Autism spectrum disorder (ASD) is a neurodevelopmental disorder that affects 1 in 68 children in the United States. Even though it is a common disorder, only two medications (risperidone and aripiprazole) are approved by the U.S. Food and Drug Administration (FDA) to treat symptoms associated with ASD. However, these medications are approved to treat irritability, which is not a core symptom of ASD. A number of novel medications, which have not been approved by the FDA to treat ASD have been used off-label in some studies to treat ASD symptoms, including medications approved for Alzheimer’s disease. Interestingly, some of these studies are high-quality, double-blind, placebo-controlled (DBPC) studies. This article systematically reviews studies published through April, 2014, which examined the use of Alzheimer’s medications in ASD, including donepezil , galantamine , rivastigmine (one study reporting improvements), tacrine (one study reporting improvements), and memantine . An evidence-based scale was used to rank each medication. Collectively, these studies reported improvements in expressive language and communication, receptive language, social interaction, irritability, hyperactivity, attention, eye contact, emotional lability, repetitive or self-stimulatory behaviors, motor planning, disruptive behaviors, obsessive–compulsive symptoms, lethargy, overall ASD behaviors, and increased REM sleep. Reported side effects are reviewed and include irritability, gastrointestinal problems, verbal or behavioral regression, headaches, irritability, rash, tremor, sedation, vomiting, and speech problems. Both galantamine and memantine had sufficient evidence ranking for improving both core and associated symptoms of ASD. Given the lack of medications approved to treat ASD, further studies on novel medications, including Alzheimer’s disease medications, are needed. Autism spectrum disorder (ASD) is a heterogenous neurodevelopmental disorder that is defined by behavioral observations and characterized by developmental delays in communication and social interaction and by repetitive behaviors and/or restricted interests. The most recent prevalence of ASD in the United States (U.S.) is now 1 in 68 children, including 1 in 42 boys . Since i®, 1993), Donepezil , Rivastigmine , Galantamine , and Memantine . Donepezil, galantamine, rivastigmine, and tacrine are cholinesterase inhibitors, and work by preventing the breakdown of acetylcholine. Galantamine also stimulates nicotinic cholinergic receptors and therefore can increase the release of acetylcholine   –30, α-ame (NMDA) , and kaie (NMDA) , 33 recee (NMDA) –36. Genee (NMDA) . Interese (NMDA) .Lastly, post-mortem studies have also implicated glutamate abnormalities. Cerebellum samples from individuals with ASD have demonstrated an increase in mRNA for AMPA 1, 2, and 3 receptors and glutamate/aspartate transporter 1 and 2, an increase in protein levels of glutamate/aspartate transporter 1 and 2 and AMPA 1 and NMDA 1 receptors, but a decrease in AMPA receptor density . In addiMemantine is an NMDA receptor antagonist and regulates the activity of glutamate, a neurotransmitter involved in memory and learning. Normally glutamate attaches to NMDA receptors allowing calcium to then enter freely into cells. Memantine prevents this by partially blocking NMDA receptors. Memantine has been reported to help obsessive–compulsive disorder (OCD) as well as impulsive behaviors in open-label –48, singGiven this background, this article reviews studies that have been published to date, which have reported on the use of these Alzheimer’s medications in ASD individuals. Overall, this review demonstrates that there is significant scientific support for some of these medications in the treatment of core ASD symptoms, suggesting that further clinical trials may be helpful to help define the role for some of these medications for the treatment of individuals with ASD.http://www.ncbi.nlm.nih.gov/entrez)] and Google Scholar databases from inception through the end of April, 2014 was conducted to identify pertinent articles using the search terms “autism,” “autistic,” “ASD,” “pervasive developmental disorder,” “PDD,” and “Asperger” in all combinations with the terms: “donepezil,” “galantamine,” “memantine,” “rivastigmine,” “tacrine,” “Alzheimer,” “cholinesterase inhibitor,” and “NMDA antagonist.” The references cited in identified articles were also searched to locate additional studies . Figure A computer-aided search of PUBMED [website (Studies were included if they: 1) involved individuals with ASD, and (2) administered a medication approved for Alzheimer’s disease to at least one individual with ASD. Articles that did not present new or unique data (such as review articles or letters to the editor) and animal studies were excluded. Studies on Rett syndrome and Childhood Disintegrative Disorder were also excluded. One reviewer screened titles and abstracts of all potentially relevant studies. After screening all records, 34 publications were identified and independently reviewed by both reviewers; 21 studies met inclusion criteria  for each treatment based on the level of evidence (LOE). Using a well-established scale , each stSeven studies reported on the use of donepezil in individuals with ASD, with five studies (71%) reporting improvements. Five studies were open-label or retrospective case-series. The first identified study was an uncontrolled, retrospective, open-label study of eight children (LOE 4) with autistic disorder (mean age 11.0 ± 4.1 years) and reported improvements in irritability and hyperactivity with donepezil (up to 10 mg/day) after at least 2 months of use; side effects included mild irritability (one patient) and gastrointestinal problems (nausea and vomiting in 1 patient) . AnotherTwo studies were double-blind, placebo-controlled (DBPC) studies. The first DBPC study contained 43 children (LOE 1b) with autism (mean age 6.8 years) and administered donepezil or placebo over 6 weeks and reported improvements in expressive and receptive speech and a decrease in overall autistic behaviors in the treatment group; side effects included diarrhea and stomach cramps . In the Overall, the evidence for donepezil is inconsistent for improvements in ASD symptoms as one of the two DBPC studies was negative and one of the five case-series reported detrimental effects rather than beneficial effects of donepezil. As two case-series reported improvements in ADHD symptoms and one case-series demonstrated improvements in REM sleep, donepezil may have favorable effects on subsets of children with ASD who have these specific symptoms. Clearly, several studies have demonstrated favorable effects of donepezil, suggesting that further studies focused on specific symptoms may be warranted in the future.Four studies reported on the use of galantamine in individuals with ASD, with all four studies reporting improvements. Two of the studies were uncontrolled, open-label, or case-series (LOE 4). In the first case-series (LOE 4) of three adults with autism (21 to 42 years old), galantamine 4–16 mg/day was reported to increase expressive language and communication; however; one individual had a regression when put on donepezil . The secTwo studies were DBPC. The first DBPC study of 20 children (LOE 2b) with autism (mean age 7.4 ± 3.2 years) reported a significant improvement with galantamine (dose not noted) compared to placebo on the ABC in irritability, eye contact, hyperactivity, and inappropriate speech; side effects were minimal . The secGiven that galantamine has been shown to improve both core and associated ASD symptoms in both open-label and DBPC studies , it is given a GOR of B. Given this preliminary positive evidence, it is clear that large multicenter high-quality controlled trials should be conducted to provide efficacy data to further define the role of galantamine in the treatment of ASD.An open-label study of 32 children (LOE 4) with autism used rivastigmine (0.4–0.8 mg twice a day) for 12 weeks and reported improvements in expressive speech and overall autism symptoms . Given tAn open-label study of three individuals (LOE 4) with ASD (mean age 17.4 ± 33.2 years) administered 20 mg of tacrine daily and reported mild improvements in irritability, hyperactivity, eye contact, and inappropriate speech as rated by combined parent and teacher scales (ABC); no significant side effects were reported . Given tNine studies reported on the use of memantine in individuals with ASD, with eight studies (89%) reporting improvements. Eight studies were open-label or retrospective case-series. In the first study, Chez et al.  administp < 0.05 for all three); no side effects were reported  examined the effects of memantine  in 18 children with ASD (age 6–19 years) and reported improvements in social withdrawal and inattention; 7 patients (39%) had side effects including irritability (4 patients), rash (1 patient), excessive sedation and vomiting (1 patient), and an increase in seizures (1 patient); 4 patients had to discontinue memantine . An uncoreported . An uncoreported . A case reported . One casreported .p < 0.001), stereotypy (p < 0.01), and hyperactivity/non-compliance (p < 0.01); side effects were similar in both groups (Only one study was DBPC and was a 10-week study of 40 children (LOE 1b) with ASD (ages 4–2 years). This study administered memantine  compared to placebo and reported significant improvements on the ABC in irritability  it might be of use.Given the fact that there is no FDA approved medication for the core symptoms of ASD and considering the limited proven effective treatments for ASD, studies are needed to identify novel treatments. Because several of the medications reviewed here show promising evidence for effectiveness for treating core and associated ASD symptoms, such medications should undergo further study in clinical trials to confirm their effectiveness for treating individuals with ASD.