Radix Dipsaci (RDD), Pyrola Herb (PHD), and Cynomorium songaricum decoction (CSD), on osteoporotic rats induced by ovariectomy (OVX).The objective of this study was to evaluate the effects of herbal medicines, such as in situ hybridization.OVX or sham operations were performed on 69 virgin Wistar rats that were divided into six groups: sham , OVX control group , and OVX rats with treatments . Non-surgical rats served as normal control . The treatments began four weeks after surgery and lasted for 12 weeks. Bone mass and bone turnover were analyzed by histomorphometry. Levels of protein expression and mRNA of OPG and RANKL in osteoblasts (OB) and bone marrow stromal cells (bMSC) were evaluated by immunohistochemistry and Compared to NC and sham rats, trabecular bone formation was significantly reduced in OVX rats, but restored in E2-treated rats. Treatment with either RDD or PHD enhanced trabecular bone formation remarkably. No significant change of bone formation was observed in CSD-treated rats. OPG expression of protein and mRNA was reduced significantly in OB and bMSC of OVX control rats. RANKL expression of protein and mRNA was increased significantly in OB and bMSC of OVX control rats. These effects were substantially reversed (increased in OPG and decreased in RANKL) by treatment with E2, RDD, or PHD in OB and bMSC of OVX rats. No significant changes in either OPG or RANKL expression were observed in OB and bMSC of OVX rats treated with CSD.RDD and PHD increased bone formation by stimulating overexpression of OPG and downregulation of RANKL in OB and bMSC. This suggests that RDD and PHD may be used as alternative therapeutic agents for postmenopausal osteoporosis.Our study showed that  Osteoporosis, the most serious bone remodeling disease, is defined by low bone mass and a high risk of fractures. It mainly affects postmenopausal women and elderly men. Osteoporosis is caused by abnormal bone remodeling , resulting in an increased risk of hip and vertebral fracture . Bone reThe development of bone fragility in postmenopausal women results from changes in bone remodeling that leads to alteration of trabecular bone volume and architecture . In ratsHormone replacement therapy (HRT) is an established regime for prevention of postmenopausal bone loss , but recRadix Dipsaci (RD), Pyrola Herb (PH), and Cynomorium songaricum (CS) have been used to reinforce the kidney-yang and strengthen bone. Although the therapeutic effects of these herbs on osteoporosis induced by estrogen deficiency are yet unknown, recent study suggests that RD crude extract could increase bone mass and alter bone histomorphology in rats [Radix Dipsaci decoction (RDD), Pyrola Herb decoction (PHD), and Cynomorium songaricum decoction (CSD) on postmenopausal osteoporosis.Chinese herbal medicine has been widely used in clinical practice to treat bone disease for thousands of years and will undoubtedly continue to be used as a cost-effective alternative medicine in China. Three Chinese herbs,  in rats . The objRadix Dipsaci (RD) is the dried roots of Dipsacus asperoides C. Y. Cheng et T. M. Ai produced in China, and was collected by the Zhonghong Herbal Drug Co. Ltd.  in March 2007, and identified and authenticated by an expert herbalist at the Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences (CACMS).Pyrola Herb (PH) is the dried aerial parts of Pyrola calliantha H. Andr produced in China, and was collected by the Huanghe Herbal Drug Co. Ltd.  in March 2007, and identified and authenticated by an expert herbalist at the Institute of Chinese Materia Medica, CACMS.Cynomorium songaricum (CS) is the dried fleshy stem of Cynomorium songaricum Rupr produced in China, and was collected by the Taizhou Herbal Drug Co. Ltd.  in March 2007, and identified and authenticated by an expert herbalist at the Institute of Chinese Materia Medica, CACMS.After collection, all the dried herbs were stored in a dry and sealed container at 4°C to protect them from moisture and moths.Decoctions of RD, PH, and CS were extracted from the herbs by boiling 300 grams of the dried RD, PH, and CS in 6 liters of water at 100°C for 2 h. Each decoction was then concentrated to a final concentration of 1 crude drug gram per milliliter.ad libitum.Sixty-nine virgin Wistar rats (3 months old) in a body weight range of 250 ± 20.0 grams were obtained from The Experimental Animal Center of the Academy of Military Medical Sciences  and housed in cages at the experimental animal room at the Institute of Basic Theory of Traditional Chinese Medicine in CACMS. These rats were maintained at 22°C with a 12 h light/dark cycle and fed with a standard rodent chow diet  containing 0.9% calcium and 0.7% phosphate, with distilled water available RDD at 5.6 ml per kg body weight a day, PHD at 5.6 ml per kg body weight a day, and CSD at 5.6 ml per kg body weight a day, respectively. This dose, about 40-fold lower than LD50 of these herbs (222.84 g/kg body weight), was optimized in our pre-experimental studies (data not shown). Rats in the NC, sham, and OVX control groups were administrated with an equal volume of distilled water to that in the decoction treatment groups. All the treatments were started four weeks after the surgery and continued for 12 weeks. The body weight of each rat was monitored weekly to assess the effect of the treatments.The acclimatized rats either underwent sham surgery   or bilaterally ovariectomized  using the dorsal approach . BrieflyOn the fifteenth and third days before sacrifice, all the rats received tetracycline at 30 mg/kg by intraperitoneal injection. After sacrifice, the success of ovariectomy was confirmed at necropsy by failure to detect ovarian tissue and by observation of marked atrophy of the uterine horns.Guide for Care and Use of Laboratory Animals, with the approval of the Institutional Ethics Committee of the CACMS.All animals were maintained according to the in situ hybridization.After sacrifice, the tibiae were dissected for histomorphometrical analysis. Proximal right tibiae were fixed in 4% paraformaldehyde for 24 h, then dehydrated in an ethanol gradient of 80%, 90%, and 100% for two days at each step. Dehydrated samples were defatted in xylene for two days before embedding in plastic polymer solution I , solution II , and solution III  for three days at each step. Each undecalcified tissue was sliced into two longitudinal sections at 5 μm thickness using microtome. One section was stained with toluidine blue, and the other was used for fluorescence morphology observation. Proximal left tibiae were fixed in 4% paraformaldehyde for 24 h, then decalcified in a decalcifying solution of 10% EDTA (pH7.3) at 4°C for three weeks. After that, the decalcified samples were dehydrated in 15% sucrose solution for 10 h. Each sample was sliced into sections of 5 μm thickness each by freezing microtome. Frozen sections were fixed in acetone and ready for use for analysis of immunohistochemistry and Undecalcified tibial slides were used for analysis of bone remodeling activity. All measurements were performed using the Qwin Image analysis system . Six bone histomorphometric parameters were analyzed, including percentage of trabecular bone volume (TBV%), percentage of trabecular resorption surface (TRS%), percentage of trabecular formation surface (TFS%), mineralization rate of trabecula (MAR), mineralization rate of bone cortex (mAR), and osteoid mean width (OSW)The undecalcified tibial sections were coated in Methyl Methacrylate Monomer solution for 2 h, then dehydrated in xylene solution for 1 h twice to remove resin. The samples were then rehydrated in an order of ethanol gradients, 100%, 90%, 80%, and 0% (in water). The rehydrated samples were stained with 1% toluidine blue, then rinsed with water. After that, the stained slides were dehydrated in an ethanol gradient of 80%, 90%, and 100%. Dehydrated slides were defatted in xylene, then sealed for microscopic analysis.d-arabinitol-diaminobenzidine; Sigma, USA) for 3 min and rinsed with running water, then counterstained with Harris hematoxylin and sealed for microscopic examination. Slides incubated with nonimmune goat serum, instead of the primary antibody, served as negative control.Frozen sections were mounted onto glass slides and used for immunohistochemical analysis. Protein expression level of RANKL and OPG in tissue sections was estimated using rat antibodies of either anti-RANKL (1:1000) or anti-OPG (1:1000) . The sections were rinsed with TBS, then immersed in 0.3% hydrogen peroxide for 5 min. The slides were then incubated with specific antibodies for 1 h at 37°C and rinsed with TBS three times for 3 min. Sections were then incubated with the appropriate unbiotinylated secondary antibody  for 30 min at 37°C. Slides were then incubated with a solution containing DAB . Five images from each tibial section of a rat were randomly captured with 400× magnification. The relative level of the osteoblasts (OB) and bone marrow stromal cells (bMSC) in trabecular area was calculated as a percentage based on the positive stained area of either OB or bMSC over the trabecular area in the selected images.After a brief warm-up at room temperature, frozen sections were immersed in solution of 30% hydrogen dioxide and methanol for 30 min, then incubated with pepsin diluted by 3% citric acid at 37°C. After that, the sections were postfixed in 1% paraformaldehyde for 10 min. Sections were then incubated with the DIG-labeled antisense cRNA probes at 38°C-42°C overnight in a humidified chamber. After multiple washes in 4 × SSC at room temperature, slides were incubated in a blocking reagent for 30 min at 37°C, then with a biotinylated anti-digoxin antibody for 60 min, SABC for 20 min, and the biotinylated peroxydase for 20 min, at 37°C, followed by staining with DAB (Sigma-Aldrich Corp.). Finally, sections were covered with glycerol-gelatin and coverslips, and were ready for microscopic examination. The primer sequences used are listed below:OPG:Forward: 5'-TGGACAACCCAGGAAACCTTTCCTCCAAAA-3'Reverse: 5'-TTTGCCTGGGACCAAAGTGAATGCAGAGAG-3'Probe: 5'-AGAAATGATAGGGAATCAGGTTCAATCAGT-3'RANKL:Forward: 5'-GCCAGCCGAGACTACGGCAAGTACCTGCGC-3'Reverse: 5'-GGCCAGGTGGTCTGCAGCATCGCTCTGTTC-3'Probe: 5'-TTTATAGAATCCTGAGACTCCATGAAAACG-3'All values were expressed as mean ± standard deviations. All analyses were carried out using SPSS 12.0 . The difference between the groups regarding the evaluated parameters was tested using the ANOVA test followed by the Tukey test. The data of all groups passed the Kolmogorov-Smirnov test of normality. All groups were carried out in triplicate and significance was accepted when P ≤ 0.05.To investigate the effects of the herbal medicines on overall bone formation, we analyzed trabecular bone formation in the undecalcified tibial sections from tibiae of OVX rats treated with RDD, PHD, and CSD using methylene blue staining Figure . Trabecup < 0.01) in rats with estrogen deficiency induced by ovariectomy. Yet, TRS%, TFS%, MAR, mAR (p < 0.01) and OSW (p < 0.05) were increased significantly in these OVX rats.Bone remodeling is a lifelong process in which two counter-balanced processes (bone resorption and bone formation) are involved. Bone remodeling activity can be monitored by measuring histomorphometric indices and bone remodeling markers. To further estimate the effects of RDD, PHD, and CSD on bone remodeling activity, we analyzed six bone histomorphometric parameters, including TBV%, TRS%, TFS%, MAR, mAR, and OSW in OVX rats treated with the Chinese herbs and E2 using histomorphometry Table . TBV% wap > 0.05)  Table .p < 0.01, Figure p < 0.05, Figure p > 0.05).To monitor bone remodeling activity in OVX rats treated with the herbs, we analyzed biomarker expression of bone remodeling using immunohistochemistry Figures A, B. OPGp < 0.01, Figure p < 0.01, p < 0.05, and p < 0.05, respectively, Figure p > 0.05).We also analyzed RANKL expression in OB or bMSC of tibia from OVX rats treated with RDD, PHD, CSD, or E2 for 12 weeks of treatment using immunohistochemistry. Ovariectomy caused a significant increase in RANKL expression in OB or bMSC of tibia compared to that from the sham group  or PHD treatment (p < 0.05); however, no significant changes in the ratio of RANKL/OPG were observed in OB and bMSC of tibia from OVX rats treated either with RDD or CSD  or PHD (p < 0.05); however, no significant changes in the ratio of RANKL/OPG were observed in OB and