Aim. This paper aimed to examine the chemoprotective actions of aqueous black tea extract (BTE) against nonalcoholic steatohepatitis- (NASH-) induced skeletal changes in rats. Material. Wistar rats (body wt. 155–175 g) of both sexes, aged 4–5 months, were randomly assigned to 3 groups; Group A (control), Group B , and Group C (HFD + 2.5% BTE). Methods. Several urinary  serum , and molecular markers of bone turnover (receptor activator of NF-κB ligand (RANKL), osteoprotegerin (OPG), and estrogen) were tested. Also, several bone parameters  and calcium homeostasis were checked. Results. Results indicated that HFD-induced alterations in urinary, serum, and bone parameters as well as calcium homeostasis, all could be significantly ameliorated by BTE supplementation. Conclusion. Results suggest a potential role of BTE as a protective agent against NASH-induced changes in bone metabolism in rats. Nonalcoholic steatohepatitis (NASH) is a condition, which is described as accumulation of fat in liver with inflammation. This disorder may be observed in patients with no history of significant alcohol consumption, though histological examination may resemble alcohol-induced liver injury. It indeed may be the first clinical indication of insulin resistance, with complication of high blood pressure, coronary heart disease (CHD), and type 2 diabetes , 2. It iCamellia sinensis) is rich in polyphenolic compounds, collectively known as the tea flavonoids. Its therapeutic value come forefront with numerous reports on tea as antioxidants, hypolipidemic, antineoplastic, and hypocholesterolemic [Tea , Ministry of Environment and Forests, Government of India. Upon arrival at our institute, all animals were kept under standard conditions  for a week with free access to drinking water and standard laboratory diet composed of 71% carbohydrate, 18% protein, 7% fat, and 4% salt mixture . All aniEighteen animals were randomly assigned to three groups: Group A (regular diet), Group B (high-fat diet), and Group C (high-fat diet + BTE). Each group contained six animals, three male and three female. Group A animals were fed standard laboratory diet  containiCamellia sinensis) extract was prepared from CTC  BOP- (Broken Orange Pickoe-) grade black clonal tea. It was processed and supplied by Tocklai Experimental Station, Jorhat, Assam, India to the Drug Development Division, Indian Institute of Chemical Biology, Jadavpur, Kolkata, India. We received a generous gift from that institute. A fresh 2.5% aqueous BTE was prepared everyday following the method of Wei et al. [The black tea  of high fat diet (HFD) and black tea extract (BTE), dose response studies were undertaken. In case of high fat, it was observed that although significant changes could be seen from 30% of high fat onward, but both biochemical and histological changes were most prominent with 60% HFD. Similar changes with 60% HFD were reported earlier . As for Fasting urine was collected for 24 hours (9 AM to next day 9 AM) according to the standard laboratory procedure  as descrμL of urine 1 mL CPC reagent and 1 mL DEA buffer was added. Colour intensity was measured at 575 nm against a blank by using a UV-double beam spectrophotometer . Calcium carbonate (CaCO3) solution pH-3.0 was used as standard. All the reagents were prepared with calcium-free triple distilled water.Urinary calcium was measured according to the method of Adeniyi et al. . The assμL of urine was diluted 50 times with distilled water and to it 1 mL molybdate reagent and 200 μL amino naphthol sulfonic acid (ANSA) was added, mixed well, and kept in dark for 10 minutes. Reading was taken at 630 nm against blank. The concentration of phosphate in urine was calculated using a standard curve of monopotassium phosphate (KH2PO4).Urinary phosphate was measured according to the method of Lowry and Lopez . For uriμL of urine was diluted 10 times and to it 2 mL saturated picric acid and 150 μL 10% sodium hydroxide solution were added, mixed and kept for 15 minutes at 37°C incubator. To it, 5 mL of water was added. Reading was taken at 520 nm against blank. The concentration of creatinine in urine was calculated using a standard curve of creatinine.Urinary creatinine was measured according to the method of R. L. Nath and R. K. Nath . For uriBlood was collected directly from the heart under urethane anesthesia (1.7 mg/g body weight). Serum was obtained by using standard laboratory protocol.Serum alkaline phosphatase was measured using p-nitrophenyl phosphate as substrate . Alkalinα-naphthyl phosphate, 6 mM Fast Red TR, and 2 mM sodium tartrate in 50 mM sodium citrate buffer, pH-5.2) and the absorbance of the sample was read at 405 nm at 1 minute interval thereafter for 3 minutes. The difference of absorbance and the average absorbance difference per minute (ΔA/min) was calculated [Serum TRAP activity was estimated by kinetic method by using reagent kit . Briefly, unhemolyzed serum was mixed with the test reagent (10 mM lculated .Serum was obtained by using standard laboratory protocol. Serum estrogen level (pg/mL) was determined by using the ELISA EIAgen Estradiol kit . All samples were assayed in duplicate. The intra-assay coefficient of variation was 9.08%. To avoid inter-assay variation all samples were run at one time.Serum RANKL was estimated by using the ELISA kits . All samples were assayed in duplicate. The intra-assay variation was 11.75%. All samples were run at one time to avoid inter-assay variation. Optical density of each well was determined by using a microplate reader .Serum OPG was estimated by using the ELISA kits . All samples were assayed in duplicate. The intra-assay variation was 10.09%. All samples were run at one time to avoid inter-assay variation. Optical density of each well was determined by using a microplate reader .3 bone volume).The right femur, eighth thoracic rib, eighth thoracic vertebra, and fourth lumbar vertebra were freed off soft tissue and cleaned using small scissors, tweezers, and cotton gauze. Before measurement of the density, femur was cut at the middiaphysis and the marrow was washed out. Bone density was measured according to the method described by Arjmandi et al.  by usingAfter sacrifice of the animal, left femur was excised and cleaned off adhering soft tissues. Bone tensile strength was measured as described by Shapiro and Heaney  using a Right femur, eighth thoracic rib, eighth thoracic vertebra, and fourth lumbar vertebra were removed and cleaned of adhering tissue. The whole bone was extracted two times with a 1 : 1 mixture of ethanol and diethyl ether for 48 hours and one time with diethyl ether for 24 hours. The dehydrated and defatted bones were turned into ash at 600˚C for 48 hours and hydrolyzed in 6 N HCl for determination of calcium and phosphate . CalciumAfter the experimental period was over, the animals of all groups were fasted for 16 hours. The preparation of animals and intestinal loops for the study of calcium transference in situ was made by following the method as described elsewhere by Islam et al. . Briefly2 was injected with a 25-gauge needle in each ligated segment. The intestinal loops were placed in their usual position and the abdomen was closed. After one hour, animals were sacrificed, the preselected loops were removed and the fluid from each loop was collected separately, together with a few washings of the lumen with triple distilled water. The collected fluid was then increased to a definite volume with calcium free triple distilled water. A fraction of this fluid was then used for the estimation of Ca2+ using a double beam spectrophotometer  according to the method described by Adeniyi et al. [2+ introduced and the amount left unabsorbed was used to estimate the amount of Ca2+ absorbed. The intestinal part constituting the loop was dried on a watch glass in an electric oven at 90°C to attain a constant weight, which was recorded as the weight of the dried loop. Intestinal transference of calcium was expressed as μM of calcium/g of dry weight/hour.For the measurement of intestinal calcium transference, 1 mL of prewarmed (37°C) Tris-HCl buffer solution containing 0.2 mM CaCli et al. . The difAfter sacrificing the animal and opening of the abdomen, the whole of the small intestine was quickly removed. The portion comprised of the duodenum, jejunum, and ileum were separated and chilled in ice. Intestinal mucosa was collected as described by Maenz and Cheeseman , and theThe activity of alkaline phosphatase of intestinal mucosa was estimated by using p-nitrophenyl phosphate as substrate . The pro2+-ATPase was also studied from the mucosal extract using the method of Rorive and Kleinzellar [2+ or Mg2+. For the assay of mucosal Ca2+, activated ATPase, 250 μL 0.4 M Tris-HCl buffer pH-7.4 and 250 μL 40 mM ATP  were added into three of the test tubes placed on ice. To tubes 1 and 3, 250 μL of 40 mM CaCl2 were added. At time zero, the reaction was started by addition of 250 μL of mucosal homogenate to tubes 1 and 2. The volumes in all tubes were adjusted to 2 mL with calcium free distilled water. The three tubes were then incubated at 37°C with gentle shaking for 30 minutes. Under this condition the release of Pi is linear unto 60 mins. The reaction was stopped by transfer of the tubes to ice and addition of 0.4 mL ice-cold 35% (w/v) trichloro acetic acid (TCA). The tubes were then centrifuged for 10 minutes at 10,000 rpm in a refrigerated centrifuge. The protein pellet was dissolved in 1 mL of 1 N sodium hydroxide (NaOH), and protein content was determined following the method of Lowry et al. [2+ ATPase enzyme activity was estimated by the method of Lowry and Lopez [The activity of the enzyme Cainzellar . The assy et al. . Phosphand Lopez .