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Cnicus arvensis is used by many ethnic groups for inflammation, pain, and other ailments. In this study, reducing sugar, carbohydrate, alkaloid, steroid, tannin, flavonoid, and saponin groups were identified using standard chromogenic method. In high-performance liquid chromatography, vanillic acid and epicatechin were identified in the extract. Antinociceptive test by acetic acid induced writhing inhibition resulted 43.17 and 95.08% inhibition for 100 and 200 mg/kg body weight, comparing with standard diclofenac Na with 74.86% inhibition for 25 mg/kg body weight. In formalin induced paw licking test for antinociceptive activity, the extract inhibited 69.87 and 75.55% licking for 150 and 300 mg/kg body weight comparing with the inhibition (68.56%) of diclofenac Na for 10 mg/kg body weight at first phase. At late phase, the extract showed 73.12 and 87.46% licking comparing with licking inhibition (71.69%) by diclofenac Na at the same dose. In open field test for CNS depressant activity, the extract showed depression of locomotor activity for 150 and 300 mg/kg body weight comparing with diazepam for 10 mg/kg body weight. All results were statistically significant (P < 0.01). The identified polyphenols are reputed for antinociceptive and CNS depressant activity. The present findings support the use of this plant in pain. Cnicus arvensis (L.) Scop. Roth. (Hoffm.), synonym: Cirsium arvense (L.) Scop. , is an erect herb which is found in Bangladesh [ C. arvensis are reported in the books of ethnopharmacological relevance [ngladesh . The flongladesh . The rhingladesh . It is ungladesh . Medicinelevance , 4, 5. Nelevance . The whoelevance . The plaelevance . Cytotoxelevance .A food supplement (milk thistle 3000 mg) in the tablet form is marketed by Simply Supplements, Le Bourg, Forest, Guernsey, and claimed to contain the key ingredient of milk thistle, silymarin, a flavonoid compound extracted from the seeds. Though this plant is a common species and is well documented for folk uses in the reference ethnopharmacological compendia, it is yet not extensively studied. To the best of our search, no previous phytochemical and pharmacological investigation was conducted on this plant till now.The current study was designed to provide scientific evidence for its use as a conventional folk remedy by examining the antinociceptive and CNS depressant properties. Cnicus arvensis were collected from Khulna University Campus, Khulna, Bangladesh, in March 2013 (coordinate: 22.35°N 89.08°E). The samples of the plant were mounted on paper and the species was taxonomically confirmed by Sarder Nasir Uddin, Principle Scientific Officer, Bangladesh National Herbarium, Mirpur, Dhaka. A voucher specimen of the plant has been deposited and preserved in the library of the same institution for further collection and reference .The aerial parts ofThe collected plants were separated from undesirable materials and shade-dried for two weeks. The shade-dried plant materials were ground into a coarse powder with the help of a suitable grinder . The powdered sample was stored in an airtight container and kept in a cool, dark, and dry place until analysis commenced. About 500 g of powered material was taken in a clean, flat-bottomed glass container and soaked in 1900 mL of methanol. The container along with its contents was sealed and kept for a period of 15 days with occasional shaking or stirring . The who Mus musculus, of either sex of 3-4 weeks of age, weighing 20–25 g of either sex were purchased from the Animal Research Branch of the International Centre for Diarrhoeal Disease and Research, Bangladesh , and were used for in vivo antinociceptive and CNS depressant screening. The animals were housed in plastic cages in standard environmental conditions at the animal house in Department of Pharmacy, Northern University Bangladesh for adaptation after their purchase under standard laboratory conditions . They were fed with standard vital feed  and water was available ad libitum throughout the period of acclimatization. The research was carried out according to the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals and Organization for Economic Cooperation and Development (OECD) guidelines. The experimental protocol was approved by the Animal Ethics Committee, Northern University Bangladesh (Ref.: NUB/Pharm/AEC-2013).Swiss-albino mice, p-coumaric acid (PCA), rutin hydrate (RH), ellagic acid (EA), and quercetin (QU) were purchased from Sigma–Aldrich . HPLC grade acetonitrile, methanol, acetic acid, and ethanol were obtained from Merck .Methanol  was used as solvent for maceration of the plant material. Lead acetate, potassium dichromate, ferric chloride, hydrochloric acid, sulphuric acid, Mayer's reagent, Dragendorff's reagent, Wagner's reagent, Hager's reagent, Molisch's reagent, Benedict's reagent, and Fehling's solutions were purchased from Himedia  for preliminary phytochemical screening. The standard drugs diclofenac Na used for antinociceptive screening and diazepam for CNS depressant activity test were purchased from Square Pharmaceuticals Ltd., Bangladesh. Tween 80 used as suspending agent for the extracts was purchased from Merck . Acetic acid, the pain inducer, was also purchased from Himedia . Formalin (Ranbaxy (Rankem)) and carboxymethyl cellulose for antinociceptive test were purchased from the local drug market. Gallic acid (GA), (+)-catechin hydrate (CH), vanillic acid (VA), caffeic acid (CA), (−)-epicatechin (EC),μm) column  which was controlled at 30°C using a temperature controlled column compartment (TCC-3000). Data acquisition, peak integration, and calibrations were performed with Dionex Chromeleon software (Version 6.80 RS 10).Electronic balance  and vortex mixer  were used for this study. Chromatographic analyses using high performance liquid chromatography (HPLC) of the extract were carried out on Thermo Scientific Dionex Ultimate 3000 Rapid Separation LC (RSLC) systems , coupled to a quaternary rapid separation pump (LPG-3400RS), Ultimate 3000RS autosampler (WPS-3000), and rapid separation diode array detector (DAD-3000RS). Phenolic compounds were separated on an Acclaim C18  column . It was controlled at 30°C using a temperature controlled column compartment (TCC-3000). Data acquisition, peak integration, and calibrations were performed with Dionex Chromeleon software (Version 6.80 RS 10).Chromatographic analyses were carried out on a Thermo Scientific Dionex Ultimate 3000 Rapid Separation LC (RSLC) systems , coupled to a quaternary rapid separation pump (LPG-3400RS), Ultimate 3000RS autosampler (WPS-3000), and rapid separation diode array detector (DAD-3000RS). Phenolic compounds were separated on an Acclaim C18  of each phenolic compound was prepared in methanol by weighing out approximately 0.0050 g of the analyte into 50 mL volumetric flask. The mixed standard solution was prepared by dilution of the mixed stock standard solutions in methanol to give a concentration of 5 μg/mL for each polyphenol except (+)-catechin hydrate, caffeic acid, rutin hydrate (4 μg/mL), and quercetin (3 μg/mL). All standard solutions were stored in the dark at 5°C and were stable for at least three months.A stock standard solution  with methanol to yield 1.0–5.0  C. arvensis at a concentration of 5 mg/mL was prepared in methanol by vortex mixing  for 30 min. The samples were stored in the dark at low temperature (5°C). Spiking the sample solution with phenolic standards was done for additional identification of individual polyphenols.A solution of methanol extract ofμm nylon syringe filter  and then degassed in an ultrasonic bath  for 15 min.Prior to HPLC analysis, all solutions  were filtered through 0.20 R2 exceeding 0.995. Data are reported as means ± standard deviations of triplicate independent analyses.The compounds were identified by comparing with standards of each identified compound using the retention time and the absorbance spectrum profile and also by running the samples after the addition of pure standards. Quantification was performed by establishing calibration curves for each compound determined, using the standards. Linear calibration curves for standards (peak area versus concentration) were constructed with  Cnicus arvensis using the acetic acid induced writhing model in mice. The animals were divided into control, positive control, and test groups with six mice in each group. The animals of test groups received the plant extract at the dose of 100 and 200 mg/kg body weight each. The positive control group was treated with diclofenac Na (standard drug) at a dose of 25 mg/kg body weight and the vehicle control group was treated with 1% tween 80 in distilled water at a dose of 10 mL/kg body weight. Test samples, standard drug, and control vehicle were administered orally 30 minutes before intraperitoneal administration of 0.7% (v/v) acetic acid solution (0.1 mL/10 g body weight) to induce abdominal contractions or writhing. Five minutes after the administration of acetic acid, the number of writhing  for each animal was counted for 15 minutes. The number of writhing in the control was taken as 100% and percent inhibition was calculated as follows:The method of Hossain et al.  was adopμL of 5% formalin was injected into the dorsal surface of the right hind paw of all the group of mice 60 minutes after administration of test samples and 30 minutes after administration of diclofenac Na . Control group received only 5% formalin. The mice were observed for 30 minutes after the injection of formalin, and the amount of time spent licking the injected hind paw was recorded. The first 5 minutes of postformalin injection is referred to as the early phase and the period between 15 and 30 minutes as the late phase. The total time spent licking or biting the injured paw (pain behavior) was measured with a stopwatch.The antinociceptive activity of the test samples was also determined using the formalin induced paw licking test in mice . Formalin = 6 per group). The control group received vehicle (1% tween 80 in water at the dose of 10 mL/kg p.o.). The test group received the crude extract (at the doses of 150 and 300 mg/kg body weight p.o.) and standard group received diazepam at the dose of 1 mg/kg body weight orally. The animals were placed on the floor of an open field (100 cm × 100 cm × 40 cm h) divided into a series of squares. The number of squares visited by each animal was counted for 3 minutes on 0, 30, 60, 90, and 120 minutes during the study period.The open field test was carried out as described by Gupta et al. . The anit-test. The differences were considered significant at P < 0.01.Data were presented as mean ± SEM, and comparisons were made using one-way analysis of variance (ANOVA). Statistical differences between control and treated groups were compared by Student's  Cnicus arvensis aerial parts showed the presence of reducing sugar, carbohydrate, alkaloids, steroids, flavonoids, tannin, and saponins -epiquercetin .The experimental results indicated that methanol extract of Cnicus arvensis was tested on acetic acid induced writhing and formalin induced paw licking in mice.Antinociceptive activity of the methanol extract ofP < 0.01) and were comparable to the standard drug (diclofenac Na) which showed 74.86% inhibition of writhing at a dose of 25 mg/kg body weight.At the dose of 100 and 200 mg/kg of body weight, the plant extract produced 43.17 and 95.08% inhibition of writhing, respectively, in test animals in both dose-dependent manners in the acetic acid induced writhing test . The resC. arvensis extract significantly (P < 0.01) suppressed the licking activity in either phase of the formalin induced pain in mice inhibiting 69.87 and 75.55% of paw licking, respectively, at the dose of 150 and 300 mg/kg of body weight in comparison with standard diclofenac Na (68.56% licking inhibition) at the dose of 10 mg/kg body weight in the first phase. The extract inhibited 73.12 and 87.46% of licking and standard diclofenac Na inhibited 71.69% licking at the previously stated dose  for all doses with a dose-dependent response  is the major inhibitory neurotransmitter in the central nervous system [A receptors in the central nervous system [Extract ofs system . Earliers system , which lHPLC analysis showed the presence of vanillic acid and epicatechin in the plant extract, so it might be possible that these phytoconstituents are responsible for its antinociceptive and CNS depressant activities. On the other hand, this study supports the ethnomedicinal use of this plant in pain and inflammation. Mechanism of action of the compounds responsible for antinociceptive and CNS depressant activities may be identified on the basis of this study.


