Crocin (CRO), chlorogenic acid (CGA), geniposide (GEN), and quercetin (QUE) are all natural compounds with anti-obesity properties, in particular, hypolipidemic effects, which have been widely used for the treatment of obesity-related metabolic diseases. However, it is not yet known whether these compounds interact synergistically. Here, we investigated the effects and molecular mechanisms of CRO, CGA, GEN, QUE, and a combination of all four compounds (CCGQ), on lipid accumulation in human hepatoma (HepG2 cells).The optimal concentration of CRO, CGA, GEN, QUE to stimulate HepG2 cells proliferation was determined using MTT assay. HepG2 cells were pretreated with 10 μmol/L simvastatin, 1 μmol/L CRO, 30 μmol/L CGA, 10 μmol/L GEN, 10 μmol/L QUE, and CCGQ  for 24 or 48 h. Oil red O staining and extracellular TC and TG levels were detected. The RT-PCR was used to observe on cholesterol metabolism-related gene expression. Immunocytochemistry and western-blot assayed the 3-hydroxy-3-methylglutaryl-coenzyme (HMGCR) protein expression in HepG2 cells.Compared to those of control, we demonstrated that treating HepG2 cells for 48 h with CCGQ resulted in a strong synergistic effect, causing a marked decrease in lipid deposition in comparison to individual treatments, in both triglyceride and total cholesterol , and Oil red O staining assays. In addition, CCGQ treatment increased ATP-binding cassette transporter (ABCA1), cholesterol 7α-hydroxylase (CYP7A1), and AMP-activated protein kinase 2α (AMPKα2) mRNA expression, while decreasing sterol regulatory element binding protein 2 (SREBP2), and liver X receptor alpha (LXRα) mRNA expression. Notably, CCGQ was more effective in decreasing HMGCR expression than the individual treatments.The CCGQ combination has potential, both as a complementary therapy for hyperlipemia, and in preventing further obesity-related complications. Obesity has become a global epidemic which is strongly associated with many metabolic diseases, such as coronary heart disease, type 2 diabetes mellitus, and non-alcoholic fatty liver disease , 2. EffeIn the last decade, many studies have focused on a variety of natural products, including botanical extracts or phytochemicals, for the prevention and treatment of obesity. In these studies, phenolic compounds and carotenoids have attracted a great deal of interest. Phenolic compounds, such as phenolic acids and flavonoids, have pharmacological properties involved in the regulation of plasma lipids, and are increasingly being studied . Crocin Gardenia jasminoides Ellis (gardenia) fruit, of the Rubiaceae family, was reported to decrease body weight, fat percentage, and BMI, lower high insulin, GLP-1, and leptin levels, and reduce insulin resistance in middle-aged obese women. It has been reported that CRO, CGA, rutin, and GEN are the main components of gardenia fruit [ia fruit . The antia fruit  and cellCGA, GEN and QUE  were purchased from the National Institute for the Control of Pharmaceutical and Biological Products . CRO was extracted and purified as previously reported  . All dru2. Cells were subcultured upon reaching approximately 80–90% confluency.The HepG2 human hepatoma cell line was purchased from the China Center for Type Culture Collection. HepG2 cells were grown and maintained in RPMI-1640 medium  supplemented with 10% fetal bovine serum, 100 U/mL penicillin, and 100 μg/mL streptomycin at 37 °C in a humidified atmosphere of 5% CO4) were seeded into 96 well micro-titer plates containing 100 μL complete culture medium per well, and incubated for 24 h at 37 °C. Cultures were transferred into serum-free medium 12 h prior to performing the assay. A range of concentrations of CRO , CGA , GEN , and QUE  were added. Cells were incubated for a further 24 or 48 h in an incubator (37 °C at 5% CO2), and then treated with MTT for 4 h. DMSO (150 μL) was added to each well, and plates were then shaken for 10 min. Absorbance was measured at 490 nm using a microplate reader (Bio-Rad). Cell viability in each group was presented as a percentage of the control.HepG2 cell growth was determined using the MTT assay. Cells (2 × 104) were seeded into 6- or 24-well plates and cultured. After incubation in the presence or absence of the different drugs for 48 h, cells were fixed with 4% polyformaldehyde, and then incubated in Oil red O working solution for 30 min. Microscopic images were taken to visualize red oil droplets with an Olympus microscope . After incubation in the presence or absence of the different drugs for either 24 or 48 h, cell supernatant was used for analysis of total cholesterol (TC) and triacylglycerol (TG) levels. TC and TG kits  were used according to the manufacturer’s instructions.HepG2 cells (2 × 10Expression of the following genes was studied using semi-quantitative RT-PCR: ATP-binding cassette transporter ABCA1), sterol regulatory element binding protein 2 (SREBP2), cholesterol 7α-hydroxylase (CYP7A1), liver X receptor alpha (LXRα), AMP-activated protein kinase 2α (AMPKα2) and 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGCR). Primer pairs  are listed in Table , sterol HepG2 cells were plated into 6-well chamber slides. After treatment with the different drugs for 48 h, the slides were fixed in 4% neutral buffered paraformaldehyde for 30 min at room temperature, subsequent HMGCR detection. Immunostaining was carried out using a kit . The endogenous peroxidase was inactivated by treated the slides with 0.3% hydrogen peroxide for 10 min. After washed with PBS, the slides were added into normal goat serum to incubate for 10 min at 37 °C. Rabbit polyclonal HMGCR antibody (1:50)  was used as the primary antibody to incubated cells at 4 °C for overnight after washed. The slides were washed and incubated with biotinylated secondary antibody for 30 min. And then slides were added into peroxidase-labeled streptavidin for another 30 min. Slides were stained with 3,3′-diaminobenzidine for 25 min. Cells were incubated with nonimmune serum or PBS to control antibody specificity, and then the nuclei were counterstained with H&E.Total cell protein from each sample was extracted using a kit  and quantified using a Bio-Rad protein kit . Equal quantities of protein were separated using 10% SDS-PAGE and transferred to PVDF membrane  overnight at 4 °C: the membrane was then blocked in 5% non-fat milk. The membrane was probed separately with the primary antibodies for GAPDH-HRP  and rabbit antihuman HMGCR , and secondary antibodies . Signal was detected using an ECL Kit  and developed on X-ray film. Protein bands were detected by ChemiDoc XRS systems and Quality One 4.6.1  software, and GAPDH antibody was used as the internal control.P < 0.05 was considered significant.All data were presented as means ± SEM, obtained from at least three separate experiments, and one representative image was selected for presentation in the figures. One-way analysis of variance followed by Dunnett’s test was used for comparison among groups (SPSS computer software version 13.0). We examined the cytotoxicity of CRO, CGA, GEN, and QUE to determine suitable concentrations for the treatment of HepG2 cells. Results showed that 100 μM CRO, CGA, and GEN, and ≤ 10 μM QUE had no inhibitory effect on HepG2 cell viability after 24 or 48 h of treatment . These results indicate that combined treatment with CCGQ results in a much greater decrease in lipid accumulation in HepG2 cells than treatment with the drugs individually.To test whether drugs affected lipid deposition, we examined lipid droplets in HepG2 cells using Oil red O staining. Compared with the control group, we found prominent lipid accumulation in the cytoplasm of HepG2 cells, using 0.1 mmol/L of oleate. Compared with the oleate group, treatment with SIM, CRO, CGA, GEN, and CCGQ significantly attenuated the accumulation of lipid droplets, however, QUE alone did not induce notable inhibition Fig. . To furtIn order to further clarify the mechanism responsible for the changes in cholesterol levels of HepG2 cells, we next examined the mRNA expression levels of cholesterol metabolism-related enzymes  leaf mainly consists of CGA, in addition to GEN and QUE [Similar to gardenia fruit, HPLC analysis showed that  and QUE . We prev and QUE . We therThe liver plays a central role in cholesterol metabolism. Cholesterol homeostasis is governed by several genes, including HMGCR, CYP7A1, ABCA1, SREBPs, LXR, and AMPK. These genes maintain a complex network involving cholesterol uptake, synthesis, intracellular transport, and excretion , 24. HMGOur data show that ABCA1, CYP7A1, and AMPK2α expression in HepG2 cells significantly increased in CCGQ-treated cells; however, HMGCR, SREBP2 and LXRα mRNA levels were significantly lower than those of the control group. Gene expression in the SIM group was similar to that of the CCGQ group, aside from up-regulated LXRα and a lack of increase in AMPK2α expression in SIM-treated cells. Again, similar effects were observed for ABCA1, AMPK2α, and LXRα mRNA expression in HepG2 cells, after treatment with CRO, CGA, QUE, and GEN. Nonetheless, we observed that CRO, CGA, and QUE also significantly decreased HMGCR mRNA expression, although this was not observed for the GEN group. Interestingly, out of all four phytochemicals, only CRO induced dramatically lower SREBP2 mRNA expression than the control group. On measuring the abundance of the CYP7A1 gene, responsible for the synthesis of bile acids, we observed significant differences between CRO-, CGA-, GEN-, and QUE-treated cells. CYP7A1 expression was repressed in the QUE group, CGA and GEN increased CYP7A1 expression, and no change was noted in the CRO group.The role for QUE in suppressing LXRα gene expression and modulating lipid accumulation has previously been described in Huh7 cells . The curIn our study, HMGCR protein expression, measured by immunocytochemistry and western blot and confirmed by PCR, was inhibited to a greater extent in the CCGQ group than in cells treated with the individual drugs, suggesting that the four phytochemicals may be more efficient in combination than separately. These data are consistent with the reduction in hepatic lipid accumulation observed in Oil red O staining and lipid content analysis. Analysis of the relative expression of related genes, based on hepatic lipid concentration, also showed that CCGQ dramatically decreased hepatic lipid accumulation to a greater extent than the individual drugs.Based on our observations, the preventative effect of CCGQ on lipid accumulation in HepG2 cells was considerably greater than that of SIM. Statins are the preferred drugs for treatment of hyperlipemia. However, evidence that they cause hazardous side effects, and are effective in only one-third of patients, highlights the need for alternative therapies .The experimental findings of the present study primarily revealed that synergistic interactions between combined CRO, CGA, GEN, and QUE demonstrated positive effects against hepatic lipid accumulation. The anti-hyperlipidemic effect of these phytochemicals appears to be mediated by the modulation of AMPK, LXRα, and associated genes engaged in cholesterol synthesis, metabolism, and efflux. To our knowledge, this is the first report demonstrating the synergistic lipid-lowering actions of CRO, CGA, GEN and QUE. Although results from in vitro experiments cannot be directly extrapolated to anti-obesity and lipid-modulating clinical effects of gardenia fruit  and euco