μm sections and stained with haematoxylin-eosin. Representative sections were observed and photomicrography was performed with the help of a bright field microscope equipped with a digital camera .The left proximal tibia and fourth lumbar vertebra were removed, dissected free of soft tissue, and fixed with 4% paraformaldehyde for 16–18 hours at 4°C. After fixation, specimens were washed for 12 hours at 5°C in each of the following series of solutions: 0.01 M PBS containing 5% glycerol, 0.01 M PBS containing 10% glycerol, and 0.01 M PBS containing 15% glycerol. The specimens were then decalcified in EDTA-G solution  for 10–14 days. The decalcified tissues were washed sequentially at 5°C for 12 hours in (a) 15% sucrose and 15% glycerol in PBS, (b) 20% sucrose and 10% glycerol in PBS, (c) 20% sucrose and 5% glycerol in PBS, (d) 20% sucrose in PBS; 10% sucrose in PBS, (e) 5% sucrose in PBS, and (f) 100% PBS. Then the tissues were washed with PBS and dehydrated in graded series of alcohols, followed by clearing in xylene, and finally embedded in paraffin . The speP < .05.Data were expressed as mean ± S.E.M. obtained from a particular group comprising both male and female animals. Data were compared using Kruskal-Wallis nonparametric ANOVA followed by Mann-Whitney “U” multiple comparison test . Differences were considered significant if P < .01). Elevated response of all these parameters was significantly counter regulated in the HFD + BTE-supplemented group of rats (Group C).The urinary calcium, phosphate, and creatinine excretion profiles together with Ca : Cr ratio of control group (Group A), HFD (Group B), and HFD + BTE (Group C) are shown in P < .01) (Group A). This increase in AP activity was significantly lowered (P < .01) in rats receiving BTE (Group C). Likewise, the significant increase (P < .01) in TRAP activity in HFD-fed group (Group B), compared to control (Group A), could be effectively reduced by aqueous BTE supplementation  activity profile of rats of control, HFD, and HFD + BTE supplemented groups are shown in Group C; .P < .01), eighth thoracic rib (P < .01), eighth thoracic vertebra (P < .01), and fourth lumbar vertebra (P < .01), compared with the control group (Group A). BTE supplementation could produce significant increase (P < .01) in bone density of all bones: right femur (P < .01), eighth thoracic rib (P < .01), eighth thoracic vertebra (P < .05), and fourth lumbar vertebra  had significantly lower densities of the right femur . BTE supplementation in these animals was found effective in recovering (P < .01) this tensile strength back to control group level.P < .01; phosphate: P < .01), eighth thoracic rib , eighth thoracic vertebra , and fourth lumbar vertebra . When these HFD-fed animals were supplemented with BTE (Group C), significant recovery in content of both minerals was seen.Results of bone calcium and phosphate levels are shown in P < .01). This could be recovered significantly (P < .01) when BTE was supplemented (Group C).P < .01) level, which was found significantly (P < .05) counter regulated on BTE supplementation. In contrast, compared to control, serum RANKL level was increased significantly (P < .01) in HFD-fed animals. On BTE supplementation, a significant decrease (P < .01) in the RANKL level was noted.P < .01 for duodenum, jejunum, and ileum). BTE supplementation could significantly correct such alterations in calcium transference in HFD-fed rats .Results in P < .01 for duodenum, jejunum, and ileum), while BTE supplementation could significantly increase this enzyme activity .P < .01 for duodenum, jejunum, and ileum). BTE supplementation was found significantly effective in restoring this enzyme activity in all these segments .in vivo effects of BTE in the HFD-fed animals. HFD-fed rats (Group B) showed a decrease in cortical thickness and empty bone marrow space at the proximal tibia  against HFD fed (60%) NASH skeletal changes. Bone manifestations are well-known as extrahepatic complications of chronic liver disease. Patients with chronic liver disease are at increased risk of developing hepatic osteodystrophy manifested as osteomalacia or osteoporosis . OsteopoRecent research also suggests that there is a strong positive correlation between osteoporosis and hyperlipidemia. Excess lipid consumption may lead to lipid accumulation and subsequent oxidation within bone vasculature, wherein osteoblastic differentiation may be altered. In addition to their inhibitory role in osteoblastic differentiation, it is believed that because oxidized lipids induce endothelial expression of monocyte chemotactic factors, as well as other potent inducers of osteoclastic differentiation, oxidized lipids may also promote bone resorption .Biochemical markers of bone turnover have been widely used as research tool to measure the effects of drugs on bone remodeling . ElevateResults of studies of two other specific markers of bone turnover, namely, urinary calcium-to-creatinine (Ca : Cr) ratio and serum alkaline phosphatase (AP) further support our speculation Tables , resp.. Such speculations were cross-examined in our studies by measuring parameters like bone density  and boneChronic liver disease accelerates the development of hypogonadism due to both reduced hypothalamic release of gonadotrophins and primary gonadal failure. A decline in circulating estrogen may be a mediator of bone loss in women and men with chronic liver disease . In the β (RANK), stimulates osteoclastogenesis and osteoclastic bone resorption. Factors that stimulate bone resorption increase RANKL expression and, with some exceptions, decrease OPG expression [Osteoprotegerin (OPG) is a novel receptor that blocks osteoclast formation. Either in a cell membrane-bound or in a soluble form, RANKL, receptor activator of NF-kappa pression . In the 2+-ATPase, has been proposed frequently in this phenomenon because the activity of these enzymes correlates with the degree of calcium absorption in different parts of intestinal tract under different circumstances [2+-induced ATP hydrolysis in the intestine is the result of two enzymatic activities, namely, alkaline phosphatase present in brush border as well as basolateral membranes and a more specific Ca2+-ATPase exclusively located in basolateral plasma membrane [2+ was reduced in HFD-fed rats. Result indicated that deficiency of estrogen had a negative influence upon intestinal transference of calcium, as these animals showed a greater degree of decrease in the transference of calcium than the control animals (in vitro and in vivo [Calcium absorption has been reported to occur in body throughout the length of the small intestine, the absorption being greater in the duodenum and proximal jejunum than in the ileum . The invmstances , 71, 72.membrane . Evidencmembrane , 75. In  animals . Such ob in vivo , 77. BTETo ascertain the mechanism of such decrease in intestinal transference of calcium, the activities of two most relevant mucosal calcium transferring enzymes, namely, alkaline phosphatase and calcium-ATPase were examined. Activities of both of these enzymes were found inhibited in HFD-fed group of animals Figures  and 6. THistological  observatIt is recognized that animal studies contain some inherent design limitations. First, restrictions of animal use and therefore sample size; second, the doses required for demonstrating the disease prevention effects are usually higher than the amounts consumed by humans. Furthermore, because of infrastructure limitations it was necessary to rely on biochemical and histological measures rather than using peer reviewed DEXA-derived indices for examining bone profiles. To minimize the error associated with such limitations, we took extreme care while selecting the parameters, collecting data and analyzing them by well-recognized statistical methods followed by us , 35 earlIn conclusion, results of this study strongly suggest a protective role of BTE against NASH-induced changes in bone metabolism in rats.


Background: Although cadmium and lead are known risk factors for hearing loss in animal models, few epidemiologic studies have been conducted on their associations with hearing ability in the general population.Objectives: We investigated the associations between blood cadmium and lead exposure and hearing loss in the U.S. general population while controlling for noise and other major risk factors contributing to hearing loss.Methods: We analyzed data from 3,698 U.S. adults 20–69 years of age who had been randomly assigned to the National Health and Nutrition Examination Survey (NHANES) 1999–2004 Audiometry Examination Component. Pure-tone averages (PTA) of hearing thresholds at frequencies of 0.5, 1, 2, and 4 kHz were computed, and hearing loss was defined as a PTA > 25 dB in either ear.p-trends < 0.05).Results: The weighted geometric means of blood cadmium and lead were 0.40 [95% confidence interval (CI): 0.39. 0.42] µg/L and 1.54  µg/dL, respectively. After adjusting for sociodemographic and clinical risk factors and exposure to occupational and nonoccupational noise, the highest (vs. lowest) quintiles of cadmium and lead were associated with 13.8%  and 18.6%  increases in PTA, respectively (Conclusions: Our results suggest that low-level exposure to cadmium and lead found in the general U.S. population may be important risk factors for hearing loss. The findings support efforts to reduce environmental cadmium and lead exposures. Hearing loss is one of the most common chronic disabling conditions among older adults . More thExperimental studies suggest that lead exposure induces degeneration in the inner ear receptor cells and latency in auditory nerve conduction velocity  and thatThe aim of this study was to investigate the associations of environmental cadmium and lead exposure with hearing loss in a representative sample of U.S. adults who participated in the National Health and Nutrition Examination Survey (NHANES) 1999–2004, while controlling for important potential confounding factors, including exposure to loud noises at work , from firearms, and during recreational activities. We also estimated the joint effects of cadmium and lead, as well as effect modification by noise and other potential risk factors.Study population. NHANES, which is conducted by the Centers for Disease Control and Prevention (CDC), National Center for Health Statistics (NCHS), is an ongoing series of cross-sectional surveys designed to obtain information from a representative sample of the civilian noninstitutionalized U.S. population. Data are collected through extensive household interviews to obtain information on health risk factors, including health behaviors, personal environment, and lifestyle. In addition, physical examinations and medical history interviews were conducted at a specially equipped mobile examination center (MEC) (er (MEC) .In the 1999–2004 NHANES, half of the participants 20–69 years of age were randomly assigned to the Audiometry Examination Component at MEC. Participants who used hearing aids that could not be removed for testing or who could not tolerate test headphones were excluded . The inin = 452) and those with missing information on blood cadmium or lead measurements (n = 183), longest job-related Occupational