Endosulfan (ES) is a widely used organochlorine pesticide and is speculated to be detrimental to human health. However, very little is known about mechanism of its genotoxicity. Using mouse model system, we show that exposure to ES affected physiology and cellular architecture of organs and tissues. Among all organs, damage to testes was extensive and it resulted in death of different testicular-cell populations. We find that the damage in testes resulted in qualitative and quantitative defects during spermatogenesis in a time-dependent manner, increasing epididymal reactive oxygen species levels, affecting sperm chromatin integrity. This further culminated in reduced number of epididymal sperms and actively motile sperms. Finally, we show that ES exposure affected fertility in male but not in female mice. Therefore, we demonstrate that ES exerts pathophysiological changes in mice, induces testicular atrophy, affects spermatogenesis, reduces quantity and vigour of epididymal sperm and leads to infertility in males. Many a time, these benefits come at the cost of adverse effects on the environment and non-target organisms including humans.http://chm.pops.int/default.aspx). Due to its extensive usage spanning over 50 years, it is one of the most commonly detected pesticide in surface waters in USA without a declining trend and a much abundant OCP in the air.9 Although phased out in some countries, ES is still widely used in several Asian countries including India and China, subjecting ~40% of world population to its direct effects10–13 and is of worldwide concern due to increasing global trade of farm produce. Despite its association with many putative abnormalities and birth defects in humans in areas of extensive use, there are no molecular studies to investigate its mechanism of action in causing cellular damage, genomic instability and ill-health.World health organization (WHO) has classified ES as a Class II pesticide (moderately toxic) and it is listed under the Stockholm convention as a persistent organic pollutant, given its environmental impact  for a period of 20 days showed remarkable fluctuations in weight in a concentration-dependent manner in case of male mice, while there was no significant change in females . Thus, oμg/l and then showed a gradual decrease and by 24 h, dropped to 23 μg/l  after oral ingestion (3 mg/kg) . ES-spik 23 μg/l . The pea 23 μg/l . Analysi 23 μg/l . Hence, Liver function tests were performed to evaluate further biochemical changes in the organs. Results showed decreased levels of alkaline phosphatase (ALP), a hydrolase enzyme of liver, on days 1 and 21 after ES treatment indicating acute hepatotoxicity . Besides20 Interestingly ES treatment led to increased levels of WBC, possibly due to an inflammatory reaction.We have investigated the effects on RBC, WBC and platelets following exposure to ES in mice (3 mg/kg). The quantitative analysis at 1, 11, 21 and 31 days post-ES treatment showed decreased levels of RBC and platelets, which was also reported in humans following accidental ES ingestion .19,20 In+ (T cell marker) cell population in thymus and CD19+ (B cell marker) cell population in bone marrow, following treatment . Although the mean percentage did not vary for thymic and bone marrow populations, there was a marked difference in the CD19+ population, following treatment with ES as compared with controls, suggesting toxicity in a sub group of animals . Results showed that liver, testes and lungs were affected maximally on ES treatment  while brSince we observed the significant difference in mice testes physiology on ES treatment, we wondered whether this observation holds true in case of other model systems. Histological analysis of the wistar rat testes at identical ES concentration and time period showed significant reduction in the area of seminiferous tubules with fully or partially depleted spermatogonial mother cells and spermatids, indicating testicular damage .As ES-induced hepatic toxicity is associated with the altered metabolic activities following pesticide exposure, lungs and testes were chosen for further investigation to understand the mechanism of ES action.21 TUNEL-positive cells were repeatedly observed in testes, when experiment was repeated 11 days after ES treatment, suggesting persistent DNA damage . Results showed TUNEL-positive cells among spermatogonial mother cells, spermatocytes and spermatids 24 h post-ES treatment . InteresA damage . Brain sA damage .22 Since spermatogenesis is a time-dependent event, perturbation in any of its cell population in the seminiferous tubule would result in a whiplash response in the succeeding cell populations. Therefore, we were interested in investigating different testicular-cell populations in a time-dependent manner, following ES treatment . FACS analysis was performed on testicular cells using propidium iodide (PI) staining , subjected to Eosin Y staining and analysed. Compared with untreated control, we could not observe any difference in the morphology of hook, head and tail of the sperms , revealiTo check whether ES treatment affects chromatin integrity of epididymal sperms, sperm chromatin structure assay (SCSA) was performed. Results showed that the percentage of red-high sperms increased significantly on 1st day post treatment with ES (3 mg/kg). However, there was no significant difference between treated and untreated groups observed on day 11, 21 and 31 , suggest23 One of the important factors responsible for compromised sperm DNA integrity is elevated levels of ROS. Since we observed reduced sperm chromatin integrity within epididymal sperm, we wondered whether this could be due to ROS. We performed DCFDA-mediated FACS analysis of epididymal sperm collected on 1st day post-ES treatment (3 mg/kg), and results showed that the ROS level in treated group was significantly higher than that of control (Reactive oxygen species (ROS) are potential threats to the genome and are responsible for generating DNA single- and double-strand breaks. control . This su control . Taken tTo check the effects of spermatogenesis-dependent testicular-cell depletion on epididymal sperm following treatment with ES , clinical analysis of epididymal sperm was carried out post-ES treatment. The time points were selected based on the completion of the mating windows used in the experiment explained below. We found a reduction in epididymal sperm count on day 11 post treatment. The difference was prominent and significant when the sperm count was taken on day 26 and 36 post-ES treatment, suggesting that effect of ES on spermatogenesis contributed to a dramatically reduced sperm count . AlthougTo test whether the effect on sperm count and motility affects the fertility in mice, mating experiments  were set up, post-ES treatment in males. Three mating windows  covering one spermatogenesis cycle were selected. In all three mating windows, percentage of infertile males increased as opposed to no infertile males in control groups, indicating that the reduced sperm count and motility contributed towards reduced infertility in males . InteresSince ES could affect male fertility, we wanted to test the synergistic effect of ES when both males and females are treated (3 mg/kg). Mating experiments were conducted in 1 : 1 ratio of males and females at 5–10 days post treatment. Numbers of infertile males were calculated from the number of non-impregnated females. Further increase in the number of infertile males  was not observed as compared with the corresponding mating window, where only males were treated . This suIn the present study, using a variety of approaches we demonstrate that coupled with pathophysiological changes, ES induced tissue damage at an organismal level, exerting its effect maximally on testes causing cell death and depleting testicular-cell populations. ES-induced changes in testes were spermatogenesis dependent and led to the reduction in sperm count and motility resulting in male infertility.μg/l of ES within 24 h of treatment when 3 mg/kg of ES was administered to mice. This effective concentration is well within the range of concentration of ES or its derivatives found in human blood in the areas of active ES exposure  antagonist nature of ES. Several of the OCPs are found to be endocrine disruptors, acting as androgen or oestrogen antagonists. Previous studies have provided biochemical evidences to suggest ES as an AR antagonist, affecting gene expression in reporter-based assays.Drosophila melanogaster.27 Consistent to this, we did not detect any noticeable difference in body weight or physiology in females on exposure to ES, unlike males, suggesting that impact of ES in males is much more pronounced than females.Being an organ comprised of actively dividing cells and having high extent of pathophysiological damage induced by ES, we extensively analysed the fitness of spermatogenesis and its physiological consequences in testes from ES-exposed mice. Our results suggest that ES-induced damage in testes caused cell death in a spermatogenesis-dependent manner, affecting haploid, diploid and dividing mother cells over a complete spermatogenesis cycle, which was also reflected in the sperm quantity and quality and culminating in male infertility. Interestingly, unlike males, females did not show significant effect on exposure to ES, suggesting male-specific action of ES. Our results also provide an explanation for the observation of sperms having reduced competence in ES-exposed In conclusion, this is the first study that examines the mechanistic actions of a pesticide exerting pathophysiological changes, altering organ physiology, casing testicular damage and affecting testicular cells in a complete spermatogenesis cycle. A probable AR specificity further increases ES’ impact by its testes centric action and this damage further lead to qualitative and quantitative defects in spermatogenesis and results in male infertility.Chemicals and reagents were obtained from Sigma , Amresco  and Cisco Research Laboratories . ES  was obtained from Sigma.BALB/c mice or wistar rats of 6–8-weeks old were purchased from central animal facility, Indian Institute of Science, Bangalore, India and were maintained as per the guidelines of the animal ethical committee in accordance with Indian National Law on animal care and use. The animals were kept in polypropylene cages and provided standard pellet diet  and purified water. The standard pellet diet is composed of 21% protein, 5% lipids, 4% crude fibre, 8% ash, 1% calcium, 0.6% phosphorus, 3.4% glucose, 2%vitamin and 55% nitrogen-free extract (carbohydrates). The animals were maintained under controlled conditions of temperature and humidity with a 12 h light/dark cycle.ES (3 mg/kg) was dissolved in 0.05% of methyl cellulose and mice were orally fed using a gastric gavage. The treatment consisted of four doses on every alternative day. All the experiments were performed with the consent from Indian Institute of Science animal ethics committee (Project No. CAF/Ethics/244/2011).BALB/c mice were orally fed with ES . Animals were killed at 0.25, 0.5, 1, 2, 6, 12 and 24 h post administration and the sera were used for HPLC analysis.BALB/c mice, at the end of ES treatment (3 mg/kg) along with controls were killed, and bone marrow and thymus were collected . Blocking was done in 2% BSA and probed with anti-CD3-FITC, for thymus and CD19-APC for bone marrow. Cells were washed and analysed by flow cytometry . Using forward and side scatter, lymphocyte populations were gated and histogram was plotted using Flowing software  and the percentage of the gated cells was calculated and further used to plot vertical dot plots.BALB/c animals were killed along with controls after ES treatment  and blood was collected in a heparin-coated syringe by heart puncture and stored at 4 °C till use. RBC, WBC and platelet numbers were analysed within 2 h at Vijay Diagnostics, Bangalore, India.Blood samples were collected from control and treated groups of BALB/c animals following treatment with ES  using a non-heparinised syringe and allowed to clot at room temperature. After 1 h, sample was spun , plasma was collected and assayed for ALP, ALT, creatinine and urea using Autospan Kit .28–30 Briefly, tissues were fixed in 4% paraformaldehyde, processed in xylene, embedded in paraffin wax, sectioned to 5–10 μm in a rotary microtome  and stained with haematoxylin and eosin.31 Sections were then observed and photographed with light microscopy . Luxol blue staining was also performed on brain sections as described earlier.33Testes, liver, kidney, intestine, brain and lung tissues were dissected out from control and treated mice following treatment with ES (3 mg/kg) on 1st day after treatment completion. Tissues were processed for histological sectioning as described.TUNEL assay was performed  on testes and lung sections on 1st and 11th day after ES treatment (3 mg/kg). The tissue sections were de-paraffinized, rehydrated and permeabilized with proteinase K (2 mg/ml). Labelling reaction and detection was performed as per manufacturer’s directions. Images were obtained at ×20 and ×40 with Carl Zeiss Axioscope A1 light microscope (Germany).34–36 Briefly, ES-treated (3 mg/kg) and control animals (n=5 per group) were killed at respective time points  and the testes were washed in Hank’s Balanced Salt Solution and single cell suspension was prepared. Later, the cells were fixed at a dilution of ~5×106 cells/ml using 70% chilled ethanol. Cells were washed, resuspended in PBS, added 1 μg/ml of PI and analysed in BD FACSCanto (BD Biosciences), 10 min later. The data were analysed using Flowing software, and from the histogram, percentage of cells belonging to G0, 1n, G1, S and G2/M phases were calculated. Relative cell death was calculated by normalising G0 population of the treated group from a particular time point to the G0 population of respective control.Testicular FACS analysis was performed as described with modification.37–39 Following ES treatment , the animals were killed, epididymis was dissected out and placed in microfuge tube containing 1 ml TNE buffer  and mechanically disrupted to allow the sperms to swim out. After 2 min, the turbid