However, only sub-therapeutic plasma concentrations of GPs were detected in human and mouse pharmacokinetic (PK) studies. Intriguingly, a significant portion of GPs occurred as phase II metabolites (mainly glucuronide conjugates) in plasma. To evaluate the disposition of GPs and understand the real players responsible for efficacy, we performed a PK and tissue distribution study in mice. Plasma exposure of GPs was similar on day 1 and 7, suggesting no induction or inhibition of clearance pathways. Both free and conjugated GPs accumulated in all tissues including tumors. While non-cytotoxicity of 6-ginerol glucuronide precluded the role of conjugated GPs in cell death, the free forms were cytotoxic against prostate cancer cells. The efficacy of ginger was best explained by the reconversion of conjugated GPs to free forms by β-glucuronidase, which is over-expressed in the tumor tissue. This previously unrecognized two-step process suggests an instantaneous conversion of ingested free GPs into conjugated forms, followed by their subsequent absorption into systemic circulation and reconversion into free forms. This proposed model uncovers the mechanistic underpinnings of ginger’s anticancer activity despite sub-therapeutic levels of free GPs in the plasma.Anticancer efficacy of ginger phenolics (GPs) has been demonstrated in various  However, their clinical development has suffered perhaps due to sub-therapeutic concentrations of constituent phytochemicals in the plasma. Ginger is an example. An almost instantaneous conversion of the ingested free ginger phenolics (GPs) into their pharmacologically-inactive phase II metabolites  in the plasma has perpetuated a diminished interest in their development. However, this highlights a knowledge gap as to how constituent phytochemicals contribute physiologically to the observed pharmacological effects of NMs. Undoubtedly, NMs are a complex concoction of chemically-diverse phytochemicals containing one to several bioactive compounds, which mostly are plant secondary metabolites. Recently, a unique approach called ‘reverse pharmacokinetics’ has helped to draw possible reasons underlying the discordance between pharmacokinetics (PK) and pharmacodynamics (PD) of NMs3. In this study, we examined how the free (active) and conjugated (inactive) species interconvert on the physiological time scale to ultimately pinpoint the bioactive species responsible for the PD effect. For almost a decade now, our laboratory has been studying the pharmacology of ginger root extract. Thus, ginger extract was our obvious choice as the probe NM to investigate the pharmacokinetic-pharmacodynamic (PK-PD) relationships between free and conjugated forms of GPs to precisely define the role of Phase II metabolites in the anticancer activity of ginger extract.Recently, there has been a resurgence of whole food-based therapeutics categorized as Natural Medicines (NMs) in prevention and treatment of chronic diseases like cancerZingiber officinale), a widely consumed spice worldwide, is an excellent source of phenolic compounds including gingerols, shogaols, paradols, gingerdiones etc.6. Literature has extensively demonstrated that 6-gingerol (6G), 8-gingerol (8G), 10-gingerol (10G) and 6-shogaol (6S) collectively referred to as ginger phenolics  in prostate cancer models as well as reported the in vitro and in vivo synergistic interrelationships among various ginger constituents when administered in their original matrix14. Furthermore, we have shown that GPs undergo enterohepatic recirculation and their glucuronide conjugates circulate in the system15. We also demonstrated that ginger extract  upon daily oral administration for 28 days in mice showed 68% tumor growth inhibition compared to controls. A quasi-mixture of 6G, 8G, 10G and 6S  showed 28% tumor inhibition and GE devoid of these components showed 35% tumor inhibition. These experiments confirmed the presence of other active constituents in GE which have anti-cancer potential and that whole ginger is better than its individual constituents. In a follow up study, we dosed GE with ketoconazole in mice, which confirmed that phase II metabolism is critical for clearance of GE phenolics than Phase I, as concentration time profile of GPs was more or less similar with or without concomitant ketoconazole administration16. However, studies by Zick et al. revealed that sufficient (or therapeutic) plasma concentrations of these bioactive GPs are not achieved in humans despite a high dose of 2 g per day. Instead of free gingerols, glucuronide and sulfate conjugates of 6G, 8G, 10G and 6S were observed in plasma17. There is a widely-held notion that phase II conjugates are water-soluble, eliminated from the system quickly and most of them are pharmacologically inactive. There are no available reports that describe whether these conjugates contribute to or modulate the anticancer efficacy of the whole ginger extract. The disconnect between the observed efficacy and sub-therapeutic plasma concentrations of free GPs generate compelling grounds to further investigate the fate of the GPs and their metabolites in vivo.Ginger  in cancer cells compared to normal cells facilitates the conversion of conjugated glucuronides to free forms in tumor tissue. In vitro studies confirmed that the free forms are more cytotoxic compared to the glucuronide conjugates.To gain insights into the interrelationships between free and conjugated forms of GPs, we conducted pharmacokinetic study in healthy mice and tissue distribution in mice bearing human xenografts. In addition, last) of 6G, 8G, 10G and 6S was similar on day 7 compared to day 1  of 10G (1244.40 ng.h/mL) was twice that of 6G (614.97 ng.h/mL), plasma exposure of 8G (116.84 ng.h/mL) was 5 times lower than 6G. Notably, the plasma exposure of 6S was lowest at (AUClast) 47.39 ng.h/mL, indicating its low bioavailability compared to other GPs.The serving size of ginger in humans varies from 250 mg to 4.8 g per day. Ginger extract is generally regarded as safe (GRAS) ingredient in accordance with US FDA15 that majority of free GPs are converted to their respective glucuronides, the tissue samples were treated with β-glucuronidase to determine the concentration of conjugated forms of 6G, 8G, 10G, and 6S. Essentially, the concentration of glucuronide conjugates was calculated by subtracting the free analyte concentration from the total concentration obtained from β-gd treated samples  of free 6G, 8G, and 6S, in various organs was more than that in the plasma suggesting extensive tissue distribution. Conversely, the AUClast of free 10G was higher in plasma compared to all other tissues (Supplementary Table last suggested a higher accumulation of conjugated GPs in heart (10.22–18.54) and lung (2.50–5.57). In all other tissues, the values of conjugated to free forms ranged from 0.2 to 2 (Supplementary Table last of free and conjugated form) to organ weight  compared to cancerous tissue (median 133) measured using liberation of 4-Methylumbelliferyl-β-D-glucuronide20. Similarly, in bladder cancer patients the values of β-gd were significantly different ; patients in remission ; patients with disease )21. Further, it is also reported that dosing rats bearing colon cancer with GE , reduced the activity of β-gd22. Our in silico analysis showed statistically significant over-expression of β-gd in stomach, intestine, lung, brain, liver, kidney, prostate, pancreatic and breast cancer tissues compared to normal ones  is overexpressed in most cancer cells with high variability reported in its expression in patients. In human prostate cancer, its expression was 3.6-fold higher than normal tissue median: 7 comparedInterestingly, accumulation of conjugated 6S was 9-fold higher compared to its free form in the tumor, while in the case of 6G, 8G, and 10G, the conjugates exposure was about 20–50% lower compared to the free forms. The exposure (free + conjugated form) of 6G, 8G, 10G and 6S was 123, 33, 32 and 31 ng·h/mL (Supplementary Table in-vitro cell proliferation assays using PC-3 and DU145 prostate cancer cells. We found no cytotoxicity of 6G-phenolic glucuronide on PC-3 or DU145 prostate cancer cells up to the highest concentration tested (200 µM). All GPs showed IC50 values ranging from 30 µM for 10G to 196 µM for 6G  of the 6S conjugate in tumors was 9-fold higher compared to the free form, while 6G, 8G, 10G displayed higher concentration of free form. Notably, both 10G and 6S showed highest anticancer efficacy in vitro among tested GPs. Thus, our results reveal an interesting scenario where the most cytotoxic GPs accumulate in tumors as both free and conjugated forms. From the Tmax and Cmax values, it is apparent that 6G, 8G, and 10G accumulate in the tumors first. This is supported by the fact that the peak levels of 6G, 8G, and 10G free forms occur at 10–15 min. Given the concentration and the length of time, these conjugates are converted to free forms in the tumor as 6G, 8G, and 10G peaked up first, followed by 6S  necessitates identification of the responsible UGTs if glucuronidation is responsible for greater than 25% of total metabolism. Typically UGT1A1, 1A3, 1A4, 1A6, 1A9, 2B7 and 2B15 can be assessed using recombinant UGT isozymes or inhibitors of UGTs like atazanavir for UGT1A1 and probenecid and valproic acid for others. Furthermore, the characterization of UGTs responsible for biotransformation of GPs is necessary to consider any DDIs during polypharmacyunbeatable fortress, wherein cancer cells show high expression of p-glycoprotein (Pgp) - the efflux protein44, βgd (angiogenesis and metastasis)45, high kinase activity (angiogenesis)46 and glutathione transferases 47. While current strategies somewhat rely on engaging a single target, we propose the notion of “pharmacokinetic engineering”, wherein customized combinations of various NMs, marketed drugs and new chemical entities can be tailored to effectively eradicate cancer by targeting the entire tumor landscape- tumor cells as well as the stromal milieu.Tumor environment is like an To summarize, this study examined the role of GP conjugates, the optimal PK analyte “chemical markers” such as 6G, 8G, 10G and 6S along with their conjugates, and “biomarkers” like β-gd in facilitating enhanced accumulation within target tissues to exert favorable PD effects. This strategy can be extrapolated to other NMs for bridging the PK-PD gap for rational development.Ginger extract (GE) was a generous gift from Sabinsa Corporation, NJ, USA. We used 6G, 8G, 10G and 6S as markers and their concentrations were 30.03, 6.86, 7.77 and 5.96 μg/mg of ginger extract, respectively. The sum of all concentrations equals 5% of the whole extract, which is similar to what was employed in the clinical studies. Acetonitrile (ACN) and methanol were purchased from Fisher Scientific . Luciferase-expressing PC-3 cells (PC-3-luc) were from PerkinElmer  and were maintained in modified Eagle’s medium with 10% fetal bovine serum obtained from Hyclone . The PC-3-luc cells were immediately expanded and frozen down for future use  of the same batch of cells. We used the Universal Mycoplasma Detection Kit from ATCC  to ensure that all the cell lines used were devoid of mycoplasma contamination. Carboxyl methylcellulose (CMC), nicotinamide adenine dinucleotide phosphate (NADPH), and β-glucuronidase were from Sigma . 