Tumour growth rates were measured in 105 patients using in vivo incorporation of bromodeoxyuridine (BrdU) and investigated for any relationship to tumour factors or survival. The median labelling index (LI) was 8.7%, the duration of S-phase (Ts) was 14 h and the potential doubling time (Tpot) was 5.9 days. The labelling index in aneuploid tumours was significantly higher than that in diploid tumours. However the total labelling index (TLI) did not differ significantly between aneuploid and diploid tumours, and so it would seem likely that the difference in LI is due to the dilutional effect of benign tissue upon the calculation of LI in diploid tumours. The total labelling index, duration of S-phase and potential doubling time were not related to the tumour factors examined . Interim survival analysis was carried out and there was no difference in survival between those patients with high values for TLI, Ts, and Tpot and those with low values.


The two techniques of flow cytometry analysis (FCM) and immunohistochemical localisation of bromodeoxyuridine (BrdUrd) incorporation after in vivo administration, were combined to study proliferation in squamous cell carcinoma of the head and neck region. Care was taken in this study to ensure that similar material was processed using both techniques such that comparisons could be made. FCM underestimated the labelling index (LI) in tumours classified as diploid compared to the histological evaluation of the tumour cells within those tumours (4.6% vs 17.1%). However, in aneuploid tumours, the FCM LI (10.7%) was similar to that obtained from histology (13.5%). Indeed, proliferation assessed by the combination of histology LI and FCM duration of S-phase (Ts) indicated that diploid tumours had a shorter median potential doubling time (Tpot) of 2.1 days compared to aneuploid (2.8 days). Despite the heterogeneity of proliferation evident histologically within the specimens, there was not a wide variation in the results of FCM analysis when multiple samples from resections were studied. Using FCM data alone, 46% of the tumours showed a Tpot of less than 5 days. When the Ts from the FCM data was combined with the average histological LI, 84% were less than 5 days and with the maximum LI, 99% were within this time interval. Compared with previous estimates, the proportion of tumours possessing proliferative characteristics which may indicate the need for acceleration of treatment seems to be much larger.


The prognosis of colorectal cancer has not significantly changed during the last 30 years. While evaluation of tumour cell proliferation may provide prognostic information, results obtained so far have been contradictory Heterogeneity in tumour cell proliferation may explain these contradictions. With in vivo injection of iododeoxyuridine (IdUrd), estimation of labelling index (LI), S-phase transit time (Ts) and potential doubling time (Tpot) may be performed from a single sample. A total of 109 colorectal cancers were studied after in vivo injection of IdUrd before surgical removal. From each cancer, four to eight samples were processed for both flow cytometrical (FCM) and immunohistochemical (IHC) visualization of IdUrd incorporation. LI/IHC was morphometrically quantified at both the luminal border and the invasive margin of these tumours. LI was significantly higher at the luminal border compared with the invasive margin, although they were correlated with each other. Using combined IHC and FCM methods, rapidly growing colorectal cancers (high LI and/or low Tpot) showed an increased survival  in the entire unselected material and for radically removed Dukes' B tumours. FCM data alone did not discriminate for survival, with the exception of Ts in diploid and radically removed Dukes' B tumours.


The proliferative potential of human solid tumours, in vivo, was investigated using bromodeoxyuridine (BrdUrd) incorporation and flow cytometry (FCM). Patients with solid tumours from a variety of sites were injected with 500 mg BrdUrd, intravenously, several hours prior to biopsy or surgical excision. The labelling index (LI), duration of S-phase (Ts) and thus the potential doubling time (Tpot) could be measured within 24 h of sampling. The results show that both the LI and Ts vary greatly between tumours (Ts ranges from 5.8 to 30.7 h). However, within this study of 26 evaluable patients, tumours of the same tissue origin tended to have similar Ts values. Melanomas had the shortest Ts (8.8 h), nine patients with head and neck cancer had Ts values ranging from 5.8 to 18.8 h (median 12.5 h). The longest Ts values (24 h) were found in lung and rectum. The estimates of Tpot ranged from only 3.2 days in an oat cell carcinoma to 23.2 days in a lymphoma. The striking feature of the study was that 38% of the tumours had a potential doubling time of 5 days or less. We found no relationship between proliferation and histopathological differentiation or DNA ploidy. It should now be possible to assess the prognostic significance of pretreatment cell kinetic measurements which may, in the future, aid in the selection of treatment schedules for the individual patient.


ADH1B) genotype was not an independent risk factor for breast cancer, athough the possibility was raised that it modifies risk associated with high levels of alcohol consumption  0.8–1.6 for ADH1B*1/*1 genotype vs 0.2, 95% CI 0.1–1.0 for ADH1B*2 carriers).In a population-based study of 613 cases and 1082 controls, alcohol dehydrogenase 1B ( Of all 2257 eligible controls, 1381 (61.2%) participated. Detailed information on demographic characteristics and various risk factors was elicited by means of a self-administered questionnaire. Alcohol consumption was assessed for three time periods, that is, 15–20, 20–30 and 30–50 years, and for different types of beverages . The detailed method for the calculation of average daily alcohol intake has been described previously .ADH1B gene. Two minor-groove-binding (MGB) DNA probes were synthesised. The probe corresponding to the wild type (5′-FAM-TCTGTCGCACAGATG-MGB-3′) was labelled with 6-FAM, and the probe corresponding to the mutation (5′-VIC-AATCTGTCACACAGATGA-MGB-3′) was labelled with VIC at the 5′-end. The genotypes were analysed in software ‘Sequence Detector’ version 1.7 by procedure allelic discrimination after PCR.Genomic DNA was extracted from peripheral blood using the QIAamp DNA Blood Kit . Primer and hybridisation probes were designed with Primer Express. Sense primer 5′-CTCTTTATTCTGTAGATGGTGGCTGTAG-3′ and antisense primer 5′-GGGTCACCAGGTTGCCACTA-3′ were used to amplify a 76 bp fragment containing the G47A polymorphism of the μl containing 40 ng of DNA, 300 nM of each primer, 200 nM of each probe and 12.5 μl of TaqMan Universal PCR Master Mix .Amplification was performed in a final volume of 25 In every assay, negative controls as well as controls for the wild type, mutant type and heterozygote were included. The PCR conditions were as follows: 2 min at 50°C plus 95°C for 10 min, followed by 40 cycles of denaturation at 92°C for 15 s, annealing and extension in one step at 60°C for 1 min. Genotyping was performed blinded to case–control status of the blood sample providers.χ2 tests were used to assess deviation from Hardy–Weinberg equilibrium. Multivariate conditional logistic regression analysis with 5-year age strata was carried out using the PHREG procedure of the statistical software package SAS release 8.2 .ADH1B genotype and alcohol consumption was tested by using multiplicative interaction terms and evaluated by the likelihood ratio test.In the multivariate model, we included several relevant variables influencing breast cancer risk see . VariablADH1B*2 allele, corresponding to allele frequencies of 0.06 and 0.05, respectively. The distribution of ADH1B genotypes was in Hardy–Weinberg equilibrium (P=0.4 for cases and P=0.6 for controls).Selected characteristics of the study population are depicted in ADH1B genotype and several risk factors, including first-degree family history of breast cancer, body mass index, parity, breastfeeding or smoking status (data not shown). However, alcohol consumption was found to differ significantly by ADH1B genotype among controls, with a mean average daily alcohol intake of 7.2 g (standard deviation (s.d.) 9.1) in women with ADH1B*1/*1 genotype and 5.8 g (s.d. 7.8) in carriers of the *2 allele (Wilcoxon's rank-sum test P=0.01). Results were virtually identical when all study subjects were considered (data not shown).We did not observe an association between ADH1B genotype on breast cancer risk  0.7–1.4). However, multivariate analysis separately for carriers and noncarriers of the ADH1B*2 allele yielded differences in breast cancer risk associated with increasing levels of alcohol intake , although the odds ratio (OR) was significant only for the category of 12 g or more alcohol per day. In contrast, among women with ADH1B*1/*1 genotype, breast cancer risk increased with increasing alcohol consumption (P=0.03). The interaction between ADH1B genotype and alcohol intake was borderline statistically significant for the highest category of alcohol consumption (P=0.05) but not for low or moderate levels.The analysis revealed no main effect of l intake . Among cADH1B genotype in our study population, which was apparent only at the highest consumption category of 12 g or more alcohol per day. We showed previously that breast cancer risk increased significantly with high daily alcohol intake of ⩾19 g in this study population . However, since ADH1B genotype and alcohol intake are not independent in our study population, the modifying effect of the ADH1B genotype on breast cancer risk associated with alcohol consumption is overestimated in the case-only analysis, partly due to residual confounding by differences in alcohol consumption caused by the genotype (ADH1B genotype and alcohol consumption, alcoholism or adverse reactions such as flushing were observed in previous studies (Our data raise the possibility of an effect modification of the association between alcohol consumption and breast cancer risk by ADH1B genotype on alcohol pharmacokinetics in vivo. Most studies failed to detect differences in blood alcohol or acetaldehyde levels by ADH1B genotype (ADH1B*2 allele (There is still controversy in the literature regarding the effect of ADH1B*2 allele in our study could therefore be explained by a higher alcohol elimination rate in these subjects. Owing to the low allele frequency of the ADH1B*2 allele in Caucasians, our study had limited power to detect a gene–environment interaction for high alcohol consumption levels and our findings about ADH1B genotype as an effect modifier of breast cancer risk associated with high alcohol consumption need confirmation in larger studies.The reduction in breast cancer risk associated with high consumption levels in carriers of the


Objective. The purpose of the study was to compare the cognitive skills of young children diagnosed with autism spectrum disorder (ASD) to same-aged peers referred for possible developmental delays or behavioral concerns using the Bayley Scales of Infant Development-Third Edition. Method. A retrospective chart review was conducted of 147 children ages 16 to 38 months who were referred to a diagnostic clinic for developmental evaluation. Children with ASD were compared to those without ASD with respect to cognition and language outcomes, both overall and by age. Results. While language skills in children with ASD were more significantly delayed than language skills in children without ASD, there was less discrepancy in the cognitive skills of children with and without ASD. Conclusion. Formal cognitive assessment of children with ASD can provide guidance for developmental expectations and educational programming. Cognitive skills of children with ASD may be underappreciated. The number of children diagnosed with autism spectrum disorder (ASD) has rapidly increased in recent years. Best estimate of current prevalence of children with ASD is just over one per 100 [Autism was first described by Kanner , who obsDiagnostic and Statistical Manual of Mental Disorder-IV with 70% to 75% of children having both [The prevalence of intellectual disability  in children with autism was estimated to be 90% before 1990 . Prevaleing both . EdelsonExpanded definitions of the autism spectrum have included children without intellectual disability, and ASD now includes the subgroups of autistic disorder, Asperger syndrome, and pervasive developmental disorder . Recent Since the DSM-IV and inclusion of a broader definition of children with ASD, more studies have been conducted looking at the developmental profiles of children with this disorder, including their IQ, motor, and language skills. In her review, Edelson  found th According to Mayes and Calhoun , 67% of The Bayley Scales of Infant Development—Third Edition [ Edition  included All composite and subtest scores for the PDD group were significantly lower that those obtained by children in the matched control group. Cognition scores were one standard deviation lower for children in the PDD group, and language scores were even more significantly delayed than cognitive skills. In summary, the ability profiles of children with autism spectrum disorder have been highly variable in former studies. There is limited research on the comparison of the cognitive profiles of young children with and without autism who are referred for language and behavioral concerns. To further explore the cognitive profiles of children with and without autism spectrum disorder, the following research questions were asked: how do the cognitive profiles of young children with ASD differ from same-aged children seen for developmental evaluation who do not have ASD? Do young children diagnosed with ASD have higher cognitive scores than language scores on a standardized assessment tool? Are there age, gender, and socioeconomic differences between the children with ASD and those without ASD with respect to cognitive abilities?This study was a retrospective chart review of children referred to the Kluge Children's