solution containing sperms was taken without disturbing the sediment and diluted in TNE buffer to get a concentration of 1×107 cells per ml. Equal amount of sperm cells and 1% Eosin Y solutions were mixed, smeared on to the slide and allowed to dry. Using DPX, a cover slip was mounted and observed under light microscope  at a magnification of ×40. Morphological changes of head, hook and tail with respect to control sperms were noted for a number of 300 sperms per group.Analysis of epididymal sperm was carried out as described previously.40 (n=5 per group). Dilution of epididymal sperm was made as described above. Sperm was used from mice belonging to 1, 11, 21 and 31 days post treatment completion of 3 mg/kg of ES, and for FACS analysis 0.6 ml of acid detergent  was added to 0.3 ml of diluted sperm in TNE buffer and incubated for 30 s. 1.2 ml of staining solution ) was added to the mixture, incubated for 3 min and acquired in FACS. AO emits green fluorescence when bound to dsDNA, and red when bound to ssDNA or RNA. Since sperm head contains no RNA, a shift in red-high cells gives the measure of fragmented DNA in sperm. The red-high population was scored for the percentage of cells and plotted the cumulative percentage of red-high sperm in treated against that of control.SCSA was performed as described previously41 On the 1st day, 9 controls and 11 ES-treated (3 mg/kg) animals were killed, epididymis was dissected out and made dilution of 1X107 sperms per ml in TNE buffer. DCFDA was added to a concentration of 10 μM, incubated for 20 min at room temperature and analysed in BD FACSVerse (BD Biosciences).ROS level in epididymal sperm was assayed using DCFDA staining followed by FACS analysis.42 Briefly, 30 μl of sperm, diluted to 1X106/ml was mixed with 70 μl of low melting agarose and coated onto slides pre-coated with agarose. After solidification, the slides were subjected to 0.08 N HCl treatment for 17 min, lysis solution I  for 20 min, followed by lysis solution II  for 15 min. The slides were then washed in TBE buffer, dehydrated in ethanol, stained using PI and visualised under Apotome fluorescence microscope (Carl Zeiss). Sperm halo was analysed as described.43 Quantification of the sperm halo area was done using ImageJ software ).Sperm dispersion assay was performed for the sperm sample which showed highest ROS levels  in epididymal sperm ROS analysis and for a control.44 Briefly, at respective time points , treated animals were killed along with controls, the epididymis dissected out and homogenised in TNE buffer (1 ml), and allowed 3 min for the sperms to swim out . The sample was analysed immediately by the clinician at Infant laboratories, Bangalore, India, and percentage of actively motile, sluggish motile, non-motile sperms along with total sperm count were calculated.Clinical analysis of epididymal sperm was performed as described.45 For experiments involving treatment of males alone, males were treated and kept till the desired time window of mating . Females of 6–8 weeks of age were used for experiments. At 5, 20 or 30 days post treatment, 1 : 3  mating was set up in separate cages for 5 days. At the end of 5 days, males were separated from females and the females were observed for pregnancy in following days. A male was considered infertile if all three females in the cage failed to get impregnated. Average litter size was calculated, taking only the impregnated females into consideration. For experiments involving treatment of both males and females, animals were kept separately till 5 days, post treatment . At 6th day post-treatment completion, 1 : 1 mating was set up in separate cages and animals were separated after 5 days. Here also, infertile male is defined by the number of non-impregnated females in the respective cage.Mating experiments in BALB/c mice were performed as described.t-test was applied. If the data set did not follow normal distribution, a two-tailed Mann–Whitney test was applied for. Significance values were calculated as follows: *P<0.05, **P<0.01, ***P<0.001.All statistical analyses were performed using GraphPad Prism 5 software . For the analysis, if the data set followed normal distribution, a two-tailed, unpaired, student’s


Objective. To evaluate the effect of calorie restriction-induced weight loss with or without aerobic exercise on liver fat. Methods. Thirty-three adults with visceral adiposity were divided into calorie restriction  or CR and aerobic exercise  groups. Target energy intake was 25 kcal/kg of ideal body weight. The CR + Ex group had a targeted exercise time of 300 min/wk or more at lactate threshold intensity for 12 weeks. Results. Reductions in body weight , fat mass , and visceral fat  were not statistically different between groups. Liver fat decreased significantly in both groups, with no difference between groups. Change in maximal oxygen uptake was significantly greater in the CR + Ex group than in the CR group . Conclusion. Both CR and CR + Ex resulted in an improved reduction in liver fat; however, there was no additive effect of exercise training. Nonalcoholic fatty liver disease (NAFLD) is recognized as an independent predictor of insulin resistance and cardiovascular disease and is regarded as a liver phenotype for metabolic syndrome . The appRegular aerobic exercise reduces liver fat despite minimal or no weight loss –5. This Excess visceral fat accumulation is widely known as a risk factor for impaired glucose tolerance, decreased insulin sensitivity, and coronary heart disease and is thought to increase the release of free fatty acids and inflammatory adipokines into the portal vein for transport to the liver . In addi2), (2) were not taking any medications affecting glucose metabolism to avoid potential confounding effects on weight change, and (3) had no thyroid disease. Before baseline measurements, subjects were randomized to receive one of two interventions, with each lasting 12 weeks: diet-induced weight loss through calorie restriction  or diet-induced weight loss combined with aerobic exercise  ) or CR + Ex group . The flow of participants through the study is shown in The present study was conducted between March 2007 and August 2011 and was approved by the Ethics Committee of Fukuoka University. Males and females aged 40–75 years were recruited by advertisements in newspapers, on television, and on public transportation. Overall, 146 subjects were contacted to participate, and 89 subjects were eligible to be enrolled in the Metabolic Syndrome Prevention/Improvement Intervention Program. The participants enrolled in the study (1) had visceral adiposity , equivalent of BMI 22, by 25. Once a week, the participants received guidance from experienced dieticians (face-to-face), who recommended appropriate daily nutrition via lectures and counseling. The sessions included adjustments of caloric intake and behavioral therapy. Food diaries were reviewed every week, and the participants were given instructions on food intake based on the prescribed energy intake. The participants were instructed to measure their weight daily.Exercise training consisted of 20 min each of step exercises, bicycle ergometry, and walking or running (60 min per session), three times per week under the supervision of exercise trainers, and a further 120 min/wk (nonsupervised) at home with the aim of achieving at least 300 min/wk of moderate exercise. The International Obesity Task Force (IOTF)  and AmerTo calculate energy intake  and macronutrient breakdown , each participant's self-recorded dietary intake was evaluated before and during the intervention (weeks 10 to 12 of the intervention period). The self-recorded food intake was conducted for a 3-day period including two weekdays and either Saturday or Sunday. All the meals were photographed to increase the accuracy of the measurement.Physical activity (the number of steps) was assessed using a small uniaxial accelerometer  . The sub2) was calculated as BW (kg) divided by the square of body height (m2). Percent body fat (%FAT), body fat mass (FM), and lean body mass were determined using the underwater weighing method, and body density was estimated after correction for residual air by the O2 rebreathing method during underwater weighing. All anthropometric measurements and body composition were conducted after a ≥12-hour fast.BW was measured to the nearest 0.01 kg using electronic scales . Height was measured to the nearest 0.1 cm using a stadiometer. Body mass index (BMI: kg/mCT protocol was performed as previously described . Viscera2peak) was determined using an incremental exercise test on a bicycle ergometer . The test was continued until subjective exhaustion was achieved. We assumed that participants had reached maximal oxygen uptake (VO2max) when at least two of the following criteria were met: (1) a plateau in VO2 with an increase in work load; (2) blood lactate levels ≥8.0 mmol/L; (3) respiratory exchange ratio ≥1.10; (4) HR within 10 beats of the predicted maximum HR; and (5) ratings of perceived exertion ≥19. Respiratory gas analysis was conducted using the mixing chamber method to evaluate the volume of expired air, and the O2 and CO2 fractions were analyzed by mass spectrometry . Lactic acid was analyzed using the Biosen 5040-lactate analyzer . To take into account individual differences in BW, oxygen uptake was expressed as kilograms of BW.Peak aerobic capacity , triglycerides, high-density lipoprotein (HDL) cholesterol, leptin, and hemoglobin A1c (HbA1c). The analyses of these parameters have been previously described [Blood samples were obtained from the antecubital vein in the morning after a 12-hour overnight fast. Serum biochemistry analysis was conducted by SRL Inc. . The biochemical parameters included interleukin-6 (IL-6), insulin, high-sensitive C-reactive protein (hsCRP), high-molecular-weight adiponectin, tumor necrosis factor- (TNF-) escribed .t-tests were used to evaluate baseline differences between groups. Changes in variables from baseline to postintervention were determined by two-way time (baseline and postintervention) by-group (CR and CR + Ex) repeated measures analysis of variance. Within-group differences  were assessed by paired t-tests. In addition, because the age of the CR group was significantly younger than CR + Ex at baseline, changes in body composition , liver fat, and liver enzymes between groups were compared using analysis of covariance (ANCOVA) adjusted for age. hsCRP was logarithmically transformed to approximate normal distribution. Values of P < 0.05 were considered statistically significant. Data are expressed as means ± standard errors. SPSS version 12.0 software  was used for all statistical analyses.Unpaired P < 0.01), there was no correlation between age and change in liver fat. The fat intake of the CR group was significantly lower than that of the CR + Ex group (P < 0.05), but there was no correlation between change in fat intake and change in liver fat.The values for the anthropometric parameters, liver fat, blood pressure, metabolic parameters, calorie intakes, cardiorespiratory fitness, and step counts were not significantly different between the CR and CR + Ex groups at baseline. Although the CR group was significantly younger than CR + Ex group  than in the CR group (n = 15) , and the improvement in VO2max was only significant in the CR + Ex group (P = 0.016).As shown in α decreased significantly in the CR+Ex group.As shown in β-adrenoreceptors in the VF depot, resulting in greater FFA release and oxidation [P = 0.075). In contrast, Ross et al. reported that when weight loss through exercise or calorie restriction is equivalent, reduction in abdominal visceral fat is similar [≈2 kg) or subcutaneous adiposity (≈20 kg) [The results of the present study suggest that calorie restriction-induced weight loss, with or without aerobic exercise, may provide effective reductions in liver fat and liver enzymes. However, contrary to our expectations, the results of this study showed no additional reductions in liver fat and liver enzymes when aerobic exercise was prescribed together with calorie restriction. Previously, we reported that 12 weeks of aerobic exercise may reduce liver fat and liver enzymes . These rxidation . The pre similar , 23. The similar . Further similar . However similar , 27. The(≈20 kg) . ExercisP = 0.032). Furthermore, the estimated energy expenditure attributable to the exercise in the CR + Ex group was 260 ± 127 kcal/day, which would account for a loss of fat mass of 3.0 ± 1.5 kg over 12 weeks . While there are conflicting reports [In the present study, the CR+Ex group did not lose more weight than the CR group. This result might be influenced by nonadherence to the lifestyle intervention. The subjects who achieved the target energy intake were lower in the CR+Ex group compared with the CR group  [Earlier studies have shown a significant correlation between total fat and saturated fat intake and liver fat. In addition, Straznicky et al. underscored the important contribution of dietary factors, particularly saturated fat to liver function (ALT) . Howeversis 26%) . In addisis 26%) .The present study found that cardiorespiratory fitness increased significantly in the CR + Ex group only. Furthermore, HDL cholesterol significantly increased in the CR + Ex group compared with the CR group. Exercise is a widely recognized modality for raising plasma HDL cholesterol levels and is one of the metabolic adaptations contributing to a reduced risk of coronary heart disease , 40. RegThis study had some limitations. First, we were unable to use a noninvasive and highly accurate method of identifying the amount of fat in the liver such as that achieved through using MRS. However, it has been shown that liver fat measured by CT correlates well with liver fat obtained through needle biopsy . Second,Calorie restriction-induced weight loss, with or without aerobic exercise, may provide an effective reduction in liver fat and arteriosclerosis risk factors in adults with visceral adiposity. In addition, the results demonstrated that there were no additional benefits relating to liver fat and liver enzymes when aerobic exercise was prescribed together with calorie restriction.