6-gingerol glucuronide standard was procured from Santa Cruz Biotechnology . The active ginger constituents, 6G, 8G, 10G and 6S, were isolated from GE by column chromatography and were characterized for >98% purity by HPLC (High-performance liquid chromatography). All other reagents used in the study were of analytical grade.ad libitum throughout the study period. Animals were marked and housed  in polypropylene cages and maintained in controlled environmental conditions with 12 h light and dark cycles. The temperature and humidity of the room were maintained between 22 ± 3 °C and 30 to 70%, respectively, and approximately 10–15 fresh air change cycles per hour. A sparse sampling design was used to collect blood samples (150 μL) from animals at 0, 0.08, 0.16, 0.25, 0.5, 1, 2, 3, 4, 6, 8 and 12 h into K2EDTA coated tubes. At each time point, 3 animals were used for blood sampling and from each animal, 3 blood samples were withdrawn, the last being terminal sampling by cardiac puncture. Plasma was harvested from blood by centrifugation of samples at 8000 g for 10 min. All samples were stored below −80 °C until bioanalysis.All animal experiments were done with approval from Georgia State University’s Institutional Animal Care and Use Committee (IACUC) guidelines and under animal research protocol No. A14031. All guidelines were strictly adhered to while performing animal experiments. Pharmacokinetic studies were performed in male C57BL6J  mice following oral (PO) administration of GE at 250 mg/kg (suspended in 0.5% sodium CMC) for seven days, where blood samples were collected on day 1 and day 7 post dosing. All animals were acclimatized for 5 days before dosing in the experimental area. Food and water were provided 6) on the right flank. After mice (25–30 g body weight) developed palpable tumors (tumor volume of 80–100 mm3), were fed with 250 mg/kg GE  every day by oral gavage daily for 7 days. The dose volume used was 5 mL/kg. On Day 7, terminal blood was collected through cardiac puncture and tissue samples  were collected at 0, 0.8, 0.25, 0.5, 1, 2, 4, 6, and 8 h by euthanizing the mice. Georgia State University’s Institutional Animal Care and Use Committee (IACUC) guidelines and animal research protocol (No. A14031) were strictly adhered to while performing animal experiments.Six-week-old male nude C57BL6J mice  were subcutaneously injected with PC-3-luc cells . To confirm the presence or accumulation of GP glucuronide conjugates, plasma and tissue samples  were treated with β-gd  and incubated at 37 °C for 1 h. These samples were further processed by protein precipitation before bioanalysis.https://www.oncomine.org). Reporter ID and platform for datasets used were as follows: gene rank 1723/18823, 2688/19574, 842/12624, 2275/18823, 2609/19574, 997/19574, 941/19189, 3345/19273 and 756/18823 analyzed on human genome U133 plus 2.0 array for gastric, colon, brain, liver, pancreas, lung, prostate, breast and renal, respectively. We selected a p value of < 0.01 as a threshold for analysis.The expression level of β-gd in healthy or cancerous tissues in humans was analyzed using Oncomine database . After 4 h, the media was aspirated and MTT solution (0.2 mg/mL) was added and further incubated for 2 h. To each well, 100 µL of DMSO was added to lyse the cells. The cell lysates were aspirated and centrifuged. The supernatant was collected and read at 570 nm in a spectrophotometer. Doxorubicin and blank buffer samples were used as controls in this study. No test inhibitor sample was considered as 100%, to calculate percentage inhibition. Each experiment was run in duplicate.Cytotoxicity of GPs 6-gingerol (6G), 8-gingerol (8G), 10-gingerol (10G), 6-shogaol (6S), and 6G-phenolic glucuronide conjugate were assessed in prostate (PC-3 and DU145) cancer cell lines. Cells were cultured in T-75 flasks, and 5000 cells per well were plated in a 96-well plate. The plate was incubated overnight at 37 °C/5% COg for 10 min, and an aliquot of 200 μL supernatant was transferred into 1.5 mL HPLC injection vials for analysis. The stock solutions of 6G, 8G, 10G, 6S and dihydrocapsaicin  were prepared in ACN: water (95:5) at 1 mg/mL. A calibration curve range of 0.1–500 ng/mL was employed for the quantification of analytes and internal standard (IS) concentration was 50 ng/mL for each analysis. The calibration curve consisted of blank, blank with internal standard and seven non-zero calibration standards. The calibration standards were within ± 15% of the nominal concentration, and lower limit of quantification was within ± 20% of nominal. All samples were analyzed using LC-MS/MS method (Agilent 6410 series) employing calibration curve and interspersed with quality control samples. All other analytical parameters are similar as reported earlier15.Tissue samples were weighed and homogenized with phosphate buffer saline . All plasma and tissue samples were processed by using protein precipitation method. An aliquot (20 μL) of the sample was spiked with 200 μL of acetonitrile containing dihydrocapsaicin as the internal standard and vortex mixed for 3 min. The tubes were centrifuged at 12000 last and AUCinf) was calculated using the linear trapezoidal rule. Following oral administration, peak concentration (Cmax) and time for to peak concentration (Tmax) were the observed values.The pharmacokinetic (PK) parameters were calculated from the concentration-time data using the non-compartmental analysis tool of Phoenix software . The area under the concentration-time curve (AUCmax and AUClast are reported as mean ± SEM and concentration-time profiles have mean ± SD. Student’s t-test was used to compare the day 1 and day 7 exposures, and a p value of less than 0.05 was considered significant.Pharmacokinetic parameters CSupplementary Information


The NCs composition is well controlled through varying molar ratios of Se to Te precursors. Through changing the composition of the CdSexTe1−x NCs, the spectral absorption of the NC thin film between 570–800 nm is proved to be tunable. It is shown that the bandgap of homogeneously alloyed CdSexTe1−x active thin film is nonlinearly correlated with the different compositions, which is perceived as optical bowing. The solar cell devices based on CdSexTe1−x NCs with the structure of ITO/ZnO/CdSe/CdSexTe1−x/MoOx/Au and the graded bandgap ITO/ZnO/CdSe(w/o)/CdSexTe1−x/CdTe/MoOx/Au are systematically evaluated. It was found that the performance of solar cells degrades almost linearly with the increase of alloy NC film thickness with respect to ITO/ZnO/CdSe/CdSe0.2Te0.8/MoOx/Au. From another perspective, in terms of the graded bandgap structure of ITO/ZnO/CdSe/CdSexTe1−x/CdTe/MoOx/Au, the performance is improved in contrast with its single-junction analogues. The graded bandgap structure is proved to be efficient when absorbing spectrum and the solar cells fabricated under the structure of ITO/ZnO/CdSe0.8Te0.2/CdSe0.2Te0.8/CdTe/MoOx/Au indicate power conversion efficiency (PCE) of 6.37%, a value among the highest for solution-processed inversely-structured CdSexTe1−x NC solar cells. As the NC solar cells are solution-processed under environmental conditions, they are promising for fabricating solar cells at low cost, roll by roll and in large area.CdSe Jsc (20–28 mA/cm2) evidently limits the performance of CdTe-solar cells due to the limited bandgap and bulk/interface recombination, while this value is ~40 mA/cm2 for single crystalline Si solar cells [N-type CdS is typically adopted as the window layer for efficient CdTe-solar cells and another CdSxTe1−x window layer is established at CdS/CdTe interfaces when depositing CdTe and heat-treating with CdCl2. However, it was reported that the formed CdSexTe1−x layer was not photoactive and no photo-currents were generated by the photons absorbed by the CdSxTe1−x window layer [xTe1−x layer can be formed easily when growing and post-treating the CdTe layer in the case of CdTe solar cells using CdSe as the window layer [xTe1−x alloys exhibit a narrower bandgap than that of CdTe, elevating the external quantum efficiency (EQE) response to a longer wavelength [Jsc of 26.3 mA/cm2. It was further optimized by Poplawsky et al. [Jsc of 27.0 mA/cm2 (without a reflective layer) and PCE of 15.4%. It is indicated that CdSexTe1−x alloy is formed through diffusing Se or Te at the CdTe/CdSe interface in foregoing cases, which shall downgrade carrier mobility and not uniformize the CdSexTe1−x layers, consequently declining the device performance [xTe1−x NCs composited differently can be easily controlled through varying the Se and Te content in the precursor before forming CdSexTe1−x alloy NC [xTe1−x alloy layer. Furthermore, NC solar cells fabricated in the wake of solution processing are numerously advantageous, such as being low in cost, having a high chemical yield and having an easy-to-sinter property in virtue of the low melting point. There are several ways to introduce the CdSexTe1−x alloy NC as an active layer to attain illumination, which can be clarified from several perspectives. First and foremost, the CdTe and CdSe NCs are deposited in sequence and annealed at high temperature to form CdSexTe1−x, consequently diffusing Se or Te at the CdTe/CdSe interface. Gur et al. [w/o)/Au, the solar cells performance can be further optimized [xTe1−x as active layer is to sinter thin films containing mixtures of CdTe and CdSe NC. MacDonald et al. [xTe1−x/ZnO/Al and ITO/ZnO/CdSe/CdSe:CdTe/CdTe/Au. Unfortunately, although Jsc has been optimized, relative low Voc (~0.6 V) is attained in the foregoing case. Alloy NC film can also be fabricated through directly depositing a solution containing CdSexTe1−x alloy NC onto the substrate. In this case, the active layer properties can be tuned through adjusting the composition of the alloy NC. Yang et al. exhibited a new device structure of ITO/TiO2/CdSexTe1−x/MoOx/Au through adopting CdSexTe1−x alloy NC as active layer and a low PCE ~4% was attained because of low Jsc and Voc, which was primarily originated from large defect in active layer [Solution processed perovskite solar cells ,2, polymar cells ,20. N-tyow layer . Conversow layer ,23. Undevelength ,25,26. Fvelength  proved CJsc 20– mA/cm2 eJsc 20– mA/cm2 ealloy NC ,30. The r et al.  first deptimized ,33. Receptimized . Anotherd et al.  and our d et al.  generateve layer .xTe1−x alloy NC with entire compositional range via hot injection method. The transmission electron microscopy (TEM), energy-dispersive X-ray spectroscopy (EDS) and X-ray diffraction (XRD) are adopted to resolve morphology, structure and ascertain compositions. The composition of CdSexTe1−x alloy is ascertained to be well controlled through varying the ratio of the Se to Te ratio in the precursor, while the optical properties denote a non-linear response to Se content, known as the “light bowing” effect. Devices with graded bandgaps of ITO/ZnO/CdSe/CdSexTe1−x/MoOx/Au and ITO/ZnO/CdSe(w/o)/CdSexTe1−x/CdTe/MoOx/Au are fabricated through sintering layer-by-layer. The effects of the NC alloy composition, thickness and annealing temperature on the graded bandgap solar cells are ascertained and disserted specifically in line with experimental results. Our works here illuminates that high Jsc and Voc can be attained simultaneously through tuning the device structure and device preparing process. By replacing the CdSe window layer as CdSe0.8Te0.2 alloy film, the FF, Voc and PCE can be further optimized in the meantime and a PCE as high as 6.37% is attained for NC solar cells with a ITO/ZnO/CdSe0.8Te0.2/CdSe0.2Te0.8/CdTe/MoOx/Au structure, owing to the improved in-band alignment, film quality and low series resistance.Here, we report on the synthesis of CdSexTe1−x NCs were prepared under environmental conditions. Trioctylphosphine selenide (TOP-Se) and TOP-Te precursor were prepared through dissolving 8 mmol Se and 8 mmol Te into 10 mL TOP solvent, respectively. When typically preparing CdSe0.2Te0.8 NCs, 0.2 mL TOP-Se was mixed with 0.8 mL TOP-Te in a fume hood under environmental conditions. The mixed TOP-Se/Te solution was then stirred for six hours to attain a