Information Network (O*NET) score (n = 376), firearm or recreational noise exposure (n = 6), or other demographic or hearing-related variables (n = 548), leaving a total of 3,698 observations. Compared with the participants with audiometric data who were excluded from our analysis, participants with audiometric data who were included had lower hearing thresholds, were less likely to be classified as having hearing loss, and were more likely to have completed high school and to be non-Hispanic white . We excluded an additional 76 participants (2.1%) who had hearing thresholds ≤ 0 dB  from the linear regression models  to better interpret regression results and to avoid adding a constant before log-transformation of hearing thresholds.For the present analysis, we excluded participants with unilateral hearing loss  with standard headphones (model TDH-39) and insert earphones (Etymotic EarTone 3A) (n = 476). As an additional measure of the quality of participants’ responses, the 1-kHz frequency was tested twice in each ear, and audiograms with ≥ 10 dB difference between the tests were classified as unreliable responses (n = 3) (Tone 3A) . Pure-to (n = 3) .We computed the hearing thresholds (decibel) at speech frequencies as a pure-tone average (PTA) of 0.5, 1, 2, and 4 kHz . HearingBlood cadmium and lead measurements. Blood cadmium and lead were measured at the Environmental Health Sciences Laboratory of the CDC, National Center for Environmental Health  after confirming the absence of background contamination in all collection and storage materials  may be important confounding factors in the associations of blood cadmium and lead with hearing loss. Direct measures of personal noise exposure are not available in the NHANES. Participants were classified as exposed (vs. unexposed) to nonoccupational firearm noise if they indicated that they had ever been exposed to the noise of a firearm at least once a month for 1 year, and were classified as exposed to recreational noise if they indicated exposure to loud noise outside of work  at least once a month for 1 year.Occupational noise exposures were classified based on occupational noise estimates according to the job title for the longest job held by each participant. These estimates were based on a new occupational noise exposure assessment tool that uses the O*NET survey database . In brieDemographic and hearing-related variables. Information on other demographic and hearing-related variables was obtained during household interviews or at the MEC. Body mass index (BMI) was calculated as dividing measured weight in kilograms by measured height in meters squared. Use of ototoxic medication was defined based on self-reported use of any aminoglycosides, loop diuretics, antineoplastic drugs, or nonsteroidal anti-inflammatory drugs during the past month. Smoking pack-years were computed, and participants were classified as nonsmokers, smokers with < 20 pack-years, or smokers with ≥ 20 pack-years. Hypertension was classified based on a self-reported physician diagnosis, current use of antihypertensive medication, or systolic blood pressure ≥ 140 mmHg or diastolic blood pressure ≥ 90 mmHg at the time of examination. Diabetes mellitus was classified based on a self-reported physician diagnosis or current use of antihyperglycemic medication, consistent with a previous study of diabetes and hearing loss  and the R survey package  to account for the complex survey design and NHANES sample weights ]. Therefore, we also modeled quintiles of blood cadmium and lead to better capture nonlinear relationships. We estimated the percent change in hearing threshold for a doubling of the blood cadmium and blood lead as (e(ln2 × β) –1) × 100%, with 95% confidence intervals (CI) estimated as (e[ln2 × (β ± 1.96 × SE)] –1) × 100%, where β and SE are the estimated regression coefficient and standard error. For quintiles, percent changes were estimated by comparing each of the upper four quintiles to the lowest quintile. p-Values for a linear trend were computed by fitting the exposure quintile as an ordinal categorical variable coded using integer values (0–4). Logistic regression was used to estimate odds ratios (ORs) for the hearing loss defined as PTA ≥ 25 dB in one or both ears.Hearing thresholds for PTA and individual frequencies were log-transformed to normalize distributions, and linear regression was used to model these outcomes. Blood lead and cadmium were log-transformed based on graphical evaluations suggesting improved fit compared with model estimates for nontransformed exposures, although estimates based on fully adjusted natural spline models indicated nonlinear associations with both log-transformed and nontransformed exposure variables , education , BMI (continuous), ototoxic medication use (yes/no), cigarette smoking , hypertension (yes/no), type 2 diabetes (yes/no), and either blood lead or blood cadmium (for the corresponding cadmium or lead model); b) model B further adjusted for occupational noise exposure ; and c) model C further adjusted for any nonoccupational firearm noise (yes/no) and any recreational noise (yes/no). We kept hypertension and type 2 diabetes in model A, although these variables could be potential intermediates between lead or cadmium exposure and hearing loss , education, ototoxic medication use, cigarette smoking/pack-years, hypertension, diabetes, and occupational , firearm, and recreational noise exposure.We also evaluated fully adjusted associations with log-transformed PTA for subgroups defined by age , sex, BMI , high cadmium and low lead, low cadmium and high lead, or high cadmium and high lead, with high and low categories defined based on median values for all study participants. Departures from additive joint effects [relative excess risk due to interaction (RERI)] and multiplicative joint effects were computed as recommended by http://dx.doi.org/10.1289/ehp.1104863)]. Overall, 441 participants (11.9%) had hearing loss (PTA ≥ 25 dB in one or both ears). The age-adjusted geometric means (95% CIs) of blood cadmium and lead in the entire population were 0.40  µg/L and 1.54  µg/dL, respectively (p < 0.001).Weighted means ± SE of age and PTA were 42.06 ± 0.28 years and 12.78 ± 0.24 dB, respectively .p = 0.78) or multiplicative joint effects .We estimated the individual and joint effects of exposures to cadmium and lead on the hearing thresholds . Particip = 0.02). For blood lead, the association was stronger in other race/ethnicity participants than in non-Hispanic whites (p = 0.01). There were borderline significant differences in associations between blood lead and PTA when we compared Mexican-Americans with non-Hispanic whites (p = 0.05), and participants who were not exposed to firearm noise with those who were exposed (p = 0.06).http://dx.doi.org/10.1289/ehp.1104863)].In a representative sample of U.S. adults who participated in NHANES 1999–2004, environmental cadmium and lead exposures were associated with hearing loss, even after adjusting for socioeconomic factors, noise exposures, and other potential risk factors. In addition, we observed positive dose–response relationships of hearing thresholds with blood cadmium and lead at levels currently observed in the general U.S. population. Compared with the lowest quintiles of cadmium and lead exposure, PTAs for participants in the highest quintiles were increased by 13.8% and 18.6%, respectively. These differences were consistent with estimated differences in PTAs associated with sex , having diabetes (19.9%), and with a two-unit increase of O*NET occupational noise scores  [see Supplemental Material, Few epidemiologic studies have evaluated associations between low-level lead exposure and hearing outcomes. Our results extend limited evidence from occupational settings or studies of children  to the gTo our knowledge, this is the first epidemiologic study to evaluate associations between hearing loss and cadmium exposure in adults. A few experimental studies have suggested possible mechanisms for cadmium ototoxicity. Studies using rats exposed to cadmium-contaminated drinking water suggested that cadmium can induce the generation of reactive oxygen species, loss of mitochondrial membrane depolarization, release of cytochrome c, activation of caspases, apoptosis, and the increase of extracellular signal-regulated kinase activation in auditory cells leading to an elevation in auditory thresholds . A recenThe present study suggests that low-level exposures to cadmium and lead currently observed in the U.S. general population may influence hearing health and supports efforts to reduce environmental cadmium and lead exposures. Compared with participants with exposures in the first quintiles, those in the fifth quintiles of blood cadmium (0.80–8.50 µg/L) and blood lead (2.80–54.0 µg/dL) are at risk for poorer hearing ability. Occupational Safety and Health Administration safety standards are currently 44.5 nmol/L (5 µg/L) for cadmium and 1.93 µmol/L (38.6 µg/dL) for lead in whole blood , 2010b. p = 0.003), based on the model that included the product of the two log-transformed metals. Another study that examined the associations of urinary cadmium and blood lead with reproductive hormones in > 700 young women in the NHANES III also found a stronger estimated effect by both high cadmium and lead in reducing inhibin B level, compared with high lead alone   and affeThe important strengths of this study include the use of data from a representative sample of the U.S. general population, which supports generalization of the observed findings; adjustment for potentially important confounding factors, including occupational noise classified using a newly developed assessment tool based on O*NET , and nonSeveral limitations in this study should be considered. The present study was conducted in a cross-sectional design that may raise an issue of validity of causal inferences between lead and cadmium exposures and hearing loss. Blood lead mainly reflects recent exposure, and therefore may not be a good biomarker to predict the long-term effect of low-level lead exposure, such as chronic diseases including hearing loss . InsteadIn conclusion, the present study supports the hypothesis that environmental cadmium and lead exposures at levels currently observed in the United States may increase the risk of hearing loss, the third leading chronic condition experienced by adults ≥ 65 years of age . Our fin(307 KB) PDFClick here for additional data file.