bMSC of tibia from OVX rats treated with either RDD or CSD  have been widely used for thousands of years to treat bone diseases in China. The theory of Chinese medicine is to restore balance at all effective levels. Although these herbal medicines are considered a cost-effective alternative by Chinese people, lack of knowledge about their mechanisms limited their application as an alternative therapeutic regime to treat osteoporosis in most Western countries.Previous studies showed that ovariectomy induced bone loss, resulting in an increased risk of osteoporosis. Our results from this study confirm this notion, indicating that the mature OVX rat is a good animal model of osteoporosis for evaluating the efficacy of herbal medicines as therapies for early postmenopausal osteoporosis.Cissus quadrangularis (CQ) to treat an ovariectomized rat model of osteoporosis, and found that CQ ethanol extract of the plant had a definite anti-osteoporotic effect [Salvia miltiorrhiza (SM), showing that SM ameliorated the decrease in BMD and trabecular bone mass of OVX-induced osteoporosis rats and decreased the serum level of osteoclast markers and oxidative stress markers. Based on the property of anti-oxidative stress, SM may be developed as a promising natural therapy for osteoporosis [Radix Dipsaci (RDD), Pyrola Herb (PHD) and Cynomorium songaricum decoctions (CSD), as used in this study, in which the effects of these medicines on an ovariectomized rat model of osteoporosis were evaluated.Recently, several studies of herbal medicines have been performed to evaluate their antiosteoporotic effects, suggesting that herbal medicines may be developed as useful alternative medicines for treatment of osteoporosis ,10. Shirc effect . Chae’s Although E2 is no longer used in clinical practice due to its side effects, use of E2 as control in this study is to help us understand the mechanism of action of the herb medicines in OVX rats. As expected, OVX rats treated with E2 for 12 weeks ameliorated the loss of bone mass, which was reflected by the increased TBV% and the decreased levels of TRS%, TFS%, MAR, OSW, and mAR. The effect of either RDD or PHD on TBV% and TRS% was similar to that of E2; however, the effect of either RDD or PHD on mAR and OSW of tibia from OVX rats was not significant, suggesting that either RDD or PHD may have a limited effect on bone cortex mineralization and osteoid formation. In addition, OVX rats treated with PHD did not show significant decreases in TFS% and MAR, suggesting that a possible anti-osteoporotic mechanism of PHD may be through inhibition of bone resorption.It is well known that the RANKL/RANK/OPG signaling pathway plays a key role in the regulation of bone remodeling . RANK isRadix Dipsaci and Pyrola Herb extract have the potential to be used for the treatment of postmenopausal osteoporosis.By comparison, this study demonstrated for the first time the potential protective effects of RDD and PHD on OVX-induced osteoporosis in rats. RDD and PHD could prevent the OVX-induced loss of bone mass; their preventive effects depended on modulating protein and mRNA expression of OPG and (or) RANKL in OB and bMSC. Our study provides evidence that Cynomorium songaricum); CSD: (Cynomorium songaricum decoction); bMSC: ; MAR: ; mAR: ; OB: (Osteoblasts); OPG: (Osteoprotegerin); OSW: (Osteoid mean width); OVX: (Ovariectomy); PH: (Pyrola Herb); PHD: (Pyrola Herb decoction); RD: (Radix Dipsaci); RDD: (Radix Dipsaci decoction); TBV: (Trabecular bone volume); TFS: (Trabecular formation surface); TRS: (Trabecular resorption surface).CS: (The authors declare that they have no competing interests.ML carried out rat experiments and drafted the manuscript. GGX and DJ were heavily involved in experimental design, and GGX was also mainly involved in scientific correction of the draft manuscript. PR, ZZ, JD, HZ, HL, YL, JP, HL, WW, and PZ were involved in sample collection and measurements of bone morphometry, as well as bone markers. All authors were involved in drafting the manuscript and revising it for critically important content. All authors have read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1472-6882/12/67/prepub


However, there are limited data correlating FDG-PET with the chemotherapy resistant tumor markers. The purpose of this study was to determine the correlation of chemotherapy related tumor marker expression with FDG–PET SUVmax in NSCLC.The chemotherapy resistance of non-small cell lung cancer (NSCLC) remains a clinic challenge and is closely associated with several biomarkers including epidermal growth factor receptor (EGFR) ( Drugs 72(Suppl 1):28–36, 012.), p53 ( Med Sci Monit 11(6):HY11–HY20, 2005.) and excision repair cross complementing gene 1 (ERCC1) ( J Thorac Oncol 8(5):582–586, 2013.). Fluorodeoxyglucose positron emission tomography (FDG–PET) is the best non-invasive surrogate for tumor biology with the maximal standardized uptake values  and immunohistochemical analysis was performed for EGFR, p53 or ERCC1 on the intraoperative NSCLC tissues. Each tumor marker was assessed independently by two pathologists using common grading criteria. The SUVmax difference based on the histologic characteristics, gender, differentiation, grading and age as well as correlation analysis among these parameters were performed. Multiple stepwise regression analysis was further performed to determine the primary predictor for SUVmax and the receiver operating characteristics (ROC) curve analysis was performed to detect the optimized sensitivity and specificity for SUVmax in suggesting chemotherapy resistant tumor markers.FDG–PET SUVmax values were observed. The tumor differentiation is significantly correlated with SUVmax (R = -0.327), tumor size (R = -0.286), grading (R = -0.499), gender (R = 0.286) as well as the expression levels for p53 (R = -0.605) and ERCC1 (R = -0.644). The expression level of p53 is significantly correlated with SUVmax (R = 0.508) and grading (R = 0.321). Furthermore, multiple stepwise regression analysis revealed that p53 expression was the primary predictor for SUVmax. When the cut-off value of SUVmax was set at 5.15 in the ROC curve analysis, the sensitivity and specificity of SUVmax in suggesting p53 positive NSCLC were 79.5% and 47.8%, respectively.The significant tumor type (P = 0.045), differentiation (P = 0.021), p53 (P = 0.000) or ERCC1 (P = 0.033) positivity dependent differences of SUVmax of primary tumor on FDG-PET might be a simple and good non-invasive method for predicting p53-related chemotherapy resistance in NSCLC when we set the cu-off value of SUVmax at 5.15.The current study suggests that SUV Lung cancer is the most frequently diagnosed cancer and leads to the most cancer mortality worldwide which accounts for almost 1.3 million deaths a year .Although significant therapeutic advances have been achieved, poor prognosis and short survival time of patients, as well as the limited value of any sort of conventional therapy are the current dilemma for NSCLC therapy . Platinumax) of primary tumors have been shown to correlate with both stage and nodal disease in NSCLC . LSD post hoc test revealed that the difference was derived from the significantly higher SUVmax from the NSCLC patients with poor (P = 0.017) differentiation. There was no significant difference in the SUVmax from poorly and moderately differentiated tumors (P = 1) or moderately and well differentiated tumors (P = 0.132). On the other hand, no difference in the SUVmax of patients at different clinical stages  was observed  and false negativity  in the evaluation of primary tumor [max larger than 5 leads to an optimized diagnosing sensitivity and specificity of NSCLC [The SUVry tumor . A recenof NSCLC . We thusmax was set at 2.55, the sensitivity and specificity of suggesting p53 positive NSCLC were 100% and 13%, respectively. However, when we set the cut-off value of SUVmax at 5.15, the sensitivity and specificity of suggesting p53 positive NSCLC were 79.5% and 47.8%, respectively  ranging from 0.654 to 0.884 (p = 0.000). When the cut-off value of SUVmax. Our study offers further evidence that the SUVmax of FDG-PET may be a predicting parameter for some chemotherapy resistant NSCLCs, especially for the p53 or ERCC1 related chemotherapy resistance. Furthermore, SUVmax may be the most relevant parameter for p53 related chemotherapy which suggests the future clinical application to design the therapeutic plan.FDG-PET, one of the current-available non-invasive imaging methods, has long been used to determine the enhanced metabolism in malignant tumor indicated by increased glucose uptake which is represented by an increased SUVmax and EGFR expression in esophageal cancer specimens. Shimizu et al [max than negative cases in lung adenocarcinoma, but without statistical significance. Our finding is quite consistent with theirs in that there is no relationship between EGFR expression and SUVmax in NSCLCs. Furthermore, we did not reveal any difference in the SUVmax between adenocarcinoma and squamous cell carcinoma. Our study, together with previous one [EGFR is a cell surface receptor found primarily on cells with epithelial origin. EGFR overexpresses in both cell lines and samples of NSCLC, and contributes to the increased tumor proliferation, poor differentiation, higher incidence of metastases to lymph nodes and a worse prognosis . Previouzu et al  reportedious one , suggestmax of ERCC1-positive cases were significantly higher than that of ERCC1-negative cases, there was statistical correlation between SUVmax and ERCC1 level, but failed to detect robust correlationship when the multiple stepwise regression was performed. It is still inconclusive whether SUVmax can be used to determine ERCC1 related chemotherapy resistance based on our current study.The anti-cancer mechanism for the platinum compounds is to form adducts and covalent cross-links between DNA double strands and thus effectively block DNA replication and transcription. ERCC1 can recognize and remove these adducts and covalent cross-links, thus resistant to platinum agents . A recenmax of p53-positive cases was statistically higher than that of p53-negative cases. Besides, we also offered evidence that p53 expression is the primary predicting factor for the SUVmax. Our findings lead to the concept that FDG-PET can be used to represent p53 expression status, thus predict the p53-related chemotherapy sensitivity. In the clinic settings, we should set the cut-off value of SUVmax at around 5 to get the optimized sensitivity and specificity However, this is a more like bench study even if we used the clinical imaging technique. Using p53 as the biomarker for chemotherapy resistance in NSCLC is risky. Thus, cautions should be taken when using the SUVmax of FDG as an alternative or reliable marker for p53, not to mention the prognosis of NSCLC. To really apply the SUVmax of FDG in the clinic settings, more bench studies and clinic trials are needed. Further efforts are needed to reveal the underlying reasons for the inconsistency between the findings of ours and others, the study to fill the gap between our experimental findings and future clinical applications should also be considered.As a tumor suppressor gene, p53 is capable of either arresting the cell cycle or inducing apoptosis. Tumors expressing p53 were less resistant to cisplatin, carboplatin, paclitaxel, and gemcitabine , probablmax on FDG-PET in NSCLCs. Of the two markers, p53 expression is the primary predictor for the SUVmax. Based on our findings, FDG-PET might be a simple and good non-invasive method for predicting p53-related chemotherapy resistance in NSCLCs. But cautions should be taken when using this method in the clinical settings.In conclusion, the expressions of p53 and ERCC1 are associated with the SUVThe authors declare that they have no competing interests.XYD and YMG designed research; XYD, WW, JSW and SJ performed the research; XYD, WW and JGG analyzed data, XYD and WW wrote the paper. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2407/13/546/prepubmax.Multiple stepwise regression analysis of the primary predictor for SUVClick here for file