Rehabilitation Center Infant and Young Child Clinic for developmental assessment during an 18-month period in 2007 and 2008. A total of 147 children ages 16 to 38 months were included in the study, with a median age of 27 months. There were 107 males (73%) and 40 females (27%). Most of the children were referred to the diagnostic clinic by either their primary physician or a family member and were seen for concerns regarding their development in areas such as language, behavior, possible autism, or global developmental delay. Each child was seen by an early childhood special educator and a developmental pediatrician. The majority of the children were from central Virginia and surrounding areas. The family's health insurance status—whether they had public insurance (Medicaid) or private insurance—was used as a proxy measure of socioeconomic status (SES). During the clinic visit, each family reported whether the child was receiving early intervention and/or therapy services, and the child was referred to appropriate services if they were not already enrolled in a local program. The study was approved by the Human Investigation Committee of the University of Virginia.Bayley Scales of Infant Development—Third Edition (BSID-III) by a person certified in their administration. Other tests of the BSID-III such as fine and gross motor and social and adaptive tests were not administered during the clinic visit due to time constraints. The third edition of the BSID [Each child was given the cognitive and language tests from the the BSID  was publChildhood Autism Rating Scale (CARS) [For children referred for screening of possible autism spectrum disorder, the e (CARS)  was admie (CARS) . P-value <.05. Overall comparisons between the two groups (those with ASD versus those without ASD) were made via chi-square tests for categorical variables and t-tests for continuous outcomes. Linear models were fit to the cognition and language outcomes, with age and group (and their interaction) as predictors. Comparisons between the two groups were then made at each of four standard ages  based on estimates from these models.All analyses were performed utilizing SAS Version 9.1; statistical significance was defined as a More children referred to the diagnostic clinic were male (73%) than female. Socioeconomic status as determined by public or private insurance was not significantly different for children with ASD and without ASD, although fewer children with ASD (26%) were insured by Medicaid than children without ASD (37%). Sixty percent of the children referred to the clinic were receiving early intervention and/or therapy services at the time of the clinic visit. Of the 147 children referred to the clinic, 64 children were administered both the BSID-III and the CARS compared to 72 children who received only the BSID-III assessment because they were not observed to exhibit any behavioral characteristics associated with ASD. There were 54 children who were diagnosed with ASD using the CARS criteria, with a higher number of males (42) than females (12). In regards to the age of diagnosis of ASD, 42 were over 24 months of age and 12 were under 24 months of age. In addition, eleven children were uncooperative during the testing session in order to complete the BSID-III tests and were not included in the analyses. These eleven children did receive a CARS evaluation by observation and parental report. Of the 11, all but one child met criteria for ASD. P = .0006). The mean ASD language composite score was over 16 points lower (P < .0001). Developmental age equivalents were also lower for children with ASD in cognitive, receptive, and expressive language . Significant cognitive differences between children with and without ASD were observed only for older children (over two years old) . Including gender in the model did not impact the results.However, significant interactions existed between age and ASD status with respect to the cognitive outcomes (interaction ars old) . Childrears old) ; no signIn the current study, the majority of young children referred to a diagnostic clinic were able to receive a standardized assessment using the BSID-III, including children who were found to have characteristics of ASD. As seen in other studies, males were more likely to be diagnosed with ASD than females. While the cognitive skills of children with ASD were generally lower than the children without ASD, many of the children with ASD scored in the average and low-average range in cognition. This relative strength in cognitive or nonverbal reasoning ability that is seen in children with ASD may provide valuable information for families or intervention agencies struggling to meet the needs of this challenging population. M = 78) and younger age (M = 42 months) were both predictive of placement of children with ASD in a regular classroom rather than a special education classroom following a preschool program that provided treatment using intensive applied behavioral analysis (ABA) [For example, the assessment of cognitive skills by a standardized tool such as the BSID-III may assist with future prognosis. Harris and Handleman found that a higher IQ (is (ABA) . Cognitiis (ABA) . Childreis (ABA) .An additional finding in the current study was the discrepancy in the cognitive scores of children with ASD by age, with older children having lower cognitive scores than children below two years of age. One reason for the difference in cognitive scores between younger and older children with ASD may be that the cognitive measure of the BSID-III incorporates increasingly difficult verbal directions and responses for children two years of age and older. Language-based concepts, such as size and color discrimination and number concepts, are included in items on the cognitive test that are asked of older children. Children with ASD may have more difficulty providing the requisite verbal responses than their peers without ASD. As would be expected, children with ASD scored significantly lower in language skills as a group than children without ASD. Individualized assessment of language skills may assist in selecting the appropriate communication approach to use with a child. The development of a functional communication system  or augme There is a growing consensus that critical components of an effective intervention program for children with ASD include early entry into a program following diagnosis, inclusion of parent training, incorporation of a high degree of structure, implementation of strategies for generalization, and low student-to-teacher ratio including one-on-one time . StandarChildren who have difficulty approaching tasks may require the intensive behavior methods of the ABA approach . In a reEarly diagnosis and intervention services may help to ameliorate the parenting stress involved with having a child with ASD. Parents and siblings of children with ASD report experiencing more depression than those of typically developing children or even children with other disabilities . ParentiEarly identification of children with ASD will need to be addressed by a variety of health care providers, including primary care physicians, pediatricians, and public health nurses. Referral for differential diagnosis should be completed as soon as concerns are raised. Because of the ongoing needs of families with children with ASD, health care providers will need to have knowledge regarding community resources that may be able to assist the family including early intervention programs, early childhood special education programs through the local public schools, child and family counseling services, and parent support groups.The current study is limited by the retrospective nature of the data collection. The subjects were children who were referred to a diagnostic developmental clinic and therefore may not represent a broader sample of the population. The assessment of the children was limited to cognitive and language domains of the BSID-III so that other areas of development were not included that may have been useful for instructional or diagnostic purposes. The children were seen only once by the educator so that longitudinal information was not available. The children were young and may have significantly different developmental profiles in the future due to maturation or intervention. It should be noted that the educator conducting the evaluations is also one of the authors of this study.The current study found that young children being screened for possible ASD were able to be evaluated using a standardized measure such as the BSID-III. As observed in other studies, males were much more likely to be diagnosed with ASD than females. Children who were over two years of age were more likely to be diagnosed with ASD and demonstrated lower cognitive scores than younger children or children without ASD. While the cognitive skills of children with ASD were slightly lower than children without ASD, specific abilities were observed that could be used to match a child's skills with treatment options. Language skills were significantly more impaired and predictive of a diagnosis of ASD, but children without ASD also exhibited a high rate of language delay and would benefit from early intervention services. The developmental profiles from standardized assessments may be used for referral to appropriate educational programs and provide valuable information for intervention strategies. While all children with language delays will benefit from referral to services, children with ASD will require more intensive services tailored to their specific strengths and challenges.


CD36) variants with polypoidal choroidal vasculopathy (PCV) and compare them with those in typical neovascular age-related macular degeneration (tAMD).To clarify the association of cluster of differentiation 36  and 198 age-matched controls. Four tag single-nucleotide polymorphisms (SNPs)—rs10499862, rs3173798, rs3211883, and rs3173800—in the Although none of the SNPs tested were associated with PCV, the allelic frequencies of rs3173798 and rs3173800 were significantly different between PCV and tAMD patients. Genotype association analysis demonstrated different associations of these two SNPs between PCV and tAMD in the genotype model. Haplotype analysis revealed that the association of the major haplotype (T-T-T-T) at four selected SNPs in CD36 differed significantly between PCV and tAMD patients.CD36 region may be associated with the difference in genetic susceptibility for PCV and tAMD.The  Age-related macular degeneration (AMD) is a leading cause of central vision loss in the elderly in industrialized countries . AlthougCD36 region with the incidence of neovascular AMD (corresponding to tAMD in the present report) in the Japanese population ).None of the SNPs reported in the present study showed any significant deviations from the Hardy–Weinberg equilibrium over the entire sample (p>0.05). nt model . In addiand tAMD . There wCD36 region, and found that the minor allelic frequencies at SNPs rs3173798 and rs3173800 and the haplotype at four selected SNPs in the CD36 region were significantly associated with the difference in genetic susceptibility to PCV and tAMD. Namely, T, T alleles were less frequent than C, A alleles in PCV than in tAMD patients at rs3173798 and rs3173800, respectively. Moreover, the T-T-T-T haplotype at rs10499862, rs3173798, rs3211883, and rs3173800 on introns 3 and 4 in the CD36 region was significantly less frequent in PCV than in tAMD.We genotyped four tag SNPs in the CD36 region with neovascular AMD [CD36 in genetic susceptibility for PCV and tAMD, which may contribute to the different clinical characteristics of PCV  from tAMD [rs10499862, rs3173798, rs3211883, and rs3173800 were not covered by the gene chips used in previous genome-wide association studies [CD36 with the incidence of tAMD. However, these SNPs did not remain significant in the prevalence of PCV. Moreover, a statistically significant difference was detected in the association of this region between tAMD and PCV. This suggests a different association of the CD36 region with the phenotype of neovascular AMD, although the details have not yet been clarified. A recent in vivo study demonstrated that a downregulation of CD36 in capillary sprout endothelial cells facilitated angiogenesis [CD36 have been found to be more susceptible to light-induced retinal damage, and are more likely to develop age-related retinal degeneration and choriocapillary rarefaction [Although our previous report demonstrated an association of SNPs in the ular AMD , the pherom tAMD ,6-9. Sin studies ,29, they studies , furtherogenesis . Rats caefaction -22. In pefaction  and in tefaction . A recenefaction . An immuefaction . Moreoveefaction .CD36 is currently unknown, because the haplotype in the present study does not reside in the coding sequence of CD36. FASTSNP [rs3173798 is located at splicing site with medium-high effect, but it was not shown whether it is located at splice donor site or acceptor site [rs3173798 tended to increase CD36 expression while reducing high-density lipoprotein levels [rs3173798 was less frequent in the tAMD group than the PCV and control groups, the reduced expression of CD36 might be correlated with tAMD but not PCV pathogenesis. Moreover, Picard et al. have recently demonstrated an accumulation of oxLDL in Bruch’s membrane among aged CD36 knockout mice [CD36 variants with tAMD and PCV.The biologic basis of the association with the haplotype in  FASTSNP  gave thetor site . Thus, tn levels ,42. Sincout mice . The oxLout mice . HoweverCD36 variants may indirectly contribute to AMD via abnormal lipid metabolism. However, the results of this study suggest a possible role of lipid metabolism in the different pathogeneses between PCV and tAMD.The limitation of this study was a possible influence of lipid metabolism on AMD pathogenesis ,46. SincIn conclusion, the present study suggested some clinical possibilities for genetic association analysis that can be further investigated to determine the specific pathogenesis of PCV as distinct from that of tAMD.