Bezafibrate therapy has been shown to improve beta-oxidation of fatty acids and to reduce episodes of rhabdomyolysis in patients with carnitine palmitoyltransferase type-2 (CPT2) deficiency. We report the efficacy of fenofibrate in a patient with CPT2 deficiency, in whom beta-oxidation was improved but an episode of rhabdomyolysis nevertheless occurred. This suggests additional methods to avoid rhabdomyolysis in patients with CPT2 deficiency should accompany fibrate therapy, including avoidance of muscular overexertion, dehydration, and heat exposure. Carnitine palmitoyl transferase type 2 (CPT2) deficiency is a rare autosomal recessive disorder of mitochondrial fatty acid oxidation . It maniin vitro. The number of episodes of rhabdomyolysis was reduced from 3–24 per patient over a 6-month period before treatment to 0–6 per patient over a 6-month period after treatment. Levels of creatine kinase (CK) were reduced from a mean of 10,900 IU/L before treatment to 4,700 IU/L after treatment, indicating a reduced rate of muscle damage [In 2009, Bonnefont and colleagues found that bezafibrate therapy restored the capacity for normal fatty acid oxidation in muscle cells from patients with CPT2 deficiency by stimulating the expression of the mutated gene [Unlike gemfibrozil and fenofibrate, bezafibrate is not widely available in many countries, necessitating therapy with the former two agents for patients with dyslipidaemia, the common indication for fibrate use. In this paper we report the case of a patient with CPT2 deficiency who responded to fenofibrate therapy with regard to improvement in plasma acylcarnitine and lipid levels, as well as improvement in muscle symptoms. While on fibrate therapy, he nevertheless experienced an episode of rhabdomyolysis after heat exposure and a viral infection. As in the experience of Bonnefont et al., fibrate therapy did not completely abolish episodes of rhabdomyolysis in this patient in spite of evidence for improved CPT2 activity. Therefore, it appears that traditional means to prevent rhabdomyolysis in CPT2 deficiency such as avoidance of overexertion and dehydration continue to be required during fibrate therapy for this rare condition. J. W., a 59-year-old immigration officer, was referred by his general practitioner for private specialist management of dyslipidaemia. From a young age, J. W. had experienced virtually constant muscle stiffness and recurrent episodes of severe muscle aches and pains. His past medical history included hypertension treated with lisinopril 20 mg daily for 5 years. A diagnosis of CPT2 deficiency was made by histochemical analysis of a muscle biopsy at the age of 39 years. Mutation analysis is yet to be performed. His only sibling also had similar symptoms, with a presumptive diagnosis of CPT2 deficiency in the absence of a muscle biopsy, which she has refused. There was no other family history suggestive of CPT2 deficiency. On examination, the body mass index was 28.55 , waist circumference 105.5 cm, blood pressure 123/78, and pulse rate 64 and regular. There was no evidence of arcus senilis, or xanthoma. He belonged to the intermediate risk category for a coronary event over the next 5 years (10–15% risk) according to current Australian cardiovascular risk calculator [3, between the 50th and 75th percentile for asymptomatic men of the same age [In view of his intermediate risk, the presence of subclinical atherosclerosis was investigated with coronary artery calcium score (CACS) and carotid duplex ultrasonography . There wR < 190), TSH , and Lp (a)  were normal. Plasma homocysteine was elevated . ApoA1  and ApoB100  were normal. The urate level was increased . Plasma acylcarnitine levels were measured by ultraperformance liquid chromatography electrospray ionization tandem mass spectrometry [R < 0.65 umol/L); see Fasting lipids were remeasured. Levels of total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), and triglycerides (TG) were 7.4, 4.4, 1.5, and 1.5 mmol/L, respectively . Levels In view of the finding of elevated LDL-C levels and significant subclinical atherosclerosis, J. W. was treated initially with low-dose rosuvastatin 5 mg daily in view of his underlying metabolic myopathy , 9. A taTwo months later his lipid profile had improved considerably to TC 4.9 mmol/L, LDL-C 2.5 mmol/L, HDL-C 1.5 mmol/L, and TG 1.1 mmol/L , but theR < 190). Ezetimibe was ceased and fenofibrate continued with resolution of the rash and of symptoms to pretreatment levels. After 6 months of fenofibrate therapy, acylcarnitine levels showed evidence of improved beta-fatty acid oxidation (presumably as a result of increased CPT2 activity), as observed for bezafibrate therapy in patients with CPT2 deficiency by Bonnefont et al. , aspartate transaminase (AST) 1450 (R < 35 U/L), and alanine transaminase (ALT) 465 (R < 45 U/L). Four months later, J. W. went on holiday and spent considerable time exposed to the sun while undertaking vigorous snorkelling and swimming. The day after returning from his holiday he had an upper respiratory tract infection followed one day later by mild generalised muscle aches and cramping. Within 48 hours his urine became dark and he complained of severe generalised muscle pain and stiffness. He was admitted to the local hospital with rhabdomyolysis. Blood levels on admission were CK 34,600 . The urate level was also elevated . Fenofibrate, allopurinol, and lisinopril therapies were recommenced at doses of 96 mg, 300 mg, and 5 mg daily, respectively, with subsequent improvement in urate level, and lipid and acylcarnitine profiles . Instead, a lower dose (5 mg rosuvastatin) was used in view of the known history of CPT2 deficiency and the propensity of any underlying myopathy to be aggravated by statin therapy , 9. ThisAggravating and precipitating factors for myalgia should then be sought, including overexertion, hypothyroidism, low vitamin D levels, and drugs that can interfere with statin catabolism such as cyclosporine, macrolide antibiotics, and antifungals , 9. OnceIt was decided to treat J. W. with fenofibrate therapy in view of the data of Bonnefont et al., showing subjective and objective improvement in lipid and CPT2 parameters with bezafibrate therapy . In ordeWith regard to acylcarnitine levels, therapy with fenofibrate resulted in significant improvement, particularly for oleoylcarnitine (95% reduction) and palmitoylcarnitine (80% reduction). This suggests improved mitochondrial beta-oxidation of fatty acids through increased CPT2 activity, consistent with previous results of Bonnefont et al. with bezafibrate . In spitThe acylcarnitine results  were obtOur data when considered with those of Bonnefont et al. suggest a class effect for fibrates in improving CTP2 activity, and a potential use for fibrates in patients with CPT2 deficiency , 11. TheIt is important to achieve optimal LDL-C reduction in asymptomatic patients with significant asymptomatic atherosclerosis . A targeMuscle adverse effects are the most frequently encountered adverse effect of statin therapy, and the most common cause for statin intolerance. Patients with underlying metabolic myopathies are particularly prone to statin muscle adverse effects for reasons that remain unclear, as the mechanism(s) of statin myopathy are currently poorly defined and are probably multifactorial. Other patients prone to statin myopathies are those with hypothyroidism and vitamin D deficiency, in whom replacement of thyroxine or vitamin D usually ameliorate the adverse effects of statins. There have been few reports of statin adverse effects in patients with CPT2 deficiency, but J. W.'s experience with increased CK levels and muscle symptoms was not unexpected and necessitated withdrawal of statin therapy. Whether lower statin doses in patients with statin myopathy  would have been successful in J. W. remains problematic. The use of additional agents such as bile acid sequestrants and/or niacin could be considered in future, but these agents are generally poorly tolerated. Another important aspect of J. W.'s management is monitoring of subclinical atherosclerosis, which is likely to progress in the presence of an elevated LDL-C and renal impairment. Annual carotid ultrasonography and stress echocardiography is planned, with repeat calcium scoring after 2-3 years to assess the rate of progression of subclinical coronary atherosclerosis. Hopefully means to lower LDL-C currently in development will be available in Australia in the near future. These include mipomersen  and triple therapy with niacin-laropiprant-simvastatin (as available in Europe).


Abnormal posture (AP) is often seen in Parkinson's disease (PD), and marked forms known as dropped head syndrome and camptocormia encumber daily living activities. Unlike other motor disabilities such as bradykinesia or muscular rigidity, AP is not always improved but rather deteriorated by PD medication.To clarify factors associated with neck and thoracolumbar AP.Neck flexion (NF) and thoracolumbar (TL) angles were measured in 216 consecutive PD patients and 175 elderly healthy controls. The differences in NF and TL angles between PD patients and controls were designated as ΔNFA and ΔTLA, respectively. The association of ΔNFA or ΔTLA and predictable factors such as age, sex, duration of PD, Hoehn Yahr (H–Y) stage, Unified Parkinson's Disease Rating Scale Part 3 (UPDRS-3), daily dose of dopamine agonists, and comorbid orthopedic spinal lesions was investigated in PD patients. Patients were divided into quartiles according to ΔNFA or ΔTLA. The association between predictable factors and ΔNFA or ΔTLA was estimated as odds ratio (OR), comparing with the lowest quartile as the reference by multivariate regression analysis.Compared with controls, distributions of all three posture angles were significantly shifted rightward in PD patients. Although there were no difference in UPDRS-3 scores in the quartiles of ΔNFA, the highest quartile was associated with H–Y stage ≥3  after adjustment for age, sex and comorbid orthopedic spinal lesions. The highest quartile of ΔTLA was associated with comorbid orthopedic spinal lesions , and UPDRS-3 score .Thoraco-lumbar AP was associated with UPDRS-3 scores and orthopedic spinal lesions, and in contrast, neck AP was not associated with these factors, suggesting that they had different pathomechanisms. Abnormal posture (AP) is a frequent complication of Parkinson's disease (PD), and severe AP such as dropped head syndrome and camptocormia (marked bending of thoracolumbar spine) encumber the activities of daily living. To explore the pathogenesis of AP, the following issues should be addressed: disease specificity, definition, coexistence of two or more types of AP, and relationship to other extrapyramidal motor disturbances of PD. Bending posture, including a marked form called bent spine syndrome, is seen in elderly people without PD, suggesting that AP could occur in people without PD In the present study, three body angles, neck flexion (NF), fore-bent (FB) and lateral-bent (LB) angles were surveyed in PD patients and in healthy controls. The distribution of the body angles was compared between PD patients and controls. Clinical characteristics were investigated by the quartiles of body angle difference from the mean angle in the controls. The association of predictable variables was investigated using multinomial regression models. With reference to the lowest quartile of body angles, odds ratios (ORs) of predictable variables were estimated using multivariate multinomial regression.From March to July 2010, we enrolled 221 consecutive patients with PD who were able to keep a standing position without any assistance, and were treated at the Center for Parkinson's Disease and Related Disorders, Utano National Hospital, Kyoto, Japan. A diagnosis of PD was made according to Steps 1 and 2 of the United Kingdom Parkinson's Disease Society Brain Bank Clinical Diagnostic Criteria As healthy elderly controls, we recruited people registered with the Employment Service Center for Elderly People in Kyoto  and spouses of PD patients who gave informed consent for the study. Posture angles were measured using the same procedures as for the patients.The study was approved by the National Hospital