Aim. The aim was to compare body mass index (BMI), waist circumference (WC), waist hip ratio (WHR), and waist height ratio (WHtR) to identify the best predictor of metabolic syndrome (MetS) among Qatari adult population. Methods. A cross-sectional survey from April 2011 to December 2012. Data was collected from 1552 participants followed by blood sampling. MetS was defined according to Third Adult Treatment Panel (ATPIII) and International Diabetes Federation (IDF). Receiver operating characteristics (ROC) curve analysis was performed. Results. Among men, WC followed by WHR and WHtR yielded the highest area under the curve (AUC) . Among women, WC followed by WHtR yielded the highest AUC . Among men, WC at a cut-off 99.5 cm resulted in the highest Youden index with sensitivity 81.6% and 63.9% specificity. Among women, WC at a cut-off 91 cm resulted in the highest Youden index with the corresponding sensitivity and specificity of 86.5% and 64.7%, respectively. BMI had the lowest sensitivity and specificity in both genders. Conclusion. WC at cut-off 99.5 cm in men and 91 cm in women was the best predictor of MetS in Qatar. Metabolic syndrome (MetS) is known to be a cluster of interrelated risk factors of metabolic origin such as elevated blood pressures, glucose metabolism disturbances, dyslipidemia, and obesity , which aIt has been widely considered that central obesity as a marker of body fat can be conveniently and cost effectively estimated by measuring body mass index (BMI) and waist circumference (WC) that in turn might effectively predict the risk of MetS , 8. ReceWe conducted a cross-sectional study among the adult Qatari population above 20 years of age over a period from April 2011 to December 2012. The study protocol was approved by the Ethics committee of Hamad Medical Corporation. Each participant was provided with brief information about the study and was assured of strict confidentiality. Only participants who agreed to participate and gave written consent were included in the study.We developed a multistage stratified cluster sampling design using the administrative divisions of the primary health centres (PHC) in Qatar that had approximately equal population coverage. In order to secure a representative sample of the study population, sampling was stratified with a view to obtain proportional representation from urban and semiurban areas. The sample size was determined based on a priori assumption for the prevalence of MetS in Qatar and Eastern Mediterranean region to be 17–20%; with the 99% confidence interval and 2% bound on error of estimation, a minimum sample size of 2,182 would be required for this study. Out of total 22 PHCs available, 13 were selected at random. Of these 10 were located in urban and 3 in semiurban areas of Qatar. Lastly, subjects were selected systematically 1-in-2 sampling procedure. During the study period, 2,182 subjects were approached, of whom 1,552 (71%) consented to participate.We used a well designed and pilot tested questionnaire to collect the data. The questionnaire was tested among 100 subjects as a pilot study before the initiation of the main survey. We made necessary corrections and modifications in the questionnaire based on the findings from the pilot study. The first part of the questionnaire included information about sociodemographic and anthropometric characteristics including age, sex, marital status, education level, occupation, height, weight, and waist and hip circumference. The second part consisted of life style habits such as physical activity, fast food consumption and smoking habits. Last part of the questionnaire comprised of information about systolic and diastolic blood pressures, serum triglyceride, total cholesterol, high-density lipoprotein (HDL) cholesterol, low density lipoprotein (LDL) cholesterol, Hemoglobin A1c (HbA1c), and fasting plasma glucose levels (FPG).  National Cholesterol Education Program: Third Adult Treatment Panel (ATP III) .According to ATPIII, presence of at least three of these risk factors diagnose the MetS: (a) FPG ≥ 100 mg/dL (5.6 mmol/L); (b) blood Pressure ≥ 130/85 mmHg; (c) triglyceride ≥ 150 mg/dL (1.7 mmol/L); (d) HDL Cholesterol: Men < 40 mg/dL (1.03 mmol/L); women < 50 mg/dL (1.29 mmol/L); (e) men with waist circumference >102 cm and women with waist circumference >88 cm. International Diabetes Federation (IDF) .According to IDF, a participant has the MetS if she/he has a waist circumference (≥94 cm in men and ≥80 cm in women) plus any two of these risk factors: (a) FPG ≥ 100 mg/dL (5.6 mmol/L) or previously diagnosed impaired fasting glucose (b) blood pressure ≥ 130/85 mmHg or treatment for hypertension; (c) Triglyceride ≥ 150 mg/dL (1.7 mmol/L); (d) HDL Cholesterol: Men < 40 mg/dL (1.03 mmol/L); Women < 50 mg/dL (1.29 mmol/L) or treatment for low HDL.We used two different international criteria as given below to diagnose MetS among the participants.Igaal and Guttra). Weight was measured in kilograms using a weight scale . The subjects were asked to remove any objects from their pockets and to stand on the weight scale bare feet with light clothing. BMI was calculated as the ratio of weight (kilogram) to the square of height (meters). Obesity and overweight were classified according to WHO criteria [2, overweight if BMI ≥25 kg/m2 and <30 kg/m2. Physical examination and measurements were performed by a trained nurse. Height was measured in centimetres (cm) using a height scale  while the subject was standing bare feet and with normal straight posture. Male subjects were requested to remove their head cover  was calculated by taking the waist circumference (cm) and dividing by the hip circumference (cm) while on the other hand Waist to Height ratio (WHtR) was calculated by taking waist circumference (cm) and dividing by height (cm). Two readings of the systolic (SBP) and diastolic (DBP) blood pressure were taken from the subject's left arm while seated and his/her arm at heart level, using a standard zero mercury sphygmomanometer after at least 10–15 minutes of rest. Then the average of the two readings was obtained. A blood sample of 10 mL was collected through venipuncture from each participant after fasting for 10 hours, into vacutainer tubes containing EDTA. The samples were kept at room temperature and transported within 2 hrs to a central certified laboratory at Hamad General Hospital, HMC, Doha, Qatar. Plasma glucose, total cholesterol, triglyceride, HDL-cholesterol, and LDL-cholesterol were measured by an autoanalyser . Information on cigarette and sheesha smoking was obtained separately by asking questions regarding the smoking status ; if smoker then further questions were asked regarding number of years smoked and number of cigarettes smoked per day. Information on physical activity/exercise was also obtained subjectively by asking question regarding any activity causing light perspiration or slight to moderate increase in breathing or heart rate for at least 30 minutes which is performed regularly (yes/no). Information about fast food consumption was measured through a single binary response question “do you eat fast foods? Yes/No.” t-test for continuous scale variables. The receiver operating characteristic (ROC) curve was generated to obtain the values of area under the curve (AUC) with 95% CI, and also sensitivity and specificity for each obesity index as a predictor of MetS. To determine the locally appropriate sex-specific cut-off point for each obesity index, the Youden index (sensitivity + specificity − 1) was calculated and the corresponding cut-off value for the highest Youden index was considered as the optimal cut-off value. Data were analyzed using the Statistical Package for Social Sciences version 20  software. Continuous variables were tested for normality using histograms and Kolmogorov-Smirnov test. Continuous variables were expressed as mean with standard deviation and categorical variables were expressed as frequency with percentage. Comparison of sociodemographic variables, lifestyle habits, and anthropometric and biochemical measurements between subjects with and without MetS was made using Pearson chi square for categorical variables and independent samples Student's In addition, multivariable logistic regression analysis was conducted for total population, males and females separately to identify the strength of association of different obesity indices  and MetS.P < 0.001, 57% versus 49%; P = 0.005, and 47% versus 39%; P = 0.024, resp.); however, no such difference was observed between the groups based on IDF criteria (P > 0.05). Level of education, consumption of fast food, and physical activity were significantly different between groups with and without MetS using both the diagnostic criteria. Overall, the prevalence of MetS was 26.2% according to ATPIII and 36.9% according to IDF. P < 0.001, resp.). Average WC, WHR, WHtR, BMI, FPG, triglycerides, SBP, and DBP were significantly higher among the participants with MetS as compared to those without MetS irrespective of the diagnostic criteria. In contrast, average HDL cholesterol was significantly lower in MetS than without MetS   as compared to slightly higher than one third of the metabolically healthy obese individuals  .2 and the traditional cut-off value of 30 kg/m2 were found to be having the lowest Youden index and corresponding sensitivity and specificity. WC at a cut-off point of 99.5 cm among men and 91 cm among women happened to be the best predictor of metabolic syndrome in Qatari population.Among men, WC at a cut-off value of 99.5 cm resulted in the highest Youden index with corresponding sensitivity of 81.6% and 63.9% specificity. At a traditional cut-off value of 102 cm of WC for men, the sensitivity dropped to 75.9%, and specificity slightly raised to 67.3%. Similarly among women, WC at a cut-off point of 91 cm resulted in highest Youden index with the corresponding sensitivity and specificity of 86.5% and 64.7%, respectively. At a traditional cut-off point of 88 cm WC among women, the sensitivity steeply increased to 94.4%, but this happened at the expense of significant drop in the specificity from 64.7% to 53.2%. Among both men and women, the BMI at a cut-off value of 28 kg/m2 and 28.4 kg/m2 in men and women, respectively. In this cross-sectional survey of Qatari nationals aged 20 years and above, we found that the overall prevalence of MetS was 26% according to ATPIII and 37% according to IDF criteria which was consistent with the previous study conducted among Qatari adult population [ Which measure of obesity should be used for predicting MetS is widely debated. A recent cross-sectional survey among adult Iranian population found that WC was superior to BMI and WHR in discriminating MetS among healthy individuals . Similar Irrespective of all these ethnic variations regarding appropriateness of obesity indices, studies have shown that BMI poorly discriminates between excess adipose tissue and high lean muscle mass and that it does not account for body fat distribution , 18. On  In this study we found that WC at a cut-off value of 99.5 cm for men and 91 cm for women has the highest sensitivity and specificity to predict the development of MetS. When we applied the WC cut-off value of 102 cm for men and 88 cm for women as recommended by ATPIII criteria . Another2 than the recommended cut-off point by WHO [ Our results also show that, after the WC, WHR at a cut-off point of 0.90 for men and 0.88 for women yields the highest sensitivity and specificity to differentiate MetS. This finding is consistent with previous studies from Oman, Korea, and Iran , 11. We t by WHO  for bothOur study has two important limitations. Firstly, the cross-sectional design of this study has inherent limitation of temporality and hence inability to draw causal inferences. Secondly, we could not adjust our sample size calculation for the potential nonresponse bias. However, despite these limitations we were able to recruit a large number of representative samples with a response rate of 71% from both rural and urban areas of Qatar and our results are generalizable to the entire Qatari adult population. Waist circumference at a cut-off point of 99.5 cm among men and 91 cm among women happened to be the best predictor of metabolic syndrome in Qatari population. Using the traditional cut-off values of 102 cm for men and 88 cm in women as recommended by ATPIII criteria for the Arab region might result in underestimation of MetS among men and overestimation among women. Looking at the discrepancies we recommend that WHO, IDF, and ATPIII criteria for obesity might not be appropriate for predicting the risk of MetS among Qatari adult population.


