Aspirin resistance (AR) is a prevalent phenomenon and leads to significant clinical consequences, but the current evidence for effective interventional strategy is insufficient. The objective of this systematic review is thus to assess the efficacy and safety of Chinese herbal medicine (CHM) for AR. A systematical literature search was conducted in 6 databases until December 2012 to identify randomized controlled trials (RCTs) of CHM for AR. As a result, sixteen RCTs with a total of 1011 subjects were identified, suggesting that the interests of the medical profession and the public in the use of CHM for AR have grown considerably in the recent years. Tongxinluo capsule and Danshen-based prescriptions were the most frequently used herbal prescriptions, while danshen root, milkvetch root, Leech, and Rosewood were the most frequently used single herbs. Despite the apparent reported positive findings, it is premature to determine the efficacy and safety of CHM for the treatment of AR due to poor methodological quality and insufficient safety data. However, CHMs appeared to be well tolerated in all included studies. Thus, CHM as a promising candidate is worthy of improvement and development for further clinical AR trials. Large sample-size and well-designed rigorous RCTs are needed. Cerebral infarction and myocardial infarction can be fatal and seriously affect patients' living quality, and their incidence will increase greatly in recent decades, especially in developing countries, due to more work pressure and population aging , 2. Antiin vitro platelet reactivity is not properly blocked despite the use of aspirin, ranged from the specific failure to inhibit thromboxane A2 (TXA2), failure to inhibit a test of platelet function that is dependent on TXA2 production, and the very broad failure to inhibit one or more platelet function assays. (3) “High on-treatment residual platelet reactivity” found in vitro in patients with antiplatelet therapy does not necessarily mean “resistance” to the antiplatelet drug from a pharmacological point of view. (4) Some authors distinguished types of “aspirin resistance” [in vivo addition of aspirin; (b), pharmacodynamic resistance: platelet aggregability continued when in vitro aspirin was added, with the persistent formation of TXA2; (c), pseudoresistance resistance: platelet aggregability was continued even when in vitro aspirin was added, but there was successful inhibition of TXA2 formation.Aspirin resistance has been defined by clinical and/or laboratory criteria, but a generally accepted definition based on valid diagnostic criteria has not been established. The possible first description of AR has been debated since the 1980s; FitzGerald et al.  demonstristance” , 18 as fThe key laboratory parameters are used to examine platelet responses to aspirin in a laboratory study using measures such as: (a), Bleeding time: measurement of the time necessary for bleeding to stop after standardized skin incision; (b), light transmission aggregometry: analysis of light transmission after stimulation of platelets with arachidonic acid, collagen, ADP, or other agonists in anticoagulated platelet-rich plasma; (c), Impedance aggregation (whole blood aggregometry): Measurement of electrical impedance between two electrodes in whole blood after stimulation with arachidonic acid, collagen, ADP, or other agonists; (d), platelet function analyzer-100 (PFA-100), assessment of aggregation under high shear; whole blood is aspirated through an aperture coated with collagen and either epinephrine or ADP; (e), rapid platelet function assay (VerifyNow Aspirin system): analysis of light transmission in a test cartridge containing fibrinogen-coated beads in whole blood; (f), levels of serum TXB2: radioimmunoassay or ELISA; TXA2 is rapidly converted into the stable metabolite TXB2; (g), urinary 11-dehydro-TXB2: measurement using radioimmunoassay or ELISA; systemic TXB2 undergoes hepatic transformation into 11-dehydro-TXB2; (h), flow cytometry: automated laser detection of platelet activation markers  using antibodies; (i), thromboelastography: analysis of clot strength from formation to lysis after stimulation with arachidonic acid or ADP , 19–21.The current evidence for effective intervention of AR is insufficient. Clinical treatment of AR such as an increase in aspirin dose and the addition of other antiplatelet drugs is usually not effective , 22. AspFaced with the limitations of the presently available treatments, complementary and/or alternative medicine (CAM) is thus increasingly being sought to treat AR worldwide. Currently, the usage of CAM has increased between the general population and medical personnel in many countries . One recRheum palmatum and Erigeron breviscapus and some tonic herbs such as Codonopsis pilosula, Astragalus membranaceus, Angelica sinensis, and ginsenosides play an important role in the inhibition of TXA2 synthesis in vitro in the porcine lung microsoma as donor of enzymes [Ginkgo biloba leaves is an inhibitor of collagen stimulated platelet aggregation by reducing TXA2 formation in rat blood sample with a dose-dependent effect [Atractylodis lanceae rhizoma can inhibit collagen- or TXA2-induced platelet aggregation by suppressing the collagen-induced signal pathway, which is upstream of the release of TXA2 in rabbit blood sample with a concentration-dependent effect, and Poria(Indian bread) inhibits TXA2 receptor-mediated platelet aggregation by suppressing Gq-mediated signaling pathway in rabbit blood sample with a concentration-dependent effect [Pharmacological studies have found the multitarget intervention effects of CHM on the pathophysiology of AR . For exa enzymes . Ginkgolt effect . The ingt effect . Panaxtrt effect , 57. Atrt effect . Ilexonit effect . Ilexonit effect . Danshent effect  with thet effect  and witht effect . Thus, COwing to the significant health risk of AR and the limitations of currently available conventional therapies, there have been a number of controlled studies over the past decade to evaluate the efficacy and safety of CHM for AR. However, to date, there was no systematic review available to determine the efficacy of CHM for AR. In addition, many of the current clinical trials of CHM for AR are thought of as “not so good” studies according to Cochrane criteria. In a TCM reviewing process, researchers may need to include such papers to identify current problems and areas worthy of improvement and future development . TherefoThis systematic review is conducted according to the preferred reporting items for systematic reviews and meta- analyses: the PRISMA statement .Types of studies: studies have to be randomized clinical trials (RCTs) concerning specifically the effectiveness of CHM for AR, regardless of blinding, publication status, or language. Studies of quasi-RCTs which are allocating participants by date of birth, day of the week, medical record number, and month of the year were excluded.μmol/L ADP and >20% aggregation rate induced by 0.5 g/L arachidonic acid (AA). The diagnostic criterion of aspirin semiresistance (ASR) is meeting one of the two conditions above. The exclusion criteria of AR are the following: (1) aspirin allergy or asthma; (2) all kinds of blood disease, hemorrhagic disease, or bleeding tendency; (3) platelet count >450 × 109/L or <100 × 109/L; (4) using ticlopidine, clopidogrel, dipyridamole, unfractionated heparin, low molecular heparin, and other nonsteroidal anti-inflammatory drugs during the last 2 weeks of observation period; (5) active gastric ulcer patient; (6) gout patient.Types of participants: criteria for the diagnosis of AR are as follows: >70% platelet aggregation rate induced by 10 Types of interventions: any intervention using CHM monotherapy or adjunct therapy for AR in the treatment group was included regardless of the dose, frequency, and intensity of CHM. The interventions of control groups were regular low dose aspirin treatment (aspirin 100 mg/d), high dose aspirin treatment (aspirin 300 mg/d), no intervention, or other antiplatelet drug treatment such as dipyridamole, clopidogrel, and cilostazol. The treatment course was at least 2 weeks in these studies.Types of outcome measures: the primary outcome measurement was platelet aggregation rate induced by AA and/or ADP at the end of treatment course. The secondary outcome measurement was the clinical efficacy rate and adverse events at the