Salvia miltiorrhiza radix, has been reported to have antioxidant effects. We examined the effects of LAB on the prevention of diabetic retinopathy in Otsuka Long-Evans Tokushima Fatty (OLETF) rats, an animal model of type 2 diabetes.Lithospermic acid B (LAB), an active component isolated from LAB (10 or 20 mg/kg) or normal saline were given orally once daily to 24-week-old male OLETF rats for 52 weeks. At the end of treatment, fundoscopic findings, vascular endothelial growth factor (VEGF) expression in the eyeball, VEGF levels in the ocular fluid, and any structural abnormalities in the retina were assessed. Glucose metabolism, serum levels of high-sensitivity C-reactive protein (hsCRP), monocyte chemotactic protein-1 (MCP1), and tumor necrosis factor-alpha (TNFα) and urinary 8-hydroxy-2′-deoxyguanosine (8-OHdG) levels were also measured. Treatment with LAB prevented vascular leakage and basement membrane thickening in retinal capillaries in a dose-dependent manner. Insulin resistance and glucose intolerance were significantly improved by LAB treatment. The levels of serum hsCRP, MCP1, TNFα, and urinary 8-OHdG were lower in the LAB-treated OLETF rats than in the controls.Treatment with LAB had a preventive effect on the development of diabetic retinopathy in this animal model, probably because of its antioxidative effects and anti-inflammatory effects. However, the causes of these problems and therapeutic options have yet not been fully uncovered. Among the side effects, diabetic retinopathy is the most common microvascular complication of individuals with both type 1 and type 2 diabetes, with nearly all patients with type 1 diabetes and more than 60% with type 2 diabetes being estimated to develop diabetic retinopathy 20While laser treatment at best permits preservation of visual acuity in cases of diabetic retinopathy, many agents such as fenofibrate, ruboxistaurin, renin-angiotensin system blockers, and peroxisome proliferator-activated receptor gamma agonists have been used in attempts to delay or prevent diabetic retinopathy It is currently believed that retinopathy is an inflammatory process, because various studies have shown that the circulating leukocytes activated in diabetic patients play an important role in the increased adhesion of leukocytes, macrophages and vascular adhesion molecules, and in the process of vascular occlusion and retinal ischemia Today, antioxidants are known to be effective against microvascular complication such as diabetic nephropathy as well as macrovascular complications of diabetes such as myocardial infarction, atherosclerosis and stroke Salvia miltiorrhiza radix is a traditional Chinese herbal medicine that has been used for many years in East Asia for the treatment of diabetic complications. Lithospermic acid B (LAB), a recently isolated component of S. miltiorrhiza, is known to have multiple pharmacological actions such as antihypertensive in vivo and platelet-derived growth factor-induced vascular smooth muscle cell proliferation and migration in vitroTreatment with LAB showed protective effects against micro- and macrovascular complications of diabetes Reactive oxygen species have been reported to be associated with microvascular complications in patients with diabetes weeks of age, when obesity and insulin resistance develop. The OLETF rats were held in the Preclinical Laboratory of Seoul National University Bundang Hospital, South Korea, for the duration of the study.All animals were handled in compliance with the Guide for Experimental Animal Research of the Laboratory, Seoul National University Bundang Hospital. Seoul National University Hospital Ethics Committee for Animal Study approved this study (63-2010-020). Thirty five-week-old male Otsuka Long-Evans Tokushima Fatty (OLETF) rats, an obese animal model of insulin resistance, were donated by the Otsuka Pharmaceutical Co. . They were allowed to grow to 24mL normal saline per day and the experimental rats were given 10mg/kg or 20mg/kg of LAB per day. Rats were administered with LAB or normal saline using an oral Zonde needle  at 9–10 am for 52weeks. All rats were fed a regular chow diet and had free access to water; they were maintained in plastic cages in an air-conditioned room at 22±2°C and 55±10% humidity.We divided the rats into three groups (n = 10 each). Controls received 5Salviae miltiorrhizae Radix, and subsequently purified by normal silica gel column chromatography with polar-eluent  to give a 1.01% yield. Acidification of magnesium lithospermate B with 0.5N-HCl afforded lithospermic acid B at an 84% yield Lithospermate B was isolated from 80% MeOH extract of weeks. All retinal photographs and fluorescein angiography images were independently reviewed twice by one ophthalmologist (SJW), and the retinal vascular caliber was measured in each blood vessel 1mm distant from the optic disc and averaged for each eye.Fundus photography on both eyes of each animal was performed by an experienced technician using a 45° fundus camera  at week 0, 12, 24, 36, and 52 of the dosing phase. Fundus fluorescein angiography was performed on both eyes at 52Eyeballs were enucleated from rats (both control and LAB groups) after euthanasia with a lethal dose of pentobarbital and fixed in 10% neutral buffered formalin solution. They were embedded in paraffin wax. Sections (4 µm thickness) perpendicular to the optic disc were stained with hematoxylin and eosin (H&E) for light microscopy. Digital images were obtained using a high-resolution digital camera system  linked to a microscope  and desktop computer. For all eyes, retinal images were obtained at the same distance (1500 µm) from the optic nerve.h at room temperature. Slides were washed with PBS.Sections were deparaffinized in xylene, rehydrated in a graded ethanol series, and stained using a BenchMark XT automated immunostainer . Immunohistochemistry was performed using an anti-VEGF antibody . Sections were washed with phosphate buffered saline (PBS) and incubated with a secondary antibody , prepared in PBS containing 1% horse serum, 1% bovine serum albumin and 0.3% Triton X-100, for 1mL syringes and 30 gauge needles. Samples were stored at −70°C until assayed. The VEGF concentration was measured with a rat VEGF-specific polyclonal antibody using a commercial enzyme-linked immunosorbent assay (ELISA) . The detection limit of the method was 31.2–2000 pg/mL, based on a standard curve from the manufacturers. All measurements were carried out in duplicate. The coefficients of variation in intra- and interassay variations were 7.2% (79.4±5.7 pg/mL) and 9.4% (87.6±8.2 pg/mL), respectively.Immediately after the rats were euthanized, the VEGF concentration was measured in ocular fluid–a mixture of the aqueous humor and vitreous fluid–by puncturing the ocular globe using disposable 1M cacodylate buffer (pH7.4) for 1h, and then postfixed in 2% osmium tetroxide. They were then dehydrated in ethanol series, and embedded in epoxy resin. Semithin sections (1 µm) were stained with toluidine blue for orientation and identification of the capillaries. Thin sections (60nm) were cut with a diamond knife, placed on 300-mesh copper grids, and stained with uranyl acetate and lead citrate. The sections were viewed and photographed using a JEOL JEM-100SX transmission electron microscope . Each micrograph was analyzed using a commercial image analysis program . Basement membrane thickness was measured on four distinct capillaries for each eye and five measurements were taken per capillary with 10 independent measurements from each eye. One researcher (SJW) examined all eyes in the study and was blinded to the treatment groups.Electron microscopy was performed to determine the retinal basement membrane thickness. The eyes were enucleated, opened at the equator, fixed in 3.5% glutaraldehyde in 0.1mM Tris-HCl pH7.6, 150mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS), and the proteins (10mg/sample) were immediately heated at 100°C for 3min. Total cell lysates were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis on gels containing 15% (wt/vol) acrylamide with each lane receiving 20 µg of the sample. After electrophoresis, proteins were transferred onto a nitrocellulose membrane for 2h. The membrane was blocked by treatment with 5% skim milk in Tris-buffered saline (TBS) supplemented with 0.1% Tween-20 (TBST) for 1h. It was then incubated with a primary polyclonal antibody  at a final dilution of 1:1000, overnight in TBST. After three washes in TBST, the membrane was incubated with peroxidase-conjugated secondary antibody  in TBS for 1h and subsequently washed. An enhanced chemiluminescence reaction agent was reacted with the membrane for 3min. Band density was quantified using a densitometer .Tissue samples were homogenized in lysis buffer, and the protein concentration was determined using a protein assay kit . Extracted retinal tissues were lysed in RIPA buffer (252  = 0.994) over the concentration range of 1.0–1,000 µg/mL. The intra- and interassay coefficients of variation were 6.2% and 2.7%, respectively. The mean serum concentrations of LAB were 12.3±2.4 and 17.1±3.1 µg/mL in the rats treated with 10 and 20mg/kg of LAB, respectively. LAB was not detected in the control rats.Levels of LAB were measured using a liquid chromatography-tandem mass spectrometry (LC-MS/MS) system from plasma samples collected at the end of the study. The standard curve was linear  was done at baseline and after 24 and 52weeks of LAB treatment. After the 12h fasting glucose concentration had been measured, each animal was injected intraperitoneally with 1.5 g/kg of a 50M glucose solution. Blood samples (about 10 µL) were collected from an incision in the tail at 30, 60, 90 and 120min after the glucose load. Plasma glucose concentration was measured using reagent strips read in a glucose meter . Insulin was measured in duplicate using a commercial liquid phase radioimmunoassay kit . The homeostasis model assessment of the insulin resistance (HOMA-IR) was calculated using fasting insulin and glucose levels. In addition, the area under the curve for glucose (AUCglucose) was calculated using the trapezoid rule for glucose data from 0 to 120min. High sensitivity C-reactive protein (hsCRP) was measured using ELISA kits developed by BD Biosciences Pharmingen . Monocyte chemotactic protein-1 (MCP1), tumor necrosis factor-alpha (TNFα), and plasminogen activator inhibitor-1 (PAI1) activity were also measured using a multiplex kit .Possible relevant factors affecting the degree of diabetic retinopathy, such as glucose homeostasis, cytokines, and inflammatory status were evaluated. For glucose homeostasis, postprandial glucose concentrations were measured 2weeks of treatment of LAB or control, urine samples were collected directly from the urinary bladder with a 10mL syringe on the day of euthanasia after fasting the rats overnight. The oxidative DNA damage marker, 8-OHdG was measured in the urine samples using an established high performance liquid chromatographic method based on anion exchange chromatography, fraction collection, and electrochemical detection After 52post hoc test, and P values <0.05 were considered statistically significant. Analysis was done using SPSS for Windows .Results are reported as the mean ± standard error (SE). Mean values were compared for the LAB-treated groups and control group by analysis of variance (ANOVA) with Tukey's b mg/kg LAB group, 53.2±8.8 µm in the 20mg/kg LAB group . Fluorescein angiography allowed more precise quantitative analysis of diabetes-induced microvascular dysfunction. At the end of treatment, the diabetic rats (control group) showed multiple diffuse leakages from retinal capillaries in the late phase of angiography . The high dose (20mg/kg) LAB-treated group showed lower VEGF expression than did the low dose group (P<0.05).Immunohistochemical staining showed greater VEGF expression in the control diabetic rats than in the LAB-treated rats Fig. 4. mg/kg LAB-treated, and five 20mg/kg LAB-treated rats. The concentration of VEGF in the high dose treated group was significantly lower than in control rats .Ocular fluid was obtained from five control, six 10rol rats Fig. 5mg/kg LAB group, and 0.09±0.02 µm in the 20mg/kg LAB group, P<0.05 among groups).Retinal capillary basement membrane thickness was assessed by electron microscopy. For each animal, a minimum of 40 separate measurements of the basement membrane thickness was obtained. A representative micrograph of the basement membrane from each of the three groups is shown in h glucose concentrations from weeks 24 to 50  revealed that control rats had higher glucose concentrations at weeks 46 and 48 than did the LAB-treated rats. There was no difference in glucose excursion for the IPGTTs performed at week 24 among the three groups . After 52weeks of LAB treatment, the 90min and 2h postload glucose concentrations and the AUCglucose during the IPGTT decreased significantly compared with that of the controls .Postprandial 2mg/kg LAB group was not significant. LAB treatment also reduced the level of 8-OHdG, an oxidative stress marker, in a dose-dependent manner  for 52weeks alleviated the development of diabetic retinopathy. Systemic LAB treatment prevented vascular leaking, reduced the expression and concentration of VEGF, and attenuated the disruption of the neurovascular units in the retina. The antioxidative and anti-inflammatory properties of LAB seem to account for this protective role. A significant improvement in postload glucose excursion with LAB treatment can be considered to contribute to this beneficial effect on the development of retinopathy.In this OLETF rat model of type 2 diabetes, treatments with LAB Click here for additional data file.File S1Supporting Figures.Figure a, Fundoscopic findings in 76-week-old OLETF rats after 52weeks of treatment with LAB. A: Control group. B: 10mg/kg LAB group. C: 20mg/kg LAB group. Figure b, Postprandial 2h glucose concentrations from weeks 26 to 50 in the LAB-treated and control rats. Figure c, Glucose concentrations from the intraperitoneal glucose tolerance test (IPGTT) at week 24 in the LAB-treated and control rats. Figure d, Glucose concentrations from the intraperitoneal glucose tolerance test (IPGTT) at week 52 in the LAB-treated and control rats.(DOCX)Click here for additional data file.