clear and homogeneous solution. In a three-necked flask equipped with a thermometer, 905.6 mg (1.6 mmol) of cadmium myristate, 114 mg myristate acid (0.5 mmol) and 2.40 g trioctylphosphine oxide (TOPO) were added. The mixtures were heated up to 240 °C under N2 flow. When heating, cadmium myristate was dissolved progressively and a faint yellow homogeneous solution was generated. The mixture was kept at this temperature for 10 min and then 1 mL TOPSe-Te solution was quickly injected into the reaction flask and the whole reaction lasted for 30 min. To fabricate alloy NCs with compositions, TOP-Se/TOP-Te of different ratios was conducted while the other conditions remained unchanged. After the reaction was cooled to room temperature, the mixture was washed three times with methanol/toluene and centrifuged. The final outcomes were refluxed in pyridine for 24 h and centrifuged with n-hexane. The CdSexTe1−x NCs products were re-dispersed into pyridine/1-propanol (1:1 v/v) with a concentration of 45 mg/mL. The ZnO precursor, CdSe NCs and CdTe NCs were prepared following our previous works [x and 100 nm Au were evaporated through a mask at a pressure below 10−5 Torr, achieving an active area of 0.16 cm2.CdSeus works ,39. The us works . It is nxTe1−x materials composited differently are compounded through injecting hybrid TOP-Se/TOP-Te into the Cd2+ carboxylic precursor and adopting TOPO as the coordinate solvent at a moderate temperature of 240 °C. It is ascertained that the CdSexTe1−x alloy NCs indicate a morphological rod shape when x < 0.8 . The NC indicates an irregular dot shape with high Se content, similar to that reported of pure CdSe NC [xTe1−x NCs are further characterized by powder XRD. The XRD pattern (xTe1−x NCs with different Se contents bespeak similar diffraction peaks which can be indexed as (100), (002), (101), (110), (103) and (200) or (111), (220) and (311) facets of wurtzite CdTe/zinc blend CdSe. It is ascertained that the diffraction peaks shall be progressively converted into larger angles with the rising Se content. In contrast with pure CdSe or CdTe NC, the XRD patterns of CdSexTe1−x alloy NC denotes an evident size-broadening effect as the lattice constant varies. No phase separation or separated nucleation of CdTe/CdSe in the CdSexTe1−x is detected, indicative of the formation of a homogeneous alloy. The alloy NC composition is dependent on energy disperse spectroscopy (EDS) measurements , as exhibited in 0.2Te0.8 and CdSe0.4Te0.6 NC is presented in xTe1−x as the Cd precursor is excessive in this reaction (with Cd/(Se + Te) = 2:1 in all reactions).CdSe x < 0.8 a–f, whilondition ,39. In tondition ,42. The  CdSe NC . More ho pattern a of the x = 1 or x = 0), indicating the formation of NCs with homogeneous size and narrow in size distribution. For the ternary alloy CdSexTe1−x NCs, the variation in absorption edge is ascertained to be composited differently. For NCs containing a low Se content, the absorption edge can be differentiated. Conversely, no peaks are distinguished for NC with a high Se content and a long tail in the absorption spectrum because of the extensive size distribution and the alloy structure. In contrast with the absorption edge of CdTe NCs, the absorption edge shifted from 660 to 780 ± 30 nm in the case of x = 0.1 to 0.6. The spectral shifts demonstrated in xTe1−x alloy, which refers to “light bowing” effects [2 versus the photon energy have a linear onset, indicative of a direct bandgap  = (1 − x)Eg(CdSe) + xEg(CdTe) − bx(1 − x), where b = 0.75 is the optical bowing coefficient and the values of Eg(CdSe) and Eg(CdTe) are 1.73 eV and 1.50 eV, respectively. The experiment results conform well to compatible results at high Se content (x > 0.6), with a slight shifting at low Se content (x = 0.1 to 0.4).The absorption peak for pure CdSe and CdTe is 570 nm and 660 nm, respectively, as presented in  effects ,44. As t bandgap b. We canxTe1−x (160 nm)/MoOx(5 nm)/Au(70 nm). All alloy NC thin films are annealed at 350 °C with CdCl2 treatment for 30 min. The hole carrier mobility of CdSexTe1−x NC thin film is ascertained via the space-charge-limited-current (SCLC) method. The carrier mobility is attained in line with the following equation [xTe1−x, L is the thickness of alloy NC, pμ is the hole mobility, V is the applied voltage and Vs is the voltage drop due to contact resistance, while Vbi is the built-in voltage. As exhibited in x = 0, 0.1, 0.2, 0.4, 0.6, 0.8 are 2.02 × 10−4, 3.48 × 10−4, 4.05 × 10−4, 4.34 × 10−4 and 3.07 × 10−4 cm2/Vs, respectively. The value of ternary alloy NC is basically two orders higher than those ever reported [To ascertain the charge transport properties of alloy NC with composition, we fabricate hole-only devices with a structure of ITO/CdSeequation :J=98ε0εrreported .0.2Te0.8 NC film is adopted as the active layer and devices with an inverted structure of ITO/ZnO (40 nm)/CdSe (30 nm)/CdSe0.2Te0.8 (400 nm)/MoOx (8 nm)/Au (80 nm) are fabricated through depositing five NC layers on CdSe film. The cross-section SEM image of NC device is exhibited in J-V curves for alloy NC solar cells with different annealing temperatures are exhibited in Jsc) of 16.04 mA/cm2, an open circuit voltage (Voc) of 0.63 V, a fill factor (FF) of 36.68% and a PCE of 3.71%. It is ascertained that device performance degrades with the increase of temperature, which is primarily ascribed to the reduced Voc (below 0.5 V). For CdTe NC-based solar cells, high annealing temperatures shall partially resolve the NC film and make a device shunt, which has been confirmed previously [Jsc and FF, similar results is also attained by Yang et al. [CdSe NC had been proved to be a good partner for CdTe NC solar cells in our previous report . To asceeviously . For thig et al. . The devJsc and FF attained in NC solar cell devices with a single-alloy active layer, the vertically-graded bandgap is formed by using the layer-by-layer solution processed method. In the case of devices with single bandgaps as the active layer, they can absorb only the photons with energy larger than, or equal to, the bandgap of the material. Conversely, in devices with two or more semiconductor active layers, the absorption range is extended and most of the photons available within the solar spectrum can be attained through designing an optimized device structure [w/o)/CdSexTe1−x/CdTe/MoOx/Au is presented in xTe1−x alloy NC active layers consisting of one or more bandgaps are prepared through depositing layer by layer. The optimized active layer is constituted by one layer of CdSe NC (~30 nm), one layer of CdSexTe1−x NC (~80 nm) and four layers of CdTe NCs (~320 nm). The introduction of CdSexTe1−x alloy NC film is anticipated to extend the optical response to longer wavelengths. The thickness of the CdSexTe1−x NCs/CdTe NCs shall evidently exert influence on the band alignment and spectrum response of NC solar cells. Therefore, the influence of thickness of alloy NC film on device performance is ascertained with the CdSe0.2Te0.8 NCs. It is attained that the device performance degrades with the increase of alloy NC thickness  of 16.54 mA/cm2, an open circuit voltage (Voc) of 0.63 V, a fill factor (FF) of 54.93% and a high PCE of 5.75% . This value is higher than the controlled devices without alloy an NC layer (with PCE of 5.45% for devices with the structure of ITO/ZnO/CdSe/CdTe/MoOx/Au). From the J-V curve  for devices with different alloy NC films is presented in Jsc result obtained from J-V curve measurement. As the carrier mobility of CdSexTe1−x NC alloy has a similar value, the change in device performance with different NC alloy active layers cannot be attributed to the change in carrier transfer properties alone. We speculate that, in the case of CdSexTe1−x with low Se content, the lattice constant is near the value of CdTe and the interface (CdTe/CdSexTe1−x) defects are low after annealing. The decreased defects between the alloy NC layer and the CdTe NC layer shall optimize carrier departure and collection efficiency. Therefore, high device performance is obtained in the case of using an alloy NC layer with low Se content. On the contrary, large lattice mismatch between the alloy NC layer with high Se content and the CdTe NC film shall reduce carrier collection efficiency and device performance.To surmount the drawback of low tructure . Otherwitructure ,49. Herehickness f. The PC-V curve c and TabxTe1−x NC alloy active layers, more CdSexTe1−x layers shall be adopted in the graded bandgap system. From another perspective, the existing large bandgap CdSe film shall accelerate the parasitic absorption and degrade the device performance. Here, for the sake of further tuning the ITO/ZnO/CdSe/CdSe0.2Te0.8/CdTe/MoOx/Au device performance, we selected CdSe0.8Te0.2 as the front contact for ZnO and designed a new device structure of ITO/ZnO/CdSe0.8Te0.2/CdSe0.2Te0.8/CdTe/MoOx/Au without the CdSe layer, which consists of one layer of CdSe0.8Te0.2, one layer of CdSe0.2Te0.8 and three layers of CdTe NC. For comparing, controlled devices without CdSe0.8Te0.2 (ITO/ZnO/CdSe0.2Te0.8/CdTe/MoOx/Au) are also prepared /CdSexTe1−x/CdTe/MoOx/Au. The performance of the graded bandgap NC solar cells is primarily influenced by the lattice matching degree between the alloy NC layer, band alignment and the mobility of the NC thin film. The introduced alloy active layer can extend the spectrum response to longer wavelengths due to the low bandgap nature of the alloy NC thin film. Through adopting a structure of ITO/ZnO/CdSe0.8Te0.2/CdSe0.2Te0.8/CdTe/MoOx/Au, high Voc and high Jsc are effected, attaining a power conversion efficiency up to 6.37%. This work also indicates routes to further tune performance by controlling the annealing strategies and the design of new graded bandgap device architectures.In summary, CdSe


Discordance in estrogen receptor alpha (ERα), progesterone receptor (PR), androgen receptor (AR) and human epidermal growth factor receptor 2 (HER2) status between primary breast cancers and solid distant metastases (“conversion”) has been reported previously. Even though metastatic spread to the peritoneal and pleural cavities occurs frequently and is associated with high mortality, the rate of receptor conversion and the prognostic implications thereof remain elusive.in situ hybridization. Data were coupled to clinical variables and treatment history.We therefore determined receptor conversion in 91 effusion metastases  of 69 patients by immunohistochemistry (IHC) and vice versa in 25-30%, 30-35% and 46-51% of cases for the 1% and 10% thresholds for positivity, respectively. 