end of treatment course. The clinical efficacy rate was defined as the decreased value of platelet aggregation rate before and after treatment. The standard of clinical efficacy rate met any one of the following: (1) AR becomes ASR or aspirin sensitive; (2) ASR becomes aspirin sensitive; (3) the decreased value of platelet aggregation rate is more than 10% and platelet active value is normal. The diagnosis criteria of aspirin sensitive are as follows: ≤70% platelet aggregation rate induced by 10 mL ADP and ≤20% aggregation rate induced by 0.5 g/L AA.We electronically searched CENTRAL, PubMed, EMBASE, Chinese National Knowledge Infrastructure (CNKI), VIP information database, and Wanfang Data Information Site until December 2012 to identify RCTs involving CHM for AR patients. In addition, we hand-searched a list of medical journals relevant to this topic and references from relevant articles. Using free text and the medical subjects headings (MeSH) terms combined “aspirin resistance” and “Chinese herbal medicine or traditional Chinese medicine or Chinese materia medicine or integration of Chinese herbal and Western medicine.”Two investigators independently reviewed the titles and abstracts to select potential references. Then, the two investigators read the selected articles independently and made a final decision for selection or not. Key data were extracted according to standardized criteria, including patients, methods, interventions, and outcomes. Disagreements were settled through discussion or consultation with one correspondence author.The methodological quality of RCTs was assessed independently by two investigators using the 12-item criteria from Cochrane Back Review Group . All datWe identified 106 potentially relevant articles. After removal of duplicates, 103 records remained. After going through the titles and abstracts, 78 papers were excluded on the basis that they were nonclinical trials, case report, or lack of comparison group. By reading the full text of the remaining 25 articles, 7 were excluded for not being RCTs or not real RCTs; 1 paper was removed due to plagiarism; 1 paper was excluded as a result of not carrying out random method. Ultimately, 16 eligible studies were selected out for this systematic review. The screening process is summarized in a PRISMA 2009 flow diagram .μmol/L ADP as an agonist and/or ≥20% induced by 0.5 g/L AA [There were a total of 1011 subjects in the 16 included trials of CHM for AR published between 2006 and 2012. Among them, 398 participants received CHM adjunct therapy; 217 participants received CHM monotherapy; 396 participants received aspirin alone therapy, the addition of other antiplatelet drug to aspirin therapy, or no treatment. The age of participants ranged from 40 to 84 years old, and the treatment duration ranged from 2 weeks to 3 months. All of the studies were performed in a single center and conducted in China. These studies related to a wide range of conditions including cardiovascular disease , 46–48, 5 g/L AA . Two stu5 g/L AA , 47. Onl5 g/L AA . Five st5 g/L AA , 39–41. 5 g/L AA , 40. Bot5 g/L AA . In two 5 g/L AA , 45, 67;5 g/L AA . Clinica5 g/L AA , 67, but5 g/L AA , 67, andSalvia miltiorrhiza Bge.), Huangqi  Bge. var. Mongholicus (Bge.) Hsiao or Astragalus membranaceus (Fisch.) Bge.), Shuizhi , and Jiangxiang  were the most frequently used single herbs, which were used for more than 3 times in all the trials. Nine of 11 preparations were Chinese patent medicine and have a rigorous quality control of herbal preparations, whereas the other 2 preparations were homemade [Seven Chinese herbal prescriptions were tested in 12 (72.5%) of the 16 included studies, while the other 4 studies used single herb or active ingredients . Tongxinhomemade , 49 . Although platelet aggregation rate was partly reduced after CHM monotherapy, there was no significant difference between CHM monotherapy group and no treatment control group (P > 0.05), One RCT  reportedP < 0.05). Chai et al. [P < 0.05) but has no significant effect in reducing platelet aggregation induced by AA (P > 0.05)  .P < 0.05). Based on the testing of TXB2 and TXB2/6-K-PGF 1 α, Su [P < 0.05). However, Luo et al. [There are 2 RCTs comparing CHM monotherapy with high dose aspirin therapy group (aspirin 300 mg/d) , 49. Stu 1 α, Su  indicateP < 0.05) [There were 6 RCTs comparing CHM plus aspirin with aspirin 100 mg/d therapy. All 6 trials indicated more significant effect of CHM plus aspirin for reducing platelet aggregation rate compared with aspirin 100 mg/d therapy ( < 0.05) –41, 43.P < 0.05, P < 0.01). However, Song et al. [P > 0.05).There were 5 RCTs that compared CHM plus aspirin therapy with dipyridamole or cilostazol or clopidogrel plus aspirin therapy , 45, 67.g et al.  and Zhang et al.  indicateP < 0.05, P < 0.01) [P > 0.05).There were 7 RCTs which compared CHM plus aspirin therapy with CHM monotherapy , 46–48 . Six RCT < 0.01) , 46–48,  < 0.01)  showed nP < 0.05). Liu et al. [P < 0.05). Su [P < 0.05).Clinical effective rate was reported in 4 out of 16 included studies , 49, 67..05). Su  and Luo P < 0.05 or P < 0.01). A number of mild adverse events were observed in 3 studies [Gingko biloba plus aspirin group and 17 cases in dipyridamole plus aspirin group (P < 0.01). Su [P < 0.01). Luo et al. [P < 0.01); see Among the 16 included studies, adverse events were reported in 7 studies , 49, 67. studies , 49, 67,.01). Su  reportedTo our knowledge, this study is the first systematic review to determine the efficacy and safety of CHM for AR patients. A total of 1011 subjects in the 16 included trials of CHM for AR have emerged between 2006 and 2012, suggesting that the interests of the public and the medical profession in the use of CHM for AR have grown considerably in the recent years. Most of herbal preparations were Chinese patent medicine, which have a rigorous quality control. The current evidence is insufficient to recommend the routine use of CHMs for AR because of the poor methodological quality and lack of safety data.Several methodological limitations in the primary studies should be addressed. First, although all the included trials claimed to be RCTs, only 4 trials , 40, 67 The available evidence from present systematic review is insufficient to recommend the routine use of CHM for AR because of the poor methodological quality, high clinical heterogeneity, and lack of safety data. However, CHMs appeared to be well tolerated in all included studies. Therefore, the efficacy and safety of CHM therapy for AR remain to be further determined. However, it should be remembered that a lack of scientific evidence does not necessarily mean that the treatment is ineffective . TXLC 3–41, 47 aCHMs are widely used in the treatment of AR. There are too many unanswered questions, making it impossible to draw positive or negative conclusions. The most frequently used standardized Chinese patent medicine such as TXLC and Danshen-based prescriptions as well as the most frequently used herbs, including danshen root, milkvetch root, Leech, and Rosewood that may contribute in composing a fundamental prescription, could be promising candidates for further clinical trials of AR. Owing to serious attention to methodological quality, we recommend that the CONSORT 2010 statement , RCTs inAngelica sinensis (Oliv.) Diels), Jiang , Yinxingye , Gancao , and Jianghuang , could increase bleeding risks in those patients with cardio/cerebrovascular diseases [The safety of herbal medicines has become a major concern to both national health authorities and the general public. Thus, World Health Organization (WHO) published WHO guidelines on safety monitoring of herbal medicines in pharmacovigilance systems in 2004 . Howeverdiseases . Howeverdiseases , 49, 67.diseases . Therefodiseases .Sixteen RCTs were identified in this systematic review for evaluating the efficacy and safety of various CHMs in the treatment of AR. Despite the apparent reported positive findings, it is premature to determine the efficacy and safety of CHM for the treatment of AR due to poor methodological quality and insufficient safety data. However, CHMs appeared to be well tolerated in all included studies. Therefore, CHM as a promising candidate are worthy of improvement and development for further clinical AR trials. Large sample-size and well-designed rigorous RCTs are needed to accurately determine the benefits and risks of CHM for AR.