Magnetic nanostructures of two ferromagnetic metals have been combined within porous silicon, and the magnetic switching behavior of the resulting porous silicon/metal nanocomposite has been modified by varying the arrangement. The two magnetic materials are Ni and Co, whereas Co is the magnetic harder one. These “hard/soft” magnetic nanocomposites have been achieved by two different routes. On the one hand, double-sided porous silicon has been used whereas one side has been filled with Ni nanostructures and the other one with Co nanostructures. On the other hand, Ni and Co have been deposited within one porous layer alternatingly. The filling of the pores has been carried out by electrodeposition with varying the deposition parameters. In systems which offer two distinct slopes of the hysteresis curves due to the different saturation behavior of the two types of deposited metal, magnetic exchange coupling is not present. For samples which show smooth hysteresis curves exchange, coupling between the Ni and Co nanostructures seems to be present. The aim is to control especially the structure size of the soft and the hard magnetic materials and the distance between them at the nanoscale to optimize exchange coupling resulting in a maximum energy product. The utilization of low-dimensional structures becomes more and more important due to the miniaturization of devices and also due to the novel arising nanoscopic properties. The fabrication of such nanostructures is often carried out by nanopatterning using lithographic methods. On the other hand, self-assembling techniques are of great interest due to the uncomplex and low-cost fabrication process. Quite common are nanoparticles grown on a substrate by self-organization. Also, three-dimensional arrays of nanostructures  have been formed without prestructuring in using hexagonal arranged porous alumina as matrices . Also, qConsidering the achieved nanocomposites, magnetization reversal processes with the concomitant domain wall motion within the deposited metal nanostructures, the interactions among them, and also transport phenomena like magnetoresistance in spin valves are of great interest. Magnetic materials in the nanometer scale exhibit changed properties compared to bulk material and therefore offer great potential for novel nanotechnological applications. The nanoscopic systems consist either of particles or wires with magnetic properties dependent on their geometry and arrangement. For technical application of the system, the magnetic nanostructures should be ferromagnetic at room temperature. In some cases, a high anisotropy between the two magnetization directions, perpendicular and parallel to the surface, is of interest and thus needle-like structures are favorable due to their high demagnetizing field. One method to achieve low-dimensional structures is the deposition of metal nanostructures on patterned surfaces or into porous membranes with channels perpendicular to the surface, and therefore, the metal structures exhibit a high density with respect to the sample surface. Templates like porous alumina or polycarbonate foils are usually electrochemically fabricated and afterwards filled with a magnetic material by electrochemical deposition. In commercial microelectronics, most devices are based on silicon technology and thus for process compatibility, a silicon substrate is a good precondition for applicability.In the present work, a combined porous silicon/“hard-soft” magnetic nanocomposite is investigated and the results concerning the magnetic properties with respect to the switching behavior are figured out.+ silicon wafer. On the one hand, a silicon wafer has been porosified on one side with an average thickness of the porous layer of 40 μm. On the other hand, ultrathin wafers have been etched on both sides (front and back) in using an electrolytic double tank cell. The thickness of the porous layers was around 20 μm on each side. As anodization electrolyte, an 10 wt% aqueous hydrofluoric acid solution has been used. To achieve pore diameters of about 60 nm, a current density of 100 mA/cm2 has been applied. The etching rate was around 4 μm/min.Two kinds of porous silicon templates have been fabricated by anodization of an n2 with 0.05 Hz and for the Co deposition, a current density of 20 mA/cm2 with 0.1 Hz has been applied. In the case of the one-sided sample, both metals have been electrochemically deposited alternately inside the pores resulting in a stacked arrangement of Co and Ni nanostructures. As electrolytes, a Ni and Co metal salt solution  has been used. The deposition time for each metal was up to 10 min. Magnetic characterization of the samples has been performed by SQUID (superconducting quantum interference device) magnetometry .The prepared porous silicon templates offer a morphology in the mesoporous regime with a quasi-regular pore arrangement. Within the pores of these templates, nanostructures consisting of two different magnetic materials have been incorporated by electrodeposition. The double-sided porous silicon has been filled with Ni on one side and with Co on the other side, also using a double tank cell. Depending on the polarity of the applied current, either Ni on the one side or Co on the other side is deposited. For the Ni deposition, a current density of 25 mA/cmThe deposited magnetic nanostructures consist of Co and Ni, whereas Co is the magnetic “harder” material. Considering the magnetization measurements of double-sided porous silicon containing Co nanostructures on one side and Ni nanostructures on the other side, one sees that the magnetic behavior is composed of two terms. The first one is due to the softer magnetic material, Ni, which exhibits a saturation magnetization of 0.6 T (bulk Ni). The second one is due to the harder magnetic material, Co, which exhibits a saturation magnetization of 1.7 T (bulk Co). Thus, the hysteresis curves of the investigated nanocomposite systems show two different slopes corresponding to the two different materials. Figure HC of the nanocomposite depends mainly on the size and shape of the deposited metal structures. In the case of a dense spatial distribution with magnetically coupled deposits, additionally, the interaction influences HC. The magnetic anisotropy is mainly due to shape anisotropy and increases with the elongation of the deposited metal structures. However, the deposition of densely packed structures results in a wire-like behavior caused by magnetic coupling between them. Generally, the coercivity decreases with the elongation of the metal deposits. In Table MR/saturation magnetization MS) of Ni deposited within porous silicon is given. Considering double-sided samples containing two metals, the magnetic characteristics are a combination of both metals. Considering single-sided samples consisting of stacked Co/Ni nanostructures, the magnetic behavior depends on the kind of deposition. In the case of using one combined electrolyte bath, consisting of the Ni- and the Co-salt, different voltages have been applied for the two metals. The samples show also a clear kink in the hysteresis curve demonstrating that exchange coupling between the two materials is excluded which can be ascribed to a distance greater than 2 nm between the different nanostructures or an oxide formation separating them. Details of this circumstance are currently under investigation. An example of such a sample with combined filling can be seen in Fig. In the case of double-sided samples filled with Ni and Co, magnetic interactions can only occur between nanostructures within one layer. The distance between the two porous layers is in the range of 30 μm which makes dipolar coupling negligible. For non-interacting deposits, the coercivity Figure 4 solution), field-dependent magnetization shows a smooth hysteresis which indicates the presence of exchange coupling between the Ni and Co deposits  can be modified by varying the size and shape of the Ni and Co deposits. Therefore, the magnetic characteristics of the specimens can be broadly tailored, as desired. The magnetic properties of the resulting nanocomposites are not only correlated with the size, shape, and spatial distribution of the deposited metal structures but also strongly depend on the filling ratio between the “softer” (Ni) and “harder” (Co) magnetic materials as well as on the way how the electrodeposition is performed (from a combined or separate solutions).The presented “hard/soft” magnetic nanocomposite is fabricated during a low-cost two-step electrochemical process whereas double-sided as well as one-sided porous silicon acts as template. Considering the magnetic behavior of double-sided systems and single-sided systems gained from a combined electrolyte solution, two characteristic terms are observed. The first one is caused by the magnetic properties of the “softer” magnetic metal (Ni) and the second one is caused by the higher saturation magnetization of the deposited “harder” magnetic nanostructures (Co). In the case of depositing Co and Ni alternatingly from a Watts-solution and CoSO


We show that FoxP3hiCD45RA−CD4+ Treg cells [activated Treg (aTreg) cells] are the predominant cell population among tumor-infiltrating FoxP3+ T cells, and that high aTreg cell-infiltrating content is associated with reduced survival in patients with head and neck squamous cell carcinoma (HNSCC). In vitro studies have demonstrated that aTreg cells can suppress tumor-associated antigen (TAA) effector T cell immune responses in HNSCC. Moreover, C-C chemokine receptor 4 (CCR4) was specifically expressed by aTreg cells in the peripheral blood of HNSCC patients. Using a RayBiotech human chemokine antibody array, we showed that monocyte chemoattractant protein-1 (MCP-1), an endogenous CCR4-binding ligand, was specifically upregulated in the HNSCC microenvironment compared to the other four CCR4-binding ligands. Blocking MCP-1/CCR4 signaling-induced aTreg cell recruitment using a CCR4 antagonist evoked antitumor immunity in mice, and lead to inhibition of tumor growth and prolonged survival. Therefore, blocking aTreg cell trafficking in tumors using CCR4-binding agents may be an effective immunotherapy for HNSCC.FoxP3 In addition, both a monoclonal antibody against human MCP-1 and a CCR4 antagonist significantly blocked tumor extract-induced aTreg cell migration (P < 0.01 for each). In support of these results, recombinant MCP-1 induced significant aTreg cell migration (P < 0.01 versus control) that was efficiently blocked by the anti-MCP-1 antibody and CCR4 antagonist (P < 0.01 for each) . After administration of a CCR4 antagonist, we observed inhibition of SCC-VII tumor growth at day 33 , suggesting that the CCR4 antagonist inhibited aTreg cell migration in vivo   Figure .