Organization Utano Hospital Review Board, and all eligible individuals were informed of the purpose and methods of the study, and all of them provided informed consent. According to the comment from the committee, written consent was not required but oral consent was obtained because of the observational nature of the study and the photographs were not recognizable. The study investigators documented the oral consent of the participants in their medical records, and the committee approved the procedure to document oral consent.NF, FB and LB angles were measured on photographs of the lateral and back views of the participants in an upright position (in an “ON” period if the participants had motor fluctuations). On lateral view photographs, the angle between the two crossing lines—the line connecting the external acoustic foramen and the acromion, and the line connecting the acromion and the grater trochanter—was defined as the NF angle. Similarly, FB angle was the angle between the line connecting the acromion and the greater trochanter and a vertical line. On back view photographs, the angle between the line connecting the posterior process of the seventh cervical vertebra and that of the fifth lumbar vertebra and a vertical line was defined as the LB angle . NF and Clinical data including age, sex, duration of PD, PD onset age, initial symptoms (tremor-dominant or not), Hoehn and Yahr (H–Y) stage, history of psychosis, and history of agonist-related AP were collected as possible predictors of AP. History of agonist-related AP was defined when AP occurred after dose escalation of dopamine agonists and recovered after discontinuation or de-escalation. In addition, scores on the Unified Parkinson's Disease Rating Scale Part 3 (UPDRS-3) were collected. Comorbid orthopedic spinal lesions  were diagnosed on plain X-ray or MRI performed in all participants within 6 months before the acquisition of body angle photographs. Information about regular physical exercise prior to entry was extracted from clinical records.Daily doses of dopamine agonists and use of selegiline and amantadine at entry were also recorded. L-Dopa dose was calculated using the following formula: 1.0× regular levodopa dose or 1.25×regular levodopa dose if taking entacapone. The dose of dopamine agonists was calculated as the levodopa equivalent dose (LDED) according to the formula: LDED (mg)  =  pramipexole (mg) ×67 + ropinirole (mg) ×25 + pergolide (μg) ×67 + cabergoline (mg) ×67 + bromocriptine (mg) ×10 + talipexole (mg) ×67.The differences in NF angle (ΔNFA) and TL angle (ΔTLA) between PD patients and healthy controls were evaluated according to the following formulae:In addition FB angle (ΔFBA) was also investigated because there may be quite different mechanisms in lateral bending and antero-posterior bending APs.vs. 3–4), history of psychosis, history of agonist-related AP, orthopedic spinal lesions, and use/non-use of selegiline and amantadine were regarded as dichotomous variables.Age, duration, onset age, daily dose of L-Dopa, dopamine agonists, and UPDRS-3 scores were regarded as scale variables. Sex, initial symptoms, H–Y stage .NF, FB and LB angles were compared between patients with PD and healthy elderly controls by Mann–Whitney As controls, 91 men and 84 women aged 70.0 (7.1) and 69.7 (8.2) years, respectively, gave informed consent and their body angles were measured. In the controls, NF angle was 21.1° (10.6) in men and 11.8° (10.5) in women, and FB angle was –5.5° (4.0) in men and –3.8° (3.3) in women. There were significant differences in NF and FB angles between male and female controls (p<0.001 and p = 0.005). Due to significant sex differences in the angles in the controls, ΔNFA, ΔTLA and ΔFBA were calculated after separation by sex.Among 221 enrolled patients with PD, five were eliminated because of deep brain stimulation (n = 2) and missing data (n = 3) and the remaining 216 patients were analyzed. In 216 PD patients (83 men and 133 women) with a mean age of 69.0 (9.0) years, PD duration was 8.0 (4.9) years, and onset age was 61.0 (9.7) years. Ninety-nine patients (45.8%) had tremor-dominant onset, and 116 (53.7%) were H–Y 3 or 4 and UPDRS-3 score was 20.6 (10.8). Orthopedic spinal lesions on X-ray or MRI were found in 41 (19.0%) patients. Sixty-one patients (28.2%) experienced psychosis and 22 (10.2%) had a history of agonist-related AP. Mean daily dose of L-Dopa and dopamine agonists (LDED) was 392.7 (187) mg and 103.7 (135.3) mg, respectively. Mean NF, FB and LB angle was 22.9° (14.9), 3.9° (10.0) and 3.2° (3.5) degrees, respectively.Distribution of NF, FB and LB angles in patients with PD and healthy controls is shown by sex in There was no correlation between ΔNFA and ΔTLA . The demographic data by the quartiles of ΔNFA or ΔTLA are shown in In There was strong multicollinearity between age and PD onset age; therefore, PD onset age was excluded. L-Dopa+agonist dose was eliminated from predictable variables because there was multicollinearity between L-Dopa dose and L-Dopa+agonist dose, . All theAll of the NF, FB and LB angles in PD were significantly larger than those in healthy controls. Distributions of NF and FB angles were bell-shaped both in PD patients and controls, and were shifted to the right in PD patients . AlthougPrevious studies have identified many clinical factors as risk factors for camptocormia, such as duration of PD There are no reports of risk of neck AP such as anterocollis or dropped head in PD. In the present study, ΔNFA was associated only with H–Y stage ≥3. As shown in Unexpectedly, age did not contribute significantly to AP. Spinal lesions were significantly associated with thoracolumbar AP, and this finding was consistent with a series of previous reports about camptocormia Although several antiparkinsonian drugs such as dopamine agonistsPossible association of other factors such as contaminant myopathy or disturbance of body angle sensation could not be assessed in the present study, and these points are limitations of the study. Although this study had another limitation owing to its cross-sectional observational nature, the results suggest that body angles are shifted rightward in most patients with PD, and it is difficult to establish a clear cut-off point for body angles. TL AP is independent of NF AP and the significant risk factor for TL AP was comorbid orthopedic spinal lesions and motor disturbance of PD.Figure S1Schematic representation of body angles. NF angle was defined as the angle between the two crossing lines: a line connecting the external acoustic foramen and the acromion, and another line connecting the acromion and the greater trochanter. Similarly, FB angle was the angle between a line connecting the acromion and the greater trochanter and a vertical line. On back view photographs, the angle between the line connecting the posterior process of the seventh cervical vertebra and that of the fifth lumbar vertebra and a vertical line was defined as LB angle.(TIF)Click here for additional data file.Figure S2Scattered plots of scale predictable variables. The relationship between possible predictable variables  was investigated in scattered diagram. There was a linear correlation between age and onset age, and therefore PD onset age was excluded from statistical analysis. There was multicollinearity between L-Dopa dose and L-Dopa+agonist dose, and therefore L-Dopa+agonist dose was eliminated from predictable variables.(TIF)Click here for additional data file.Table S1(DOCX)Click here for additional data file.Table S2(DOCX)Click here for additional data file.


Autism spectrum disorder (ASD) refers to a range of neurodevelopmental conditions characterized by social communication deficits, repetitive behaviours, and restrictive interests. Impaired inhibition has been suggested to exacerbate the core symptoms of ASD. This is particularly critical during adolescence when social skills are maturing to adult levels. Using magnetoencephalography (MEG), we identified the location and timing pattern of neural activity associated with inhibition in adolescents with autism, compared to typically developing adolescents.The MEG data from 15 adolescents with ASD and 15 age-matched controls (13 to 17 years) were collected during a go/no-go task with inverse ratios of go/no-go trials in two conditions: an inhibition condition (1:2) and a baseline condition (2:1). No-go trials from the two conditions were analyzed using beamformer source localizations from 200 ms to 400 ms post-stimulus onset. Significant activations were determined using permutation testing.Adolescents with ASD recruited first the right middle frontal gyrus (200 to 250 ms) followed by the left postcentral gyrus (250 to 300 ms) and finally the left middle frontal and right medial frontal gyri (300 to 400 ms). Typically developing adolescents recruited first the left middle frontal gyrus (200 to 250 ms), followed by the left superior and inferior frontal gyri (250 to 300 ms), then the right middle temporal gyrus (300 to 350 ms), and finally the superior and precentral gyri and right inferior lobule (300 to 400 ms).Adolescents with ASD showed recruitment limited largely to the frontal cortex unlike typically developing adolescents who recruited parietal and temporal regions as well. These findings support the presence of an atypical, restricted inhibitory network in adolescents with ASD compared to controls. It is theorised that impaired inhibitory control exacerbates the social deficits or repetitive behaviours and restricted interests -3 characThe extent of inhibitory impairment in ASD is not well understood, as the existing literature has yielded inconsistent findings. Some behavioural studies have reported inhibition deficits in ASD ,7-11, whet al. found more variability of overall brain activity in children aged 8 to 12 years compared to adults [Neuroimaging studies have attempted to clarify the variability seen in behavioural studies by identifying possible endophenotypes within the brain. In typically developing individuals, particularly adults, the anterior cingulate cortex (ACC) and the right inferior frontal cortex have been implicated in inhibition tasks, likely related to their respective roles in error processing and top-down control. For example, Tamm o adults . Typicalo adults .et al. examined the functional connectivity with fMRI of the right and left inferior frontal gyri with regions in the frontal, striatal and parietal cortices in children aged 8 to 12 years as well as adults [Imaging studies on inhibition in ASD are limited, and are mainly completed in adults -28. The s adults . A trends adults . In thess adults . Decreass adults ,33, conss adults .Thus, studies have implicated the ACC and inferior frontal cortex in inhibitory control both in individuals with and without ASD. However, regions such as the parietal lobes, striatum and premotor cortex have been reported as well, but there is less of a consensus on the relevance of these regions to inhibition versus processing associated with other demands of the experimental task. As such, studies are needed to carefully characterize the inhibitory network, as well as to examine differences between adolescents with and without ASD.et al. [et al., 2013; in submission).Given the importance of timing in inhibition studies , neuroimaging modalities that allow measurement of the timing of brain activity add an invaluable dimension; MEG allows the determination of both the spatial and temporal patterns of brain activity . Using Met al. . This paThe aims of the current study were twofold:1. to examine differences in inhibitory control (performance measures) between adolescents with ASD and those without. We hypothesized poorer inhibition-related task performance would be seen in the ASD group compared to the control group2. to determine the spatial and temporal brain activity associated with inhibition in adolescents with and without ASD and to compare the neural activity pattern between groups. We hypothesized that ASD adolescents would show spatially atypical and temporally delayed neural activity patterns compared to controls.et al., in submission). None of them were on any psychotropic medications. Exclusion criteria were history of neurological