Giardia lamblia , hookworm  and Strongyloides stercoralis . The proportion of infected children increased, and the number of disease episodes effectively treated with a single dose of mebendazole decreased, over the 5-year period.We studied gastrointestinal parasites in symptomatic Cambodian children attending a provincial hospital in Siem Reap, Cambodia between 2006 and 2011. A total of 16 372 faecal samples were examined by direct microscopy. Parasites were detected in 3121 (19.1%) samples and most common were  Intestinal helminths are a major global public health concern, particularly in developing countries . ApproxiCambodia has some of the poorest health indicators in Southeast Asia, with high rates of malnutrition . Less thCryptosporidium spp. if requested. Data were extracted retrospectively from the hospital’s database. Sample results from a patient within a 2-month window-period were defined as a single disease episode. Proportions were compared using chi-squared, Fisher’s exact and Mantel–Haenszel (MH) tests (adjusted for month each year) as appropriate using Stata v10.0 . Ethical approval was given by the AHC Institutional Review Board.Angkor Hospital for Children (AHC) is a government provincial children’s hospital providing free medical care to children aged 0–16 years from Siem Reap and surrounding provinces. We studied faecal samples routinely submitted from children attending AHC between January 2006 and September 2011. Indications for sampling were diarrhoea, unexplained abdominal pain, malnutrition and/or anaemia. Direct microscopy of a wet preparation of faeces was undertaken with a modified Ziehl–Neelsen stain for p = 0.63) but the proportion of positive samples varied significantly by age (chi-squared p < 0.0001). Giardia lamblia , hookworm  and Strongyloides stercoralis  were most common. In 309 children with mixed infections, hookworm/S. stercoralis , hookworm/G. lamblia  and G. lamblia/S. stercoralis  infections were predominant and all three parasites were found in 16 (5.2%) children.Of 16 372 faecal samples analysed, 12 622 (77.1%) were single samples from individuals and 3750 (22.9%) were multiple samples. In the 2756 disease episodes associated with gastrointestinal parasites, 2447 (88.8%) featured a single parasite type, 272 (9.9%) two types and 37 (1.3%) three types. G. lamblia  and hookworm  but there was no significant change for S. stercoralis [MH OR 1.01, all years (95% CI 0.98–1.04); There was an overall increase in the proportion of infected children over the whole 5-year period (compared with negative samples) [MH adjusted OR 1.03 for all years (95% CI 1.01–1.04); Ascaris and Enterobius) [p < 0.0001).Single-dose mebendazole has cure rates of ≥90% for only two of the parasites identified (erobius) , 9. UsinG. lamblia and hookworm infections although the number of S. stercoralis infections remained stable. Laboratory staff and methods remained unchanged during the study period as did the indications for sampling, with no significant differences in the proportion of patients attending the hospital sampled and no change in their age distribution. It is impossible to assess whether an increased awareness of parasitic diseases and a concomitant change in health-seeking behaviour occurred during the study.This first published report of gastrointestinal parasitic infections in symptomatic children from Siem Reap Province in Cambodia suggests a growing problem. There were annual increases of 1–4% for G. lamblia, hookworm and S. stercoralis [Single-dose mebendazole has limited efficacy against rcoralis , 9 and trcoralis . IncreasAn important limitation of this study is that direct microscopy was the only method used for parasite detection and there was no external quality control programme. This is the method currently in use in most hospitals in the resource-limited diagnostic setting of Cambodia and national estimates of parasite burden may be under-representative. Concentration techniques and/or agar plate cultures would have increased parasite detection . A furthSystematic and prospective surveillance of faecal parasite infections in Cambodian children could help confirm if these observed increases are real and provide a baseline to monitor the impact of future interventions. Alternative approaches might include mass drug administration with other anti-parasitic drugs, such as albendazole (improved hookworm treatment)  and/or tthe Wellcome Trust of Great Britain and the Li Ka Shing-University of Oxford Global Health Program.Funding for the study was provided by


Pseudomonas species, between August 2007 and July 2008. The annual mean total Pseudomonas count (TPC) was 1.20 × 104 (cfu/100 mL), 1.08 × 104 (cfu/100 mL), and 2.66 × 104 (cfu/100 mL), for the Alice, Dimbaza, and East London WWTPs respectively. The effluents were generally compliant with recommended limits for pH, temperature, TDS, DO, nitrite and nitrate; but fell short of target standards for turbidity, COD, and phosphate. The tested isolates were highly sensitive to gentamicin (100%), ofloxacin (100%), clindamycin (90%), erythromycin (90%) and nitrofurantoin (80%); whereas high resistance was observed against the penicillins (90–100%), rifampin (90%), sulphamethoxazole (90%) and the cephems (70%). MAR index ranged between 0.26 and 0.58. The study demonstrated that MAR Pseudomonas species were quite prevalent in the final effluents of WWTPs in South Africa; and this can lead to serious health risk for communities that depend on the effluent-receiving waters for sundry purposes.The final effluents of three  wastewater treatment plants (WWTPs) were evaluated to determine their physicochemical quality and prevalence of multiple antibiotics resistant (MAR)  Pseudomonas species are prominent members of this category of emerging waterborne pathogens [Pseudomonas aeruginosa) are opportunistic pathogens often associated with infections of the urinary tract, respiratory system, soft tissue, bone and joint, gastrointestinal infections, dermatitis, bacteremia, and a variety of systemic infections, particularly in patients with severe burns, cancer and AIDS [Pseudomonas species have been incriminated in a number of waterborne outbreaks including those associated with use of recreational waters [Globalization of trade, changing population demographics and changes in treatment technology are driving factors in the emergence of new pathogens, including those associated with the water systems . Majoritathogens . The Pseathogens . Membersathogens . Most meand AIDS . Althougl waters ; showersl waters ; thus maPseudomonas pathogens is particularly worrisome to stakeholders in the public health sector for two reasons. First, environmental or non-pathogenic forms of the bacteria may serve as a storehouse of genetic determinants which, if transferred to other bacterial strains, may confer novel virulence capabilities [Pseudomonas strains is on the increase, whereas few antibacterial agents are being developed in parallel. In the United States D’Agata [Pseudomonas species during a 9-year period, and Jung et al. [P. aeruginosa isolates were resistant to any anti-pseudomonal agent in 1998, 32% of isolates were resistant to at least three agents by 2002. Despite the rising threat of MAR Pseudomonas species, no new classes of drugs have been introduced since the advent of imipenem in the early 1980s, and none are expected to appear for commercial use in the near future [The emergence of waterborne bilities . Secondl D’Agata  observed D’Agata ) Pseudomg et al.  noted thr future . Thus liPseudomonas species as emerging waterborne pathogens is based primarily on their ability to live in biofilms  which often serves as protective cover for the bacteria against biological, physical, chemical and environmental stresses [Pseudomonas species are increasingly getting entrenched in the water system even after disinfection of water resources. Pseudomonas survival of chlorine disinfection was recently reported by Samie et al. [et al. [et al. [et al. [Pseudomonas aeruginosa. Given the significant correlation between effluent quality  and that of the receiving waters [Pseudomonas species into receiving surface water bodies portend great danger for the South African public health. This is more so as a significant number (about 80%) of the South African population were reported to depend on these surface water bodies for drinking, domestic, recreational and agricultural purposes [Pseudomonas species. The importance of stresses . Growth stresses . This exe et al. ; while X [et al.  corrobor [et al.  suggeste [et al.  found thg waters , it woulpurposes ,20. Furtpurposes ; and thePseudomonas species isolated from chlorinated municipal wastewater effluents in the republic. Given the prevalence and survival strategy of Pseudomonas species in water systems, coupled with their opportunistic nature, it is very likely that these pathogens are present in wastewater effluents in South Africa even after disinfection. It is therefore imperative that the presence of these pathogens in wastewater effluents meant to be discharged into South African waters be monitored in the interest of public health. This study was therefore designed to investigate the prevalence and antibiogram profiles of Pseudomonas species isolated from chlorinated effluents of three wastewater treatment plants in the Eastern Cape Province of South Africa.Whereas a considerable number of studies have been carried out on various pathogens isolated from wastewater effluents in South Africa, there is little or no report in the literature on the prevalence and antibiogram of The study sites were located in the Eastern Cape Province of South