Clostridium difficile is the leading cause of antibiotic-associated diarrhea in hospitals worldwide, due to hypervirulent epidemic strains with the ability to produce increased quantities of the large toxins TcdA and TcdB. Unfortunately, accurate quantification of TcdA and TcdB from different toxinotypes using small samples has not yet been reported. In the present study, we quantify C. difficile toxins in <0.1 mL of culture filtrate by quantitative label-free mass spectrometry (MS) using data-independent analysis (MSE). In addition, analyses of both purified TcdA and TcdB as well as a standard culture filtrate were performed using gel-based and gel-independent proteomic platforms. Gel-based proteomic analysis was then used to generate basic information on toxin integrity and provided sequence confirmation. Gel-independent in-solution digestion of both toxins using five different proteolytic enzymes with MS analysis generated broad amino acid sequence coverage (91% for TcdA and 95% for TcdB). Proteomic analysis of a culture filtrate identified a total of 101 proteins, among them TcdA, TcdB, and S-layer proteins.  Clostridium difficile is a gram-positive, anaerobic, spore-forming, rod-shaped bacterium that can produce at least three toxins including two Rho GTPase-glucosylating toxins (TcdA and TcdB) and the binary C. difficile transferase (CDT) toxin. The organism can cause C. difficile infection (CDI) in humans and animals. C. difficile is considered an important cause of healthcare-associated infection in humans and is the leading cause of antibiotic-associated diarrhea in hospitals worldwide [C. difficile through cultivation, detection of Tcd A and Tcd B in stool samples using immunoassay and DNA-based methods [orldwide , 2. CDIsorldwide . LaboratReflecting its changing epidemiology, the incidence and severity of CDI have increased significantly in the past ten years. Among the possible causes of increasing morbidity is the emergence of strains considered to be more virulent . These sAlthough it has been demonstrated , 5 that C. difficile in which proteins released in vitro during high toxin production [C. difficile reference strain 630, including a surface protein and insoluble protein fraction analysis [C. difficile reference strain 630 were compared to two hypervirulent strains (CD196 and CDR20291), and five secreted proteins were identified exclusively in the supernatants of the hypervirulent strains [Proteomics has been described as a key technology in the postgenomic era that provides information complementary to that provided by genomics. Proteins can be analyzed rapidly, accurately, and with high sensitivity using mass spectrometry (MS). Proteomic examinations have been performed for oduction  were ideanalysis , 10, spoanalysis , and a panalysis . In an a strains . E) that does not require labeled compounds and is amenable to sample-limited experiments [E, data are acquired in a data-independent fashion using an alternating low/high-energy scan mass analysis and can be used to perform both protein identification and quantification in one MS experiment [Absolute protein quantification by MS is a well-studied technique typically performed using stable isotope dilution –16. Howeeriments  is a newperiment , 19. C. difficile toxin production by different isolates. We expect that such efforts will contribute to a better understanding of these toxin-mediated illnesses and will lead to new preventive measures and therapies against C. difficile infection. We have previously described MS-based proteomics studies of different bacterial toxins , includiC. difficile VPI 10463—ATCC 43255 and were provided in vials containing 100 μg of TcdA or 20 μg of TcdB lyophilized in 0.05 M Tris. Three vials of each toxin  were assigned numbers in house and designated as distinct biological samples . The vials containing TcdA and TcdB were reconstituted as per the manufacturer's instructions, aliquoted, and used immediately as the designated BioS. Aliquots (20 μL) from the first biological sample (BioS1) were used to run SDS-PAGE gels and for three separated fast trypsin in-solution digestions to verify method reproducibility [μg and ending at 0.125 μg) and used to determine the sensitivity of the MSE method used in this study. In addition, a commercially available lyophilized C. difficile culture filtrate (CFil) control reagent  was used for both the quantitative method and qualitative proteomic analyses. A schematic flow diagram of the procedures used in this work can be found in Purified TcdA (one lot—lot 1-15215A1C) and TcdB (two lots—lot 1-1551A1B and lot 2-15518A1B) used in this study were purchased from List Biologicals Laboratories Inc. . The toxins were purified from cibility . Ten micμL of trypsin (0.5 μg/μL) diluted in 25 μL of a 50 mM ammonium bicarbonate solution, pH 8.5, containing 1 mM calcium chloride (digestion buffer), was added to each sample. After 5 min incubation at room temperature (RT), 25 μL of digestion buffer was added and the samples were incubated at 37°C overnight (ON). Following ON incubation, the digests were quenched with 0.1% formic acid (FA), sonicated for 3 min, and centrifuged at 1200 g for 10 min. The supernatants were used for nanoscale ultrapressure liquid chromatography (nUPLC)-MS/MS analysis. Duplicates of purified TcdA lot 1 (BioS1) and two lots of TcdB lot 1 and lot 2 (BioS1) were treated with NuPAGE  sample buffer and the proteins were separated by 1D SDS-PAGE using the NuPAGE Novex system  as per the manufacturer's instructions (Invitrogen). In addition, BioS1 in-solution tryptic digests were separated using Tricine gels (Invitrogen). The gels were then either stained with Colloidal Coomassie Blue  or with Pierce Silver Stain Kit for Mass Spectrometry (Thermo Scientific). After being scanned, each gel band was cut into slices of approximately 0.4 cm. In-gel digestion was performed with sequence grade trypsin  as previously described . Brieflyμg/μL) and CFil (10 μg/μL). Ten microliters of 0.2% RapiGest (RG), an acid-labile surfactant , in-digestion buffer, was added to each 10 μL-aliquot of TcdA, TcdB, and CFil and the tubes were incubated at 99°C for 5 min using a thermocycler . The solution was rapidly cooled to RT, and trypsin (~50 pmol) in-digestion buffer, was added. The samples were incubated at 52°C for 3 min. To hydrolyze the RG, 10 μL of 0.45 M HCl was added and the samples were incubated at 37°C for 30 min. The suspension was further diluted to a final volume of 50 μL with 0.1% FA and centrifuged (1200 g) for 10 min. The supernatant containing the peptides was frozen at −70°C if not used immediately. For calibration of the quantification method, yeast alcohol dehydrogenase (ADH) standard tryptic digest solution (Waters Corporation) was added to the supernatant before analysis by MSE, at a concentration required to give 100 fMol ADH on column. Each toxin was digested in triplicate  and submitted to the MS instrument in triplicates  and the results of all MS runs were compared. To visually evaluate digestion effectiveness, BioS1 tryptic digests were analyzed using SDS-PAGE tricine gels and silver stained. Four additional enzymes, AspN, chymotrypsin, GluC, and LysC , were separately used to digest aliquots of BioS2 in order to maximize proteome coverage. Enzymatic digestions were performed as per the manufacturer's instructions.In-solution detergent-based 3 min tryptic digestions were performed as described , using tE method on the QTof Premier MS, were used as previously described [Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was carried out using an nUPLC coupled either to a QTof Premier MS system , or to a linear ion trap (LTQ)-Velos Orbitrap tandem MS instrument . All the conditions for nUPLC separation, (including flow rate and solvent concentrations) as well as the MSescribed . E were further processed using the ProteinLynx Global Server v2.4 software , for protein identification and quantification. Database searches were performed using the PLGS IdentityE database search algorithm against either a UniProt protein database  or a modified NCBInr database created with the term “difficile”  to which the ADH 30,030 Da amino acid sequence was added. The PLGS software package provided statistically validated peptide and protein identification along with the determination of the stoichiometry of the protein constituents of the mixture (relative quantification) along with the expected amounts of protein present in the mixture (absolute quantification). The remaining parameters, including mass accuracy for precursor (10 ppm) and product (20 ppm) ions and criteria for protein identifications, were defined as before [E and the Expression software [Each digest was analyzed in triplicate , and the respective raw data files obtained using data-independent LC-MSs before . Similarsoftware , 19. Thesoftware . The repAdditionally, protein digests were analyzed using an LTQ Velos Orbitrap tandem MS instrument. Peptides were separated using an nUPLC system directly coupled online to the MS instrument through an Advance Captive Spray source from Michrom Bioresources . The spray voltage was set at 1500 V, and the capillary temperature was 200°C. nUPLC separation was performed as previously described . Brieflym/z 400 to 1400 at a nominal resolution setting of 60,000 FMHM for parent ion acquisition in the Orbitrap. Tandem mass spectra of doubly charged and higher charge state ions were acquired for the top 15 most intense ions in each survey scan. All tandem mass spectra were recorded by use of the linear ion trap. This process cycled continuously throughout the duration of the nUPLC gradient. All tandem mass spectra were extracted from the raw data file using Mascot Distiller  and searched using Mascot (version 2.2.0). Mascot was setup to search using the entire NCBInr database or a modified NCBInr database created to search “C. difficile” recognized proteins, or to search for C. difficile strain ATCC 43255 in which trypsin is used as the digestion agent. Mascot and Scaffold search parameters were used as described before with stringent parameters so the probability of a wrong assignment was below 0.1% [http://www.psort.org/psortb/) [http://www.genome.jp/kegg/kegg3.html as described before [The MS was programmed to perform data-dependent acquisition by scanning the mass range from low 0.1% , 23. PSOpsortb/) . NCBI gi1D SDS-PAGE followed by silver stain revealed intense bands corresponding to purified TcdA and TcdB proteins . InteresC. difficile toxins. As revealed by nUPLC-MS/MS analysis, in-solution digestion of the toxins generated a large peptide pool. Additionally, to confirm completeness of digestion, analysis of the digests using a silver-stained Tricine gel (not shown) revealed no bands, suggesting completeness of digestion.First, three separate aliquots of BioS1 (TcdA lot1 and TcdB lot2) were used to verify the effectiveness of the 3 min digestion method for the large Secondly, BioS2 aliquots were digested in parallel using multiple enzymes. The amino acid percent coverage, for TcdA and TcdB for each enzyme used, was 37% and 34% for AspN, 28% and 35% for chymotrypsin, 40% and 61% for GluC, 8% and 57% for LysC, and 80% and 66% for trypsin. The two enzymes that delivered the most complementary sequence coverage were trypsin and GluC (combined sequence coverage of 83% for TcdA and 86% for TcdB). Summation of the entire MS peptide analysis revealed broad amino acid sequence coverage for TcdA (91%) and TcdB (95%).E (μg/mL) and 1.25 