19-25% of patients converted clinically relevant from “ERα+ or PR+” to ERα-/PR- and 3-4% from ERα-/PR- to “ERα+ or PR+”. For HER2, conversion was observed in 6% of cases. Importantly, receptor conversion for ERα (p = 0.058) and AR (p < 0.001) was more often seen in patients adjuvantly treated with endocrine therapy. Analogous to this observation, HER2-loss was more frequent in patients adjuvantly treated with trastuzumab (p < 0.001).ERα, PR and AR receptor status converted from positive in the primary tumor to negative in the effusion metastases or Alike solid distant metastases, receptor conversion for ERα, PR, AR and HER2 is a frequent phenomenon in peritoneal and pleural effusion metastases. Adjuvant endocrine and trastuzumab therapy imposes an evolutionary selection pressure on the tumor, leading to receptor loss in effusion metastases. Determination of receptor status in malignant effusion specimens will facilitate endocrine treatment decision-making at this lethal state of the disease, and is hence recommended whenever possible. Each year, around 550.000 women die from the consequences of breast cancer , largelyImmunohistochemistry (IHC) plays a valuable role in effusion cytology for the identification of metastatic malignancy. Inclusion of hormone receptor status assessment could direct treatment decision-making. This is underlined by the finding that tamoxifen treatment showed a therapeutic benefit in patients with ERα-positive malignant pleural effusions –11.In the clinical management of metastatic breast cancer, the choice of systemic treatment is traditionally based on the tissue characteristics of the primary tumor. Several previous studies have however shown that the expression of predictive tissue markers including ERα, PR and HER2 may differ between the primary breast tumor and solid distant metastases (“receptor conversion”) in a significant proportion of patients –14. Prolhttp://www.cancer.gov/about-cancer/treatment/clinical-trials/, trial IDs NCI-2015-02043 and NCT02605486). However, the role of ERα-inhibitor induced selective pressure on AR receptor status in distant metastases remaines to be elucidated.Androgen receptor (AR) is expressed in 60% of breast cancers and is more frequently expressed in ERα-positive than in ERα-negative tumors. AR signaling pathways show a distinct pattern, depending on the breast cancer subtypes. In ERα-positive breast cancer, AR is thought to antagonize the proliferative effect of ERα and in ERα-negative tumors, AR signaling has a proliferative role . In a coTo our knowledge, there have been no studies on the influence of adjuvant endocrine therapies on receptor conversion between primary breast tumors and their corresponding malignant effusions so far, while this is a frequent metastatic site , 21. Furin situ hybridization in 69 patients with primary breast carcinomas and their matched malignant peritoneal and/or pleural effusions and solid distant metastases. We furthermore investigated the influence of adjuvant therapies on receptor conversion. Extensive knowledge of possible receptor conversion in malignant effusions could facilitate optimizing patient tailored therapy strategies for metastatic breast cancer patients.Here we report IHC staining of ERα, PR, AR and HER2 complemented with HER2 In total, 69 female breast cancer patients were included in this study with a median age at diagnosis of the primary tumor of 56 years Table . The prip = 0.022 for the 10% threshold and p = 0.079 for the 1% threshold; ERα positivity of 38% for both thresholds), as was the case for effusion metastases . PR positivity was generally lower than ERα in the primary tumors . AR was expressed in 60% or 71% of primary tumors and 10% or 26% of effusion metastases .ERα positivity was observed in the vast majority of primary tumors . Solid metastases were more often ERα negative . For the 10% theshold for positivity, 30% (21/69) of patients showed ERα conversion, with 26% (18/69) from positive in the primary tumor to negative in the effusions and 4% (3/69) from negative to positive. For PR, conversion rates were similar , with 25% (17/69) of samples converting from positive to negative and 5% (4/69) from negative to positive. For AR, discordance was even higher with 50% (35/69) of samples converting from positive to negative, and 1% (1/69) conversion from negative to positive. When comparing solid metastases to paired effusion metastases, ERα, PR and AR protein expression diverged in 20% of cases  of patients showed ERα conversion, with 21% (14/69) from positive in the primary tumor to negative in the effusions and 4% (3/69) from negative to positive. For PR, 35% (24/69) converted in total, with 30% (21/69) of samples converting from positive to negative and 5% (3/69) from negative to positive. For AR, 43% (30/69) of samples converted from positive to negative, and 3% (2/69) from negative to positive. When comparing solid metastases to paired effusion metastases, ERα diverged in 20% (3/15), PR in 13% (2/15) and AR in 20% (3/15) of cases  was perceived in 28% (19/69) of patients for the 10% threshold and in 23% (16/69) of patients for the 1% threshold. 25% (17/69) and 19% (13/69) of patients, respectively, converted from “ERα+ or PR+” to ERα-/PR- and 3% (2/69) and 4% (3/69) of patients from ERα-/PR- to “ERα+ or PR+”.HER2 discordance was seen in 6% 4/69) of cases, were 3% (2/69) shifted from positive to negative and 3% (2/69) from negative to positive  of the patients (of whom treatment history was known), while 63% (31/49) received adjuvant chemotherapy. For 45% (5/11) of HER2 amplified cases, trastuzumab was prescribed. In p = 0.006 or p = 0.058 for the 10% or 1% thresholds for positivity, respectively) and AR (p = 0.001 or p < 0.001), but not of PR .Patients adjuvantly treated with endocrine therapy showed more often conversion of ERα (p < 0.001). Again, this effect was not seen for chemotherapy (p = 0.117).For HER2, adjuvant trastuzumab treatment also influenced the change of receptor status  component and performed ISH on samples that scored 2+ or 3+ [Since generally less steroid receptor positivity was seen in peritoneal and pleural effusion metastases compared to their matched primary breast carcinomas, the question arises whether IHC staining on histologically processed cytology specimens is reliable. However, several studies have compared ERα, PR and HER2 status in cell blocks to tissue blocks and found high concordance rates between cytology and histology specimens , 51. To 2+ or 3+ . Since h2+ or 3+ , we inclhttps://breast-cancer-research.biomedcentral.com/articles/10.1186/bcr3599), it is conceivable that AR-loss would have a comparable effect on abrogating ERα function. Since more than 35% of hormone receptor positive primary tumors convert to ERα and/or PR negative metastases or vice versa, determination of receptor status in malignant effusion specimens may help to optimize patient tailored hormonal treatment and is therefore recommended whenever possible. Especially the new finding of treatment-induced loss of AR protein expression as shown here, might have ramifications for clinical studies addressing AR-targeted therapies in metastatic breast cancer.In summary, we have shown for the first time that ERα, PR, AR and HER2 expression in primary breast cancers is frequently lost in peritoneal and pleural effusion metastases. For ERα, PR and HER2 this is in line with previous findings in solid distant metastases, but AR conversion in late stages of tumor progression is a new observation. We demonstrate that this loss may be inflicted by the evolutionary selection pressure of adjuvant endocrine or targeted therapies, as such accounting for acquired therapy resistance. Considering Enzalutamide treatment, which blocks AR nuclear import, inhibits estrogen-driven MCF-7 cell proliferation , Rijnstate Hospital Arnhem (1 patient), Radboud University Medical Center Nijmegen (2 patients), Bronovo Hospital The Hague (1 patient), Meander Medical Center Amersfoort (2 patients), OLVG Amsterdam (1 patient), Pathology Laboratory Friesland (2 patients), Groene Hart Hospital Gouda (1 patient), Leiden University Medical Center (1 patient), St. Franciscus Hospital Rotterdam (5 patients), Isala Clinics Zwolle (1 patient), all in The Netherlands. Fourteen effusion samples from nine patients were collected prospectively in the Netherlands Cancer Institute in Amsterdam and six samples from four patients from the University Medical Center Utrecht. Selection criteria were: availability of tissue from both primary tumor and effusion metastases, more than 20 tumor cells per cell block, enough tissue to cut sections for IHC analyses on ERα, PR, AR, HER2 and potentially FISH. Original diagnoses had been made between December 1989 and February 2016.Cytology samples were initially fixed in isopropanolol and embedded in paraffin by Cellient, (Hologic). For each case, hematoxylin-eosin stained slides of the paraffin blocks were reviewed by a single experienced pathologist (PvD) to confirm the presence of malignancy in all cytology samples. Only samples containing at least 20 tumor cells were selected. Ber-EP4 monoclonal antibody staining (Monosan), labelling epithelial tissues without reacting with mesothelial cells, was used to confirm presence of tumor cells.All samples were compared with the corresponding primary tumor and, when present, with one or more paired solid distant metastases (fifteen patients). All samples were recut and restained, using current standardized techniques (see below).This study was performed in accordance with the medical ethical guidelines of the University Medical Center Utrecht. The use of anonymous or coded left over material for scientific purposes is part of the standard treatment agreement with patients and therefore ethical approval was not required .IHC for ERα, PR, HER2, AR and Ber-EP4 was carried out on full 4-μm sections with the Ventana  according to the manufacturer's instructions with mouse monoclonal antibodies against Ber-EP4/Ep-CAM  and AR  and rabbit monoclonal antibodies against ERα , PR  and HER2 . Appropriate controls were used throughout.Scoring of IHC slides was performed by consensus of two observers (PvD & WS) in random order, blinded to other data. The adequacy of staining in the primary carcinoma was checked by also evaluating the normal breast parenchyma when present.For ERα, PR and AR, the percentage of positively stained nuclei was estimated side by side with the BER-Ep4 stained slide as a reference. Samples with 10% or more immunopositive malignant cells, regardless of staining intensity, were classified as ERα or PR positive (European standard). The same was done for the 1% USA threshold.vice versa.HER2 expression was scored using the DAKO scoring system as 0, 1+, 2+ and 3+ . HER2 exin situ hybridization using a HER2/CEP17 dual FISH probe (Cytocell) on 4-μm slides. Analysis was performed on a Leica DM5500 B microscope system with Application Suite Advanced Fluorescence Software (Leica Microsystems).All cases with 2+/3+ and discordant results in primary tumors compared to paired metastases were subjected to fluorescence In short, formalin-fixed paraffin-embedded slides were deparaffinized and pretreated with citrate and protease buffers. Next, they were dehydrated and hybridized with 10μl probe in a ThermoBrite (Abbott Laboratories) at 37°C overnight. The next day, slides were washed in saline-sodium citrate buffers, counterstained with DAPI, dehydrated and mounted with Vectashield Mounting Medium (Vector Laboratories). One hundred tumor cell nuclei per tumor were assessed for HER2 gene and CEP17 probe signals at 100x magnification. The HER2/CEP17 ratio was calculated as well. A ratio below 1.8 was defined as a normal copy number, a ratio of 1.8–2.2 as an equivocal copy number and a ratio above 2.2 as gene amplification, according to the ASCO & CAP guidelines .Expression frequency of ERα, PR, AR and HER2 was compared in the primary tumors versus paired effusion and solid metastases using Wilcoxon signed-rank test. Comparison of IHC expression in peritoneal and pleural effusions was performed using Mann-Whitney U test. Dichotomized conversion data (from positive to negative and vice versa) were calculated for 1% and 10% thresholds for positivity and compared by Mc Nemar's test. As steroid receptor conversion is clinically important if a patient converts from “ERα+ or PR+” to ERα-/PR-, or from ERα-/PR- to “ERα+ or PR+”, we calculated the frequency for these conversions as well. Statistical analysis was performed using IBM SPSS Statistics version 21 and visualized using GraphPad Prism 6.We thank Stichting PALGA for the national query for cases.