Africa. Three wastewater treatment plants (WWTPs) were carefully selected to represent rural , peri-urban  and urban  settings. Monthly samples were collected between August 2007 and July 2008 from approximately 1 m below the surface of the final treated (chlorinated) effluent just before it was discharged into the receiving water bodies. Samples were collected in duplicates in sterile one litre Nalgene bottles containing 0.1% sodium thiosulphate (3% solution) and transported in cooler boxes containing ice packs to the laboratory for analyses. Sodium thiosulphate was not included in samples meant for physicochemical analyses. All samples were analyzed within 24 h of sample collection.43−), nitrate (NO3−), nitrite (NO2−), and chemical oxygen demand (COD) were determined in the laboratory by the standard photometric method [All field meters and equipment were checked and appropriately calibrated according to the manufacturers’ instructions. pH, temperature, total dissolve solid (TDS), and dissolved oxygen (DO), were all determined on site using the multi-parameter ion specific meter (Hanna-BDH laboratory supplies). Turbidity and free chlorine residual (CR) were also determined on site using a microprocessor turbidity meter  and an ion-specific meter  respectively. The concentrations of orthophosphate (POc method  using thPseudomonas species was done according to standard spread plate technique on Pseudomonas Isolation Agar (PIA agar) (BD Diagnostic Systems). Briefly, aliquots of appropriately diluted samples were inoculated onto PIA agar and incubated at 35 °C for 18–48 h. Typical Pseudomonas colonies appear blue-green on PIA agar plates. Total Pseudomonas counts (TPC) were taken, and presumptive isolates were purified and stored on nutrient agar slants at 4 °C for further analyses. The presumptive Pseudomonas species were confirmed by standard cultural characteristics and biochemical reactions and using API 20NE . Pseudomonas aeruginosa (ATCC 27853) was used as control. The cultural isolation of Nineteen clinically relevant antibiotics were utilized for the antibiogram test. The paper disc antibiotics were supplied by Mast Diagnostics  and included: ampicillin (30 μg), cefotaxime (30 μg), cephalothin (30 μg), cefepime (30 μg), chloramphenicol (10 μg), clindamycin (2 μg), erythromycin (15 μg), gentamicin (10 μg), minocycline (30 μg), nalidixic acid (30 μg), nitrofurantoin (300 μg), ofloxacin (30 μg), oxacillin (1 μg), penicillin G (10 μg), rifampin (5 μg), sulphamethoxazole (5 μg), tetracycline (30 μg), vancomycin (30 μg), and ampicillin-sulbactam (20 μg).et al. [The antibiotic susceptibility test was performed and interpreted based on the disk diffusion method as described by the Clinical and Laboratory Standard Institute  using Muet al.  as follo    MAR = a/bwhere a = number of antibiotics to which the isolate was resistant; b = total number of antibiotics against which individual isolate was tested.p values of <0.05 or <0.01.Calculation of the means was done using Microsoft Excel Office 2007. Correlations (paired T-test) and analysis of variance (one-way ANOVA) were performed using the SPSS 15.0 version for windows program (SPSS Inc.). Correlations and test of significance were considered statistically significant at p < 0.05) and sampling site (p < 0.05); while TDS, nitrate, and phosphate showed significant differences with sampling site (p < 0.01) but not with season. p < 0.05) but not with season. There was no significant correlation between CR and TPC in Alice and East London treatment plants. However, a significant (p < 0.05) negative correlation was observed between CR and TPC at the Dimbaza treatment plant.Pseudomonas counts (TPC) during the study. TPC ranged from 0 to 4.9 × 104 cfu/100 mL. The highest TPC was observed in the Alice plant in October 2007; while the lowest counts were observed in the Alice  and Dimbaza (November 2007 and July 2008) treatment plants. The annual average TPC for Alice, Dimbaza and East London plants were respectively 1.20 × 104 cfu/100 mL, 1.08 × 104 cfu/100 mL and 2.66 × 104 cfu/100 mL. TPC varied significantly (p < 0.5) in effluents collected from Alice and East London treatment plants, but not with season. No significant difference was observed for other treatments either with season or sampling site.Pseudomonas belonging to four species  were isolated during this study. Ten of these isolates were randomly selected for the antibiogram assay against the panel of 19 antibiotics  and their potential impact on the receiving environments and public health were reported and discussed in details in our previous studies ,31,32. W4 to 106 cfu/100 mL) reported previously [4) observed by Alaoui et al. [Pseudomonas species from the effluents (p < 0.05) negative correlation between CR and TPC was recorded. The observation suggests that the Pseudomonas isolates in this study were generally resistant to chlorination even at concentrations far higher than the recommended limits (0.3–0.6 mg/L) of no risk at point of use [Pseudomonas species at CR concentrations as high as 3 mg/L. However, Mena and Gerba [P. aeruginosa has a reputation for being resistant to disinfection, most studies show that it does not exhibit any marked resistance to the disinfectants used to treat drinking water such as chlorine, chloramines, ozone, or iodine. The significance of this observation is that operators of WWTPs may be forced to increase their CR dosage; and attempts to eliminate Pseudomonas from water supply using relatively high dose of disinfectant may produce disinfection by-products more hazardous than the pathogen itself [Values for TPC in this study were lower than those  in lieu of the presence of pathogens in effluents to be discharged into the environment . The obs unknown , it is pPseudomonas belonging to four species  were isolated during this study. All four representative species have been reported in bioremediation/biodegradation studies [Pseudomonas species of both environmental and clinical significance. It is difficult to differentiate between pathogenic and non-pathogenic strains of Pseudomonas species. According to Alonso et al. [Pseudomonas species  may not show a clear delineation between virulent and non-virulent strains. Thirty-nine (39) strains of  studies ,43,44,45 studies ,47,48,49o et al.  opportunPseudomonas species were reported to be highly sensitive to gentamicin [P. aeruginosa strains due to resistance [et al. [Pseudomonas resistance to the aminoglycosides (including gentamicin) and fluoroquinolones (including ofloxacins) in clinical isolates; while Lateef [Pseudomonas strains isolated from pharmaceutical effluents. The observations were not surprising as clinical and pharmaceutical environments tend to exert more selective pressure (leading to antibiotic resistance) on bacterial populations than non-clinical/non-pharmaceutical  environments [Pseudomonas sensitivity to clindamycin and nitrofurantoin were not common in the literature, Nagata et al. [P. aeruginosa biofilm formation; while Navon-Venezia et al. [P. aeruginosa-associated chronic lung diseases, such as cystic fibrosis and diffuse panbronchiolitis. Contrary to the observation of this study, several reports have been documented on Pseudomonas resistance to clindamycin [Pseudomonas species were frequently resistant to these antibiotics [et al. [P. aeruginosa strains isolated from urinary tract infection patients in Jos, Nigeria; while Lateef [Consistent with the observation of this study ntamicin  and oflontamicin . The obssistance . Contrar [et al.  reportede Lateef  observedronments ,54,55. Ta et al.  reporteda et al.  suggestendamycin ,58 and nndamycin ,60. Wheribiotics ,60,61,62 [et al.  reportede Lateef  observedet al. [et al. [et al. [Pseudomonas species were naturally resistant to the penicillins, cephems and rifampin because they have relatively impermeable membrane, inducible efflux systems and a chromosomally encoded inducible β-lactamase. However, Murray et al. [et al. [Our tested isolates exhibited high levels of resistance to the penicillins (90–100%) the cephems (80%), rifampin (70%) and sulphamethoxazole (70%) in agreement with reports of other authors ,63,64. Cet al.  reported [et al.  observedy et al.  demonstr [et al.  suggesteet al. [Pseudomonas isolates that were resistant to only single antibiotics, all the tested isolates in this study showed multiple antibiotic resistances (MARs) ranging from five to 11 antibiotics distributed among three to seven classes. Consistent with the observation of this study, Paul et al. [Pseudomonas strains with resistance patterns varying between five and eight antibiotics; while Lateef [Pseudomonas with resistance patterns of two to seven antibiotics. Two major intrinsic mechanisms were reported to confer bacterial resistance to multiple antimicrobial drug classes: mutations in outer membrane porins resulting in reduced permeability to antimicrobials; and over expression of multidrug efflux pumps, which tend to pump out antibiotics before they (the antibiotics) have the opportunity of acting on their target [et al. [Although Malekzadeh et al.  reportedl et al.  reportede Lateef  documentr target . In addi [et al. . The obsPseudomonas species were prevalent in chlorinated municipal wastewater effluents in South Africa. Since the emergence of MAR Pseudomonas species is a public health issue, our data support the need for regular and consistent monitoring of municipal sewage effluents with a view to preventing the dissemination of these pathogens into the environment. This study demonstrated that MAR