Leishmania spp., offering new opportunities for epidemiological studies of this infection.Road-killed wild animals have been classified as sentinels for detecting such zoonotic pathogens as Leishmania spp. and Leishmania chagasi DNA by PCR in tissue samples  from 70 road-killed wild animals.This study aimed to evaluate the presence of Cavia aperea (Brazilian guinea pig), five Cerdocyon thous (crab-eating fox), one Dasypus septemcinctus (seven-banded armadillo), two Didelphis albiventris (white-eared opossum), one Hydrochoerus hydrochoeris (capybara), two Myrmecophaga tridactyla (giant anteater), one Procyon cancrivorus (crab-eating raccoon), two Sphiggurus spinosus (porcupine) and one Tamandua tetradactyla (lesser anteater) from different locations in the Central Western part of São Paulo state. The Leishmania chagasi DNA were confirmed in mesenteric lymph node of one Cerdocyon thous. Results indicated common infection in wild animals.DNA was detected in tissues of one Leishmania spp. and L. chagasi, as well as determining natural wild reservoirs and contributing to understand the host-parasite interaction.The approach employed herein proved useful for detecting the environmental occurrence of  Leishmania that infects several mammal species, including humans, and is transmitted by the phlebotomine sandfly. Leishmania species include visceral, cutaneous and mucocutaneous forms of the disease in both the Old and New Worlds [Leishmaniosis is a zoonotic, parasitic disease caused by kinetoplastid flagellate protozoan parasites of the genus w Worlds ,2.Great concern has been sparked by the contribution that global warming might be making to the recent increase in the number of reported cases and geographical areas . EnvironLeishmania spp. in wild animals have become more numerous in Brazil due to the importance of these species in the life cycle of leishmaniasis [Studies on maniasis . Studiesmaniasis -12. Howemaniasis -17.Despite the difficulty of culturing and histopathologically analyzing tissue samples from road-killed animals, molecular techniques can be used in the identification and typing of pathogens by polymerase chain reaction (PCR), which presents high specificity and sensitivity to a certain fragment of the pathogen’s specific DNA .Leishmania species [Leishmania kinetoplastic DNA (kDNA)-specific probes have been used for the detection and identification of this protozoan, and demonstrated to be useful for epidemiological field studies because a large number of samples can be handled simultaneously . A minic species .Leishmania spp. in tissues of road-killed wild mammals. Thus, research into new hosts by molecular techniques is distinctive in epidemiological studies of pathogens and represents suitable indicators of environmental contamination by Leishmania spp. [The present work aimed to describe possible new hosts for leishmaniasis by using molecular tools to detect nia spp. .Callithrix penicillata (black-tufted marmoset), four Cavia aperea (Brazilian guinea pig), one Cebus apella (capuchin monkey), 13 Cerdocyon thous (crab-eating fox), three Dasypus novemcinctus (nine-banded armadillo), one Dasypus septemcinctus (seven-banded armadillo), nine Didelphis albiventris (white-eared opossum), one Eira barbara (tayra), one Euphractus sexcinctus (six-banded armadillo), two Gallictis vittata (grison), two Hydrochoerus hydrochaeris (capybara), one Leopardus tigrinus (leopard cat), five Lepus europaeus (brown hare), three Lutreolina crassicaudata (latrine opossum), two Mazama gouazoubira (brown brocket deer), one Myocastor coypus (coypu), six Myrmecophaga tridactyla (giant anteater), three Procyon cancrivorus (crab-eating raccoon), two Puma concolor (cougar), two Rattus rattus (black rat), five Sphiggurus spinosus (porcupine) and two Tamandua tetradactyla (lesser anteater). Only recently killed animals (1–7 hours) and those with no exposed viscera were collected. This study is in accordance with the Brazilian Institute of Environment and Renewable Natural Resources’ (IBAMA) normative statement n. 119 of October 11, 2006, chapter VI, art .26, which authorizes the collection and transport of animals that were found dead for scientific or didactic purposes. This work was also approved by the Ethics Committee for Animal Experimentation at our Institution (CEEA/FMVZ n.211/2008).Seventy road-killed wild animals were studied: one The geographic positions of the road-killed animals, established through global positioning system (GPS), were plotted on a digital map using a geographic database by the TerraView 3.6.0 .et al. [Leishmania spp. PCR were also assayed for Leishmania braziliensis complex and Leishmania mexicana complex [Leishmania spp. were used in order to identify the DNA of all possible Leishmania species that cause visceral or cutaneous leishmaniasis. The cycling profile consisted of an initial denaturation at 95°C for three minutes, followed by 30 cycles at 95°C for 30 seconds, 63°C for 30 seconds and 72°C for one minute, and a final extension at 72°C for seven minutes. Positive controls were included in each assay and consisted of 10 ng of DNA extracted from Leishmania major (MHOM/SU/1973/5-ASKH) and L. chagasi (MHOM/BR/2002/LPC-RPV). Negative controls were: ultrapure water and DNA from T. cruzi (ColTryp 0032/MCAN/BR/2008/CAO) that were added to the mix-PCR. The PCR mixture was composed of 10 mM Tris HCl pH 8.0, 50 mM KCl, 1.5 mM MgCl2, 0.2 mM dNTP, 10 ρmol of each primer, 0.2 units of Taq DNA polymerase, and 10 ng DNA template.DNA extraction from the animals’ tissue samples  was carried out by using the kit Illustra™ Tissue & Cells Genomic Prep Mini Spin . PCR reactions were performed by employing the primers LinR4 (5’-GGGTTGGTGTAAAATAGGG-3’) and Lin19 (5’-CAGAACGCCCCTACCCG-3’), described by Aransay et al. , to ampl complex ,24. GenuLeishmania chagasi DNA was performed utilizing primers Lc14 (5’-CGCACGTTATATCTACAGGTTGAG-3’) and Lc15 (5’- TGTTTGGGATTGAGGTAATAGTGA-3’) on a 190 bp fragment, by using the following cycling profile: initial denaturation at 94°C for four minutes, 40 cycles of 94°C for 30 seconds, 59°C for 30 seconds, 72°C for 30 seconds, and 70°C for ten minutes. Positive controls were included in each assay and consisted of 10 ng of DNA extracted from L. chagasi (MHOM/BR/2002/LPC-RPV). Negative controls were: ultrapure water and DNA from T. cruzi (ColTryp 0032/MCAN/BR/2008/CAO) that were added to the mix-PCR. The PCR mixture was composed of 10 mM Tris HCl pH 8.0, 50 mM KCl, 1.5 mM MgCl2, 0.2 mM dNTP, 10 ρmol of each primer, 0.5 units of Taq DNA polymerase, and 10 ng DNA template.The amplification of Amplification was performed in a MasterCycler EP gradient . The sequence was analyzed by electrophoresis in 1.5% agarose with SYBR® safe DNA gel stained , and visualized in an image analyzer  by using VisonWorks®LS Software. Amplicons were purified by using ExoSap  and the sequencing reactions were carried out on both strands in a 3500 Genetic Analyzer (Applied Biosystems). The obtained sense and antisense sequences were visualized , aligned by the software MEGA 4 and compared with the NCBI database using BLASTn  .The present results draw attention to a very important source of research and emphasize the importance of using this biological resource in an epidemiological study of zoonotic infection.One of the main problems in elucidating leishmaniasis epidemiology is to identify and confirm that a vertebrate host is a natural reservoir. The natural reservoirs are widely unknown because of the difficulties in capturing a sufficient number of wild animals and due to the techniques used in isolating and identifying the parasite.Leishmania spp. may represent a useful alternative to the utilization of captured ones in research studies, as indicated by animal research ethics committees. In the present study, a great diversity of road-killed wild mammal species was found. Culture analysis and histopathology are difficult and laborious. Sensitive and specific molecular tools allow pathogens to be identified without the need of culturing.This approach of using road-killed wild animals for the molecular detection of Leishmania spp. and Leishmania chagasi was attempted from several wildlife species using PCR. Several studies have reported the presence of this parasite in mammalian species, including rodents, carnivores, primates and marsupials [In this paper, molecular detection of rsupials ,26-29.Leishmania spp. and Leishmania chagasi from deposited homologue DNA sequences, as determined by BLASTn analysis. Figure Table Cerdocyon thous (crab-eating fox). A previous report indicates seropositivity in wild non-captive Cerdocyon thous[Leishmania-free dog populations [Leishmania DNA was detected in 5/12  samples from yon thous. The impulations .Leishmania spp. DNA was detected in 1/3 Procyon cancrivorus (crab-eating raccoon). Voltarelli et al.[Leishmania antibodies in Procyon cancrivorus in Northwestern Paraná. These findings suggest that these species can act as a reservoir for Leishmania spp.In the current study, li et al. reportedDidelphis albiventris specimens (white-eared opossums), are habitat generalists and currently occur in areas near dwellings, including farms, yards and urban centers [Leishmania spp. in 2/8 group members may be common, as was already described in several regions of Brazil: Manaus, Amazonas state; in Barra de Guarituba, Rio de Janeiro state; in Amaraji, Pernambuco; and Bauru, São Paulo state [The members of Didelphidae, represented by  centers . This sp centers -37. The lo state -41.S. spinosus (porcupine), four C. aperea (Brazilian guinea pig), two Rattus rattus (black rat), two Hydrochoerus hydrochaeris (capybara) and one M. coypus (coypu). Current data show that 8/14  specimens of wild rodents were positive, a finding that corroborates the literature that considered some rodent as reservoirs of Leishmania spp.Rodents were represented by five D. novemcinctus (nine-banded armadillo), one E. sexcinctus (six-banded armadillo), one D. septemcinctus (seven-banded armadillo), two T. tetradactyla (lesser anteater) and six M. tridactyla (giant anteater). These animals present some peculiar physiological and ecological characteristics including a weak immune system and low body temperature, besides the fact that they live literally immersed in soil and organic matter, mainly in tropical and subtropical regions, under biotic and abiotic conditions that promote multiple encounters with a diverse group of pathogens and vectors.The superorder Xenarthra was represented by 13 specimens: three Leishmania spp. DNA in armadillos (one D. septemcintus) and anteaters (one T. tetradactyla and two M. tridactyla). Casadeval and Pirofski [Leishmania spp. to be a pathogen whose ability to provoke disease also depends on host immune response. Since the cellular immune response is weak in armadillos and anteaters, it is possible to detect yeast cells in many of their organs; however, this is not sufficient to cause disease as observed in human hosts. Taken together, these factors make xenarthrans suitable models for studying host-pathogen interaction [The present study confirms the occurrence of Pirofski  clarifieeraction .These animals are assumed to be sources of infection since the agent’s DNA was found in internal organs; in addition, parasitism may occur in internal and cutaneous organs, facilitating transmission from the blood meal by the vector that inoculates promastigote forms of the agent into the man while sucking.L. chagasi sequence deposited at GenBank (access number AF308682.1) (Table The identities of the amplicon were confirmed by direct double-strand sequencing which showed 100% similarity with Leishmania