ng (0.43 μg/mL). Finally, BioS3 was used for the absolute quantification of TcdA and TcdB in small samples using MSE . The minE method was to successfully determine the relative and absolute amounts of TcdA and TcdB in a commercial lyophilized C. difficile culture filtrate (CFil) control reagent. In addition, because MSE data analysis can be used to determine the relative abundance of proteins in a complex mixture, data obtained with the CFil were processed and 24 constituents of the filtrate could be quantified  sequence coverage maps. The expected unique protein band (~300 kDa MW) was visible in the TcdA lane, while two bands were observed in the TcdB lane for the first lot of a commercially available toxin. Further MS analysis of the gel-excised tryptic-digested proteins from this first lot demonstrated that the band at the ~270 kDa region was the complete TcdB, whereas the extra-band ~210 kDa) was a truncated TcdB protein missing the N-terminal peptides (~68 kDa) which are associated with enzymatic activity. Interestingly, the presence of two bands at ~210 and ~68 kDa is normally expected when TcdB is autocleaved by the cysteine-protease present in the toxin molecule during the activation process which can occur 0 kDa wasC. difficile strains. The use of multiple proteases has been reported before not only to improve AA coverage, but also to differentiate homologous proteins, detect posttranslational modifications, and identify editing events [C. difficile AA coverage. However, since high-quality proteases are expensive, the use of multiple enzymes for each sample can be cost prohibitive. Working towards this, careful analysis of TcdA and TcdB AA sequence coverage maps from all five enzymes proved that using only two enzymes, trypsin and GluC, collectively garnered enough sequence information for most of our applications.The gel-independent approach provided unique information related to toxin quantification of small samples (<0.1 mL). The first set of experiments using BioS1 confirmed the robustness and efficiency of our modified rapid trypsin digestion method . While tg events –27. For Quantification of TcdA and TcdB in samples irrespective of the volume capacity has typically been performed using enzyme-linked immunosorbent assays (ELISA) –8, cytotE to separately quantify C. difficile toxins in small sample volumes. After assessing the MSE spectrum data, standard curves were constructed for TcdA and TcdB, and the minimum amounts detected were determined for each toxin. We report here the use of data-independent nUPLC-MSE concepts and applications has been published by others [E method has been reported. The authors found that there is a linear dynamic range of three orders of magnitude and low limit of quantification when they tested complex mixtures in small volumes [E methodology is expected to be very sensitive [E method, coupling it with other known toxin concentration methods such as antibody capture prior to MS analysis [A review of MSy others , 19. In  volumes . Since tensitive , the obtanalysis . E for the quantification of botulinum toxin complexes [C. difficile large toxins reported here led us to examine a commercial culture filtrate (CFil) to determine nUPLC-MSE utility for analysis of a complex C. difficile mixture. A proteomic analysis of CFil, normally used as a positive control in toxicity assays, revealed the identification of 101 unique C. difficile proteins, of which 24 could be quantified by MSE. The proteins identified are comparable with results from previous C. difficile proteomic studies [Previously, we have used nUPLC-MSomplexes , 22. The studies . Most imE. Even more, other proteins, such as glycine cleavage system protein H, isocaprenoyl-CoA:2-hydroxyisocaproate CoA-transferase, and molecular chaperone DnaK, that have not been previously cited in proteomic studies were also detected in lower but significant amounts. This finding suggests that culture supernatants may harbor a pool of specific proteins and could be a key matrix to search for potential unique C. difficile biomarkers. Because of the high TcdA and TcdB toxin concentration detected in the CFil, we initially hypothesized that the filtrate had been enriched by the manufacturer. However, we were assured by the manufacturer that the CFil used in these studies is indeed a filtrate of the culture supernatant of the strain VPI 10463, ATCC43255, which has not undergone any type of enhancement treatment, besides filtration, that would concentrate the protein pool present in this matrix . Interestingly, a recent publication describes the proteome examination of culture supernatants of three C. difficile strains, two of them hypervirulent isolates [Interestingly, label-free quantification of CFil identified four proteins  all detected in significantly large amounts. One possible explanation is that these proteins are traditionally abundant and thus would likely be detected in greater amounts. In addition, they are large proteins, which once digested likely have a larger peptide pool compared to other less abundant proteins, resulting in a greater chance for detection by nUPLC-MSisolates . The autC. difficile toxins. The overall goal was to determine the potential of proteomics using mass spectrometry-based methods to develop a quantification method for these large toxins. Moreover, the use of MSE for C. difficile toxin quantification in small samples, and for multienzymatic digestion to increase protein amino acid sequence coverage, was performed. The gel-based work revealed basic information on toxin integrity and provided sequence confirmation. The gel-independent platform was applied to in-solution digestion of both toxins using five different enzymes followed by analysis using two different mass spectrometer instruments. Broad amino acid sequence coverage for TcdA (91%) and for TcdB (95%) was generated using this approach. These data, if coupled to in silico sequencing analysis, suggest that the method has the potential to determine subtle sequence differences of TcdA and TcdB from different C. difficile toxinotypes. Moreover, label-free proteomics using MSE data collection and analysis provided the ability potential to determine the absolute quantity of TcdA and TcdB in small samples and was applied to a culture filtrate. A proteomic study of the culture filtrate demonstrated that the most abundant proteins are S-layer protein, TcdA, TcdB, and NAD-specific glutamate dehydrogenase. Taken together, data presented in this study provide performance characteristics and the basis for future development of improved MS-based detection and quantification methods in determining the factors involved in C. difficile toxin production by different isolates.Proteomic analyses using gel-based and gel-independent platforms were used to further characterize and quantitate the large


Our findings provide a rationale for the combined use of rapamycin and imatinib, both FDA approved drugs, for the treatment of TS.Tuberous sclerosis (TS) is a common autosomal-dominant disorder characterized by tumors of the skin, lung, brain, and kidneys. Monotherapy with rapamycin however resulted in partial regression of tumors, implying the involvement of additional pathways. We have previously implicated platelet-derived growth factor-BB in TS-related tumorigenesis, thus providing a rationale for a combination of mTOR/PDGF blockade using rapamycin and imatinib. Here, we test this combination using a well-established preclinical model of cutaneous tumorigenesis in TS, tsc2ang1 cells derived from a skin tumor from a mouse heterozygous for tsc2. Treatment of tsc2ang1 cells with a combination of rapamycin and imatinib led to an inhibition of proliferation compared with either vehicle treatment or treatment with rapamycin or imatinib monotherapy. Combination therapy also led to a decrease in Akt activation. Potent  TSC1) and tuberin (TSC2). In addition, neuro-developmental complications of TS, including seizures and autism, lead to significant morbidity. While no strict genotype-phenotype relationship has been established, the disease is more severe in patients with tsc2 mutations. Loss of heterozygosity (LOH), which deletes the unaffected allele, is required for the development of some neoplastic features of TS, including renal angiomyolipomas, lymphangiomyomatosis, and skin lesions, while LOH is not observed in brain tubers which often cause epileptic foci [TS is a common autosomal-dominant disorder characterized by the development of tumors of the brain, kidney, skin and lung. The disorder is characterized by mutations or deletions in one of two large genes, hamartin TSC and tubeTSC2) was weak or absent in angiomyolipomas, but present in healthy kidney, whereas, phosphorylated p70 S6 kinase and pS6 were present only in angiomyolipomas. Activation of a mammalian target of rapamycin metabolic pathway in tuberous sclerosis lesions, which contributes to their growth [The signaling pathways implicated in TS are complex. A hotspot for mutations in tsc2 involves regions implicated in controlling rheb, although multiple other signaling pathways have also been linked to TS-related neoplasia, including mTORC1, notch, p42/44 MAP kinase, NFkB, and Akt . Tuberinr growth ,13. Thesr growth .in vivo, and combined blockade might be more efficacious than rapamycin monotherapy. To test this idea, we used the FDA-approved drug imatinib (gleevec) to treat mouse tumors formed by implanted tsc2ang1 cells, isolated from a cutaneous sarcoma that arose in a tsc2 heterozygous mouse and a well-validated model of TS-related neoplasia. Imatinib functions as a specific inhibitor of a number of tyrosine kinase enzymes leading to downregulation of MMP-2 [www.gleevec.com). We demonstrate that addition of imatinib to rapamycin decreases the levels and phosphorylation of PDGFRβ. Consistent with this, combination treatment resulted in a greatly decreased phosphorylation of Akt, and Akt is a major determinant of tumorigenesis in vivo. Finally, we show that the combination of rapamycin and imatinib has a greater antitumor effect compared to vehicle alone than either rapamycin or imatinib compared to vehicle in vivo. These findings provide a rationale for combination therapy with rapamycin and imatinib in TS.We have previously demonstrated that platelet-derived growth factor β receptor (PDGFRβ) is present and active in human and murine TS lesions -17. Otheof MMP-2 . CurrentTo assess if rapamycin and imatinib combination was superior to either agent alone, we assessd their effects on the proliferation of Tsc2 ang1 cells. Treatment of cells for 72 h with rapamycin (5nM) or imatinib (10 μM) alone reduced the level of proliferation significantly compared with control . However, combined treatment with with both agents led to significantly better inhibition of proliferation , rapamycin (5nM) or their combination on the levels of phosphorylated PDGFRβ (at Tyr-1009) in Tsc2ang1cells. Combined treatment led to downregulation of PDGFRβ phosphorylation, , rapamycin (5 nM) or their combination and the levels of Akt and p Akt were measured by densitometry scanning of the signals on western blot membrane. Total levels of Akt were unchanged by treatment and served as loading controls. We found first that overall, treatment with drug led to significantly decreased phospho Akt compared to vehicle controls . In addition, we found that treatment with rapamycin and imatinib reduced the levels of pAkt  are essential for successful cell migration through extracellular