Aponogeton madagascariensis) forms perforations between longitudinal and transverse veins in spaces known as areoles, via developmental PCD; cell death begins in the center of these areoles and develops towards the margin, creating a gradient of PCD. This gradient was examined using both long- and short-term live cell imaging, in addition to histochemical staining, in order to establish the order of cellular events that occur during PCD.Developmentally regulated programmed cell death (PCD) is the controlled death of cells that occurs throughout the life cycle of both plants and animals. The lace plant (m), nuclear shrinkage and PM collapse. Timing from tonoplast rupture to PM collapse was approximately 20 minutes. The entire process from initial chlorophyll reduction to PM collapse took approximately 48 hours. Approximately six hours following PM collapse, cell wall disappearance began and was nearly complete within 24 hours.The first visible change observed was the reduction in anthocyanin pigmentation, followed by initial chloroplast changes and the bundling of actin microfilaments. At this stage, an increased number of transvacuolar strands (TVS) was evident. Perhaps concurrently with this, increased numbers of vesicles, small mitochondrial aggregates, and perinuclear accumulation of both chloroplasts and mitochondria were observed. The invagination of the tonoplast membrane and the presence of vesicles, both containing organelle materials, suggested evidence for both micro- and macro-autophagy, respectively. Mitochondrial aggregates, as well as individual chloroplasts were subsequently seen undergoing Brownian motion in the vacuole. Following these changes, fragmentation of nuclear DNA, breakdown of actin microfilaments and early cell wall changes were detected. The vacuole then swelled, causing nuclear displacement towards the plasma membrane (PM) and tonoplast rupture followed closely, indicating mega-autophagy. Subsequent to tonoplast rupture, cessation of Brownian motion occurred, as well as the loss of mitochondrial membrane potential (ΔΨin vivo. These findings can be used to compare and contrast with other developmental PCD examples in plants.Results showed that a consistent sequence of events occurred during the remodelling of lace plant leaves, which provides an excellent system to study developmental PCD  Programmed cell death (PCD) occurs in both plants and animals, and is a highly regulated process that happens either as a part of normal development or in response to environmental influences ,2. ExampThe classification of plant PCD into categories based on morphological characteristics has been challenging in the past ,7,20. InAutophagy is known to play a major role in the regulation of death in animal cells, but the extent to which autophagy is involved in plant PCD has yet to be concretized ,24-26. Ain vivo to understand the order of organelle changes, which occur during developmentally regulated PCD in planta. Therefore, this study focused on establishing the sequence of cellular changes during plant PCD, to begin understanding cause and effect relationships between plant organelles.Processes such as leaf senescence, TE differentiation, and the deletion of the embryonic suspensor have emerged as valuable examples used to study plant cell death ,29,30. Hin vivo[The aquatic lace plant is a submerged monocot endemic to Madagascar ,32, thatin vivo,33 .in vivo long- and short-term live cell imaging. This study will also employ experiments using fixed tissue samples in order to supplement this data. As a subset of this goal, this paper will provide visual evidence for autophagy during developmental PCD in the lace plant.The process of perforation formation in lace plant leaves has been divided into five stages, as outlined in Gunawardena et al (2004) . The preThe gradient of cell death seen simultaneously in one areole of a window stage leaf was used to classify cells into groups . Thus, each cell that underwent PCD during perforation formation transitioned from EPCD to LPCD . Therefore, long-term live cell imaging was used to focus on individual cells  were in the vacuole in later stages of cell death Figure I, J. TheIn LPCD, nuclei were observed to be displaced, visibly smaller and pushed against the PM . All experiments were completed at least 5 times, unless otherwise stated. Additionally, all image plates were composed using Adobe Photoshop Elements version 6.0.Lace plants were propagated using the sterile tissue culture technique described in Gunawardena et al (2006) . In brieExperiments used window stage, or stage 2, lace plant leaves Figure B. Areolei compound microscope . Images were captured using a digital camera (DXM 1200c). NIS-Elements AR Version 3.0 software was used for both imaging and analysis on this microscope. All slides were prepared as wet mounts.All light microscopy observations were conducted using DIC optics on a Nikon Eclipse 90i microscope fitted with a digital camera (Nikon DS-Fi1). EZ-C1 3.80 imaging software was used for image acquisition and Ti Control was used for microscope control. Images were captured using DIC optics and corresponding fluorescent images were taken using either tetramethylrhodamine isothiocyanate  or fluorescein isothiocyanate  lasers. Maximum projection images were composed of approximately 30 to 50 successive z-stack images compiled using NIS-Elements AR version 3.0 software.Confocal laser scanning microscopy observations were conducted using a Nikon Eclipse TWindow stage leaves were cut from lace plants and segments of approximately 2 mm × 2 mm were fixed in 2% (w/v) gluteraldehyde in 0.05 M sodium cacodylate buffer (pH 6.9) in a vacuum at 20 psi for 24 h. These segments were rinsed in buffer and post fixed for 4 h at room temperature in 2.5% aqueous osmium tetroxide and then dehydrated in a graded ethanol series. Leaf pieces were then placed in ethanol:Spurr resin mixtures and embedded in Spurr resin and polymerized for 9 h at 70°C. A Reichert-Jung ultra-microtome was used to prepare the gold sections that were then gathered on formvar coated grids and finally stained with lead citrate and uranyl acetate. These prepared segments were viewed using a Philips Technai 12 TEM  operating at 80 kV. This microscope was fitted with a Kodak Megaview II camera , with appropriate software .Videos were acquired on a compound light microscope using the audio video interleave (AVI) capture function in NIS-Elements AR software version 3.0. Long term live cell imaging (~72 h) experiments were carried out using window stage leaves where PCD was not visibly initiated, as denoted by central cells of the areoles still possessing green colour due to the presence of chloroplasts. The leaves were mounted between a custom slide and coverslip, submerged in 400 μL of distilled water and then sealed with petroleum jelly to prevent evaporation. Every six hours, the samples were rinsed three times with distilled water, re-mounted, and refocused to the appropriate cells.Following the determination of the basic timeline of the cellular changes in PCD, it was necessary to obtain high-magnification videos for particular events. Videos were taken of carefully chosen window stage leaves, containing areoles in which the centermost cells were about to undergo membrane blebbing. The leaves were placed in between a slide and a coverslip and mounted as mentioned above. Samples were viewed for 1–6 h (short-term), with constant supervision to prevent changes in focal plane.All videos were compressed using the Radius Cinepak Codec and kept at full length and size until subsequent analysis. Videos were shortened to the desired length using either QuickTime Pro (Version 10.0), IMovie (Version 9.0.2) or Adobe Premiere Pro CS5 (Version 5.0) software. Arrows and text were inserted into the videos using Adobe Premiere Pro CS5 (Version 5.0) or QuickTime Pro (Version 10.0). Videos captured throughout individual long-term live cell imaging experiments were trimmed, compiled and edited using Adobe Premiere Pro CS5 (Version 5.0).2 and incubated overnight with 0.1 μM Alexa Fluor 488 phalloidin  with 0.1% Triton X-100, all in ASB. After rinsing, the segments were counterstained with 0.5 mg/ml PI for approximately 5 min, mounted in Fluorogel  and viewed via confocal microscopy.Window stage leaves were removed from plants and the midrib was excised. These segments were then fixed in 2% paraformaldehyde  solution containing actin-stabilizing buffer (ASB) for 3 h at 4°C. These segments were rinsed, cut into 5 mm20 to a final concentration of 0.6 μM. Window stage leaves were removed from lace plants, the mid rib was removed and the leaf was cut into segments of approximately 5 mm2. These leaf sections were incubated in CMXRos at room temperature in the dark for 60 min. Following this, the leaves were rinsed 8 times in dH20 and shaken at 100 rpm for 90 min. The leaf sections were then mounted in dH20 and viewed via confocal microscopy.CMXRos stain was dissolved initially in DMSO and further in dHWindow stage leaf segments were prepared as per the CMXRos staining protocol, fixed in FAA for 2 h, and rinsed 3 times in phosphate buffered saline (PBS). The TUNEL assay was completed according to the manufacturer’s instructions . Segments were counterstained with 3% (w/v) propidium iodide (PI) for 2 min. for co-localization of nuclei; PI fluorescence was excluded during figure preparation. Positive controls were competed with DNase 1 and negative controls were completed without the terminal deoxynucleotidyl transferase enzyme. All samples were then viewed with confocal microscopy.Window stage leaf segments were prepared as per the CMXRos staining protocol and incubated in a 5 μg/ml FM1-43 solution dissolved in ice cold PBS. Leaf pieces were then incubated in a