 α (Mm00443258_m1) according to the manufacturer's recommendations. Values are expressed relatively to control RNA obtained from motor cortex from mice that were not submitted to surgery. Brain tissue samples qPCR was performed using ABI PRISM 7500 Sequence Detector, using Sequence Detection Software 1.9 for analysis , using TaqMan probes (Applied Biosystems) for HPRT , IL-4 (Mm00445259_m1), IL-6 (Mm00446190_m1), IL-10 (Mm00439614_m1), and TNFNeurosphere samples qPCR was performed using Brilliant II SYBR Green QPCR Master Mix  using Mx3000P QPCR System using MxPro qPCR Software for analysis (Stratagene). Primers sequences were GAPDH , GFAP , nestin , GAP-43 , and SOX2 . Values are expressed relatively to RNA from neurospheres cultured in regular medium (control). −ΔΔCt method [The quantification of the target genes was normalized by an endogenous control gene (HPRT for brain tissue samples and GAPDH for neurosphere samples). The threshold cycle (Ct) for the target gene and the Ct for the internal control were determined for each sample, run in triplicates. The relative expression of mRNA was calculated by 2t method . α), CCL4 (MIP-1β), CCL5 (RANTES), eotaxin (CCL11), CXCL1 (KC), CXCL2 (MIP-2), CXCL5 (LIX), CXCL9 (MIG), CXCL10 (IP-10), G-CSF, GM-CSF, INF-γ, IL-1β, IL-4, IL-5, IL-6, IL-10, IL-13, IL-17, LIF, TNF-α, and VEGF. Samples were run in triplicate for each assay. The assay was read on the Bio-Plex suspension array system, and the data were analyzed using Bio-Plex Manager software version 4.0. Standard curves ranged from 10,000 to 3,2 pg/mL. In order to analyze alterations in cytokines expression in mice serum after TBI, a Bio-Plex assay was performed. Custom Bio-Rad . Bio-Plex cytokine analysis kits were used together with the Bio-Plex system array reader according to the manufacturer's directions. The following cytokines were quantified: CCL2 (MCP-1), CCL3  followed by Tukey's posttest by the use of GraphPad Prism software version 4.0 . Statistical significance was set at α persists significantly higher than in undamaged tissue and IL-6 expression decreases  resulted in increased proliferation  and survival, but increased proliferation, indicating that the factors secreted by MSC may contribute to enhancing the number of NSC without affecting survival (Our aim was to verify if the immunomodulatory properties observed in the survival . On the other hand, MSC-CM soluble factors modulated NSC gene expression, upregulating GFAP expression, downregulation of nestin, and no alterations in GAP43 and SOX2 expression . SOX2 isin vitro, as well as increasing the expression of GFAP, a gene related to mature astrocytes, indicating that factors expressed by MSC could induce differentiation of NSC in vivo. Altogether these results indicate that there are still open possibilities of new regeneration therapies to treat nervous tissue injuries, highlighting the importance of immunomodulatory properties of MSC and its potential to allow NSC survival and proliferation. In this study we observed that MSC are able to modulate the inflammatory response in an acute TBI model by changing the expression of pro- and anti-inflammatory cytokines, along with modulation of serum levels of several chemokines. In addition, MSC secreted soluble factors are capable of stimulating proliferation of NSC