+ Treg cells can suppress tumor-specific immunity, which provides a clear rationale for development of immunotherapies that can modulate the influence of these regulatory cells [+ T cell subsets in HNSCC tissues. Intriguingly, our data showed that the majority of tumor-infiltrating FoxP3+ T cells were aTreg cells. Although there is a large amount of evidence that FoxP3+ T cells predominantly infiltrate tumor tissues [+ T cells have not been characterized. Our finding of predominant aTreg cell infiltration revealed the phenotypes of such cells. Additionally, aTreg cell numbers were significantly higher in late-stage (III and IV) HNSCC tumor tissue than in early-stage tissue (I and II), and were inversely correlated with patient prognosis. Given the clinical importance of tumor-infiltrating aTreg cells, inhibition of aTreg cell recruitment in HNSCC may be an effective immunotherapeutic strategy.Accumulating evidence indicates that FoxP3ry cells , 28. Her tissues , 29, 30,in vitro. These findings provide a rationale for the development of immunotherapies to control the effects of aTreg cells in HNSCC.We recently confirmed that aTreg cells suppress proliferation of non-TAA T cells , 25. How+/−CD4+ T cell subsets and other types of immune cells. This result was consistent with a previous study, which showed that CCR4 was predominantly expressed in peripheral blood aTreg cells from melanoma patients [Since chemokine receptors have central roles in the recruitment of immunoregulatory cells , 32, 33,patients . These d+ Treg cells express CCR4 and migrate in response to TARC and MDC [Circulating FoxP3 and MDC –38. Howe and MDC –41. Howein vivo studies showed that inhibition of tumor growth and prolonged mouse survival could be attributed to inhibition of aTreg cell recruitment resulting in expansion of tumor infiltrating CD4+ and CD8+ T cells. These data demonstrate that aTreg cells are involved in the progression of HNSCC via their suppressive effects on TAA immune responses. Therefore, therapeutics that bind aTreg cells could be an effective immunotherapy for HNSCC. Several recent clinical trials have shown that depletion of CCR4-expressing FoxP3+CD4+ Treg cells by anti-human CCR4 monoclonal antibody is a promising approach to augment antitumor immune responses in cancer patients [Our patients , 43. TheIn conclusion, our data suggest that a combination of a CCR4-binding therapeutic that can block aTreg cell trafficking in tumors, tumor antigen immunization, and monoclonal antibodies may enhance the clinical efficacy of immunotherapies for HNSCC.PBMCs were obtained from healthy donors and HNSCC patients who had not received any previous oncological treatments. Tumor- and nontumor-infiltrating lymphocytes were obtained from tumor and adjacent nontumor tissues after surgical debulking. Informed consent was obtained from all participants prior to enrollment in the study. The study was approved by the Ethics Committee of The First Affiliated Hospital of Sun Yat-sen University, Guangzhou, China .Infiltrating lymphocyte suspensions were prepared by enzymatic digestion . DissectThe SNU899 LSCC cell line was provided by Professor Ja-Lok Ku . The mouse SCC VII OSCC cell line was provided by Professor Si-Xi Liu . Cells were cultured under standard conditions.Eight-week old male C3H-HeN mice were purchased from Slac Laboratory Animal Co., Ltd. . All mice were housed under specific pathogen-free conditions. Experimental procedures were approved by the Institutional Animal Studies Committee and conducted in accordance with Institutional Animal Care and Use Committee guidelines.+ cells were isolated from PBMCs using CD14 Microbeads . The cells were then seeded into a 6-well plate at a density of 1.5 × 106 cells/well and cultured as described previously [CD14eviously . On day After stimulation for 5 h with a cocktail of phorbol 12-myristate 13-acetate, ionomycin, and Golgi stop (brefeldin A and monensin) (eBioscience), cells were stained for cell surface markers and intracellular cytokines  using anti-hTNF-α-Alexa Fluor 700, anti-hIL-2-PE-Cy7, and anti-hIFN-γ-APC-eFluor 780 antibodies as well as the Fixation/Permeabilization Buffer and Permeabilization Buffer from eBioscience.+ and CD8+ T cell proliferation and cytokine production of CD4+ T cells were assessed by flow cytometry using anti-hCD3-eFluor 450, anti-hCD4-FITC, anti-hCD8a-PE-Cy7, anti-hIFN-γ-APC-eFluor780, anti-hIL-2-PE-Cy7, and anti-hTNF-α-Alexa Fluor 700 antibodies (eBioscience). SNU899-derived, lysate-pulsed immature DCs were labeled with 5 μM 5,6-carboxyfluorescein diacetate succinimidyl ester (eBioscience) and mixed with T cells that had been cultured for 13 days at a ratio of 1:10 in 96-well microplates for 4 h in a 5% CO2 atmosphere at 37°C. Cytotoxicity was assessed by flow cytometry using APC-annexin V and 7-amino-actinomycin D (7-AAD) (BD Biosciences).Antigen-loaded DCs were added to autologous T cells as stimulators at a ratio of 1:20 in a round bottom 96-well microplate. The aTreg cells were prepared as described previously , 25 and +CD4+ T cell subsets, T cells obtained from tumor tissues, adjacent non-tumor tissues, and blood were stained with anti-hCD3-eFluor 605NC, anti-hCD4-FITC, anti-hCD25-APC, anti-hCD45RA-eFluor 450, and anti-hFoxP3-PE antibodies (eBioscience).To identify FoxP3th edition of the Union for International Cancer Control (UICC 2008) tumor-node-metastasis classification of malignant tumors. Detection of FoxP3  and CD25  was performed on 4 μm-thick, paraffin-embedded sections of tissue samples with the indicated antibodies using a double staining kit . For chemokine receptor ligand staining, sections of tissue samples were stained with anti-MCP-1, -MDC, or -TARC antibodies using an ABC kit .The main clinical and pathological characteristics of LSCC patients are presented in The expression of CCR2, CCR4, CCR5, CCR6, CCR7, and CXCR4 on lymphocytes was analyzed by flow cytometry using anti-hCD3-eFluor 605NC or anti-hCD3-eFluor 450, anti-hCD4-FITC, anti-hCD8-PE-Cy7, anti-hCD25-APC, anti-hCD45RA-eFluor 450, anti-hFoxP3-PE, anti-hCD14-FITC, anti-hCD19-PE-Cy7, anti-hCD11c-Alexa Fluor 700, anti-hCD16-PE, anti-hCD56-APC, anti-hCCR6-PerCP-eFluor710, anti-hCCR7-APC-eFluor780, and anti-hCXCR4-PE-Cy7 antibodies (eBioscience). The anti-hCCR2-Alexa Fluor 647, anti-hCCR4-PerCP-Cy5.5, and anti-hCCR5-APC-Cy7 antibodies were purchased from BD Pharmingen .A Human Chemokine Antibody Array  was used according to the manufacturer's instructions. After development, films were scanned and the images processed and quantified using the National Institutes of Health ImageJ software. The intensities were normalized to internal positive controls for comparison.4 aTreg cells. Human recombinant MCP-1 , a human anti-MCP-1 antibody , or tumor tissue extract were added to the lower chamber. The aTreg cells were preincubated with a CCR4 antagonist  for 30 min at 37°C. Migrated cells were counted using an automated cell counter .Migration was assessed as described previously  using 5 5 tumor cells were injected subcutaneously into the right flank. Either a CCR4 antagonist (87.5 μg) or saline were injected intraperitoneally three times per week for 3 weeks from day 5 after tumor cell injection. In parallel, a group of six mice was used as a blank control. Tumor size was measured every 3 days using fine calipers. Tumor volumes were calculated as length × (width)2 × 0.52. All mice were sacrificed 7 days after the last injection.Eighteen C3H-HeN mice were randomly and equally divided into three groups and ear tagged prior to treatment. On day 0, 1 × 10After sacrifice, tumors were removed and single cell suspensions were prepared by enzymatic digestion. The types of tumor infiltrating cells were analyzed by flow cytometry using anti-mouse CD3-FITC, anti-mouse CD4-eFluor 450, anti-mouse CD8-Alexa Fluor 700, anti-mouse CD25-APC, anti-mouse IL-2-PE, anti-mouse IFN-γ-PE-Cy-5, anti-mouse TNF-α-PE-Cy7, anti-mouse FoxP3-PE, and anti-mouse CD45RA-FITC antibodies , and a fixation/permeabilization kit. All antibodies were purchased from eBioscience unless otherwise specified. An additional three randomly divided groups of mice were used for survival analysis. The observation period was 50 days.U-tests, Student's t-tests, or Kruskal-Wallis tests. Comparisons of aTreg cell infiltration levels between groups were performed using the Wilcoxon matched-pairs signed-ranks test. Survival variables were estimated using the Kaplan-Meier method and compared using log-rank tests. Multivariate analysis using the Cox proportional hazard model was used to determine the influence of each variable, when adjusted to the others, on overall survival. A P-value of less than 0.05 was considered statistically significant.Statistical analysis was performed with SPSS Standard version 13.0 software . Differences between groups were assessed using Mann-Whitney


Then we evaluated their anticancer activities in vitro and in vivo by utilizing human lung cancer cell line A549. The in vitro results showed that among the compounds, 3d performed effectively anti-growth activity by inducing A549 lung cancer cell apoptosis and activating Nrf-2/HO-1 (heme oxygenase-1) pathway. In vivo, we proved that compound 3d inhibited the tumor growth effectively through inducing cell apoptosis without affecting CAM normal angiogenesis. These data suggest that our discovery of a novel Nrf-2 activator compound 3d would provide a new point of human lung cancer treatment.Increasing evidence indicates that Nrf-2, named the nuclear factor-erythroid 2-related factor, may perform anticancer function. In this study, a series of novel substituted phenyl- (3-methyl-1H-indol-2-yl)-prop-2-en-1-one  derivatives were synthesized and their effects on Nrf-2 activity were observed. We found that compounds 3a Therefore, much more attention has been paid to the discovery of new anticancer drugs2.Lung cancer is a considerable worldwide public health concern. Comparing with the survival rates of other cancers, 5-years survival rate of lung cancer is lower3. It has been reported that cell death plays a crucial role in the progress of cancer. As we know, apoptosis and programmed necrosis are two major types of cell death which show different cell morphologies and pathways. Furthermore, as a new alternative target in tumor treatment, autophagy needs us to pay more attention4. Discovering new agents in anticancer therapy by inducing different cell death types is meaningful.Cytotoxicity induced by xenobiotic can cause cell death5. There are a lot of chalcone derivatives that have been synthesized and identified by researchers in the laboratory through different chemical methods6. Many reports have displayed that minor structural transformation of chalcones could induce considerable difference in the effect of anticancer, anti-inflammatory or autoimmune diseases7. For example, compound II2, a novel dithiocarbamate–chalcone derivative could apparently inhibit the growth of SK-N-SH cells by triggering apoptosis and blocking the cell cycle8. Chalcones could prevent cancer by inhibiting p53 degradation9. In human breast cancer, Cathepsin-K contributes to tumor spread. Chalcones agents can suppress Cathepsin-K enzyme activity and effectively inhibit tumor invasiveness in body10. As we know that PI3K/Akt/mTOR pathway which modulates cell proliferation, metabolism, apoptosis, autophagy and other cellcular biological activities is important in tumorigenesis12. A novel quinazolinone chalcone derivative (QC) has been reported to inhibit PI3K/Akt/mTOR signaling pathway and trigger human HCT-116 cells apoptosis13. A pyrrole derivative of chalcone, (E)- 3-phenyl-1-(2-pyrrolyl)-2-propenone (PPP) performs anti-inflammatory effect through inhibiting the activity of Syk, Src, and TAK114. It has been also reported that the dysfunction or abnormal proliferation of immune cells may cause autoimmune diseases, atherosclerosis, and tuberculosis16. Chalcones as immunomodulatory drugs show different effects on various immune cells, including triggering apoptosis in dendritic cells17, inhibiting superoxide anion production by weakening the activity of PKC in PMA-induced rat neutrophils18, performing anti-inflammatory potential in monocytes and macrophages19, suppressing rabbit platelets aggregation caused by arachidonic acid or collagen21, inhibiting the generation of functional cytotoxic T cells from mouse spleen and so on22.Chalcones, are diffusely existing in natural plant products. It has been reported that chalcones have many pharmacological and biological activities, such as anti-oxidative, anti-cancer, anti-mutagenic, anti-inflammatory, etc. The biological activities of chalcones maybe changed through the interaction with different compounds23. Indole alkaloids with biological activity are plentiful in the nature, such as strychnine and lysergic acid diethylamide. Various indole alkaloids isloated