vacuum at 20 psi for 4 h. These leaf segments were not rinsed, but counterstained immediately with 3% (w/v) PI for 3 min and subsequently mounted in Fluorogel . These segments were viewed via confocal microscopy within 1 h of staining.To conduct the vesicle counting trials, window stage leaves were stained as above. Cells from each stage of PCD were captured with z-stack imaging and compiled into a maximum projection image, to simultaneously show all planes. The total number of whole cells seen in each image was counted. The number of cells in each image that contained at least one vesicle was counted. The number of cells containing vesicles was then divided by the total number of cells and multiplied by 100 in order to obtain a percentage of cells containing vesicles per stage. This value was averaged over at least 120 cells per stage. Data was analyzed using a general linear model of variance ANOVA and the average percentages were compared using the Tukey test at 95% confidence intervals (P < 0.05). Statistical analysis was carried out using Minitab 16 statistical software .Three-dimensional image analysis was performed on window stage leaves stained with FM1-43, as described above. Confocal microscopy was employed to capture 30–50 successive z-stack images of cells from an areole of a window stage leaf. Files from the EZ-C1 software were exported and opened with NIS-Elements AR (Version 3.0), containing the Deconvolution Demonstration from Nikon. Images were compiled and edited with the Volume View function and the Deconvolution Demonstration Package.20. Leaves were then washed in distilled water, mounted on a slide and viewed with the compound light microscope.Window stage leaves were cut from lace plants and had their midribs removed. The leaf segments were then stained for one hour in a 0.5% (w/v) solution of Evans Blue dissolved in dHm: Mitochondrial membrane potential; CMXRos: MitoTracker Red CMXRos; EPCD: Early-Programmed cell death; FITC: Fluorescein isothiocyanate; LPCD: Late-Programmed cell death; NPCD: Non-Programmed cell death; PCD: Programmed Cell Death; PI: Propidium Iodide; PM: Plasma membrane; TE: Tracheary elements; TEM: Transmission Electron Microscopy; TRITC: Tetramethylrhodamine isothiocyanate; TUNEL: Terminal Deoxynucleotidyl Transferase dUTP Nick End Labeling; TVS: Transvacuolar strands.ΔΨThe authors declare that they have no competing interests.JW and CENL both contributed equally to manuscript preparation. JW carried out experiments including: selected short-term imaging, all FM1-43 staining and 3D analysis, selected CMXRos and all Evans blue staining, as well as TUNEL staining and imaging. CENL carried out experiments including: selected short-term imaging, cytoskeleton staining, TUNEL staining and imaging, selected CMXRos staining, statistical analysis, as well as light microscopy for cell wall degradation images. AND completed selected CMXRos staining, long-term live cell imaging, video editing and flow-chart compellation. JW drafted the first manuscript while JW, AND and CENL all contributed to final manuscript revisions. AHLANG, conceived the study, participated in its design and coordination, and helped in manuscript revisions as well as supervised all experimental work.Video clip compilation showing the development of a window over a period of 72 hours. Note that this video focused initially on early-PCD (EPCD) stage cells. Chloroplast pigmentation, movement and size differ between cells in different stages of PCD. Small aggregates become larger as PCD advances and individual organelles are seen attaching to the aggregate. Transvacuolar strands (TVS) can be seen within cells and are most frequent in EPCD. Note the advancement of EPCD visibly denoted by a decrease in chloroplast size and number, resulting in a reduction in chlorophyll pigmentation. The two cells of interest show a loss of chloroplast pigmentation, individual organelles and aggregates undergoing Brownian motion, and near the final stages of PCD, nuclear displacement followed by PM collapse. Approximately 100X sped up.Click here for fileTransvacuolar strands (TVS) in EPCD stage lace plant cells. Video displays chloroplasts and mitochondria  within lace plant cells, in addition to the occurrences of visible TVS. Note the instances of organelle movement along TVS. Also note the perinuclear accumulation of some chloroplasts. Note the visible reduction in anthocyanin from the top right corner to the bottom left corner. Approximately 100X sped up.Click here for fileAggregate cessation of Brownian motion. This video shows a gradient of cell death: control cells on the left, early-PCD (EPCD) stage cells in the center and late-PCD (LPCD) stage cells to the right. Video depicts perinuclear accumulation of chloroplasts. Note the moving organelle aggregates at the beginning of the movie. Note visible vacuole swelling, causing displacement of the nucleus and subsequent tonoplast rupture. Following tonoplast rupture, the organelle aggregate stops Brownian motion; PM collapse quickly follows this process. Approximately 200X sped up.Click here for fileMitochondrial aggregates stained with CMXRos. Video displays that the aggregate of organelles contains CMXRos stained, mitochondria. This video demonstrates that the aggregate is moving within the cells vacuole via Brownian motion. Refer to Figure Click here for fileLPCD stage cells displaying tonoplast rupture and subsequent events. This video depicts vacuolar swelling, displacing the nucleus toward the PM. Following this, the tonoplast ruptures, allowing the nucleus to be temporarily liberated. The nucleus then condenses. Following this, the tonoplast shrinks and PM collapse follows closely. Approximately 200X sped up.Click here for fileVisualizing the process of PM collapse. Note that the mesophyll cells’ PMs begin collapse prior to the epidermal cells. Also note the synchronicity of PM collapse. Approximately 200X sped up.Click here for file


Purpose. To evaluate in chronically haemodialysed patients (CHPs), if: (1) the vascular access (VA) position (upper arm or forearm) is associated with differential changes in upper limb arterial stiffness; (2) differences in arterial stiffness exist between genders associated with the VA; (3) the vascular substitute (VS) of choice, in biomechanical terms, depends on the previous VA location and CHP gender. Methods. 38 CHPs  were studied. Left and right carotid-brachial pulse wave velocity (PWVc-b) was measured. In in vitro studies, PWV was obtained in ePTFE prostheses and in several arterial and venous homografts obtained from donors. The biomechanical mismatch (BM) between CHP native vessel (NV) and VS was calculated. Results/Conclusions. PWVc-b in upper limbs with VA was lower than in the intact contralateral limbs (P < 0.05), and differences were higher (P < 0.05) when the VA was performed in the upper arm. Differences between PWVc-b in upper limbs with VA (in the upper arm) with respect to intact upper limbs were higher (P < 0.05) in males. Independently of the region in which the VA was performed, the homograft that ensured the minimal BM was the brachial artery. The BM was highly dependent on gender and the location in the upper limb in which the VA was performed. We have recently demonstrated that, in chronically haemodialysed patients (CHP), the vascular accesses determine significant changes in the upper limb native arteries' stiffness . More prNative vessels are the conduits of first choice in performing vascular accesses for haemodialysis, arterial reconstruction, and bypassing. However, in CHP, native vessels are often damaged or not available; therefore, the only current alternative is expanded polytetrafluoroethylene (ePTFE) prostheses. This choice is very far from the ideal solution because of their poor patency rates , 5. In tIntimal hyperplasia stenosis in the native vessel-vascular substitute anastomotic region is the Achilles' heel for most vascular accesses in CHP. It is determined, among other factors, by native vessel-vascular substitute biomechanical mismatch (BM) . Hence, The purposes of this work were to evaluate, in CHPif vascular access position (upper arm or forearm) is associated with variations in upper limb arterial stiffness,the potential gender-associated differences in arterial stiffness changes related to the vascular access position, and,if the vascular substitute of choice, in terms of its BM with respect to native arteries, depends on (a) previous vascular access location and (b) CHP gender.This study was approved by our Institutional Review Board and Ethics Committee. Patients gave written agreement for noninvasive studies and documented consent was obtained for the vascular homograft procurement from deceased human multiorgan donors according to no. 14005 and 17668 legal rules of the Uruguay.n = 18), and forearm (n = 20) level. The term upper limb was used to define the anatomic region between the shoulder and the wrist joints. The terms upper arm and forearm were used to define the part of the upper limb between the shoulder and the elbow joints, and between the elbow and the wrist joints, respectively. Haemodialysis was performed in the morning three times a week during 4 to 5 hours, on standard bicarbonate bath. Patients had been submitted to renal function substitutive therapy for  66 ± 56  months (Pulse wave velocity (PWV) was measured in 38  ambulatory patients (age:  53 ± 17  years old) who had AVF for haemodialysis in the upper limbs, at upper arm  were obtained by the same observer previous to the PWV measurements. Body mass index (BMI) and waist-to-hip ratio were calculated for all patients.The carotid-femoral and right and left