spp. are interesting. Of the 20 species described in the New World, five have never been reported to have caused visceral human leishmaniasis: Leishmania enriettii, Leishmania hertigi, Leishmania deanei, Leishmania aristidesi and Leishmania forattinii[L. forattinii was isolated from pooled liver and spleen of opossum Didelphis marsupialis captured in Conchas, SP, Brazil [Even without the DNA detection of the cutaneous leishmaniasis agents, the positive results for orattinii-48. The , Brazil .Leishmania forattinii and that the evaluated site is close to that where the parasite was first isolated, since the species nucleotide sequence deposited at the GenBank was not found. Considering the occurrence of both the cutaneous and visceral form, in the studied municipalities, it must be emphasized that the sandfly vector may be present and serve as transmitter of Leishmania to these animals and humans.It is suggested that the species is Leishmania spp., considering the wide variety of intermediate hosts that contribute to the epidemiological transmission chain of this infection.These findings corroborate the worldwide distribution of L. chagasi.It is important to emphasize that Bauru, SP, is endemic for leishmaniasis; therefore, our results indicate the need for epidemiological molecular biology research on environmental contamination by It was possible to evaluate 22 different wild species, without the necessity of exerting a laborious sampling effort. In fact, the numbers and diversity of road-killed animals are considerably higher and, in general, they are killed after their own natural habitats had been invaded by roads . In thisLeishmania spp. in addition to the significance of the environment and ecology of these positive mammals in the interaction of Leishmania with different Leishmania species that may be pathogenic to humans.These results show risk factors such as free movement of the circulating parasite and vectors, as well as the importance of road-killed animals as possible reservoirs for the transmission of Leishmania spp. and L. chagasi, as well as contributing to understand the host-parasite interaction.The presented results focus that road-killed animals may serve as an important reservoir for transmission of The authors declare that there are no competing interests.VBRP participated in the design of the study, data collection, laboratory tests, analysis and interpretation of data, writing and editing of the manuscript. PMM and EYH participated in the data collection, laboratory tests and took part in the writing. CV participated in geographical location of animals, analysis and interpretation of data, writing and revision of the manuscript. RCS participated in the analysis and interpretation of data, writing and revision. HL was responsible for the coordination, study design, analysis and interpretation of data, writing and editing of the manuscript. All authors read and approved the final manuscript.


The objective is to explore the value of preoperative magnetic resonance (MR) histogram analyses in predicting microvascular invasion (MVI) of hepatocellular carcinoma (HCC).Fifty-one patients with histologically confirmed HCC who underwent diffusion-weighted and contrast-enhanced MR imaging were included. Histogram analyses were performed and mean, variance, skewness, kurtosis, 1th, 10th, 50th, 90th, and 99th percentiles were derived. Quantitative histogram parameters were compared between HCCs with and without MVI. Receiver operating characteristics (ROC) analyses were generated to compare the diagnostic performance of tumor size, histogram analyses of apparent diffusion coefficient (ADC) maps, and MR enhancement.P <0.05), with area under the ROC curves (AUCs) of 0.66 to 0.74 for ADC and 0.76 to 0.88 for PVP. The largest AUC of PVP (1th percentile) showed significantly higher accuracy compared with that of arterial phase (AP) or tumor size (P <0.001).The mean, 1th, 10th, and 50th percentiles of ADC maps, and the mean, variance. 1th, 10th, 50th, 90th, and 99th percentiles of the portal venous phase (PVP) images were significantly different between the groups with and without MVI (MR histogram analyses—in particular for 1th percentile for PVP images—held promise for prediction of MVI of HCC. Liver transplantation was proved to be harmful in patients with resectable HCC with MVI. Noninvasive assessment of the probability of MVI could be beneficial, as preoperative prediction may allow for treatment optimization, for example, selecting appropriate patient for liver allocation due to the severe organ shortage and predicting prognosis etc.Liver transplantation and surgical resection are the potentially curative treatment options for patients with hepatocellular carcinoma (HCC).,7 Studies on methods for prediction of MVI in the patients with HCC included assessment of tumor margin by computed tomography (CT) or gadoxetic acid-enhanced MR imaging,,10 CT perfusion analysis, and apparent diffusion coefficient (ADC) measurement,,13 etc. However, assessment of tumor margin remains subject to interpretation differences. Furthermore, it does not provide a quantifiable measurement. CT perfusion requires a relatively complex image acquisition and postprocessing algorithm and also delivers relatively high radiation dose. ADC measurement provides only information on the mean value, which does not account for the underlying intra-tumoral heterogeneity.In daily clinical practice, MVI is always identified by pathology from resection or liver specimens after transplant. The value in the clinical decision-making is currently limited. Tumor biopsy is, still, a matter of debate. reported that MR enhancement histogram analysis could potentially be used to differentiate clear cell from papillary subtype of renal cell cancer with high accuracy. Studies also demonstrated that histogram analysis of ADC maps had potential value for predicting aggressiveness of endometrial cancer, prostate cancer, and bladder cancer. We hypothesized that histogram analyses of MR enhancement images and ADC maps could assess the histogram distribution of perfusion and cellularity of HCC, which might be used to predict MVI for HCCs preoperatively.Histogram analysis is a new approach for quantifying tumor heterogeneity using routinely acquired imaging data and refers to a mathematical approach to evaluate gray-level intensity variations within a region of interest (ROI), which may be used to assess intralesional heterogeneity. Chandarana et alTherefore, the purpose of this study was to assess whether there were any differences in histogram analyses of dynamic contrast-enhanced MR images and ADC maps to discriminate between the 2 groups with and without MVI in patients with HCC.22.1This retrospective study was approved by Institutional Review Board of our hospital and the requirement for informed consent was waived. We retrospectively evaluated 51 consecutive patients who were suspected with HCC and underwent hepatic MRI between February 2011 and August 2011. Inclusion criteria consisted of histopathologically confirmed HCC by hepatectomy or liver transplantation; without any preoperative cancer-related treatments, that is, trans-arterial chemoembolization and radiofrequency ablation; availability of preoperative dynamic contrast-enhanced (DCE) MR imaging and DWI within 10 days before surgery; without macrovascular invasion on MR imaging.2.2b = 0.500 s/mm2) in the axial plane with a single-shot, echo-planar sequence was performed before DCE-MRI. A total of 20 to 24 slices were obtained during a 15- to 20-second breath-hold by using generalized autocalibrating partially parallel acquisition (GRAPPA) with R factor of 2. ADC maps were calculated for each diffusion study by the standard console software of the system. The parameters were as follows: 2600/66 (repetition time msec/echo time msec), 128 × 112 matrix, 380–400 × 300–324-mm field of view, 7-cm section thickness with 2.1-mm gap, and 1500 Hz/pixel bandwidth. A 3D T1-weighted gradient echo sequence (volumetric interpolated breath-hold examination [VIBE]) with fat-suppression technique was performed before and after the administration of the contrast media. The following parameters were used: 5.04/2.31 (repetition time msec/echo time msec), 12° flip angle, 256 × 192 matrix, 380–400 × 300–324-mm field of view, 24-cm slab thickness resulting in an interpolated 4-mm section thickness, and 300 Hz/pixel/bandwidth. A parallel imaging technique (R factor of 2) was performed with GRAPPA. For DCE-MRI, gadopentate dimeglumine  was rapidly administered manually (at a rate of approximately 2 mL/s) by 1 investigator through a 20-gauge intravenous catheter placed in a cubital or cephalic vein. Immediately afterward, a 20-mL saline flush was administered at the same injection rate. Contrast-enhanced images were obtained 20 to 30 seconds for arterial phase (AP), 70 to 80 seconds for portal venous phase (PVP), and 180 seconds for equilibrium phase after the injection.All patients were performed with a 1.5T MR system . All images were obtained in the transverse plane with a body phased-array coil anterior and a spine array coil posterior. Three scan trace DWI (2.3www.eletel.p.lodz.pl/programy/mazda/).,19 Images were transferred from the hospital's PACS system to an offline workstation. All measurements were performed on the ADC maps, AP and PVP images. Measurements were performed by a single radiologist with 2 years specific experience in abdominal imaging (YQH). For each patient, the radiologist manually delineated free hand ROI around the largest cross-sectional area of the tumor. The largest available tumor was selected as target lesion in case of multiple tumors. Gray-level normalization of each ROI was performed, using the limitation of dynamics to μ ± 3σ , to minimize the influence of contrast and brightness variation. Then histogram data was generated for the ROI  is a normalized histogram vector , i = 1, 2, …, Ng, and Ng denotes the number of intensity levels.Histogram analyses were performed by using Mazda , and categorical variables were compared by the χ2 test or Fisher exact test as appropriate. Histogram parameters were compared between the group with MVI from that without MVI using Student t test or Mann–Whitney U test when not normally distributed. Receiver operating characteristics (ROC) analyses were further constructed to determine the potential diagnostic performance for detecting the presence of MVI. Corresponding areas under the ROC curve (AUCs) with 95% confidence intervals (95% CI) were calculated. Differences in AUCs were analyzed by comparing the ROC curves according to the method of DeLong et al. Statistical analyses were performed using the Statistical Package for the Social Sciences  and MedCalc . Differences with a P value less than 0.05 were considered statistically significant.Baseline characteristics of the patients were expressed as mean and standard deviation (SD) or count and proportion. Continuous variables were compared with Student 33.1P = 0.042) showed statistically significant associations with MVI with an AUC of 0.61 (95 CI: 0.46–0.74). For age, sex and a-fetoprotein (AFP), there were no statistically significant differences between the groups with and without MVI. All patients were classified as Child–Pugh A. The poorly differentiated HCCs had high probability of MVI compared with moderately differentiated HCCs (P = 0.01).The histopathological results revealed that 26 HCCs were positive for MVI, whereas 25 lesions were negative for MVI. The clinical characteristics and histopathological findings in patients with and without MVI are presented in Table 3.2P = 0.007–0.027, Fig. P >0.05).ADC map