disorder, including epilepsy and or acquired brain injury, known neurodevelopmental syndromes , primary psychiatric disorders , or chronic medical disorders , current psychotropic medication use, prematurity, uncorrected vision, full scale IQ <80 and standard contraindications to MEG and MRI imaging (ferromagnetic objects in the body). All participants were recruited based on known ASD diagnosis from a clinician and the diagnosis was confirmed using the Autism Diagnostic Observational Schedule - Generic (ADOS-G) [social:19.4(6.4), ADIcommunication:14.8(4.8), ADIrepetitive: 6.1(2.2); ADOScommunication:3.1(1.4), ADOSsocial:8.4(2.1), and ADOSrepetitive:1.45(1.43). The imaging study was approved by the institutional REB at Holland Bloorview Kids Rehabilitation Centre and the Hospital for Sick Children; the imaging was conducted at the Hospital for Sick Children. Informed consent or assent was obtained from all participants/guardians, as per institutional policies.Included in this study were 30 adolescents. Fifteen participants  had an ASD diagnosis, between the ages of 13 and 17 years . These participants were age- and sex-matched with typically developing control adolescents  , and the(ADOS-G) . Mean anAutism Diagnostic Observation Schedule-Generic (ADOS-G)et al., [The ADOS-G  is a welet al., ). It hasAutism Diagnostic Interview - Revised (ADI-R)This well-established semi-structured interview , validatWechsler Abbreviated Scales of Intelligence (WASI)Two subtests of the WASI (vocabulary and matrix reasoning) were used to obtain an abbreviated measure of IQ . The twoet al., 2013, in submission). Participants completed a ‘go/no-go’ task while lying in the MEG. Subjects were instructed to rapidly respond to go stimuli, solid black shapes on a white background and withhold their response to no-go stimuli, the same as Go stimuli, but with a grey ‘X’ superimposed in the centre  and condition type (inhibition versus baseline) effects and interactions on behavioural measures of the go/no-go task.The mean reaction time (RT) was measured for go trials, from stimulus onset to the initial button response. RTs under 100 ms were not included, as they probably reflected subject anticipation. False alarm rate  was calculated as the percentage of incorrect responses to no-go trials out of the total number of no-go trials. Hit rate was calculated as the percentage of correct go trials out of the total number of go trials. Statistical analyses on behavioural data were carried out using STATISTICA, Version 8, MEG data were acquired on a 151-channel CTF system, using a 600 Hz sampling rate, and an online 0 to 150 Hz bandpass filter, with third order spatial gradient noise cancellation. All participants also completed an MRI scan, on a 3 T Siemens Trio system. Anatomical T1-weighted MRIs  were used to co-register the MEG data. Functional analyses were conducted with scripts written in-house that generated global field power (GFPs), source localizations and permutation tests between conditions and between groups.MEG trials were epoched into 600-ms windows with a 100-ms pre-stimulus baseline. Only correct no-go trials were analysed. Trials with artefacts such as eye blinks and movement were manually removed on a trial-by-trial basis, based on the agreement of two research members. The no-go trials  were averaged, and then grand averaged across participants, by condition (baseline versus inhibition) and group.Global field power (GFP), which is the root mean squared power across all sensors, was calculated on our grand-averaged data, to determine MEG amplitude changes across time. To better visualize the frontal differences between groups, we generated GFP plots from the 38 frontal sensors Figure . Based oFor source localisation analyses, we used an in-house vector beamformer algorithm (SPF) . MultispP < 0.005. Permutation tests shuffled group membership of the two samples, while maintaining the cardinality, and computing mean differences between the samples. The calculated mean differences for n >2,000 permutations were plotted to create a distribution, allowing the difference from the original sample to be compared to the distribution of values, to obtain P values. Between-group differences on the beamformer images were tested as well using permutation tests.To assess significance of within-group activity, permutation tests were completed on the subtracted beamformer images , generating activation maps at P < 0.005 were visualised using MRI3DX, and only the highest 15% of peak activations were included, and with their Talairach coordinates noted; anatomical terms and Brodmann areas (BA) were verified in Talairach Client [Images with h Client ,44.Mean RT, hit rate and false alarm rate are listed in Table P = 0.825), diagnostic group (control or ASD)  = 1.937, P = 0.175) or an interaction  = 2.478, P = 0.127) for RT.There were no main effects of condition (inhibition or baseline conditions)  = 0.05, P = 0.001) where the inhibition condition (M = 22.3% ± 12.6 SD) evoked a higher false alarm rate than the baseline condition (M = 5.2% ± 4.6 SD). A trend towards a main effect of diagnostic group on false alarm rate was also seen  = 3.875, P = 0.059; eta squared = 0.122), where higher false alarm rates were seen in adolescents with ASD (M = 16.4% ± 8.7 SD) than controls (M = 11.1% ± 7.8 SD). The group by condition interaction was not significant (P = 0.110). However, because the interaction was expected to be nonsignificant for the baseline condition, we evaluated the group by condition interaction for the no-go condition. The effect of group on the false alarm rates for the no-go condition was examined and a trend towards a main effect of diagnostic group was found for the no-go condition [ = 3.737, P = 0.63; eta squared = .0118), where higher false alarm rates were seen in adolescents with ASD (M = 26.5% ± 3.1 SD) than in control teens (M = 18.1% ± 3.1 SD).A main effect of condition type was found  = 79.554, P = 0.367), diagnostic group  = 1.063, P = 0.311) or an interaction  = 2.584, P = 0.119) for hit rate.There were no main effects of condition type  = 0.839, Waveforms representing the magnitude of overall frontal brain activity occurring from 100 ms prior to stimulus onset to 500-ms post-stimulus onset are plotted in Figure P < 0.005, uncorrected; ≥5 mm from peak activation) are listed in Table P < 0.005, uncorrected, 5,000 permutations).Significant within-group activations, where the inhibition condition was significantly greater than the baseline condition  from 200 to 250 ms followed by the left superior frontal gyrus  and left inferior frontal gyrus (BA 45) from 250 to 300 ms. From 300 to 350 ms, the typically developing adolescents recruited the right middle temporal gyrus (BA 21) and the right superior temporal gyrus (BA 22) from 350 to 400 ms. The right precentral gyrus (BA 4) and right inferior parietal lobule (BA 40) were also recruited during this final time window (350 to 400 ms).The adolescents with ASD first recruited the right middle frontal gyrus (BA 45) from 200 to 250 ms followed by the left postcentral gyrus (BA 3) from 250 to 300 ms. The left middle frontal gyrus (BA 10) was then activated from 300 to 400 ms, along with the right medial frontal gyrus from 350 to 400 ms.A diagnosis of ASD in our adolescent sample was associated with poorer impulsivity measures as seen in higher false alarm rates, during the inhibition condition of our task compared to the baseline condition. Neuroimaging findings complemented these behavioural findings by revealing an inhibitory network that differed significantly from the typically developing adolescent group. Adolescents with ASD recruited predominantly the frontal cortex, while controls recruited frontal as well as supplementary regions, including the inferior parietal lobule and the temporal lobe. Additionally, the ASD group initially engaged the right rather than the left frontal cortex and activated an area in the prefrontal cortex (BA 10).P < 0.05) were found between groups for response accuracy, measured by hit rate or RT. An ASD diagnosis was associated, however, with a trend for higher false alarm rates (P = 0.059), or commission errors, suggesting poorer inhibitory skills in ASD individuals. A significant main effect between the baseline and inhibition conditions for false alarm rates (P = 0.001) confirms that the baseline condition was an effective control task, as it incurred a lower error rate.No significant differences , and typical adolescents in our current study were observed to activate the right inferior cortex at sub-threshold levels. Therefore, the significant activation of this region in adolescents with ASD, while performing more poorly on task measures of inhibition  than control adolescents, suggests the activation may be inefficient or ineffective in ASD due to reduced selectivity in recruitment [Adolescents with ASD recruited the right middle frontal gyrus in a non-homologous region (right BA 46) during the first time window of 200 to 250 ms, unlike the adolescent controls, who first engaged the left middle frontal gyrus (BA 6). The right inferior prefrontal cortex BA 45/46), or the ventrolateral prefrontal cortex, has consistently been shown to play an important role in inhibition /46, or t  at 250 to 300 ms, which then moved anteriorly to the left middle frontal gyrus in BA10 from 300 to 400 ms. Conversely, control adolescents recruited the inferior parietal lobe as well as the temporal lobe during final time window of 350 to 400 ms. Although the recruitment of the postcentral gyrus by adolescents with ASD was parietal, the BA 3 region is associated with somatosensory-related task activity, whereas the parietal area activated by control adolescents was the inferior parietal lobe, an area commonly activated during inhibition tasks -57. It iThe poor recruitment of more widespread cortical regions, such as the parietal  and tempet al. in 2006 [BA 10 activity was observed between 300 to 400 ms in our adolescents with ASD, but activity was not seen in this region in the control adolescents. The BA 10 region has been associated with maintaining a balance between rapid responding and careful or controlled responding, during cognitive flexibility paradigms ,61, as w in 2006 . We suggLimitations of this study include the small sample size, which limited our ability to detect between group differences beyond statistical doubt, as well as its cross sectional nature, which limits our ability to discuss developmental trajectories of the differences noted. In addition, comorbidity was not systematically assessed. This is important as inhibitory control defects are also a feature of ADHD . UntInhibition-related MEG activity in the adolescents with and without ASD demonstrated distinct spatiotemporal neural processing patterns. More extensive frontal activity was found in adolescents with ASD, which we suggest may be due to inefficient long-range connectivity,  as well ACC: anterior cingulate cortex; ANOVA: analysis of variance; ASD: autism spectrum disorder; BA: Brodmann area; ERB: event-related beamforming; fMRI: functional Magnetic Resonance Imaging; GFP: global field power; ISI: interstimulus intervals; MEG: magnetoencephalography; IQ: intelligence quotient; MRI: magnetic resonance imaging; ROI: region of interest; RT: reaction time; WASI: Wechsler abbreviated scale of intelligence.EA has received consultation fees from Seaside Therapeutics and Novartis, an unrestricted grant from Sanofi-Canada, and has consulted without fees to Proximagen and Neuropharm.AV participated in study coordination, subject recruitment, data acquisition, the analyses of data and the interpretation of results and drafted the manuscript. EP assisted with interpretation of results and finalizing the manuscript. KT participated in study design, participant data acquisition, and helped with drafting of the manuscript. JV contributed to study design and interpretation of results. MT contributed to the conception and design of the study and to the interpretation of results, and drafting of the manuscript. EA participated in study design and coordination, contributed to interpretation of the results and guided the drafting of the manuscript. All authors read and approved the final manuscript.