carotid-brachial PWV measurements were performed in a stable environment, as in previous works , 13. TheFinally, blood was drawn from each patient and routine chemical analyses were performed to quantify haematocrit, haemoglobin, serum albumin, serum creatinine, calcium, phosphate, parathyroid hormone (PTH), urea, total cholesterol, HDL and LDL cholesterol, and triglycerides . Serum cin vitro study [In this study we included both biological  and synthetic  vascular substitutes. We obtained vascular segments (5 cm in length) from 10 deceased human multiorgan donors aged  31 ± 5  years: saphenous vein, femoral (muscular homograft), carotid (elastic homograft), and brachial  arteries. Vascular segments were submitted to a biomechanical ro study . Vessel In vitro dynamic analyses were performed in homografts and in 6 ePTFE  segments (5 to 6 cm in length). All conduits were mounted in a mock circulation loop and were maintained, immersed in, and perfused with Tyrode's solution  in a specimen chamber. The mock circulation loop consisted of polyethylene tubing, with fluid circulation powered by a pneumatic pump  that allowed pressure value and waveform adjustments. Intraluminal pressure was measured with a solid-state transducer . The segments' external diameter was measured with ultrasonic crystals  [in vitro studies, and considering our experimental conditions , we assumed a sound speed of 1580 m/s [iameter) . The ultiameter) , 17, 21,1580 m/s , 23.R0) was computed as the radius at 0 mmHg [h) was calculated asAfter being placed in the specimen chamber, all segments were allowed to equilibrate under steady state flow and pressure. The simulated haemodynamic conditions mimicked those of the CHP included in the study, ensuring isofrequency and isobaric comparisons among groups. During each experimental session, pressure and diameter signals were measured under dynamic conditions, displayed in real time, digitised every 5 ms, and stored for offline analysis. All recorded samples included at least twenty consecutive beats. At the end of each experiment, in order to calculate the conduit strain, a recording of the nonpressured midwall radius  was calculated as:The internal radius  of 1.06 g·cm−3.PWV was calculated using the Moens-Korteweg equation:EINC is the incremental elastic modulus and R is the midwall radius [l radius .EINC, values of the conduit strain (ε) and circumferential stress (σ) were calculated asIn order to determine R0 is the non-pressured midwall radius obtained as described above and P is the intraluminal pressure. Finally, the stress-strain loop was constructed in all cases; this allowed us to obtain the σ-ε purely elastic relationship, using the hysteresis-elimination method. EINC was calculated asdσ/dε is the first derivative of σ respect to ε [ect to ε .Finally, the CHP native artery-vascular substitute biomechanical mismatch (BM), expressed as a percentage, was calculated as , 13P < 0.05 was considered statistically significant. Unpaired t-test and ANOVA plus Bonferroni tests were employed. The statistical software used was the SPSS 17.0 .Measured and calculated values were expressed as mean value ± SD. A in vitro studies, performed according to the methodology described above, provided high quality biological wave recordings. No technical mistakes occurred during data acquisition. As was expected, the studied CHP showed an increased aortic stiffness level, evidenced by a higher carotid-femoral PWV than the expected for age and blood pressure-matched healthy European [Both noninvasive and European , 25 or SEuropean  subjectsPatients with the vascular access for haemodialysis in the forearm and those with the vascular access in the upper arm showed nonsignificant differences in terms of age, height, weight, and waist and hip perimeters . SimilarP < 0.05) in terms of height, haematocrit, PTH, and haemoglobin levels  than in the contralateral one . When AVF was performed in the forearm, a statistically significant difference was observed between contralateral upper limbs (10.50 ± 0.47 versus 11.30 ± 0.36 m/s); a similar finding was confirmed when the AVF was constructed in the upper arm (9.60 ± 0.61 versus 11.30 ± 0.78 m/s) (P < 0.05) in CHP with the AVF in the upper arm than in those with the vascular access in the forearm (−17.00 ± 5.18% versus −7.10 ± 2.29%) (Regardless of the vascular access location (upper arm or forearm), the carotid-brachial PWV was lower in the upper limb with the vascular access (.78 m/s) . The men± 2.29%) .The carotid-brachial PWV measured in intact upper limbs  of CHP described above was similar, regardless of whether the AVF was performed in the upper arm (11.30 ± 0.78 m/s) or in the forearm (11.30 ± 0.36 m/s) .P < 0.05) in the limb with the AVF than in its contralateral intact limb (P < 0.05) than in the contralateral intact limb (10.00 ± 0.67 versus 10.60 ± 0.57 m/s); similar findings were observed in males (8.70 ± 1.29 versus 12.60 ± 2.09 m/s)  . The men.09 m/s) .Human brachial artery homografts ensured the best BM values with native vessels , regardlP < 0.05) regardless if the AVF was performed in the upper arm or in the forearm. Similar differences were found when comparisons were made considering gender , see P < 0.05)  . Finally < 0.05) .We have recently demonstrated that in CHP, arterial stiffness values measured in the arterial pathway of the upper limb where the vascular access was constructed were lower than those measured in its contralateral limb with intact arteries  . ContinuFirst, the reduction in the carotid-brachial PWV was greater in upper limbs with the vascular access located in the upper arm.Second, carotid-brachial PWV reduction in upper limbs with vascular access was evidenced in males and females CHP. When the AVF was constructed in the upper arm, the arterial stiffness reduction was greater in males.Third, independently of the AVF location , the vascular homograft that ensured the best BM was the brachial artery.Fourth, the native vessel-vascular substitute BM showed significant differences, depending on the gender and the upper limb considered (intact or submitted to AVF construction).PWV provides reliable information about arterial stiffness. In fact, at present, the carotid-femoral PWV is considered the gold standard technique to evaluate the regional aortic stiffness using non-invasive methods . FurtherIn this study serum PTH levels were elevated (as is common in CHP ), and diAVF have shown a beneficial impact on aortic stiffness; this improvement would be greater in patients with increased PWV before vascular access construction . AdditioStiffness differences between limbs with the AVF in the upper arm and those with the vascular access in the forearm could be related with anatomical factors since the brachial artery diameter is the highest among upper limb arteries . This isThe AVF maturation and patency rates are lower in female than in male haemodialysed patients , 3, 34. Gender and location of the vascular access  differences could be the origin of previously reported findings, in which the authors pointed out that forearm AVF have low success rate in chronically haemodialysed females .When analysing the carotid-brachial PWV changes in hemodialysed subjects, at least three factors should be considered for an adequate interpretation of our results. At least in theory, the fistulas/PTFE grafts maturation could influence PWV values, since changes in AVF or grafts blood flow impedance associated with the maturation process could modify the upper limb local hemodynamic conditions. For instance, changes in local haemodynamic conditions could determine changes in the shear-stress applied to the endothelial layer, which could modify the arterial wall biomechanics through changes in the vascular smooth muscle tone. Taking into account this, looking for stable conditions, in our work the non-invasive studies were always done in mature AVF. Second and related with that stated above, it is noteworthy that our patients' average length on renal replacement therapy was 66 months. This time could differ (being higher or lower ) with thAccording to our results, in CHP, the location of the vascular access would result in differences in arterial stiffness evaluated through PWV measurements in the carotid-brachial pathway. In addition, as previously stated, the BM between the native vessels and the vascular substitute to be used in the vascular access construction could depend on (a) gender, (b) the evaluated limb (with or without a VA), and (c) location of the previous vascular access. Hence, the patient's characteristics and arterial biomechanical parameters should also be evaluated. Nonetheless, our results show that the brachial artery homograft would be the best vascular substitute of choice, in terms of BM improvement.Prostheses made out of ePTFE are widely used as a second choice in peripheral vascular surgeries, among them, vascular access construction. However, it is well known that ePTFE vascular accesses have poor long-term outcomes. This could be attributed to the intimal hyperplasia development, associated, among other factors, to the native vessel-ePTFE high BM . We haveWe conclude that in CHP the carotid-brachial PWV reduction was greater in patients with the AVF performed in the upper arm than in those with the AVF in the forearm. Additionally, when the AVF was constructed at upper arm level, the arterial stiffness reduction was greater in males than in females. Finally, the native vessel-vascular substitute BM could be minimized considering: (a) the histological type of vascular homograft, (b) CHP gender, (c) vascular access location , and (d) existence of a previous vascular access.