matrix. In order to determine the levels of MMPs in our samples, conditioned media from equivalent numbers of imatinib- or rapamycin-treated cells along with that of control cells were collected and quantitatively analyzed using monospecific ELISAs. MMP-2 levels in conditioned media from tsc2ang1 cells treated with either imatinib p = .0243), rapamycin or combination treatment with rapamycin and imatinib (p = .0092) were significantly decreased compared to that of controls   Figure . Tumor vTS is a multisystem disorder characterized by benign or malignant neoplasia, as well as autism and seizures. No highly effective and long lasting therapy exists for TS neoplasia. Renal lesions such as angiomyolipomas may cause massive bleeding and compromise renal function, often requiring a kidney transplant. Lymphangiomyomatosis, a neoplastic complication most commonly seen in young women, can only be cured by lung transplantation. Deforming skin lesions cause significant psychological distress . FinallyThe lack of total blockade by rapamycin is consistent with the incomplete clinical response of tumors to systemic rapamycin, implying that either additional pathways are already present in these tumors, or are induced by rapamycin monotherapy. High among the candidates are the PDGFRβ mediated signal pathways, especially since mTORC1 activation and PDGFRβ signaling have been shown to have an inverse relationship .Treatment with combination of rapamycin and imatinib led to downregulation of PDGFRβ in tsc2ang1 cells, in a c-cbl-independent manner (data not shown). In vitro proliferation assays demonstrated an additive effect of rapamycin and imatinib on tsc2ang1 cells.in vivo. Given that the combination of these two approved drugs is well tolerated in vivo, and that rapamycin alone does not cause long-lasting remissions, this study provides a rationale for the combination of these two drugs in humans.Rapamycin has been used as monotherapy in patients with TS, resulting in benefit only as long as the patients are exposed to the drug . Our finTsc2ang1 (ATCC CRL 2620) is a murine cell line derived from a cutaneous sarcoma that arose in the extremity of a mouse heterozygous for tsc2; these mice develop cutaneous sarcomas at a frequency of 10 to 15%. The cells were cultured in complete DMEM medium supplemented with 10% FBS .10,000 tsc2ang1 cells per well were plated in 24-well dishes in triplicate. The next day, fresh medium containing the compounds or vehicle controls was added. Cells were incubated at 37° C for 24 h, and cell number was determined using a Coulter Counter .3VO4, and 1% aprotinin). Protein concentration in cleared lysates was determined using an Eppendorf BioPhotometer. Samples were resolved using SDS-PAGE  and transferred to nitrocellulose membranes. The membranes were blocked with 5% nonfat dry milk in 10 mmol/L Tris/0.1% Tween 20/100 mmol/L NaCl and incubated with primary antibodies followed by horseradish peroxidase–conjugated secondary antibody. The immunoreactive bands were visualized by enhanced chemiluminescence (Amersham Biosciences). The antibodies used were: Phospho-PDGFR- β antibody(Tyr 1021) ; Akt antibody (9272) , pAKT (4058) antibody  monoclonal anti-GAPDH antibody (Santa Cruz L-18 S-48167) was used as a loading control.Lysates of Tsc2ang1 cells treated with vehicle or indicated drugs were prepared in NP-40 lysis buffer . Specimens, standards and reagents were prepared according to manufacturer's instructions. Protein concentration was determined via the Bradford method using bovine serum albumin as the standard as described previously .in vivo activity of compounds that inhibit tsc2ang1 growth in vitro, groups of four nude mice per compound (or control). We injected 1 million Tsc2ang1 cells s.c. into 4 nude mice in each group. I.p. treatment with imatinib, rapamycin and imatinib and rapamycin were conducted for 30 days. Rapamycin and imatinib were obtained from LC Laboratories . Beginning 2 days later, the mice received daily i.p. injections of vehicle (control), rapamycin (12 mg/kg/day), imatinib (120 mg/kg/day) or imatinib plus rapamycin. The compounds were suspended in 0.1 ml of ethanol and 0.9 ml of Intralipid solution  [2 xL)0.52, where w(width) represents the shortest diameter of the tumor.To test the  Sweden) . No locaOne way ANOVA, and non parametric test were preformed for the tumor volume statistics. We did parametric analysis when the conditions for ANOVA were satisfied and in case where conditions are not satisfied and if the variables are not normally distributed, we conducted corresponding non parametric test and opted to present results for Wilcoxon test.United States Patent Application 20070078142 Inventor: Jack L. Arbiser, patent filed by Novartis Treatment of tuberous sclerosis associated neoplasms.BG,LW, ASC,MYB, and MGA performed experimental studies. SC and EV performed statistical analysis. HB, MAM and JLA designed experiments and wrote the manuscript.Animals studies were performed in compliance with the Emory IACUC.


Perfluoroalkyl substances (PFASs) are a group of highly persistent chemicals that are widespread contaminants in wildlife and humans. Exposure to PFAS affects thyroid homeostasis in experimental animals and possibly in humans. The objective of this study was to examine the association between plasma concentrations of PFASs and thyroid stimulating hormone (TSH) among pregnant women.th week of gestation. Linear regression models were used to evaluate associations between PFASs and TSH.A total of 903 pregnant women who enrolled in the Norwegian Mother and Child Cohort Study from 2003 to 2004 were studied. Concentrations of thirteen PFASs and TSH were measured in plasma samples collected around the 18Among the thirteen PFASs, seven were detected in more than 60% of samples and perfluorooctane sulfonate (PFOS) had the highest concentrations . The median TSH concentration was 3.5  μIU/mL. Pregnant women with higher PFOS had higher TSH levels. After adjustment, with each 1 ng/mL increase in PFOS concentration, there was a 0.8%  rise in TSH. The odds ratio of having an abnormally high TSH, however, was not increased, and other PFASs were unrelated to TSH.Our results suggest an association between PFOS and TSH in pregnant women that is small and may be of no clinical significance. Perfluoroalkyl substances (PFASs) are a group of man-made chemicals with a fully fluorinated hydrophobic linear carbon chain attached to a hydrophilic functional group . Being bThyroid hormones play an essential role in ensuring a healthy pregnancy and normal fetal growth and development. Fetuses depend on the mother’s supply of T4 entirely during the first trimester, and continue to rely on it to varying degrees throughout the pregnancy ,10. At bIn pregnant rats and their offspring, treatment with perfluorooctane sulfonate (PFOS) can cause a decrease in total plasma thyroxine (T4) and triiodothyronin (T3) -17. In hGiven the potential association between PFASs and thyroid hormones, concern arises about whether PFAS exposure may disrupt maternal thyroid hormone homeostasis during pregnancy. In the present study, our aim was to examine the relationship between PFASs and TSH as a screening measure of thyroid dysfunction in pregnant women.The Norwegian Mother and Child Cohort Study (MoBa) is a prospective population-based pregnancy cohort study conducted by the Norwegian Institute of Public Health . ParticiThe subjects in the present study were from a previous case–control analysis of subfecundity in a subset of MoBa women . These sBlood specimens were collected at enrollment , and thePlasma TSH measurements were performed on the same plasma specimen using an immunoassay  on the LiquiChip 200 Workstation  at the National Institute of Environmental Health Sciences, USA. Duplicate plasma TSH concentrations were measured for each sample and the mean was used in the analysis. The minimum detectable concentration was 0.01 μIU/mL. Both the intra-assay CV and inter-assay CV were <10%.vs 1.19 μIU/mL, student t-test P = 0.34), we combined them in the subsequent analysis. Scatter plots showed approximately linear relationships between PFASs and ln-TSH, thus we used linear regression models to examine PFASs and ln-TSH and applied weighted methods  to adjust for selection into the previous case–control study  = 4) and ranged from 18 to 44 years , and between PFOS and PFHpS . The median blood concentration of TSH was 3.5  μIU/mL.Among the seven most frequently detected PFASs, the median concentration was greatest for PFOS , followed by PFOA, PFHxS, PFNA, PFUnDA, PFHpS, and PFDA (Table P < 0.1). Among parous women, those who experienced longer inter-pregnancy interval had higher concentrations of PFASs, except PFHxS (P < 0.05). Most PFASs  were positively associated with seafood intake in times/week (P < 0.05).Based on bivariate linear regression models, all PFAS concentrations in nulliparous women were higher than in parous women (P = 0.03), with a slight attenuation in the coefficient (β). To express the coefficients as percent change in TSH , per ng/mL increase in PFOS, we calculated 100 × [exp (β) – 1]. For example, the adjusted β of 0.008 for PFOS means a 100 × [exp (0.008) – 1] or 0.8%  increase in TSH per ng/mL increase in PFOS.Among the seven PFASs, PFHpS, PFNA, and PFOS were positively associated with TSH levels Table . AdjustmNext, to examine the relationship between categorical PFASs and TSH levels we divided PFASs into quartiles and used the first quartile as the reference group. Figure vs < 7.5 μIU/mL) in logistic models, no significant association with any PFAS emerged, whether the latter was considered either as a continuous or categorical variable. In addition, there were no significant associations found between PFASs and self-reported thyroid abnormality.When TSH was dichotomized . Among women in the subfecund group, the association was inverse and non-significant .The precise classification of suboptimal thyroid status requires measurement of more than one hormone. For example, low free T4 with normal TSH reflects hypothyroxinemia . ElevateIn a study of 44 Korean pregnant women with serum measures of PFAS and several thyroid hormones in cord blood , inverseIn animal experiments, treatment with PFAS generally does not affect serum TSH levels ,34. WhenCompared with women from other western countries, the median concentrations of PFOS and PFOA in this study were slightly lower ,37,38. FWe found concentrations of PFOA and PFHxS were both correlated with PFOS, suggesting they might come from common sources. However, they were not associated with TSH levels statistically and PFOA showed a different direction of association. These substances were usually present lower at levels than PFOS, and have different binding capacities with transthyretin due to different structures .The observed association between PFOS and TSH was not present in the subfecund women. Although thyroid disorders are related to reduced fertility , we did Our study had several limitations. Unfortunately, we did not have access to sufficient plasma volume to perform any other measurements of thyroid hormones, such as total T4, free T4 and T3. TSH, however, is a sensitive marker of thyroid status and reflects the physiologic log-linear relationship of TSH to free T4 , and is Our results suggest an association between PFOS and TSH in pregnant women that is small and maybe of no clinical significance.BMI: Body mass index; CI: Confidence interval; CV: Coefficient of variation; HDL: High density lipoprotein; IQR: Inter-quartile range; LOQ: Limit of quantification; MoBa: The Norwegian Mother and child cohort study; PCBs: Polychlorinated biphenyls; PFASs: Perfluoroalkyl substances; PFBA: Perfluorobutanoic acid; PFDA: Perfluorodecanoic acid; PFDoDA: Perfluorododecanoic acid; PFHpA: Perfluoroheptanoic acid; PFHpS: Perfluoroheptane sulfonic acid; PFHxS: Perfluorohexane sulfonic acid; PFNA: Perfluorononanoic acid; PFOA: Perfluorooctanoic acid; PFOS: Perfluorooctane sulfonate; PFOSA: Perfluorooctane sulfonamide; PFTeDA: Perfluorotetradecanoic acid; PFTrDA: Perfluorotridecanoic acid; PFUnDA: Perfluoroundecanoic acid; T3: Triiodothyronine; T4: Thyroxine; TSH: Thyroid stimulating hormone.The authors declare that they have no competing interests.MPL developed the study design and conducted the study; YW performed data analysis and drafted the manuscript; WJR and APS participated in reviewing, editing, and revising the manuscript; LSH, ME, GB, and CT contributed to the assessment of PFASs; GT and DK contributed to the assessment of TSH; and JAH assisted in the biobank of MoBa. All authors read and approved the final manuscript.*.Background characteristics, pregnancy-related exposures, and pregnancy complications [N(%)] for all women giving birth in Norway, MoBa participants, and subjects in the present study Click here for file