from plants have been reported for some therapeutic value, including anticancer, the treatment of Hodgkin’s diseases24 and psychiatric disorders25, anti-inflammatory, cytotoxicity, antiviral26, being as antimicrobial agents27 and so on. Therefore, indole derivatives have attracted many researchers’ attention and a lot of indole derivatives have been synthesized or extracted from natural resources28.Indole is anther important chemical group in this series of compounds which we have synthesized. It has been reported that indole is known as its heterocyclic system, it involves in the protein synthesis in the form of tryptophan29. α-Cyano bis (indolyl) chalcone inhibits the growth of A549 lung cancer cells effectively by promoting tubulin polymerization30.It has been reported that chalcones or indoles have a lot of medicinal value in the treatment of diseases, and various chalcones or indole derivatives have been synthesized. However, to date, few indolyl-chalcone compounds have been reported. Novel indoly-chalcone derivatives (CITs) have been synthesized and identified to play a role in anti-cancer treatment through inducing cell death and inhibiting proliferation in PC3, A549, CLR2119 and PAN02 cells31. Nrf-2 maintains cellular redox balance by Kelch-like ECH-associated protein 1 (Keap1)-Nrf-2-antioxidant response element (ARE) pathway to make response to endogenous and exogenous stresses32. It has been reported that oxidative stress involves in the initiation of cancer, Nrf-2 might exert anticancer function and is implicated in chemoprevention. For example, as an Nrf-2 activator, the natural product sulforaphane (SFN) that presents in cruciferous vegetables has been researched in clinical trials of different cancers33. Moreover, a recent study shows that cisplatin as a well-known anticancer drug has been proved to induce oxidative damages and cell death in Hep-2 cells through improving the expression of Nrf-2 and HO-1. According to the current studies, intensifying Nrf-2 activity seems to be an attractive strategy in the process of cancer treatment.As we know, Nrf-2 (nuclear factor-erythroid 2-related factor) encoded by NFE2L2  gene belongs to the basic-leucine zipper (bZIP) family of transcription factors and is expressed in various tissues3d, which dramatically inhibited tumor growth in vitro and in vivo by inducing A549 lung cancer cell apoptosis and activating Nrf-2/HO-1 pathway. Additionally, compound 3d did not show any significantly autophagy or necrosis in A549 lung cancer cells. Consequently, the discovery of the novel Nrf-2 activator would provide a new point of human lung cancer therapy.Here, we synthesized a novel series of indolyl-chalcone derivatives and identified a new Nrf-2 activator named indolyl-chalcone derivative 34. Compound 3 was synthesized from compound 2. Firstly, the mixture of compound 2 (1 mmol) and NaOH (2 mmol) in ethanol (10 ml) were stirred at room temperature, and then substituted aldehyde (1.2 mmol) in ethanol (5 ml) was added dropwise. The mixture was stirred for 2–4 h. The end of reaction was detected by TLC. Then the mixture was poured into cold water and filtered. The crude product was recrystallized from ethanol to obtain compound 3 in 60-90% yield. Compounds 4 and 5 were synthesized according to previous reported method36. Compound 6 was procured by the following reactions. The mixture of substituted acetophenone (1 mmol) and NaOH (2 mmol) in ethanol (10 ml) were stirred at room temperature, and then compound 5 (1.1 mmol) in ethanol (5 ml) was added dropwise. The mixture was stirred for 2–4 h. The end of reaction was detected by TLC. Then the mixture was poured into cold water and filtered. The crude product was recrystallized from ethanol to obtain compound 6 in 60–90% yield.A series of indolyl-chalcone derivatives were designed and synthesized Fig. . Compoun33, especially in cancer treatment, we firstly analyzed endogenous Nrf-2 activity in HeLa cells which were transfected with luciferase-based Nrf-2 reporter plasmid after treatment with a series of novel substituted phenyl-(3-methyl-1H-indol-2-yl)-prop-2-en-1-one, indolyl-chalcone derivatives . The luciferase assay suggested that compounds 3a, 3b, 3c, 3d and 6c (10 μM) elevated Nrf-2 activity significantly compared with the control after treatment for 12 h or 24 h  assay suggested that compound 3d inhibited the growth of A549 lung cancer cells most efficiently  induced apoptosis of A549 lung cancer cells, we performed Hoechst 33258 staining assay. The data suggested that compound 3d (2.5 μM) could notably trigger apoptosis in A549 lung cancer cells through inducing chromatin condensation and nuclear fragmentation formation  in A549 lung cancer cells. As a major member of Poly (ADP-ribose) polymerases (Parps) family, PARP plays a crucial role in modulating DNA repair and death of cells37. Moreover, up-regulation of cleaved-PARP is one of the main characteristics of cell apoptosis38. The results showed that the level of cleaved-PARP (89 KDa) increased after treatment with compound 3d   staining assay, western blot analysis  and LDH assay. The results demonstrated that compound 3d did not cause autophagy or necrosis in A549 lung cancer cells (data not shown).To detect whether compounds 3c, ion Fig. . We furtμM) Fig. . It indi39. Cancer cells are vulnerable to high levels of reactive oxygen species (ROS)40. Evidence indicates that ROS influences proliferation and apoptosis in various cancers41. The overproduction of ROS results in oxidative stress and induces in cell apoptosis42. Therefore, we detected the level of ROS after treatment with compound 3d (2.5 μM) for 12 h and 24 h. The data demonstrated that 3d time-dependently increased the level of ROS  for 0.5, 1, 3, 6, 12, 24, 48 h. The data revealed that compound 3d elevated the expression of HO-1 remarkably in A549 lung cancer cells  has been widely used in studying tissue grafts, tumor growth, angiogenic or toxicological analysis as the chick’s immunocompetent system and the immune rejection are not fully developed44. Therefore, we investigated the effect of 3d on tumor growth and normal angiogenesis by CAM model. 5-FU was used for positive control drug. After seeding A549 lung cancer cells on the CAM surface for 2 days, the tumor tissue masses formed locally. Then from the day 3 to 8, we treated the tumor tissue masses with PBS, PBS/3d or PBS/5-FU every 2 days. The results demonstrated that 3d significantly suppressed tumor growth  for 48 h, the cell cycle of A549 lung cancer cells has not been arrested  -prop-2-en-1-one compounds, we observed a dose-dependent and time-dependent inhibition of growth in A549 lung cancer cells. Among the nine indolyl-chalcone derivatives, compound All experimental procedures and animal care in this work were performed in accordance with the ARRIVE guidelines 39 and approved by the ethics committee in Shandong University.1H NMR (300 MHz or 400 MHz) and 13C NMR (75 MHz or 100 MHz) spectra were acquired on a Bruker Avance 300 spectrometer or Bruker Avance 400 spectrometer, with d6-DMSO or d6-Acetone used as a solvent and tetramethylsilane (TMS) as an internal standard. High resolution mass spectrometry (HRMS) involved a Q-TOF6510 spectrograph (Agilent). Unless otherwise stated, all reagents were used without further purification from merchants. Twice-distilled water was used throughout all experiments.2. Cells were seeded in 24 well plates or other appropriate dishes (30000 cells/ml).Human lung cancer cell line A549 grew in RPMI-1640 medium  containing with 10% (V/V) bovine calf serum. HeLa cells which were transfected with luciferase-based Nrf-2 reporter plasmid were grown in Dulbecco’s modified Eagle’s medium  with 10% bovine calf serum. All cell lines were cultured at 37 °C in humidified air with 5% CO3d, 6a-6e and 5-FU at 0.1, 1, 2.5, 5, 10 μM for 24 h or 48 h. Cell viability was analyzed by Sulforhodamine B  assay according to the manufacturer’s instructions.A549 lung cancer cells were cultured onto 96 well plates as previously described. Next, treating cells with 0.1% DMSO or compounds 3a-Cells were washed twice with PBS and lysed in 100 μl protein lysis buffer . All cell lysates were centrifuged at 12,000 × g for 15 min by using a refrigerated centrifuge. Then the protein concentrations were analyzed by using bicinchoninic acid (BCA) protein assay kit . After SDS-PAGE at 4 °C for 2 h, transferred to PVDF membranes . At room temperature, the membrane was blocked with 5% non-fat milk in TBST (TBS containing 0.05% Tween 20) for 1 h. Thereafter the membrane was incubated with anti-PARP  and anti-β-actin  antibodies overnight at 4 °C, washed 3 times with TBST for 5 min. Subsequently incubated with secondary antibodies which are HRP-conjugated for 1 h at room temperature. The membrane was incubated with HRP substrate for 4 min after washed 3 times with TBST and the fluorescence signals were detected by using X-ray films. The protein was quantified using Image J software.3d, 6c for 24 h and 48 h. Cells were washed with PBS for twice then photographed by using an Olympus (Japan) BH-2fluorescence microscope.A549 lung cancer cells grew on 24 well were stained with 10 mg/ml Hoechst 33258 and avoid the light for 30 min at 37 °C after treatment with 0.1% DMSO or compounds 3c, As previously described, A549 lung cancer cells grew on 24 well were washed with RPMI-1640 medium for 5 min and incubated with 10 μM 2′, 7′-dichlorodihydrofluorescein  at 37 °C for 30 min. After washed 3 times with PBS, it was photographed by utilizing an Olympus (Japan) BH-2fluorescence microscope./pGL4-3 × ARE-basic luciferase reporter vector were seeded onto 96-well plates and cultured overnight, then incubated with indolyl-chalcones derivatives (3d) at the different concentrations  and times (12 h or 24 h). Luciferase activity of cells was examined by using Luciferase Reporter Gene Assay Kit  and normalized to cell viability measured by Sulforhodamine B (SRB) assay.HeLa cells which contain Nrf-2-responsive49. In brief, A549 lung cancer cells were fixed with 4% paraformaldehyde for 15 min and blocked with 3% normal donkey serum  for 20 min at room temperature. Then, the cells were incubated with primary antibody (1:100)  at 4 °C overnight and then corresponding secondary antibody (1:200) at 37 °C for 1 h. Cells were washed 3 times with 0.1 M phosphate-buffered saline . Cells were incubated with DAPI for 10 min and washed 3 times with PBS, then photographed by using confocal fluorescence microscopy Zeiss LSM700 (Germany).Immunofluorescence assay was performed as describedExtraction of total RNA use of Trizol reagent . The reverse transcription step involved use of the PrimeScript RT reagent kit with gDNA Eraser . The relative mRNA level of HO-1 was quantified by SYBR Premix Ex Taq (Tli RNaseH Plus) RT-PCR reactions. The expression of β-actin was used to normalize with a melting curve for each reaction. Primers for HO-1 were: sense TGCACATCCGTGCAGAGAAT; antisense CTGGGT TCTGCTTGCTTGTTTCGC. Primers for β-actin were: sense GAAGTGTGACGTGGACATCC; antisense CCGATCCACACGGAGTACTT.3d at the concentrations of 100 or 200 μM every 2 days, 5-FU (200 μM) was as the positive control group. After treatment with 3d or 5-FU for 6 days, samples of the CAM and tumors were taken. The size of tumors were measured and the tumor volume calculation was performed as described in literature50.The fertilized chicken eggs were incubated at 37 °C with 60% relative humidity. On embryonic day 8, a silicone ring with a 5.5 mm inner diameter was placed on the CAM, and then 8 million A549 lung cancer cells in 20 μl of medium were seeded into this silicone ring. Eggs were divided into four groups in which 5 eggs were contained. On day 3, every egg was treated with TUNEL assay was performed according to the manufacturer’s instructions  to detect DNA fragmentation of the tumor tissues. Then the apoptosis was assessed by utilizing laser scanning confocal microscopy Zeiss LSM700 (Germany).3d (100 and 200 μM) or DMSO was placed on the CAM. The treatment with 3d or DMSO was performed every 2 days. After 6 days, the CAM zones including the gelatin sponge were taken out. The biomicroscopy image and quantitative analysis were performed by Image-Pro Plus.The fertilized chicken eggs were incubated at 37 °C with 60% relative humidity. On embryonic day 9, the gelatin sponge absorbed compound A549 cancer cells were treated with compound 3d (2.5 μM) or DMSO for 48 h, then gathered by centrifugation at 400 g, 4 °C for 5 min. Cells were fixed with 75% ethanol, then stained with 2 mg/ml propidium iodide (PI) containing 1 mg/ml RNase A at 4 °C for 30 min. The stained cells were analyzed by flow cytometry .All data were presented as means ± SEM from at least three independent experiments and analyzed by SPSS  software. When p value was < 0.05, differences were recognized as statistically remarkable.Supplementary Information