P < 0.001), indicating that C18-ceramide might have a role in glioma. These roles were examined by reconstitution of C18-ceramide in U251 and A172 glioma cells via addition of exogenous C18-ceramide or overexpression of CERS1, which has been shown to specifically induce the generation of C18-ceramide. Overexpression of CERS1 or adding exogenous C18-ceramide inhibited cell viability and induced cell death by activating endoplasmic reticulum stress, which induced lethal autophagy and inhibited PI3K/AKT signal pathway in U251 and A172 glioma cells. Moreover, overexpression of CERS1 or adding exogenous C18-ceramide increased the sensitivity of U251 and A172 glioma cells to teniposide (VM-26). Thus, the combined therapy of CERS1/C18-ceramide and VM-26 may be a novel therapeutic strategy for the treatment of human glioma.Ceramide synthase 1 (CERS1) is the most highly expressed CERS in the central nervous system, and ceramide with an 18-carbon–containing fatty acid chain (C18-ceramide) in the brain plays important roles in signaling and sphingolipid development. However, the roles of CERS1 and C18-ceramide in glioma are largely unknown. In the present study, measured by electrospray ionization linear ion trap mass spectrometry, C18-ceramide was significantly lower in glioma tumor tissues compared with controls ( Gliomas are a group of heterogeneous primary tumors in the central nervous system (CNS). Malignant gliomas are the most common primary brain tumors in the CNS and account for approximately 79% of malignant CNS tumors . In geneSphingolipids perform essential functions in all eukaryotes and are major players in cell signaling, growth, apoptosis, differentiation, and proliferation , 7. CeraIn the CNS, the most highly expressed CERS is CERS1, which is particularly present in neurons of the neocortex, hippocampus, and cerebellum . Reducti2+ storage, and signaling in the ER 2,5-diphenyl tetrazolium bromide, diethylaminoethyl Sephadex A-25, methanol, dichloromethane, isopropanol, n-hexane, iodomethane and dimethyl sulfoxide were purchased from Sigma. The C18-ceramide was dissolved with DMSO at stock concentration of 50 mM, and diluted to work concentration (20 μM) with DMEM medium. The C18-ceramide treatment was adding the C18-creamide solution (diluted with DMEM medium) to the cells, and the control was the same volume of DMSO (diluted with DMEM medium).2. The cells were transfected with control vector pcDNA3.1(+) (Invitrogen), pcDNA3.1(+)/CERS1 or pcDNA3.1(+)/GFP-LC3, or negative control siRNA or CHOP RNAi siRNA using Lipofectin , according to the supplier’s instructions.Two human glioma cell lines, A172 and U251, were obtained from the Laboratory of Cellular and Molecular Tumor Immunology, Soochow University. They were cultured in Dulbecco’s modified Eagle’s medium, with 10% fetal bovine serum, 100 units/mL penicillin and streptomycin, 2 mM L-glutamine, and nonessential amino acids, and cells were incubated in a 37°C incubator in an atmosphere of 5% COTotal ceramides were extracted from previously frozen brain tissues  and glioma cell lines (A172 and U251 cell line) by extensive sonication with mixed polarity solvents. The first extraction used chloroform–methanol 1:1, v/v (solvent A), followed by extraction with isopropanol–hexane–water 65:25:20, v/v/v (solvent B) and it was replicated 4 times; the supernatant were removed before use of solvent B. Each sonication was followed by centrifugation  to remove supernatant; all the supernatants were dried under nitrogen stream at 40°C. Ceramides were separated by anion exchange chromatography on diethylaminoethyl Sephadex A-25 as previously described , except The neutral fractions were transferred into a conical glass vial, and dimethyl sulfoxide (150 μL) was added to each sample. Then finely powdered sodium hydroxide (ground in an agate mortar) was added to the sample solution and stirred at room temperature quickly until completely dissolved. Iodomethane (150 μL) was added with an injector, then the mixture was vortexed at room temperature for 1 h. The reaction was quenched after 2 mL deionized water was added. The permethylated products were extracted with dichloromethane (10×2 mL), and the combined dichloromethane was frozen on dry ice. The liquid phase dichloromethane was transferred into a new glass tube and dried in a centrifugal dryer.1 and MSn) was carried out in positive or negative ion mode on a linear ion trap mass spectrometer (LTQ), with a flow rate of 2 μL/min, isolation width m/z 1.5, spray voltage 3.5 kv, and at capillary temperature 300°C with injection time 50.00 ms. All ions were detected as sodium adducts. Each ceramide sample was dissolved in methanol (100-500 μL), then the general MS1 was measured in ESI-LIT-MS, and the MSn spectra was obtained from multistep fragmentation via the MSn pathway as previously described [Electrospray ionization mass spectrometry . After incubation for 2 h, the absorbance was measured at 450 nm with a microplate reader .Cells were seeded in culture dishes and allowed to adhere overnight. The cells were harvested, washed, and resuspended in 1× binding buffer. In the next step, the cells were stained at a ratio of per 100,000 cells; staining of cells with 5 μL of FITC-Annexin V  and 5 μL of 7-AAD (50 μg/mL) was done for 15 min. Then, 200 μL of 1× binding buffer was added to each sample. Unstained, untreated cells were also included as controls. A minimum number of 10,000 events was acquired for each replicate using flow cytometry. A quadrant statistic was used to measure the population of viable, early apoptotic, late apoptotic, and secondary necrotic cells. These different populations were determined by estimating quantitatively the cells that were stained with AnnexinV/7-AAD.Total RNA was extracted using Trizol reagent according to the manufacturer’s instructions and stored at –80°C until quality tests and microarray analyses. Gene expression microarray experiments were performed using the SurePrint G3 Human Gene Expression 8×60 K (Agilent Technologies) by CapitalBio Corporation  according to the standard protocols.www.ncbi.nlm.nih.gov/tools/primer-blast/). Then PCR reactions with three replicates for each cDNA sample were carried out using 2×SYBR Green PCR master Mix on the real-time PCR system (Applied Biosystems). The thermal cycle profile was as followings: 95°C for 5 min, followed by 95°C for 15 s, 60°C for 60 s, and 72°C for 15 s for 40 cycles. Then the melting curves were generated to confirm the presence of a single amplicon. The 2∆∆CT was calculated to assess the relative mRNA expression.Total RNA was extracted with Trizol reagent, and RNA concentration was measured by a microplate reader. For all samples, the A260/A280 ratio was between 1.8 and 2.0. The synthesis of first-strand cDNA was performed by using TaKaRa RNA reverse transcription reagents. The primers were designed by Primer-Blast . Cell extracts were quantified using the BCA protein assay kit, and equal amounts of proteins were separated by SDS-PAGE. Gels were then transferred to PVDF membranes, blocked with 5% non-fat dry milk, and incubated with the primary antibody solution. Alternatively, primary antibodies were diluted in 5% BSA in PBST overnight at 4°C. The membrane was washed with PBST and incubated with secondary antibody conjugated with infrared dyes for 2 h at room temperature. Finally, the protein bands were detected by Odyssey infrared image system , and the protein expression levels were quantified by Image Studio software .P < 0.05, **P < 0.01, ***P < 0.001). All statistical analyses were evaluated using SPSS 13.0 software .All the results were expressed as the mean ± standard deviation (SD) of data obtained from three separate experiments and determined using t-test to compare with controls. The n numbers show independent experiments. P < 0.05 was considered as statistically significant (*


Starting basic cardiopulmonary resuscitation (CPR) early improves survival. Fishermen are the first bystanders while at work. Our objective was to test in a simulated scenario the CPR quality performed by fishermen while at port and while navigating at different speeds. Twenty coastal fishermen were asked to perform 2 minutes of CPR (chest compressions and mouth-to-mouth ventilations) on a manikin, in three different scenarios: (A) at port on land, (B) on the boat floor sailing at 10 knots, and (C) sailing at 20 knots. Data was recorded using quality CPR software, adjusted to current CPR international guidelines.p = 0.01) or at 20 knots . The percentage of ventilation that achieved some lung insufflation was also significantly higher when CPR was done at port (77% ± 14) than while sailing at 10 knots (59% ± 18) or 20 knots (57% ± 21) (p = 0.01). The quality of CPR (QCPR) was significantly higher at port (43% ± 10) than sailing at 10 knots (30% ± 15;  In the event of drowning or cardiac arrest on a small boat, fishermen should immediately start basic CPR and navigate at a relatively high speed to the nearest port if the sea conditions are safe. The 2015 European Resuscitation Council (ERC) Guidelines for Resuscitation include basic cardiopulmonary resuscitation (CPR) in special circumstances of drowning . Ventilation quality (QV) was assumed when air inflow was between 500 and 600 ml per mouth-to-mouth insufflation.Global quality CPR (QCPR) was calculated using the formula [(QCC + QV)/2] .Effective cardiopulmonary resuscitation (ECPR) is the measurement of all quality parameters of resuscitation and ventilation and was calculated using the formula [(EV + QCPR)/2].The cut-off for quality was established at the arbitrary but generally accepted point of 70% .At the end of each test, fishermen were asked to rate their CPR performance through rating of perceived ability (RPA) on a scale of 0–100% and rating of perceived exertion (RPE) during CPR using the modified ten-point Borg scale .Data capture was carried out on August 12, 2016, between 16:00 and 20:00 on Rianxo's port (Northwest Spain) beginning at the following GPS location: 42°38′49′′–8°49′30′′. The weather conditions were as follows: air temperature of 27° Celsius, clear sky, wind speed of <1 knot , and calm sea with crests of vitreous appearance and without breaking. APPASPECT GPSSpedd HD, 2.2.3 for IOS , registered the movement of the boat and the measure was under 0.5 m in both axes.The vessel used was the usual inshore fishing boat in the area, with the following characteristics: arch gross tonnage (GT) of 1.55 T, arch gross register tonnage (GRT) of 2.23 T, total length of 6.7 m, and out-of-board 75-horsepower engine. The boat had enough room on deck for resuscitation of an adult victim.Sailing was planned and guided by GPS, so the same route during the same time (2 minutes) was guaranteed. Sailing speed was programmed at 10 and 20 sea knots (approximately 20 and 40 km/h) (see Supplementary Video ). One med effect size. Effect sizes with values of 0.2, 0.5, and 0.8 were considered to represent small, medium, and large differences, respectively  were expressed by absolute and relative frequencies. The QCPR variables were expressed by central tendency and dispersion measures [mean (standard deviation) (SD)]. The comparison of the dependent variables was performed using ANOVA with Bonferroni and Friedman correction. Statistical differences from parametric variables were determined using Cohen's ectively . From nor ≥ 0.50 . The pro2 ± 3.Data was available from all participants (20 fishermen); then, 60 CPR 2-minute tests were recorded. Subjects' age was 36 ± 7, weight was 86 kg ± 14, height was 177 cm ± 8, and BMI was 27.3 kg/mp = 0.01, ES = 1.04) and 20 knots  (The results of CPR variables are presented in  = 1.26) p = 0.11)R was quite similar in all conditions, ranging between 115 and 119 compressions per minute (p < 0.05) in all cases, the 2015 international recommendations were fulfilled in all scenariosAlthough D was significantly better at port versus when sailing at 10 and 20 knots  and at 20 knots .Data presented in p = 0.01, ES = 1.05) or at 20 knots . 