Coronary artery disease (CAD) is one of the most common diseases leading to mortality and morbidity worldwide. There is considerable debate on whether serum transforming growth factor β1 (TGF-β1) levels are associated with long-term major adverse cardiovascular events in patients with CAD, and to date, no study has specifically addressed levels in patients with different degrees of CAD severity.Serum TGF-β1 and mothers against decapentaplegic homolog 3 (SMAD3) concentrations were evaluated in 279 patients with CAD and 268 controls without CAD. The clinical and biochemical characteristics of all subjects were also determined and analyzed.TGF-β1 and SMAD3 concentrations in CAD patients were significantly higher than those in the controls. The serum TGF-β1 level in acute myocardial infarction (AMI) was significantly higher than that in both stable angina pectoris (SAP) and unstable angina pectoris (UAP) (p < 0.05), while there was no marked difference between levels in SAP and UAP (p > 0.05). SMAD3 levels showed no obvious difference among AMI, SAP, and UAP. TGF-β1 and SMAD3 are potential biomarkers for CAD, and may be more accurate than Lpa, ApoA1, uric acid, BUN, or triglycerides (TG).Serum TGF-β1 and SMAD3 levels are closely associated with CAD, and may become useful biomarkers for diagnosis and risk stratification. Coronary artery disease (CAD) is one of the leading causes of mortality and morbidity worldwide, especially in many developed countries,2. AccorA number of inflammatory cytokines mediate adverse cardiovascular events in patients with CAD, and transforming growth factor-β1 (TGF-β1) has garnered particular attention because of its multiple roles in important pathological changes such as enhancement of macrophage and fibroblast chemotaxis, stimulation of extracellular matrix (ECM) synthesis, and vascular cell proliferation abnormalities,10. AlthWe collected serum samples from 279 patients with CAD and from 297 controls, and assessed for any association between serum TGF-β1/SMAD3 levels and the presence and severity of CAD. We evaluated the potential clinical application of measurements of these cytokines for CAD diagnosis, comparing their utility with the currently used biochemical indicators.Written informed consent was obtained from the population involved in this study and the study protocol was approved by the Ethics Committee of Affiliated Hospital of Guangdong Medical College.A total of 279 patients who were referred for coronary angiography by their attending physicians from May to August 2013, and who were found to have at least 50% stenosis in at least one coronary artery, were included. Their ages were about 69.30 years. The 268 controls were consecutive subjects undergoing routine medical examinations at the Physical Examination Center. Those who had any clinical manifestations or a medical history of heart disorders, a family history of coronary artery disease, or abnormal ECG were excluded. Exclusion criteria also included acute or chronic infections or inflammatory diseases, severe hepatic or renal dysfunction, malignant tumors, or hematologic disorders. The CAD group was divided into three subgroups: acute myocardial infarction (AMI), unstable angina pectoris (UAP), and stable angina pectoris (SAP).All study participants underwent a standard clinical examination. Body mass index (BMI) was calculated as the individual's body mass divided by the square of their height. Smoking was defined as having smoked for at least 1 year currently. Hypertension was defined as a systolic blood pressure of ≥ 140 mm Hg or use of antihypertensive therapy. Diabetes was diagnosed according to nonfasting glucose levels ≥ 11.1 mmol/L.Approximately 2 mL of venous blood was collected from each subject, and then kept in tubes without preservatives at 4°C overnight. Serum aliquots were obtained after centrifugation at 1000 g for 40 min at 4°C.All coronary angiographies were performed using standard technique. Significant CAD was considered to be present if there was an internal luminal stenosis ≥ 50% in the left main coronary artery, right coronary artery, and/or their major branches. The degree of coronary atherosclerosis was further categorized according to the number of coronary vessels with significant stenoses as 1-, 2-, or ≥ 3-vessel disease. All catheterizations and imaging analyses were performed by two experienced interventional cardiologists who were unaware of the patients’ clinical data.Fasting blood samples were obtained and prepared for the biochemical assay of clinical indices associated with CAD diagnosis, including lipoprotein (a) (Lp(a)), blood urea nitrogen (BUN), uric acid (SUA), blood glucose (Glu), total cholesterol (CHOL), high-density lipoprotein (HDL), low-density lipoprotein (LDL), triglycerides (TG), apolipoprotein A1 (ApoA1), and apolipoprotein B (ApoB).Determination of serum active TGF-β1 and SMAD3 concentration was performed by ELISA using the ELISA-Quantikine kit  according to the manufacturer’s instructions. The detection range of the ELISA used to measure TGF-β1 was 100–1200 ng/L, and the detection limit of SMAD3 was 2.5–30 pg/mL. Their accuracy was expressed in the correlation coefficient, which was required to be greater than 0.9999.Categorical and continuous variables were expressed as percentiles and mean ± standard error of mean (SEM), respectively. Differences among groups were assessed using the chi-squared or analysis of variance tests, followed by post-hoc analysis (Bonferroni’s correction) for comparison among groups. The association between serum TGF-β1 or SMAD3 and CAD or its risk factors was estimated using a multivariate logistic regression model.The main demographic and clinical characteristics of all the studied subjects are summarized in TableThere were significant differences between CAD and control groups in important risk factors including age, BMI, smoking, hypertension, DM, atrial fibrillation (AF), stroke, TG, HDL, Lp(a), BUN, uric acid, ApoA1, and ApoB. CAD patients were divided into three subgroups by pathological types: AMI, SAP, and UAP Table. The staTGF-β1 and SMAD3 levels were both increased significantly in the CAD group compared with the controls (p < 0.0001) , while there was no marked difference between those in SAP and UAP (p > 0.05) Figure. HoweverTo explain a potential mechanism of TGF-β1 and SMAD3, we evaluated their relationship with CAD and its risk factors including Lp(a), BUN, creatinine, uric acid, blood glucose, total cholesterol, TG, HDL, LDL, ApoA1, and ApoB. There was a very significant correlation between CAD and concentration of TGF-β1 and SMAD3 (p < 0.0001). Among the other biochemical indices, TGF-β1 ROC) indicated that both TGF-β1  and SMAD3  were the more potent biomarkers for CAD, compared with Lpa , ApoA1 , uric acid , BUN , or TG   compared with controls . The area under the Receiver operating characteristic curves  and uric acid, which both are considered to be markers of atherosclerosis, indicating that this cytokine may contribute to the establishment of CAD by regulating atherogenesis. SMAD3 levels correlated closely with blood glucose, implying that SMAD3 may influence CAD occurrence and the development of DM. Interestingly, it has been reported that risk factors including Lp(a), uric acid, BUN, triglycerides, and ApoA1 can be useful biomarkers for different clinical diagnoses,32, but Serum TGF-β1 and SMAD3 levels are closely associated with CAD. The underlying mechanism may be their regulatory effects on atherosclerosis and blood sugar.A limitation of the present study is that the control population differs from the cases, in terms of age and cardiovascular risk factors. The mean age difference (~5 years) should be acceptable in an epidemiologic study of CAD according to a previous report,9. CAD pThe authors had no conflicts of interest to declare in relation to this article.WL and CC performed the study and wrote the manuscript. WC, JZ, and WL performed the study and/or contributed to data analysis and interpretation. XG, BL, and HW reviewed/approved the research protocol. CH takes full responsibility for the work as a whole, including the study design, access to data, and the decision to submit and publish the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2261/14/18/prepub


An association between low birth weight, insulin resistance (IR), dyslipidemia, and atherogenesis has been shown in girls with precocious adrenarche (PA).To evaluate whether girls with PA born appropriate for gestational age (AGA) have increased risk for metabolic complications at initial evaluation.s.d.) age 7.1±1 years) and 45 body mass index (BMI)- and waist circumference (WC)-matched prepubertal peers born AGA (mean (±s.d.) 7.5±1.9 years). A standard 2-h oral glucose tolerance test with insulin sampling was performed. Fasting plasma lipids and high-sensitivity C-reactive protein were analyzed, and blood pressure was recorded. Insulin sensitivity (IS) index (ISIcomp), homeostasis model assessment of IR, and atherogenic index (AI) (triglycerides/high-density lipoprotein cholesterol) were calculated.We conducted a cross-sectional study on 69 AGA born girls with PA , and it was correlated with BMI SDS  and WC . The significant correlation of AI with ISIcomp  disappeared after correcting for BMI . Multivariate regression analysis revealed that DHEAS was the only significant parameter influencing AI in girls with born AGA .AI was significantly lower in girls with PA than in controls (Metabolic screening in prepubertal AGA born girls with PA may yield favorable lipid profiles. AI in girls with PA is increased in relation to decreasing IS and increasing BMI and WC. DHEAS seems to have the most significant effect on AI. Precocious adrenarche (PA) refers to the concurrent presence of adrenal androgen levels increased above the age- and sex-specific range and clinical signs of an increased androgen action before the age of 8 years in girls in vitro studies with human monocyte-derived macrophages (MDMs) due to differences in androgen receptor densities between sexes DHEAS, the major biochemical hallmark of adrenarche, has been considered as an antiatherogenic steroid, though