The transformation is achieved by a merged iminium–enamine activation. The enantioselective desymmetrization reaction, leading to three adjacent stereocenters, furnished the target products in complete regioselectivity and moderate to high diastereo‐ and enantioselectivities (d.r. up to 15:1 and e.r. up to 93:7). In all cases a slight decrease of the enantiomeric ratio was observed compared to the optimized model system.With the optimized results in hand, a variety of sulfenyl chlorides and phenylselenyl chloride were subjected to the reaction Table . Varioustert‐butyl sulfenyl chloride, the system avoided the steric hindrance between the bulky tert‐butyl moiety and the aliphatic chain by forming the disulfide 3 g, and thus two equivalents of the sulfenyl chloride had to be used; lower amounts resulted in the same product and selectivity, but with lower yield. With primary alkyl sulfenyl chlorides the desired products were not observed. Additionally, commercially available methoxycarbonyl sulfenyl chloride and phenylselenyl chloride were tested as reagents. The products 3 h and 3 i were obtained, respectively, in good yield with acceptable diastereo‐ and enantioselectivity. The use of analogous sulfenyl and selenyl bromides showed no conversion of 1 a, probably because the less polarized S−Br and Se−Br bonds are not sufficiently reactive.The use of sterically demanding alkyl sulfenyl chlorides ‐enantiomer as major product of the reaction  should be addressed to the authors.SupplementaryClick here for additional data file.


Mesenchymal stem cells (MSCs) have emerged as a promising treatment for inflammatory diseases. The immunomodulatory effect of MSCs takes place both by direct cell-to-cell contact and by means of soluble factors that leads to an increased accumulation of regulatory immune cells at the sites of inflammation. Similar efficacy of MSCs has been described regardless of the route of administration used, the inflammation conditions and the major histocompatibility complex context. These observations raise the question of whether the migration of the MSCs to the inflamed tissues is a pre-requisite to achieve their beneficial effect. To address this, we examined the biodistribution and the efficacy of intraperitoneal luciferase-expressing human expanded adipose-derived stem cells (Luci-eASCs) in a mouse model of colitis. Luci-eASC-infused mice were stratified according to their response to the Luci-eASC treatment. According to the stratification criteria, there was a tendency to increase the bioluminescence signal in the intestine at the expense of a decrease in the bioluminescence signal in the liver in the “responder” mice. These data thus suggest that the accumulation of the eASCs to the inflamed tissues is beneficial for achieving an optimal modulation of inflammation. Mesenchymal stem cells (MSCs) are multipotent adult stem cells that exist in the bone marrow , adipose+ T-cells was determined at 120 h by flow cytometry. Luci-eASCs inhibited T-cell proliferation at similar levels as the untransduced eASCs as described in %) and in TNBS-colitic mice , the liver  and the spleen . In contrast, very low bioluminescence signals were detected in the lungs, heart and peripheral blood following administration of the Luci-eASCs by IP route C. The inAs shown in Overall, these data indicate that IP infusion of Luci-eASCs favors the accumulation of the Luci-eASCs in the intestine, spleen and liver being practically undetectable within the lymphatic system. When colonic inflammation was induced, an increased amount of bioluminescence signal was found in the intestine in TNBS-colitic mice with a concomitant decrease in the liver.To know whether the presence of the Luci-eASCs at the site of inflammation can be correlated with their anti-inflammatory and immunomodulatory effect in vivo, the main goal of this study, we analyzed the efficacy of the IP-administered Luci-eASCs in our model of TNBS-induced colitis. To this end, body weights A, diseasAs expected, TNBS-treated mice had a significant reduction in the body weights at 24 h (−2.1[−2.9 to −0.72] fold-change in body weight) and 48 h (−3.0[−4.2 to −1.1] fold-change in body weight) with respect to the control groups  A. ImportSimilarly, all Luci-eASC-treated colitic mice had a significant reduction in the disease activity index at 24 and 48 h  with respect to the TNBS-colitic mice  with respect to the spleen (23.4[3.5–29.8]%) and the liver (3.2[1.5–6.6]%). Moreover, the bioluminescence signal of the intestine in the “responder” mice tended to increase with respect to the “non-responder” mice (56.2[33.9–67.7]%), although this difference did not reach statistical significance. This was in parallel to a tendency to decrease the total bioluminescence signal in the liver in the “responder” (3.2[1.5–6.6]%) with respect to the “non-responder” mice (18.2[9.3–35.8]%), again not significantly. No differences were found in the spleens in “responder” mice (23.4[3.5–29.8]%) with respect to the spleens in the “non-responder” mice (24.8[8.8–29.0]%) A.When the bioluminescence signal was analyzed separately in the different LNs in the “responder” and “non-responder” mice, although low, a tendency to decrease the bioluminescence signal in the different lymph nodes analyzed in the “responder” with respect to the “non-responder” mice was observed B.Altogether, these results suggest that the slight increase accumulation of the eASCs at the inflamed colonic tissue in “responder” mice may explain, at least in part, their positive response to the cell therapy treatment with the eASCs.The biodistribution of MSCs has been studied in a variety of animal models and experimental settings , IP and IL administration has been demonstrated under different inflammatory conditions ,25,34,38and mLNs ,46 togetand mLNs . Furtherand mLNs .C57/BL6 male mice (6–8 weeks) were obtained from Charles River. All experiments were performed in accordance with the corresponding regulations regarding experimental animal welfare (RD 223/1998 and Directive 2010/63/EU protocols). The experimental protocol was reviewed and approved by the ethics committee for animal research of the CIEMAT and Comunidad de Madrid (based on the RD 53/2013).4Cl to eliminate remaining erythrocytes and suspended in culture medium (Dulbecco’s modified Eagle medium with 10% FBS). Cells were seeded in tissue culture flasks and expanded . Cells were transferred to a new flask when they reached 90% confluence. Experiments were performed with a pool of cells from three male and three female adult donors at population doublings 12–14. All the eASCs used fulfilled the release criteria of identity, purity and potency needed for their clinical use.Human samples were obtained after informed consent as approved by the Spanish Ethics Committee of reference for the site of tissue procurement . Human adipose tissue aspirates from healthy donors were processed as described elsewhere . BrieflyReporter Luciferase-EGFP bicistronic retroviral vector was constructed using standard cloning procedures as described . BrieflyLuci-eASCs were defined according to the criteria of the International Society for Cellular Therapy . Luci-eABuffy coats were provided by the National Transfusion Centre of the Comunidad Autonoma of Madrid, Spain. Peripheral blood mononuclear cells (PBMCs) were isolated by density centrifugation gradient using Ficoll-Paque Plus  following the supplier’s protocol.7) were resuspended in 10 mM of 5(6)-carboxyfluorescein diacetate N-succinimidyl ester  solution and incubated at 37 °C for 10 min. Reaction was stopped by adding ice-cold medium (RPMI + 10% FBS) and cells were washed with ice-cold phosphate buffer saline. After resting overnight, CFSE-labeled PBMCs were cultured in 24-well plates alone or with Luci-eASCs  in a RPMI+10% FBS and were activated with the Pan T Cell Activation Kit  following the manufacturer’s instructions [+/7-AAD− population (viable CD3 T lymphocytes) was determined by flow cytometry, according to the decrease in the CFSE fluorescence intensity. Data were analyzed with the use of FCSExpress 4 software  [PBMCs  .5 of Luci-eASCs per mouse were administered IP. As controls, healthy mice with the Luci-eASC treatment and preimmunized mice with intrarectal 50% EtOH (vehicle of the TNBS) with the IP Luci-eASC treatment. Colitis score  was monitored for 48 h. The fold-change in body weights were calculated by the difference with respect to the initial body weight at day 0. Disease activity score was defined as follows: (1) Body weight loss ; (2) stool consistency  and (3) the general activity .To induce colitis, anaesthetized mice were preimmunized on their shaped back with 1% of trinitrobenzene sulfonic acid . After one week, colitis was induced by intrarectal administration of 3 mg of TNBS in 50% ethanol (100 µL) per mouse. One hour after, TNBS administration, 3.2 × 10Colons were surgically removed and fixed with formalin overnight at 48 h, following the TNBS administration. 1-cm colon tissues cut were collected. Colon sections were embedded in paraffin and stained with hematoxylin/eosin. The sections were microscopically examined for histopathological changes using the following scoring system .2 per steradian using Living Imaging 3.0 . For photon quantification, a region of interest was manually selected and kept constant within each experiment.Bioluminescence imaging analysis was conducted at 48 h, following the infusion of the Luci-eASCs, with the IVIS 200 imaging system . Whole body bioimaging analysis was done in anesthetized mice. The bioimaging analysis in main organs, tissues  and secondary LNs  were determined immediately after culling the mice. Photons emitted were acquired as photons per s/cmBioluminescence signal was analyzed as percentage of light units per tissue relative to the total number of light units per mouse. Total bioluminescence signal for tissues, organs and LNs was calculated as the sum of the light units in each tissue. Values of bioluminescence signal below 10,000 light units were considered negative.Data are presented as the interquartile range,  and the mean and standard error of the mean (SEM). Normal distribution was analyzed by the Shapiro-Wilks test. Non-parametric techniques (Mann-Whitney U test) were used (with Bonferroni adjustment). Analysis was performed using the software Stata 11  and GraphPad Prism 7.00 .Our results indicate that when eASCs are administered IP in colitic mice, the IP-infused MSCs tend to accumulate in the inflamed intestine to perform their therapeutic effect. The stratification analysis based on the response to MSC treatment provides a very useful approach that may help to clarify the mechanisms mediating the therapeutic effects of MSCs and, hence, improve the use of MSC-mediated therapies in the clinic.