histogram measurements are provided in Table 3.3P = 0.007–0.014, Fig. P >0.05) for AP. For PVP, mean, variance, 1th percentile, 10th percentile, 50th percentile, 90th percentile, 99th percentile in the patients with MVI were significantly lower than that in the patients without MVI . The differences in AUCs among histogram analyses of ADC maps, AP images, or tumor size were not significant (P = 0.07–0.80).Tumor size, the highest AUCs of histogram parameters for ADC maps (1th percentile), AP (10th percentile), PVP images (1th percentile) were used to compare the diagnostic performance of presence of MVI in HCC Table . Compari4The goal of our study was to evaluate the value of MR histogram analyses for preoperatively predicting the presence of MVI of HCC. The results of our study indicated that the histogram-derived parameters for PVP images provided high accuracy for identification of MVI of HCC with AUCs up to 0.88 and showed significantly better diagnostic performance compared with that of ADC maps, AP images, or tumor size according to ROC analyses. ADC histogram analyses resulted in AUCs up to 0.74. AP images histogram analyses (AUC: 0.45–0.72) and tumor size (AUC: 0.61) showed low accuracy for predicting MVI. The diagnostic performance for histogram-derived 1th percentile of PVP images and ADC maps increased compared with mean values . which is at level beyond the resolution of routine imaging data. In theory, MVI could change perfusion of the tumor by invading the branch of portal venous. And the poorly perfusion is associated with higher tumor grades, poorer treatment response, higher probability of metastatic disease, and shorter progression-free survival.,23 Wu et al demonstrated that portal vein flow calculated from liver CT perfusion in HCC with MVI was significantly higher compared with HCC without MVI, but there was no significant difference for hepatic artery flow. Our study showed the similar results that PVP images histogram analyses outperformed AP images. The possible explanations might be that MVI mainly affected portal vein perfusion of tumor, besides tumor heterogeneity is determined by heterogeneous vascularization, cell density, necrosis, and fibrosis of a tumor. So the differences in contrast-enhancement of HCC between with and without MVI were more evident for PVP images than AP images. Previous studies reported that tumor size was supposed to be critical factors for prediction of MVI in HCC.,26 However, some studies demonstrated that tumor size was not an independent predictor of MVI by multivariate logistic regression analysis.,27 We found that tumor size showed less accuracy for detection of MVI in comparison with histogram analyses of PVP images.MVI is defined as the presence of tumor emboli in a portal vein radicle, large capsule vessel, or vascular space lined by endothelial cells only on microscopy,,29 Previous studies demonstrated that lower ADC values can be a useful predictor of MVI.,13 However, the AUCs of ADC histogram parameters for predicting MVI in our results were not very high (up to 0.73). The possible explanations might be that absolute ADC values are dependent on technical parameters and have been shown to vary between vendors; lower imaging resolution of ADC maps (matrix: 128 × 112 in our study), which might be insufficient to reveal histogram distribution features of the voxels; DWI was performed by using only 2 b values (0 and 500 seconds/mm2), which might affect the accuracy of ADC calculation. Theoretically, ADC measurement with DWI obtained with multiple b values could reduce the measurement error.DWI is a functional MR imaging technique that uses differences in the extracellular movement of water molecules to discriminate between tissues of varying cellularity. ADC can provide quantitative information related to the tissue cellularity and integrity of cellular membranes, as well as microcapillary perfusion by reflecting the molecular diffusion of water and perfusion. Our results suggested that heterogeneity of capillary perfusion and/or cellularity of HCCs were represented to a great extent by 1th percentile of PVP images and ADC maps than by mean values. This was in keeping with the results of previous study. Donati et al reported that 10th percentile ADC showed significantly higher accuracy than mean ADC in differentiating low-grade from intermediate- or high-grade prostate cancer.As compared with mean values of PVP images and ADC maps measurements, 1th percentile had higher diagnostic performance for prediction of MVI , which might be explained by different heterogenous biologic and perfusion characteristics between HCCs with and without MVI. Our findings showed that the 1th percentile PVP images and 1th percentile ADC maps for HCC with MVI showed significantly lower than that of HCC without MVI. The reason may be that HCCs with MVI have higher cellularity and/or decreased capillary perfusion.b values of 0 and 500 seconds/mm2. However, our results showed histogram of ADC maps had the potential value for prediction of MVI in HCC, despite the relative low AUC (up to 0.73). Recently, studies showed that intravoxel incoherent motion DW-MR imaging applying multiple b values might be a promising tool to improve data reproducibility and assess histopathological features of HCC. Histogram of intravoxel incoherent motion (IVIM) might hold the promise for detecting MVI.There are several limitations in our study. First, because of its retrospective nature, the possibility of a selection bias cannot be excluded; second, we performed DWI using only 2 In conclusion, histogram analysis of PVP images showed higher accuracy compared with that of ADC, AP, or tumor size for discriminating HCCs with MVI from HCCs without MVI. Moreover, the 1th percentile of PVP images might be the best parameter for risk stratification in patients with HCC at the time of diagnosis. Because of limited sample size, we did not go further on proposing and validating an algorithm with the clinical, laboratorial, and MR histogram markers. The current results are obviously still premature and warrant further validation of these preliminary data presented by large and prospective patient studies.


Purpose. To investigate the correlation between best-corrected visual acuity (BCVA), the foveal inner segment/outer segment (IS/OS) junction or ellipsoid portion of inner segment (EPIS/ellipsoid zone), and the cone outer segment tips (COST) line or interdigitation zone integrity in eyes with uveitic macular edema (ME). Method. A retrospective observational study involving all patients from January 2012 to December 2013 with uveitic ME was performed. All patients underwent BCVA using Snellen charts spectral-domain optical coherence tomography (SD-OCT) examination using Spectralis OCT . Results. Fifty-two eyes from 45 patients were included in this study. Multivariate analysis showed a negative correlation between BCVA and the central retinal subfield thickness (CST), the cystoid pattern of edema, and the interdigitation zone interruption. Univariate logistic analysis showed a strong correlation between the ellipsoid zone and the interdigitation zone integrity. Conclusions. The ellipsoid zone defect, the interdigitation zone interruption, and the CST are correlated with poor vision. Visual acuity is also strongly affected by the cystoid pattern. The interdigitation zone integrity appears to be the most important factor in the visual prognosis of uveitic ME. Macular edema (ME) is a typical, but nonspecific, complication of uveitis and occurs most frequently in those with vitreous involvement. ME is among the leading causes of decreased vision in patients with uveitis , 2.In the literature, ME is described in intermediate uveitis (25–70%), anterior uveitis (20–26%), panuveitis 35%), and posterior uveitis (20%) and it can dramatically affect vision [5%, and pAcute retinal necrosis, birdshot chorioretinopathy, Adamantiades-Behçet's disease, juvenile idiopathic arthritis, and sarcoidosis are the most common uveitis entities associated with ME .Optical coherence tomography (OCT) is a noncontact and noninvasive diagnostic technique, which is increasingly used for diagnosing macular pathology and evaluating the response to therapy –7. OCT pIn previous studies , 4, 8, 9Spectral-domain OCT (SD-OCT), the new OCT generation, was introduced recently. It provides a higher resolution and image formation up to 100-fold faster than the conventional time-domain OCT . SD-OCT The integrity of the outer retinal layers—and particularly the photoreceptor layer—has gained much interest because of its close correlation with visual function . A numbeOn SD-OCT, the outer retina has four distinct hyperreflective lines, which represent the external limiting membrane (ELM), inner and outer segments of the photoreceptors (IS/OS) junction otherwise named ellipsoid portion of inner segment (EPIS/ellipsoid zone), the cone outer segment tips (COST) otherwise named interdigitation zone, referred to as the intermediate line or Verhoeff's membrane, and the RPE. The innermost ELM is formed by the back reflection of the zonulae adherentes that joins the inner segment to the Müller cells. The EPIS is thought to represent the boundary between the inner and outer segments of the photoreceptors and is localized between the ELM and the RPE histologically. The interdigitation zone represents the outer tip of the cones. The outermost RPE line separates the photoreceptors from Bruch's membrane and choriocapillaris .The purpose of the present study was to investigate the correlation between the best-corrected visual acuity (BCVA), the foveal EPIS, and interdigitation zone integrity in eyes with uveitic ME.A retrospective observational study was performed on all patients with uveitic ME from January 2012 to December 2013. Inclusion criteria were ME diagnosed ophthalmoscopically associated with any anatomical type of uveitis .Exclusion criteria were other coexisting ocular diseases limiting visual acuity (VA): amblyopia, cataract, optic atrophy, macular epiretinal membrane (ERM), macular hole, or central scars.Informed consent was obtained from all patients involved in this research. The study was conducted in accordance with local and regional regulations, good clinical practice, and the tenets of the Declaration of Helsinki.The BCVA using Snellen charts was performed. ME was diagnosed by clinical examination.All patients underwent SD-OCT examination with Spectralis OCT . Raster scans (20 × 15 degrees) consisting of 37 high-resolution horizontal B-scans were performed. SD-OCT evaluation was performed by three ophthalmologists.Four patterns of ME, DME , CME Fi, and SRDAn evaluation of the central subfield retinal thickness (CST), the integrity or disruption of the interdigitation zone , the intR for statistical computing” environment, version 2.16 [P values were calculated using the Wald test. The CST in the multivariate analysis was intended to measure a 100 μm increase in thickness. Univariate logistic regression analysis between the interdigitation zone interruption and the EPIS interruption was performed in R using GLM.The CST measurement was considered a continuous variable and the mean ± SD was calculated. The decimal BCVA was measured on the Snellen decimal chart and considered ordinal data. As seen in the population distribution analysis of BCVA, the underlying population distribution did not exhibit a normal continuous distribution, even considering the BCVA above unity. Moreover, for retrospective studies, the truncation to 10/10 introduces a ceiling effect, making the normality assumption unsuitable  nor fixable using logMAR conversion. Thus, the median (1st–3rd