Echium amoenum Fisch. and Mey has been implemented in Iranian folk medicine for several centuries. Antioxidant, analgesic, immunomodulatory, and anxiolytic properties of E. amoenum suggest that this plant may have beneficial effects in the management of acute pancreatitis. The aim of this study was to evaluate the protective effect of petals of E. amoenum extract (EAE) on a murine model of pancreatitis. Acute pancreatitis was induced by five intraperitoneal (i.p.) injection of cerulein (50 μg/kg) with 1h intervals which was characterized by pancreatic inflammation and increase in the serum level of digestive enzymes, in comparison to normal mice. EAE  was administered i.p., 30 minutes before induction of pancreatitis. Pretreatment with EAE (400 mg/kg) reduced significantly the inflammatory response of cerulein-induced acute pancreatitis by ameliorating pancreatic edema, amylase and lipase serum levels, proinflammatory cytokines, myeloperoxidase activity, lipid peroxidation and pathological alteration. These results show that EAE attenuates the severity of cerulein-induced acute pancreatitis with an anti-inflammatory, immunomodulatory and antioxidant effects. Acute pancreatitis is a morbid inflammatory condition of pancreas with limited specific therapy. Enhanced oxidative stress plays an important role in induction and progression of acute pancreatitis. So reducing oxidative stress may relieve this pathogenic process.  Circulaammation .Among several experimental models of acute pancreatitis that are similar to human pancreatitis, cerulein-induced acute pancreatitis is one of the best known and widely used experimental models . Indeed,Echium amoenum (E. amoenum) Fisch. and Mey (Boraginaceae), an important Iranian traditional remedy, is widely used as a tonic, tranquillizer, diaphoretic, and as a remedy for cough, sore throat, and pneumonia [E. amoenum aqueous extract was effective in the treatment of obsessive-compulsive disorder [E. amoenum have been recently recognized as an important source of phenolic compounds like rosmarinic acid, cyaniding, and delphinidin [neumonia . It is bneumonia –12. Alsodisorder . Dried vphinidin . E. amoenum attenuates PGE2 production and cyclooxygenase-2 expressions by inhibiting activation and translocation of c-Jun and NF-κB factors into nucleus [Cyanidin 3-glucoside, the most common anthocyanin, which is present in petals of  nucleus . Also th nucleus . E. amoenum, inhibits TNF-alpha induced COX-2 expression by directly inhibiting Fyn kinase activity [E. amoenum has also antimicrobial, antiviral, and anti-inflammatory effects [Perilla frutescens on liver injury was examined by Osakabe et al. The results showed that hepatoprotective effect of rosmarinic acid is due to the scavenging or reducing superoxide or peroxynitrite free radicals [E. amoenum hydroalcoholic extract (EAE) in a murine model of acute pancreatitis which caused by cerulein administration. To gain access to better insight into the mechanism(s) of action of the observed anti-inflammatory effects of EAE on pancreatitis, we have investigated the effects of EAE on pancreatic edema, leukocyte infiltration, amylase and lipase level, TNF-alpha, interleukin 6 (IL-6), myeloperoxidase activity, and lipid peroxidation. Delphinidin, the other anthocyanin which is present in petals of activity . Rosmari effects . Moreoveradicals . The preE. amoenum petals (flowers) were collected from Ghazvin, Iran in summer, and authenticated by Professor Mohammadreza Rahiminejad, Isfahan University, Isfahan, Iran. The voucher specimen of E. amoenum was deposited in the Herbarium Department of our school with number 1147.For preparation of hydroalcoholic extract, dried and finely powdered petals (50 g) were soaked in adequate volume of ethanol : water (70 : 30), and the extraction was carried out for 48 h to obtain full extract using percolator apparatus. The product was then shaken, filtered, and evaporated in a rotary evaporator to obtain semisolid extract under reduced pressure and then freeze-dried .λ max of 510 nm is related to the anthocyanin content.Anthocyanins have a critical role in the color quality of many plants. With a change in pH, anthocyanin pigments undergo reversible structural conversions. Colored oxonium form prevails at pH 1.0; however, the colorless hemiketal form exists at pH 4.5. The difference in absorbance of oxonium and hemiketal form at A is the difference of anthocyanin absorbance, in two different mediums and calculated as (A510 nm−A700 nm)pH 1.0 − (A510 nm−A700 nm)pH 4.5, Mw is the molecular weight of cyanidin 3-glucoside (= 449.2 g/mol), DF is dilution factor, L is the path length of cell (1 cm), ε is the molar absorptivity of cyanidin 3-glucoside (= 26900 lit/mol.cm), and 1000 indicates the conversion factor of gram to milligram [Anthocyanin concentration was calculated as cyanidin-3-glucoside equivalent by the following equation:illigram .μg/kg body weight of cerulein  with 1 h intervals according to the method which was previously demonstrated by Mazzon et al. [Acute pancreatitis was induced by five intraperitoneal (i.p.) injection of 50 n et al. . CeruleiMale mice weighting 25–30 g and bred in animal house  were used in this study. Animals were kept in uniform environment of temperature, humidity, and light/dark cycles (12/12 h) and allowed free access to pelleted rodent chow and tap water. Before initiation the experiment animals were fasted over the night. The study was approved by the Ethics Committee for Animal Care and Uses, Isfahan University of Medical Sciences, Isfahan, Iran.n = 6).Animals were randomly assigned into the following 5 groups .Negative control groups: mice with acute pancreatitis were pretreated with normal saline (5 mL/kg and i.p). E. amoenum extracts were chosen because they were in the range of safe therapeutic doses reported by Mehrabani et al. and did not show signs of hepatotoxicity [EAE groups: mice with acute pancreatitis were pretreated with  as a single dose (i.p.). Test doses of toxicity . AppliedIntraperitoneal treatments were carried out 0.5 h before pancreatitis induction. Mice were sacrificed 6 h after the last injection of cerulein. Blood samples were obtained by directed intracardiac puncture and stored at −70° for biochemical analysis. After decapitation, the pancreas was quickly removed and fixed in formaldehyde (10%) for histological examination. Besides, portions of this organ were promptly frozen in liquid nitrogen and stored at −70°C until assayed.Serum lipase and amylase activity were determined by using commercially available lipase and amylase kits . MPO activity, an index of polymorphonuclear leukocyte accumulation, was measured according to the modified method of Bradley et al. . PancreaμL of distilled water. The mixture was heated at 95°C for 60 min and centrifuged at 4000 rpm for 10 min; its absorbance was then measured at 532 nm. The standard curve was obtained by 1,1,3,3-tetramethoxypropane and results were expressed as nanomoles of MDA per gram of wet tissue [The level of malondialdehyde (MDA) in pancreatic tissue was determined as an index of lipid peroxidation. Briefly 200 mL of tissue homogenate was added to 0.2 mL of 8.1% sodium dodecyl sulfate (SDS), 1.5 mL of 20% acetic acid (pH 3.5), 1.5 mL of 0.8% aqueous solution of thiobarbituric acid, and 600 t tissue .α ELISA kit Glory Science Co., Ltd., Hong Kong, IL-6 ELISA kit Boster Biological Technology, Ltd., China). The cytokine levels were calculated after plotting the standard curves and expressed as pg/mL.Tissue TNF-alpha and IL-6 were measured using an enzyme-linked immunosorbent assay (ELISA) commercial kit according to the manufacturer's instructions (TNF-μm), stained with hematoxylin and eosin (H&E), and examined by coworker pathologist unaware from experimental protocol.Paraffin-embedded pancreas samples were sectioned . Leukocyte infiltration was also graded from 0 to 3 . Grading of vacuolization was based on the appropriate percentage of acinar cells involved 0 = absent, 1 = less than 25%, 2 = 25–50%, and 3 = more than 50% of acinar cells .U test. The minimal level of significance was considered at P < 0.05.Biochemical results are expressed as mean ± SEM. Statistical analysis was carried out by one-way analysis of variance (ANOVA) followed by Tukey's multiple comparison test. Nonparametric data was analyzed by Mann-Whitney Anthocyanins of EAE was 3.1% of cyanidin-3-glucoside equivalent.Cerulein-induced pancreatitis in vehicle-treated mice was associated with significant rises in the serum levels of amylase and lipase. The increase in amylase and lipase was markedly reduced in cerulein-treated mice which had been pretreated with EAE in dose of 400 mg/kg by i.p. injection Figures .P < 0.05) in cerulein treated mice which had been pretreated with EAE in dose of 400 mg/kg by i.p. injection . Pancreas levels of IL-6 and TNF-alpha were significantly reduced   .P < 0.05) . MDA levels of Pancreatic tissue were obviously reduced by EAE pretreatment in dose of 400 mg/kg i.p. administration ( < 0.05) .In normal saline treated mice, pancreas did not show any tissue injuries at light microscopic level (×10 magnification). Administration of cerulein induced acute edematous with severe leukocyte infiltration pancreatitis in all mice tested. Prominent interlobular and severe intralobular edema was also accompanied with moderate perivascular and abundant diffuse inflammatory infiltration, but no vacuolization, necrosis, or hemorrhages were seen.P < 0.05). . .E. amoenum had good potential to attenuate pancreatitis in mice as indicated by biochemical, immunological, and histological evaluations. Biochemical and immunological assays confirmed that administration of EAE reduced amylase and lipase activity, TNF-alpha, IL-6, MPO activity, and lipid peroxidation which are markers of pancreatitis. Interestingly EAE, especially at the dose of 400 mg/kg i.p., showed significant protection against pancreatitis compared to control group. Regarding the histological results, administration of EAE showed an effective protection in a manner that was partly dependent to the dose because the highest dose of EAE (400 mg/kg i.p.) had significant effects compared to the control group while other two smaller doses were not effectiveIn the present study, results showed that This finding was repeated in different examinations and the results showed that the lower doses of extract (100 and 200 mg/kg i.p.) were not effective to suppress pancreatitis and neither of doses had significant effects on serum levels of amylase and lipase activities as well as inflammatory mediators and lipid peroxidation. The dose of 400 mg/kg; however, was effective for all the parameters to be declined significantly. On the other hand, the difference between the applied doses was not statistically significant and this suggests that EAE protective activity on acute pancreatitis was at least partly dependent to the dose. This is in accordance with the results obtained by Zhao et al.  which de1 and CCK2 receptors and causes activation of the Jak2/Stat3 pathway [κB) activation thereby enhance cytokine expression and lipid peroxidation [κB activation and so reduce the inflammatory response associated with cerulein induced acute pancreatitis [Cerulein is an analog of cholecystokinin, which acts on CCK pathway . The Jak pathway . On the xidation . Thereforeatitis . E. amoenum is considered as a promising source of bioactive compounds with various beneficial biological activities. Antibacterial, antioxidant, analgesic, anxiolytic, antidepressant, and immunomodulatory actions are the most important properties of E. amoenum [ amoenum –12.E. amoenum flower aqueous extract has been investigated by Ranjbar et al. in human. The results showed a significant reduction in blood lipid peroxidation after 14 days of extract (7 mg/kgs) intake. They suggested that this antioxidant potential