We aimed to investigate the effects of red ginseng extract (RGE) on the expression of efflux transporters and to study the pharmacokinetics of representative substrate. For this, rats received single or repeated administration of RGE (1.5 g/kg/day) for 1 and 2 weeks via oral gavage. mRNA and protein levels of multidrug resistance-associated protein2 (Mrp2), bile salt export pump (Bsep), and P-glycoprotein (P-gp) in the rat liver were measured via real-time polymerase chain reaction and Western blot analysis. Ginsenosides concentrations from the rat plasma were also monitored using a liquid chromatography–tandem mass spectrometry (LC–MS/MS) system. Plasma concentrations of ginsenoside Rb1, Rb2, Rc, and Rd following repeated administration of RGE for 1 and 2 weeks were comparable but significantly higher than those after single administration of RGE. These dosing regimens did not induce significant biochemical abnormalities in the liver, kidneys, and lipid homeostasis. In the RGE repeated oral administration groups, the mRNA and protein levels of Mrp2 significantly decreased. Accordingly, we investigated the changes in the pharmacokinetics of methotrexate, a probe substrate for Mrp2, following intravenous administration of 3 mg/kg methotrexate to rats in the RGE 1-week repeated oral administration group, compared to that in the control group. Biliary excretion, but not urinary excretion, of methotrexate decreased in the RGE repeated administration group, compared to that in the control group. Consequently, the plasma concentrations of methotrexate slightly increased in the RGE repeated administration group. In conclusion, repeated administration of RGE for 1 week resulted in a decrease in Mrp2 expression without inducing significant liver or kidney damage. Pharmacokinetic herb–drug interaction between RGE and methotrexate might occur owing to the decrease in the mRNA and protein levels of Mrp2. Panax ginseng C.A. Meyer) has been extensively used for more than 2000 years in East Asian countries [Ginseng  resulted in slight inhibition of CYP2D6 activity, with no significant effects on CYP3A4, CYP1A2, and CYP2E1 in elderly subjects [Similar to other herbal medicines, ginseng products are frequently co-administered with prescribed Western medications. Accordingly, the potential for pharmacokinetic herb–drug interactions between ginseng and concomitantly administered drugs should be evaluated since they may result in toxicity or treatment failure . Ginsengsubjects . Two-weesubjects .Besides inhibition or induction of CYP, the modulation of drug-transporters is also an important mechanism for herb–drug interaction ,12,13. DTherefore, in the present study, we investigated the effects of single or repeated administration of RGE on efflux transporters and examined the potential changes in the pharmacokinetics of their substrate drugs. Of these, Mrp2 drew our interest because it is mainly involved in the pharmacokinetics and renal and biliary elimination of its substrate anions as well as amphipathic drugs that conjugate with glucuronide, sulfate, and glutathione (GSH) ,18. For To measure ginsenoside concentrations, we developed analytical methods for quantification of 14 ginsenosides in diluted RGE and rat plasma samples with slight modification of a previously described method . Among t1/2). For example, Rb1 is the most abundant and stable ginsenoside with a long elimination half-life of 58.47 h [After RGE was orally administered to rats at single or multiple doses for 1 or 2 weeks, the plasma concentrations of ginsenosides were also monitored. Among the 14 ginsenosides monitored, only four ginsenosides  could be quantitated ; however 58.47 h ,29. The  58.47 h ,30.Because of the potential of drug interactions between herbal medicines and efflux transporters, we measured the mRNA expression of efflux transporters, such as Bsep, P-gp, Mrp1, and Mrp2 in the liver tissues from rats in the control, SA, 1WRA, and 2WRA groups . The expTo confirm the decrease in the mRNA expression of Mrp2 in the repeated-dose RGE-treated groups, Western blot analysis was performed using the liver tissues collected from rats in the control, SA, and 1WRA groups. As shown in The levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were not significantly affected by multiple-dose administration of RGE (1.5 g/kg/day), suggesting that it had negligible effects on liver function . AdditioNext, we investigated the effects of RGE on the biliary excretion of Mrp2 substrate drug in rats. Methotrexate was selected as a representative substrate for Mrp2 because 62% and 27% of methotrexate intravenous dose is excreted into the bile and urine, respectively, within 3 h mainly by Mrp2 .1/2, in the 1WRA group, which resulted in a significant decrease in the CLtotal and a significant increase in the area under plasma concentration-time curve (AUC) of methotrexate  was obtained from Punggi Ginseng Cooperative Association . Methotrexate was purchased from Sigma-Aldrich . Analytical standard ginsenosides were purchased from Ambo Institute .Male Sprague−Dawley rats  were purchased from Samtako Co. . All animal procedures were approved by the Animal Care and Use Committee of Kyungpook National University  and carried out in accordance with the National Institutes of Health guidance for the care and the use of laboratory animals.Rats in the control group received water (2 mL/kg as a vehicle treatment) orally at 9 a.m. for 7 days by oral gavage. RGE suspension  was administered to rats by oral gavage at 9 a.m. once (SA group), for 7 days (1WRA group), and 14 days (2WRA group), respectively. Two hours after the last treatment of RGE, abdominal arterial blood (about 5 mL) and liver tissues were collected from rats in all groups. The liver tissues were snap-frozen for analysis of the mRNA and protein expression of efflux transporters. The plasma samples were used for measurement of plasma ginsenoside concentrations and biochemical parameters. The biochemical parameters such as ALT, AST, triglycerides, plasma cholesterol levels, free fatty acids, blood urea nitrogen and serum creatinine were measured from the service of Seoul Clinical Laboratories  using UV spectrophotometric assay kits from Young-Dong Diagnostics Co. . Lyphocheck assayed chemistry control  were used as positive control and YD calibrator (Young-Dong Diagnostics Co.) was used for external calibration.Ginsenoside concentrations in RGE and plasma samples were analyzed using an Agilent 6470 triple quadrupole liquid chromatography–tandem mass spectrometry (LC–MS/MS) system  equipped with an Agilent 1260 high-performance liquid chromatography (HPLC) system according to the previously published method . Brieflym/z 1131.6 → 365.1 for Rb1 (retention time (rt) = 4.1 min), m/z 1101.6 → 335.1 for Rb2 (rt = 5.0 min) and Rc (rt = 4.2 min), m/z 969.9 → 789.5 for Rd (rt = 5.1 min) and Re (rt = 1.8 min), m/z 823.5 → 365.1 for Rf (rt = 2.7 min), m/z 824 → 643.6 for Rg1 (TR = 1.9 min), m/z 807.5 → 365.1 for Rg3 (TR = 6.0 min), m/z 661.5 → 203.1 for Rh1 (rt = 3.2 min), m/z 645.5 → 645.5 for Rh2 (rt = 6.9 min), m/z 661.5 → 203.1 for F1 (rt = 3.7 min), m/z 807.5 → 627.5 for F2 (rt = 6.1 min), m/z 645.5 → 203.1 for compound K (rt = 6.8 min), m/z 483.4 → 483.4 for protopanaxadiol (rt = 6.8 min), m/z 499.4 → 499.4 for protopanaxatriol (rt = 6.8 min), and m/z 336.1 → 320 for berberine  (rt = 3.7 min) in the positive ionization mode with collision energy (CE) of 30–65 eV.Quantification of a separated ginsenoside peak was performed using MRM mode at Total RNA was extracted from liver samples (100 mg) using Qiazol , according to the vendor’s protocol. The concentration of total RNA was determined by Nano Vue Plus .RT-PCR of Mrp1, Mrp2, Bsep, and P-gp was performed using a LightCycler 96 real-time PCR system  as previously described ,37. PrimProtein expression of Mrp2, Bsep, and P-gp was measured as previously described . BrieflyPharmacokinetic study of methotrexate was performed as previously described . BrieflyMethotrexate solution  was injected intravenously to rats. Blood samples (about 150 μL) were collected from the femoral artery at 0, 0.03, 0.08, 0.25, 0.5, 1, 2, 4, and 6 h after the methotrexate injection. Bile samples were collected at 1 and 2 h and every 2 h up to 12 h through the bile cannula. Urine samples were also collected every 4 h up to 12 h through urinary bladder.g for 10 min, an aliquot (1 μL) of the supernatant was injected directly into the Agilent 6430 triple quadrupole LC–MS/MS system, according to the previously described method [v/v) and eluted at a rate of 0.25 mL/min. Quantitation was carried out at m/z 455.2 → 308.1 for methotrexate and m/z 260 → 116 for IS in the positive ionization mode.Aliquots (50 μL) of plasma, bile, and urine samples were added to 250 μL of acetonitrile containing 2 ng/mL of propranolol (IS). After vortexing for 10 min and centrifugation at 10,000× d method . BrieflyPharmacokinetic parameters were calculated by non-compartmental analysis using WinNonlin version 2.0 software .t-test using the Statistical Package for the Social Sciences . A p value < 0.05 was considered statistically significant.Statistical comparisons were performed by