70% of the fishermen exceeded the value of 70%, which has been established as quality criteria [p > 0.05).When QCPR was analyzed, higher values were observed at port (43% ± 10) than while sailing at 10 knots .EV was slightly higher at port (83% ± 23) than while sailing at 10 knots (70% ± 36) or at 20 knots (68% ± 38), but not statistically significant  or at 20 knots . No significant differences were observed during navigation .However, ECPR was significantly higher at port (77% ± 14) than when sailing at 10 knots  than at port. For the rest of the comparisons, no significant differences were observed . However, when compared to the baseline, the perceived effort increased with sailing speed .Fishermen found it more difficult to perform CPR at 20 knots  or learning (positive). By performing the navigation tests with short pauses, fisherman can learn and become better although the improvement would be minimal as they do not have visual feedback and would become fatigued.CPR trained and refreshed fishermen are able to perform good quality CPR (chest compressions and mouth-to-mouth ventilations) on manikins both on land and while sailing at relatively low (10 knots) and high (20 knots) navigating speeds.Our results support the notion that, in the case of drowning or cardiac arrest on a small fishing boat, in calm sea conditions, fishermen should immediately start basic CPR and maintain CPR while navigating at a relatively high speed (around 20 knots) to the nearest land, where CPR would be continued with advanced procedures by expert personnel.


Calophyllum brasiliense is a plant from the Brazilian rain forests and has been used in folk medicine for the treatment of various diseases, including leishmaniasis. This infectious disease depends on the Leishmania sp. and the host immune response. C. brasiliense antileishmanial activity is well known, but the effects on immune response remain to be investigated. This study showed the leishmanicidal and immunomodulatory effects of a 30 μg/mL of hydroalcoholic extract of C. brasiliense in murine macrophages before and after Leishmania (Leishmania) amazonensis infection. The semiquantitative cytokine RNA expression was determined by RT-PCR and the anti-Leishmania activity was measured by infection index (IF). Hydroalcoholic extract of C. brasiliense reduced more than 95% of IF when used before and after Leishmania infection, with 3 and 24 h of treatment (p < 0.05). C. brasiliense inhibited or reduced significantly (p < 0.05) the TNF-α, IL-1β, IL-18, and IL-10 mRNA expression. The antileishmanial and anti-inflammatory effects showed the potential of C. brasiliense as an alternative therapy for leishmaniasis and it must be investigated. Leishmania. The parasite has a complex digenetic life cycle requiring a susceptible vertebrate host and an insect vector, which allow their transmission. Leishmania promastigote forms are inoculated by the phlebotomy vector in the skin of the host. The parasites invade macrophage cells transforming them into amastigote form [Leishmania and their transport to the regional lymph nodes, in which a microenvironment is created from immune response and parasite [Leishmaniases are endemic neglected tropical diseases caused by intracellular parasitic protozoa of the genus ote form , 2. The parasite –3. The cparasite , 2.Leishmania (Leishmania) amazonensis is characterized by skin and diffuse lesions, and the immune response of the host is essential for the resolution of leishmaniasis. The immune mechanisms involved will define the fate of the infection and its spread or control, as well as the development of the adaptive immune response [Leishmania parasites are recognized by immune cells stimulating the T helper 1 lymphocytes (Th1) to produce cytokines that induce the activation of the phagocytic mononuclear system. The macrophage cells produce the microbicidal substances leading to parasite death. The reactive oxygen species (ROS) and oxide nitric are microbicidal agents that promote the death of Leishmania sp. [The infection caused by response , 4. Leisania sp. , 4.Currently, leishmaniasis has affected 12 million people worldwide, occurring 70,000 deaths every year and there are 350 million people at risk of contracting the disease .Calophyllum brasiliense Camb. (Clusiaceae) is a tree popularly known as “guanadi-cedar,” “oil stick,” and “guarandi” [C. brasiliense leaves have been used in folk medicine against various diseases [C. brasiliense were reported such as its antibacterial, antifungal, and antiprotozoal activities, its cytotoxicity to tumor cells, and its inhibitory effect on tumor promotion and HIV-1 replication [C. brasiliense is known [C. brasiliense remain unclear [Plants are traditionally used for the treatment of many diseases and are more accessible, as they can be naturally acquired –9, leadiuarandi” , 14 and uarandi” , occurriuarandi” . The extdiseases , 17–27, diseases –13. Somelication –27. The is known –13, but  unclear .C. brasiliense played a role as potential agents to attenuate the oxidative damage produced by different prooxidants, such as ROS and lipid peroxidation [L. (L.) amazonensis can escape and survive at the immune environment, requiring the modulation of the components of the immune response as Th1 and Th2 cells and their products [C. brasiliense treatment remains to be investigated. Therefore, this study investigated the potential of hydroalcoholic extract of C. brasiliense in immunomodulating the cytokines' expression by murine macrophages infected with L. (L.) amazonensis.Blanco-Ayala and coworkers conducted an investigation suggesting that the xanthones from xidation . ROS andxidation . Besidesproducts , but itsL. (L.) amazonensis (MHOM/BR/1977/LTB0016), held in cryopreservation, were thawed and maintained at 25°C in 199 culture medium supplemented with fetal bovine serum 10% (v/v), human urine (1%), L-glutamine (20 mM), and antibiotics (penicillin 100 IU/ml and streptomycin 0.1 mg/ml). Weekly, subcultures were made of 199 medium cultures and parasite inoculum was adjusted according to each experiment.Promastigotes forms of ad libitum with 12 h light-dark cycles.BALB/c isogenic mice, female, 8–10 weeks, were obtained from the Central Animal Laboratory at the State University of Maringá, after approval by the Ethics Committee on the Use of Experimental Animals of the State University of Maringá  (warrant number 133/2012). The animals were kept in collective cages with water and food C. brasiliense were collected between January 2009 to December 2010, in Parque Estadual da Ilha do Cardoso in Cananéia, located on the southern coast of São Paulo state (Brazil). This tree is found in all coastal Atlantic forest vegetation (https://nossosparques.org.br/). The plant was identified by Dr. Maria CM Young, and a voucher specimen was deposited (SP 363818) in the herbarium of the Botanical Institute of São Paulo, SP, Brazil. The plant sample was weighed, dried in a circulating air oven  at 35°C, crushed in a Wiley mill , and subsequently stored in a dry place, away from light.The leaves of The sample (1.3 kg), previously crushed, underwent cold maceration process in ethanol : water (9 : 1) and successive extractions until all active compounds were retrieved (4 × 61) . Subsequ6 macrophages/mL  were plated on twelve-well culture plates and incubated at 37°C in an atmosphere of 5% CO2 for one hour. Subsequently, the nonadherent cells were removed, and the adherent cells were infected with 10 promastigotes of L. (L.) amazonensis by macrophage and incubated for 3 h at 37°C with 5% CO2. Hence, cultures were treated with 30 μg/mL of the hydroalcoholic extract of C. brasiliense for 3 h [μg/mL of the hydroalcoholic extract of C. brasiliense for 3 h and then infected with 10 promastigotes per macrophage, for 3 h under the conditions mentioned above. Noninfected macrophages were also treated with 30 μg/mL of hydroalcoholic extract of C. brasiliense for 3 h. Macrophages infected with promastigotes of L. (L.) amazonensis and untreated were used as positive control, and macrophage uninfected and untreated were used as negative control. All procedures were performed in triplicate.Peritoneal macrophages from BALB/c mice were collected with sterile cold RPMI 1640 medium (pH 7.6) and 1.0 × 10 for 3 h . Alterna5 macrophages/mL was added on 13 mm-diameter sterile glass coverslips , distributed into 24-well culture plates and then incubated at 37°C with an atmosphere containing 5% CO2 for 1 h. After that, nonadherent cells were removed by washing with RPMI 1640 medium, while the adherent cells were infected with promastigotes of L. (L.) amazonensis (six parasites per macrophage) [μg/mL of hydroalcoholic extract of C. brasiliense for 3 to 24 h. In another condition, macrophages were first treated for 3 h with 30 μg/mL hydroalcoholic extract of C. brasiliense, and after this period, the cells were infected with promastigotes of L. (L.) amazonensis (six parasites