unproven We hypothesized that PA in girls born appropriate for gestational age (AGA) may not be a risk factor for atherosclerosis when intrauterine growth restriction, changes in body mass index (BMI), and waist circumference (WC) as reliable indices of adiposity are excluded, as these are well-established risk factors for atherosclerosis in non-PA children as well. In order to test our hypothesis, we carried out a cross-sectional study by evaluating girls with PA born AGA and BMI- and WC-matched prepubertal peers born AGA.n=22) or were diagnosed with nonclassical congenital adrenal hyperplasia (n=1), central puberty (n=1), or adrenocortical carcinoma (n=1). Forty-five BMI- and WC-matched peers born AGA were recruited consecutively from prepubertal girls followed in our Well Child Care Clinic. Data related to family history of CAD were retrieved from medical records. The controls enrolled in the study had no chronic disease and there was no history of CAD in three successive generations. Written consent was obtained from parents and assent was obtained from children over the age of 7 years. The study was approved by the Local Ethics Committee.The study group comprised 114 girls. For the girls with PA, the inclusion criteria were presence of pubic/axillary hair before the age of 8 years. Excluded from the study were girls who were born small for gestational age (SGA), children with chronic disease, genetic syndromes, congenital adrenal hyperplasia, and other endocrine abnormalities. Between November 2010 and January 2012, all eligible girls attending our pediatric endocrine clinic were invited to participate in the study. Among 94 girls with premature onset of pubic/axillary hair, after exclusion of steroidogenic enzyme defects and virilizing tumors by biochemical analyses (standard ACTH test) and adrenal ultrasonography, 69 (73.4%) were found to be eligible for inclusion to our study. The rest of the girls were either born SGA  using a calibrated Harpenden Stadiometer and an electronic scale (sensitivity at 0.1 kg level) respectively. BMI was calculated using the formula weight/(height)comp) (Matsuda index=10 000/square root of ((fasting glucose×fasting insulin)×(mean glucose×mean insulin during OGTT))) Fasting blood samples for glucose and lipids , low-density lipoprotein cholesterol, and triglycerides (TGs)), serum insulin, LH, FSH, DHEAS, total testosterone, and high-sensitivity C-reactive protein (hs-CRP) were drawn between 0830 and 1000 h, after an overnight fast. An oral glucose tolerance test (OGTT) was performed by administering 1.75 g/kg glucose (maximum 75 g) to each subject with samples for glucose and insulin analyses taken at 30, 60, 90, and 120 min. Determination of glucose and lipids was conducted on the same day. After separation, serum samples were frozen immediately and stored at −80 °C until assayed. Serum glucose levels over 99 mg/dl were accepted as impaired fasting glucose (IFG) Evaluation of lipids included estimation of atherogenic index  The assays were routinely performed by a semiautomated Immulite System and commercial kits obtained from Diagnostic Products Corporation , were used. Serum glucose was measured using an automated glucose oxidase method. Serum LH and FSH concentrations were measured by immunochemiluminometric assay. DHEAS and testosterone levels were measured by RIA and insulin levels by ECL assay. hs-CRP levels were measured using a hs-CRP assay . The sensitivity of the assay ranged from 0.04 to 5.0 mg/l. LH and FSH assays had intra-assay coefficient of variations (CV) 4.8 and 7.5% and interassay CV of 10.7 and 5.4% respectively. Sensitivity of the testosterone assay was 0.05 ng/ml, the intra-assay CV was 4.5%, and the interassay CV was 6.4%. Values for the DHEAS assay were 4.5% (intra-assay CV) and 5.5% (interassay CV). Sensitivity of the insulin assay was 0.05 mU/l. Intra-assay and interassay CV were <5%. Plasma lipids were measured by Integra-800 autoanalyzer (Roche).Girls with PA and controls were compared with respect to anthropometric and laboratory data. The presence of significant correlations between anthropometric data, plasma lipids, hs-CRP, HOMA-IR, IS index, and hormonal measurements within each study group was assessed.s.d. or median (interquartile range). Student's t-test was used for comparison of variables between groups, and nonparametric tests were used if skewed variables do not have Gaussian distribution after transformation. Comparison of categorical variables was performed using χ2 test and Fisher's exact test. Relative risk (RR) was used to calculate the risk coefficient of significant quantitative data with 95% confidence intervals (95% CIs) given at a statistical significance of P<0.05. Correlations between continuous variables were evaluated using Pearson's correlation analyses. In controls, the nonlinear correlations between age and serum DHEAS and testosterone concentrations were calculated and graphically demonstrated by nonlinear regression analysis using GraphPad Prism Software . Multivariate regression analysis tested the independent association and contribution of changes in BMI, WC, IS index, serum DHEAS, hs-CRP, and OGTT-derived parameters with the dependent variable (changes in AI). Before regression analysis, variables that did not follow a normal distribution  were log transformed. The results were evaluated at the 95% CI and significance was granted for P<0.05. All analyses were performed using SPSS 15  and GraphPad Prism 5 Software.Normality of the variables was tested by D'Agostino and Pearson's Omnibus normality test. The results are presented as mean±P>0.05). Six of the girls with PA were older than 8 years at evaluation (range 8.3–9.2 years). The median age of the girls with PA at onset of pubarche and at evaluation was 6.6 years  6–7.2 years) and 7.1 years  respectively. BW SDS were similar in both groups as we expected , but BMI SDS and WC were similar between the groups (P>0.05), as planned in the study design (P=1), and abdominal obesity according to WC values was present in 11.6 and 15.5% of the subjects respectively (P=0.58). BA SDS was significantly higher in girls with PA than in controls .SDS values for weight and height in girls with PA were significantly higher than those of the controls . HOMA-IR was significantly higher in girls with PA than in controls (P<0.05). However, at a cutoff level of 2.5, the groups were not significantly different with respect to the presence of IR (P>0.05). ISIcomp was significantly lower in girls with PA than in controls (P=0.0009).The comparison of OGTT findings in girls with PA and controls is depicted in P>0.05; P<0.0001; P=0.0016). Serum TG concentrations ≥110 mg/dl were found in 4 (5.7%) girls with PA and 9 (13%) controls. The risk for having serum TGs ≥110 mg/dl was higher in controls than in the girls with PA (RR=2.1 (95% CI), 0.9–4.8; P=0.032). AI was significantly lower in girls with PA than in controls (P<0.001). When we compared AI levels in girls with PA having history of CAD in family with those who had no such history, the differences in AI levels between the subgroups were not significant .hs-CRP concentrations were similar in girls with PA and controls P>0.05; . HDL-C cr=0.600, P=0.0001; r=0.470, P=0.004; r=0.441, P=0.001 respectively), HOMA-IR , ISIcomp , serum TGs , HDL-C , and hs-CRP . Age at evaluation and the anthropometric measurements did not significantly correlate with serum DHEAS and testosterone concentrations (data not shown). Correlations of AI with clinical data and laboratory-related parameters are depicted in comp  disappeared after correcting for BMI SDS and weight SDS . Multivariate regression analysis including variables depicted in R2 =0.475, β=−0.018, P=0.0001).Weight SDS, BMI SDS, and WC had significant correlations with fasting serum insulin . Serum DHEAS and testosterone concentrations significantly correlated with age . Indeed, HDL-C concentrations in children have been inversely correlated with the incidence of CAD in the different countries studied R2=0.475, β=−0.018, P<0.0001). The contribution of reduced IS to increased AI disappeared after correction for changes in BMI and WC, indicating the utmost importance of change in body fat in atherosclerosis. The mechanism by which DHEAS may account for decrease in AI is not clear in our study. In vitro experiments may not represent the same conditions as in the arterial wall in vivo. In the in vivo context, the ‘net’ vascular biological effect of DHEA may be the summation of both direct and indirect effect via androgenic and estrogenic metabolites. We speculate that although adrenal androgen concentrations in girls with PA are higher than their BMI- and WC-matched peers, they cannot exert their proatherogenic effect in girls due to sparsity of androgen receptors on female human MDMs and also due to aromatization of the slightly increased androgens to estrogens and their metabolites in the periphery takes place. We also speculate that this seemingly unfavorable androgen profile in girls with PA when compared to BMI- and WC-matched peers is probably overcome by the presence of androgen-insensitive female MDMs and conversion of these ‘macrophage inert’ androgens to estrogens in the periphery, giving rise to the endocrine paradox.A novel finding of our study is that prepubertal girls with PA born AGA have reduced AI when compared with BMI-, WC-, and BW-matched controls. Girls with PA had significantly higher serum HDL-C concentrations and lower serum TG concentrations when compared with controls (The novelty of the methodology chosen in recruitment of subjects in our study is that we excluded girls with PA born SGA as it is postulated that a transient prenatal growth constraint in children born SGA may be followed by a permanent resetting of endocrine axes. Meta-analysis of all published studies revealed that low BW is indeed associated with type 2 diabetes, which is linked to atherosclerosis and metabolic syndrome Although we found that girls with PA had lower IS and higher MSI concentrations during OTT, they were not found to be more IR than controls unlike the findings in Catalonian girls, in which the presence of dyslipidemia, IR, and atherogenesis was linked to reduced fetal growth et al. We measured WC of all the girls to evaluate abdominal obesity as it is a strong correlation of total body fat hs-CRP has been shown to be a good marker of cardiovascular disease in obese children and adolescents In addition to the strengths of our study outlined earlier, three limitations of our study merit comment. First, all the girls in our study were prepubertal, and it can be speculated that metabolic changes may be observed later in the process and not necessarily at the time of diagnosis as our study is cross-sectional. However, clinical studies in children have demonstrated that an atherogenic pattern of risk factors can start in childhood In vivo studies involving end-products of peripheral metabolism of DHEAS will probably highlight this endocrine paradox. Whether the favorable impact of PA in AGA born girls on atherogenesis persists in puberty and adulthood remains to be elucidated.Our preliminary results suggest that metabolic problems in girls with PA born AGA be related to changes in adiposity. PA may be an independent negative risk factor for atherogenesis at initial assessment when compared with non-PA subjects having similar adiposity. Reduced atherogenic indices in girls with PA born AGA may be related to increased secretion of DHEAS from the adrenals.