quartile) values were reported. For the multivariate analysis, ordinal probit regression analysis  was used in the “ion 2.16 –20. The Q1–Q3  9–77) were included in this study. The patients comprised 22 males and 23 females. Demographic and clinical characteristics of the study population are summarized in Fifty-two eyes from 45 patients affected by uveitis, complicated by ME, with a median age of 32 years . The median BCVA was 0.6 (Q1–Q3  0.03–1.0). The mean (SD) CST was 411 μm (±203).The median duration of uveitis at examination was 48 months , CME in 13 eyes (25%), and foveal SRD in 11 eyes (21.15%). SRD was found in combination with other forms of ME. In particular, three eyes (5.76%) presented SRD in combination with DME and eight eyes (15.38%) presented SRD in combination with CME.Q1–Q3  0.03–1.0), that in eyes with CME was 0.6 (Q1–Q3  0.06–1.0), and that in eyes with foveal SRD was 0.4 (Q1–Q3  0.03–1.0).The median BCVA in eyes with DME was 0.7 (Q1–Q3  0.06–1.0) and it was 0.2 (Q1–Q3  0.06–0.8) in eyes with interdigitation zone interruption. The morphological features on SD-OCT with the corresponding CST and BCVA are reported in Interruption of the EPIS was observed in 26 eyes (50%), and interruption of the interdigitation zone in 13 eyes (25%). The median BCVA in eyes with EPIS interruption was 0.35 (P = 0.0009), CME (P = 0.012), interdigitation zone interruption (P = 0.0005), and age (P = 0.003). The univariate logistic analysis showed a strong correlation between the EPIS segment and the interdigitation zone integrity (P = 0.04). In the multivariate analysis the EPIS or interdigitation zone interruption had the same effect and led to generation of comparable models (ANOVA LR P = 0.29).The multivariate regression showed a negative correlation between BCVA and CST . High values of CST, which represent the increase in thickness and volume of the foveal area, negatively affect visual function (P = 0.0009). These results are in agreement with previous studies of ME secondary to uveitis [VA was strongly affected by the cystoid pattern P = 0.01. High va uveitis , 9.P = 0.0005). No previous study has investigated the correlation between VA and the interdigitation zone integrity in patients with uveitic ME. However, the importance of interdigitation zone integrity has been reported in other studies of various ocular diseases [In our study, the interdigitation zone interruption was the factor most significantly associated with poor vision , the status of the EPIS, and the status of the interdigitation zone in diabetic ME .Shimozono et al. considered the status of the interdigitation zone, in conjunction with the EPIS, to be a useful prognostic factor after ERM surgery. The photoreceptor status at 1 month, especially the interdigitation zone, was the parameter most strongly correlated with the BCVA at 6 months after ERM surgery .Itoh et al. reported a strong correlation between the interdigitation zone defect and the BCVA after pars plana vitrectomy for ERM removal. The interdigitation zone defect was significantly correlated with postoperative BCVA at 3, 6, 9, and 12 months, but not 1 month, postoperatively, suggesting continuous postoperative recovery from 1 to 12 months .Itoh et al. investigated the correlation between the recovery of foveal cone microstructure and the BCVA after macular hole surgery. Eyes with an intact ELM and EPIS at 12 months and a distinct or irregular interdigitation zone had significantly better BCVA than those with a disrupted interdigitation zone . Itoh etIt has been reported that disruption of the EPIS is associated with poor vision in uveitic ME .Maheshwary et al. reported a significant negative correlation between VA and disruption of the EPIS in patients with diabetic ME. The rate of EPIS disruption evaluated by SD-OCT was revealed to be a significant predictor of VA .In our previous study, EPIS disruption was strongly associated with CSF in uveitic ME but appears to be independent of the site of inflammation . In thisP = 0.04) which has not been reported previously in uveitic ME. EPIS disruption and the interdigitation zone defect, when considered together, showed a negative correlation with BCVA.Our statistical analysis showed a strong correlation between the EPIS and the interdigitation zone integrity (In conclusion, decreased vision has not been reported to be associated with the interdigitation zone defect in ME secondary to uveitis. Interdigitation zone integrity appears to be the most important factor in the visual prognosis of uveitic ME. An EPIS defect and interdigitation zone interruption and retinal thickness are correlated with poor vision. Also, the cystoid pattern affects VA. Further studies should investigate the prognostic role of the interdigitation zone in uveitic ME.


Background and Aims. To examine the changes in serum alpha-fetoprotein (AFP) levels after iron reduction by therapeutic phlebotomy in chronic hepatitis C patients. Methods. This retrospective study included 26 chronic hepatitis C patients. The patients were developed iron depletion by repeated therapeutic phlebotomies. Results. Iron reduction therapy significantly reduced the median level of serum AFP from 13 to 7 ng/mL, ALT from 96 to 50 IU/L, gamma-glutamyl transpeptidase (GGT) from 55 to 28 IU/L, and ferritin from 191 to 10 ng/mL (P < 0.001 for each). The rate of decline in the AFP level correlated positively only with that in GGT , although a spurious correlation was observed between the rates of decline for AFP and ALT. The AFP level normalized (<10 ng/mL) posttreatment in eight (50%) of 16 patients who had elevated pretreatment AFP levels. Normalized post-treatment ALT and GGT levels were seen in 12% (3 of 26) and 39% (7 of 18) of the patients, respectively. Multivariate analysis identified a post-treatment GGT level of <30 IU/L as an independent factor associated with post-treatment AFP normalization . Conclusions. Iron reduction by therapeutic phlebotomy can reduce serum AFP and GGT levels in chronic hepatitis C patients. Estimated 170 million people worldwide are chronically infected with hepatitis C virus (HCV) , 3. RoutEradication of HCV with interferon (IFN)-based therapy, including peginterferon alpha combined with ribavirin and telaprevir or boceprevir, is the most promising therapeutic intervention for preventing the progression to HCC in HCV patients . RecentlIron reduction by therapeutic phlebotomy was recently shown to reduce the risk for progression to HCC and improve biochemical and histological outcomes in chronic HCV patients , 19. TheAll patients provided written informed consent before participating in this study. The study protocol conformed to the ethics guidelines of the 1975 Declaration of Helsinki , as reflected in a priori approval by the institution's human research committee.C was based on the detection of anti-HCV antibodies and HCV-RNA in the serum. These patients met the following criteria: (1) adult > 20 years of age, (2) no anemia before therapy, as evidenced by a hemoglobin concentration >11 g/dL, (3) no hepatocellular carcinoma or other malignant disease, and (4) no habitual alcohol consumption.This study retrospectively investigated 26 consecutive chronic HCV patients  with persistently elevated serum alanine aminotransferase (ALT) levels. These patients were unresponsive to  or unsuitable  for antiviral therapy and achieved iron depletion by repeated therapeutic phlebotomies performed in the hepatology unit of Hamamatsu University School of Medicine Hospital between January, 2000 and December, 2009. The diagnosis of chronic hepatitis Iron reduction therapy was administered by performing intermittent phlebotomies. All patients underwent phlebotomy with the removal of 200–400 mL of blood monthly or bimonthly until iron deficiency was achieved (defined as either a serum ferritin concentration of 10 ng/mL or a blood hemoglobin concentration of 11.0 g/dL). The subjects were required to avoid an iron-rich diet during the therapy.Routine hematological parameters, including hemoglobin concentration, platelet count, ALT, gamma-glutamyl transpeptidase (GGT), ferritin, and AFP, were measured in our clinical laboratory using standard techniques.t-test or Wilcoxon signed-rank test. Correlation coefficients were calculated using Pearson's product-moment correlation analysis or Spearman's rank correlation analysis. Multiple linear or logistic regression analysis was performed to identify independent determinants. All tests of significance were two-sided, and P values <0.05 were considered to indicate statistical significance.Statistical analysis was performed using PASW Statistics, release 18.0 for Windows . All variables were tested for distribution using the Shapiro-Wilk normality test. For data not normally distributed, nonparametric statistics were used for the analyses. Quantitative pre- and posttreatment values were compared using the paired 4/μL and an aspirate aminotransferase to platelet ratio index (APRI) >1.5 for the prediction of severe hepatic fibrosis  with repeated phlebotomies. Iron depletion significantly reduced the median levels of serum AFP from 13 to 7 ng/mL, ALT from 96 to 50 IU/L, GGT from 55 to 28 IU/L, ferritin from 191 to 10 ng/mL, and hemoglobin concentration from 14.2 to 11.1 g/dL  in the ALT level was positively correlated with the decline in GGT , ferritin , and AFP  and negatively correlated with the total volume of blood removed , but not correlated with the change in the hemoglobin level (P < 0.001). The posttreatment ALT levels normalized in three patients .The ALT levels decreased in all patients after treatment. The rate of decline  and GGT , but not with decline in the hemoglobin and ferritin levels or with the total volume of blood removed (P = 0.001). The posttreatment AFP levels normalized (<10 ng/mL) in eight (50%) of the 16 patients with elevated pretreatment AFP levels. The median blood volume removed by phlebotomy tended to be less for achieving posttreatment normalization of AFP than of ALT . Multivariate analysis identified a posttreatment GGT level of <30 IU/L as an independent factor associated with posttreatment AFP normalization .All 16 patients with elevated pretreatment AFP (≥10 ng/mL) had decreased AFP levels after treatment. The rate of decline in the AFP level was positively correlated with the decline in ALT ( removed . MultiplAlpha-fetoprotein is a clinical serum marker for diagnosing HCC, especially in patients with chronic liver disease , 21. ElePrevious studies have suggested that an elevated AFP level is an independent risk factor for developing HCC , 11. TheA few studies have shown that phlebotomy can reduce serum AFP levels in patients with chronic hepatitis C –36. HoweThe mechanisms responsible for AFP reduction by phlebotomy remain unclear. In chronic HCV infection, iron-mediated oxidative stress contributes to hepatic injury  and therWith the development of potent direct-acting antiviral agents combinations, IFN-free therapy is likely to be approved in the near feature . HoweverFurther study is needed to determine if iron reduction therapy can reduce the risk of the development of HCC in HCV patients with elevated AFP. Similar intervention may contribute to the amelioration of increased risk for various iron-related cancers including HCC and colorectal cancer in patients with genetic hemochromatosis –49, consIn conclusion, iron reduction by therapeutic phlebotomy can reduce the serum AFP and GGT levels in HCV patients. This is probably mediated by the amelioration of enhanced hepatic iron-mediated oxidative stress and the resultant HPC expansion. This therapy may reduce the risk for progression to HCC in chronic HCV infection.