of E. amoenum may be due to its bioactive components, especially its flavonoids [The antioxidant activity of Iranian avonoids . Helicobacter pylori growth are implicated for the gastroprotective effects of flavonoids [Flavonoids with anti-inflammatory, antioxidant, and gastroprotective effects are widely distributed in plant kingdom . Stimulaavonoids . κB factors into nucleus suggests that suppression of cyclooxygenase-2 expressions [It could be suggested that EAE exerted its protective effects through mechanisms that are not essentially dependent to phenolic contents of extract and antioxidant effects. Indeed the presence of some bioactive compounds like rosmarinic acid with anti-inflammatory effect  and Cyanressions  and reduressions  might beressions  is one oCichorium intybus L. hydroalcoholic extracts possessed protection against cerulein-induced acute pancreatitis in mice [Ginkgo biloba extract on acute pancreatitis have been studied by Zeybek et al. [G. biloba extract at 100 mg/kg administered i.p. was able to decrease significantly in serum amylase and lipase levels as well as histopathologic scores in sodium taurocholate-induced pancreatitis. The beneficial effects had attributed to the oxygen radical scavenging potential of G. biloba flavonoids contents. Our findings indicated that various mechanisms were involved in protective activity of EAE on pancreatitis because biochemical, immunological, and histological parameters were improved. So it is plausible to accept that different active compounds of applied extract were responsible for these effects. Thus further experimental studies are necessary to isolate and identify the active principles present in E. amoenum fractions which are responsible for the protective effects on pancreatitis. Administration of medicinal herbs that possess anti-inflammatory and antioxidant properties is a new approach to attenuate inflammatory-related disorders . Our pre in mice . Similark et al. . The resE. amoenum hydroalcoholic extract possessed protective activity against cerulein-induced acute pancreatitis in mice and may suggest a therapeutic potential for therapy or prevention in this inflammatory disease condition.We demonstrated that


Nerve growth factor (NGF) is a neurotrophin and has been suggested to induce heme oxygenase-1 (HO1) expression. Although the role of HO1 in tumorigenesis remains controversial, recent evidence suggests NGF and HO1 as tumor-progressing factors. However, the correlative role of NGF and HO1 and their prognostic impact in breast carcinoma is unknown.We investigated the expression and prognostic significance of the expression of NGF and HO1 in 145 cases of breast carcinoma.Immunohistochemical expression of NGF and HO1 was observed in 31% and 49% of breast carcinoma, respectively. The expression of NGF and HO1 significantly associated with each other, and both have a significant association with histologic grade, HER2 expression, and latent distant metastasis. The expression of NGF and HO1 predicted shorter overall survival of breast carcinoma by univariate and multivariate analysis. NGF expression was an independent prognostic indicator for relapse-free survival by multivariate analysis. The combined expression pattern of NGF and HO1 was also an independent prognostic indicator of overall survival and relapse-free survival. The patients with tumors expressing NGF had the shortest survival and the patients with tumor, which did not express NGF or HO1 showed the longest survival time.This study has demonstrated that individual expression of NGF or HO1, and the combined NGF/HO1 expression pattern could be prognostic indicators for breast carcinoma patients. NTR and 121 patients received adjuvant endocrine therapy. One hundred and ten patients received both chemotherapy and endocrine therapy, and 5 patients received no adjuvant therapy. The median follow-up duration was 144.9 months . Among the 145 BRCA patients, 21 patients experienced local relapse, 33 patients had latent distant metastasis, and 44 patients died from BRCA at the follow-up endpoint. The median survival was 192.0 months and the five- and ten-year survival rates for the entire BRCA patients were 81% and 74%, respectively. All the cases were reviewed and classified by two pathologists (KY Jang and SJ Noh) according to the World Health Organization Classification, and patImmunohistochemical staining was performed using 3.0 mm tumor cores for tissue microarray (TMA). To establish the TMA, we reviewed all of the H&E slides and took two 3.0 mm tissue cores from the paraffin-embedded tissue blocks per case at the area of highest tumor grade. An antigen retrieval procedure was performed with sodium citrate buffer using a microwave oven for 20 minutes. The following markers were used: NGF  and HO1 . Immunohistochemical staining for NGF and HO1 were evaluated by the sum of the staining intensity scores and the staining area scores in each TMA core,27. The P-values less than 0.05 were considered to be statistically significant.The relationships between NGF and HO1 expression and other clinicopathological factors were determined using the Pearson’s chi-square test. The primary point of interest was overall survival (OS) and relapse-free survival (RFS). The follow-up endpoint was the date of death or the date of last contact through December 2012. OS duration was measured as the time from diagnosis to date of death from BRCA and the patients who were alive at last contact or died from other causes were treated as censored. RFS was calculated as the time from diagnosis to the date of relapse, death from BRCA, or last contact. Patients who were alive at last contact or died from other causes and who did not experience the relapse were treated as censored for RFS analysis. Univariate and multivariate Cox regression hazard analysis were performed to estimate the impact of clinicopathologic factors and expression of each marker on OS and RFS. Kaplan-Meier survival analysis with a log-rank test was used to illustrate the cumulative survival curve for OS and RFS. Statistical analyses were calculated using SPSS statistical software . P = 0.035), histologic grade (P = 0.020), presence of latent distant metastasis (P = 0.004), and the expression of HER2 (P = 0.002) and ER (P = 0.005). Especially, a strong positive correlation between NGF and HO1 was found (P < 0.001). The expression of HO1 was significantly correlated with age (P = 0.029), histologic grade (P = 0.017), presence of latent distant metastasis (P < 0.001), and HER2 expression (P < 0.001)  and 49% (71/145) of BRCA samples, respectively , HER2 expression , NGF expression , and HO1 expression  were significantly associated with shorter OS and RFS  and its expression significantly associated with shorter RFS . The expression of HO1 predicted shorter OS  and RFS . TNM stage was significantly associated with shorter OS .Univariate survival analyses of the expression of NGF and HO1 and clinicopathological factors for OS and RFS are listed in Table P < 0.001, RFS; P < 0.001), NGF expression , and HO1 expression  were significantly associated with shorter OS and RFS  and TNM stage (P = 0.003) were significantly associated with shorter OS. Among the patients who received systemic adjuvant endocrine therapy, the age of the patients , the expression of HER2 , NGF , and HO1  were significantly associated with both OS and RFS .We also performed additional survival analysis in the patients which received adjuvant chemotherapy or endocrine therapy. Among the patients who received systemic adjuvant chemotherapy, HER2 expression  and RFS    and HO1+  group  and RFS  , we analyzed the prognostic effect of the expression of one marker in two separate groups according to the positivity of another marker. In the NGF Figure A. HoweveP = 0.031) greater risk of shorter OS and a 3.042-fold  greater risk of shorter RFS. The expression of HO1  and TNM stage  were also independent prognostic indicators of OS. The expression of HER2 was an independent prognostic predictor of RFS .To test the impact of the NGF/HO1 expression pattern on OS and RFS of BRCA patients, multivariate analysis was performed with the inclusion of NGF/HO1 expression instead of the individual expression of NGF and HO1. NGF/HO1 expression was also significantly associated with OS  and RFS  , HO1 expression (P < 0.001), and NGF/HO1 expression  were the independent prognostic predictor of OS. The expression of HER2 (P = 0.024) and NGF (P < 0.001), and NGF/HO1 expression  were the independent prognostic indicators of RFS for BRCA patients. Among patients who received endocrine therapy, the age of the patients (P = 0.007), TNM stage (P = 0.022), HO1 expression (P < 0.001), and NGF/HO1 expression  were independent prognostic indicators of OS for BRCA patients. The expression of HER2 (P = 0.029) and NGF (P < 0.001), and NGF/HO1 expression  were independent prognostic indicators of RFS for BRCA patients.Among patients who received chemotherapy, TNM stage  and HER2-positive subpopulations . These results suggest the possibility that NGF has mechanism involved in the progression of BRCA which are independent of HER2-related mechanisms. Therefore, NGF-targeted therapy may be beneficial for BRCA patients in addition to the HER2-based conventional therapy.Despite recent advances in diagnosis and treatment modalities, BRCA remains the second leading cause of cancer-related death among women. Recentlreatment,35. Howereatment, and NGFreatment. HoweverIn the NGF-TrkA signaling pathway, NGF is also known as an inducer of HO1,23. NGF The expression of HO1 is increased in various cancer cells compared with normal cells,18,38,39Another interesting finding of this study is that the combined expression pattern of NGF and HO1 is helpful for the prediction of the prognosis of BRCA patients. The patients with tumors expressing NGF had the shortest OS and RFS; furthermore, the patients with tumor which did not express NGF or HO1 showed the longest survival time. Multivariate analysis revealed NGF/HO1 expression as an independent prognostic indicator of OS and RFS. Therefore, this result suggests that the combined expression pattern of NGF and HO1 might be usable as a prognostic indicator for BRCA patients.In summary, the results of this study have shown that the expression of NGF and HO1 were significantly associated with each other and that the expression of both of them significantly correlated with unfavorable clinicopathological factors and predicted shorter survival of BRCA patients. Therefore, these results suggest the possibility that the NGF-HO1 pathway may be involved in breast carcinogenesis and progression. In addition, an algorithm for the sub-grouping of breast carcinoma patients into three sub-groups according to the expression patterns of NGF and HO1 also predicted survival of BRCA patients. Therefore, this result suggests the possibility that individual expression of NGF or HO1, and the combined expression pattern of NGF and HO1 could be the new prognostic indicator of BRCA patients.BRCA: Breast carcinoma; CI: Confidence interval; ER: Estrogen receptor; HER2: Human epithelial growth factor receptor 2; HO1: Heme oxygenase-1; HR: Hazard ratio; LN: Lymph node; NGF: Nerve growth factor; NGFR: Nerve growth factor receptor; OS: Overall survival; PR: Progesterone receptor; RFS: Relapse-free survival; TMA: Tissue microarray; TrkA: Tropomyosin-related kinase A.The authors declare that they have no competing interests.SJN, JSB, SHJ, BHP, HL, MJC, WSM, MJK and KYJ participated in the study design. JSB and UJ did the immunohistochemical staining. SJN, UJ, HSP, KSK, SHJ, HJY, MJC, MJK and KYJ were involved in data collection and data interpretation. SJN, KSK and KYJ participated in the statistical analyses. SJN, HSP, KSK, HJY, HL, BHP, WSM and KYJ wrote 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/1471-2407/13/516/prepub