per macrophage) incubated for 3 to 24 hours. After these periods of treatment, the supernatant was discarded and the coverslips removed, washed, and stained with Panoptic kit Quick LB® , and after, it is fixed on glass slides (24 × 76 mm) with Entellan . The experiments were performed three times in triplicate. The macrophages infected with L. (L.) amazonensis and untreated were used as positive control and macrophages uninfected and untreated as negative control. The antiamastigote activity of C. brasiliense was determined by the infection index (IF) calculated by the percentage of infected macrophages multiplied by the average number of parasites per macrophage.A suspension of 5 × 10rophage) , and incμg of total RNA as a template in a reverse transcription reaction using Superscript III reverse transcriptase . RNA quantification (ng/ml) and the determination of purity were performed using NANODROP 2000 UV-Vis Spectrophotometer  . cDNA samples were amplified by PCR using the specific primer sequences for mRNA: tumor necrosis factor (TNF-α) [β [γ (IFN-γ) [Mus musculus) as internal control of the PCR amplification [μM of each primer, 0.2 mM dNTP MIX, 1 U Platinum Taq DNA polymerase (Invitrogen), and 1.5 mM MgCl2, and DNA (5–10 μL~100 pg/reaction) was added to each test tube in a final volume of 25 μL. The amplified DNA fragments were separated by 1.5% agarose gel electrophoresis and revealed with ethidium bromide in transilluminator. Semiquantitative RT-PCR was performed by quantifying the bands densitometrically using ImageJ software . Each cytokine gene was normalized to GAPDH as a housekeeping gene control .Total RNA was extracted from macrophages using TRIzol reagent according to the manufacturer's instructions . The complementary DNA (cDNA) was synthesized from 1.0  (TNF-α) , interleNF-α) [β , IL-12, NF-α) [β , interfe (IFN-γ) , IL-10,  (IFN-γ) , and glyfication   amazonensis and treated with 30 μg/mL hydroalcoholic extract of C. brasiliense showed a significant reduction in the infection index (IF) (p < 0.05) after 3 h  and after 24 h , when compared to untreated cells (IF: 351/3 h and 799/24 h) (C. brasiliense for promastigote and axenic amastigote L. (L.) amazonensis in a concentration between 20 and 40 μg/mL and did not observe cytotoxic activity.maniasis –13, in Bmaniasis –27. In t99/24 h) . The leiC. brasiliense extract and infected with L. (L.) amazonensis for 3 and 24 h (p < 0.05) compared to controls, from 374.5 to 15 (96%) after 3 h and from 822.3 to 11.6 (98.6%) after 24 h.In subsequent experiments, macrophages were pretreated with and 24 h . The IF L. (L.) amazonensis. The animals treated topically during 32 days after the infection revealed healing lesions, and the parasite load in the popliteal lymph nodes was significantly reduced in treated animals. The results showed that crude extract and hexane fraction of C. brasiliense have a significant in vitro and in vivo leishmanicidal activity [Honda et al.  observedactivity . These sactivity –13.C. brasiliense, such as (−) mammea A/BB and 5,7-dihydroxy-8-(2-methylbutanoyl)-6-(3-methylbutyl)-4-phenyl-chroman-2-one, may have antileishmanial activity by acting directly on the depolarization of the Leishmania mitochondria membrane potential, thus leading to parasite death [C. brasiliense Camb. extract promoted wound healing in rats with cutaneous leishmaniasis; after 14 days of the treatment start, the animals treated exhibited more than 90% reduction of wound areas. After 21 days of treatment, the treated animals exhibited a significant increase in fibroblasts. Thus, the C. brasiliense emulsion had healing properties in the topical treatment of wounds and accelerated the healing process [The compounds derivate from te death . A topic process .C. brasiliense treatment modulated the mRNA cytokine expression produced by murine macrophages infected with Leishmania and did not modify iNOS mRNA expression. From all cytokines investigated, C. brasiliense inhibited TNF-α mRNA expression (100%) and reduced in 18% IL-1β and 23% IL-10 by uninfected macrophages, compared to untreated and uninfected macrophages (negative control) (p < 0.05) , and IL-10 by macrophages, compared to the negative control (p < 0.05)  . The inf < 0.05) . TNF-α,  Figures .C. brasiliense macrophages, the hydroalcoholic extract inhibited 100% mRNA expression of TNF-α, IL-10, IL-12, and IL-18 and reduced 51% IL-1β, compared to the positive control (L. (L.) amazonensis infection induced an increase of TNF-α (10%), IL-1β (30%), and IL-18 (6%) expression, in comparison to the negative control  control . The C. reatment .C. brasiliense and after infected with Leishmania, the hydroalcoholic extract reduced TNF-α expression (9%), IL-12 (6%), IL-18 (15%), and IL-10 (7%) and increased IL-1β, compared to the negative control (p < 0.05). The parasite infection promoted the increase of IL-1β and reduced TNF-α, IL-12, IL-18, and IL-10, compared to the negative control (C. brasiliense treatment reduced significantly these same cytokines (p < 0.05) even after the infection and increased IL-12  when used after  amazonensis infection, some researchers related that this parasite species can decrease the production of nitric oxide as an escape mechanism of the immune host [C. brasiliense during infection, but other studies showed that C. brasiliense can play a role as a potential agent to attenuate the oxidative damage produced by different prooxidants, as ROS and lipid peroxidation [C. brasiliense was not able to modulate iNOS, other immune mechanisms can be investigated, like the cytokines.Interactions between parasite and immune cells lead to phagocytosis by macrophages that activate trigger mechanisms for their microbicidal response to the production of reactive oxygen metabolites (ROS) and nitric oxide (NO) . As the ishmania . The NO ishmania . In Leisune host , 28, 36.xidation . AlthougLeishmania infection. In this context, a protective immunity for leishmaniasis is related to an efficient Th1 cellular response and the production of cytokines such as IL-12 and IFN-γ [γ was not modulated by C. brasiliense, and only IL-12 was increased when C. brasiliense was tested before the infection. IL-12 is involved in cell-mediated immunity by stimulating Th1 cells and inflammation control, and it is critical for the dissemination of parasite control [Cytokines are protein molecules released by various cell types of the immune system and act on the emission of signals between the cells for immune responses. Several studies in murine models have associated a specific cytokine or a combination of cytokines as factors to susceptibility or resistance to nd IFN-γ , 38. In α expression levels were elevated in macrophages infected by L. (L.) amazonensis, but C. brasiliense was able to subvert it. Another study reported a relationship between increases in TNF-α and the susceptibility to L. (L.) amazonensis infection, inducing the persistent parasitic infection and tissue damage [α is a proinflammatory cytokine of the Th1 cellular response involved in the occurrence of an intense inflammatory reaction and damage to these tissues and results in the appearance of skin and mucosal ulcers [C. brasiliense inhibited the TNF-α mRNA expression completely when used alone or after the Leishmania infection, and it was reduced it when used before Leishmania infection. These results showed a potential of C. brasiliense to reduce the inflammatory cytokines that can lead to the worsening of the leishmaniasis lesions.Also, TNF-e damage . TNF-α il ulcers . In the C. brasiliense also inhibited IL-18 mRNA expression in infected macrophages. This cytokine is primarily produced by macrophages and dendrites cells and is considered as a cofactor for the IL-12-induced development of the Th1 immune response, which optimizes IFN-γ production by effector Th1 cells [C. brasiliense treatment can explain the IL-12 and IFN-γ inhibition. Thus, hydroalcoholic extract of C. brasiliense has shown potential anti-inflammatory effects.h1 cells . IL-18 iLeishmania sp. death that are induced by macrophages, leading to disease progression. Th2 cellular response is promoted by IL-10, IL-4, and IL-5 cytokines that are known as downregulators of the immune response [L. (L.) amazonensis presented high levels of IL-10, IL-4, and TNF-α [Leishmania infection induced TNF-α and IL-10 expression strongly, but C. brasiliense subverted these cytokines, mainly after infection. IL-10 is the main regulator cytokine of Th1 type cellular immune response and acts as a inhibitor of IL-12, IL-2, TNF-α, and IFN-γ synthesis, favoring the parasite persistence [Beyond Th1 response, Th2 immune response is also crucial to infection evolution, since it inhibits the mechanisms of nd TNF-α . In the sistence . Thus, tβ expression was also downregulated by C. brasiliense. First, following inoculation of parasites in the skin and the start of phagocytosis of promastigote forms, it activated mononuclear phagocytes releasing TNF-α and IL-1β cytokines, acting in innate immunity and inflammation, for the purpose of stimulating the recruitment of neutrophils and monocytes to sites of infection. These actions occur through the increased expression of adhesion molecules on the vascular endothelium (ligands for selectins and integrins) and by inducing chemotaxis of leukocytes [β induces the resistance to Leishmania amazonensis in infected macrophages [β can promote the proliferation and activation of Th2 cells and suppress the Th1 response [C. brasiliense is a potential IL-1β inhibitor, favoring the leishmaniasis resolution.IL-1ukocytes . Also, trophages . Thus, Iresponse . TherefoC. brasiliense at 30 μg/mL has leishmanicidal activity and immunomodulatory effects. This plant modulated the immune response in murine macrophages infected with L. (L.) amazonensis or used alone, due to inhibiting completely or reducing significantly mRNA expression of cytokines related to Th1 and Th2 cellular response. Therefore, we suggest that the other inflammatory mediators, such as prostaglandins and leukotrienes, are investigated in animal models submitted to C. brasiliense and Leishmania infection. The cytokine inhibition profile induced by C. brasiliense indicated that this plant has an anti-inflammatory activity. Thus, the antileishmanial and anti-inflammatory immunomodulatory effects showed the potential of this plant as an alternative therapy to cutaneous leishmaniasis caused by L. (L.) amazonensis.In short, our data suggest that the hydroalcoholic extract of