Previous studies have reported that pretreatment thrombocytosis is associated with poor outcomes in several cancer types. This study was designed to evaluate the prognostic significance of preoperative thrombocytosis in patients with non-small cell lung cancer (NSCLC) who undergo surgery.We retrospectively reviewed the records of 199 patients who underwent R0 resection for NSCLC between May 2003 and July 2006 at Seoul National University Bundang Hospital, Seongnam, Korea.P = 0.003) and disease-free survival  than those without thrombocytosis. In multivariable analysis, patients with preoperative thrombocytosis had a significantly greater risk of death and recurrence than those without preoperative thrombocytosis (risk of death: hazard ratio (HR) 2.98, 95% confidence interval (CI) 1.39 to 6.37, P = 0.005; risk of recurrence: HR 2.47, 95% CI 1.22 to 5.01, P = 0.012). A tendency towards a shorter OS and DFS was observed in three patients with persistent thrombocytosis during the follow-up period when compared with those of patients who recovered from thrombocytosis after surgery.The frequency of preoperative thrombocytosis was 7.5% (15/199). Patients with preoperative thrombocytosis had shorter overall survival (OS, Preoperative thrombocytosis was valuable for predicting the prognosis of patients with NSCLC. Special attention should be paid to patients with preoperative and postoperative thrombocytosis. Lung cancer is the most common cause of cancer-related death worldwide . Non-smaThe medical records of 274 consecutive patients who underwent surgical resection (lobectomy or pneumonectomy) of NSCLC between May 2003 and July 2006 at Seoul National University Bundang Hospital, Seongnam, Korea, were reviewed. Subsequently, 75 patients were excluded from the study because of the following exclusion criteria: 1) synchronous extrapulmonary malignancy; 2) apparent acute inflammatory disease in which the platelet count can rise; and 3) severe liver cirrhosis that may cause the platelet count to decline. The remaining 199 patients who underwent complete resection, either lobectomy or pneumonectomy with mediastinal lymph node sampling, were enrolled in the study. Data on patient demographics, laboratory results, and pathology based on the seventh edition of the American Joint Committee on Cancer staging system were evaluated. The study protocol was reviewed and approved by the Institutional Review Board of the Seoul National University Bundang Hospital. The study was conducted in accordance with the principles of the Declaration of Helsinki.4/mm3. A hemoglobin level of less than 13.0 g/dL in men and less than 12.0 g/dL in women was considered as anemia. A platelet count of more than 40 × 104/μL was defined as thrombocytosis, in agreement with other studies.Preoperative white blood cell count, hemoglobin level, and platelet count before invasive diagnostic procedures and within 4 weeks before surgery were determined. Leukocytosis was defined as a white blood cell count of more than 1.0 × 10Overall survival (OS) and disease-free survival (DFS) were defined as the time elapsed between the date of surgery and the date of death and the date of recurrence, respectively. Significant differences in variables in relation to thrombocytosis were tested using chi-square test or Fisher’s exact test, as appropriate. OS and DFS were evaluated using Kaplan–Meier analysis, and comparisons of survival between the two groups were made using the log-rank test.P value of more than 0.10 were removed during stepwise analysis. Two-tailed P values of less than 0.05 were considered statistically significant. All analyses were performed using IBM SPSS Statistics, version 19.0 .The relative impact of variables was analyzed by univariate analysis using the Cox proportional hazards regression method. Multivariate analysis was performed using a backward stepwise method. pT and pN stage were excluded from multivariate analysis due to multicollinearity. Variables with clinical significance and statistical significance levels of less than 0.05 were selected for covariate analysis. Variables with a Patient clinicopathological characteristics are summarized in Table P = 0.003 and P = 0.002, respectively).The relationship between preoperative thrombocytosis and clinicopathological factors is shown in Table P = 0.003; Figure P <0.001), smoking history (P = 0.030), stage (P = 0.037), and preoperative thrombocytosis (P = 0.005) as significant prognostic factors for OS. Multivariate analysis confirmed that age (P <0.001), stage (P = 0.034), and preoperative thrombocytosis (P = 0.005) were independent poor prognostic factors for OS (Table With a median follow-up period of 65 months (range: 0.7 to 102 months), the 5-year OS and DFS rates were 75.2% and 58.3%, respectively. The 5-year OS rate of patients with thrombocytosis was significantly lower than that of patients without thrombocytosis , stage (P <0.001), and preoperative thrombocytosis (P = 0.012) were independent prognostic determinants of recurrence .Platelet counts were normalized in 12 out of 15 patients with preoperative thrombocytosis. Among these 12 patients, seven patients recovered from thrombocytosis within 1 month and five patients recovered from thrombocytosis after a few months . Thrombocytosis persisted during the follow-up period in three out of 15 patients. Of these three patients with persistent thrombocytosis, two patients developed systemic recurrence and one had a local relapse after surgery. A tendency towards a shorter OS and DFS was observed in these three patients with persistent thrombocytosis when compared with those of patients who recovered from thrombocytosis (median OS, 63 months versus 15 months, Our findings demonstrated that preoperative thrombocytosis in patients with resectable NSCLC was significantly associated with an increased risk of death and disease recurrence. Several previous studies suggested that anemia or leukocytosis in patients with NSCLC might have a negative impact on survival -4. Howevet al. in 1872 [A relationship between elevated platelet counts and malignant tumors was initially reported by Reiss  in 1872 . To date in 1872 . In our et al. [et al. [Correlations between thrombocytosis and shorter survival times have been shown for many solid tumors including gynecological cancers -16, gastet al.  suggeste [et al.  demonstrin vitro and in vivo murine models have demonstrated that platelets activated by cancer cells may induce the adhesion, growth, and distant metastasis. Platelets provide a procoagulant surface to facilitate cancer-related coagulation; thus, platelet adhesion to the tumor cells may protect the tumor cells from immune responses that will lead to cancer growth and dissemination [The mechanism underlying thrombocytosis in cancer patients is not yet fully understood. It is also unclear whether thrombocytosis is a reaction to more aggressive tumors or an active disorder that aggravates cancer progression. Nevertheless, several mechanisms of both tumor-induced platelet activation and platelet-induced cancer progression have been reported. Tumor-related humoral factors, such as granulocyte colony-stimulating factor, interleukin-1, and interleukin-6, may play a role in stimulating megakaryocyte growth and platelet production -26. In amination . AlthougOur study had the limitations of retrospective study, including the relatively small number of patients from a single hospital. Because of the limited number of patients, our results should be interpreted cautiously. In addition, the impact of preoperative and postoperative treatment-related outcome was not evaluated in this study.In conclusion, preoperative thrombocytosis is an independent poor prognostic marker for resectable NSCLC. Additionally, our data showed that persistent thrombocytosis may also be a predictive marker for recurrence. Further prospective studies with a larger sample size are required to confirm the prognostic value of preoperative and persistent thrombocytosis in NSCLC patients.ADC: Adenocarcinoma; CI: Confidence interval; DFS: Disease-free survival; HR: Hazard ratio; NSCLC: Non-small cell lung cancer; OS: Overall survival; SCC: Squamous cell carcinoma.The authors declare that they have no competing interests.MK, HC, and JSL designed this study, collected data, performed analysis, and drafted the manuscript. YJK, CTL, and JHL participated in the study design, literature search, and study coordination. HCY, SJ, and KK collected data and performed data analysis. JHC participated in the study design and helped to draft the manuscript. All authors read and approved the final manuscript.


Objective(s): Green tea (GT) is one of the most popular beverages worldwide whose beneficial effects on health have been demonstrated. Recent studies suggest that GT may contribute to reduction of cancer risk and progression. The aim of this study was to evaluate the effects of whole GT on urinary bladder chemical carcinogenesis in male and female ICR mice. Materials and Methods: The GT characterization was performed using spectrophotometric methods. Urinary bladder lesions were induced using N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN) by gavage during 10 weeks and whole GT (0.5%) was provided ad libitum during 20 weeks. Results: Animals from groups BBN+GT and BBN only developed preneoplastic lesions. Conclusion: We did not observe any effects by GT infusion administration on urinary bladder cancer development. Schistosoma haematobium’s chronic infection is another factor that contributes to increase in the incidence of this disease (N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN) is a chemical carcinogen that in rodents induces the development of preneoplastic and neoplastic urothelial lesions similar to those observed in humans and accompanied by a significant inflammatory reaction  equivalent (milligrams per gram) from a calibration curve. Antioxidant activities of the GT and leaf samples were estimated by trolox equivalent antioxidant capacity assay (TEAC) according to Liebert et al  using an Ultimate 3000 Column Compartment Dionex equipped with a photodiode array detector (PDA) 100 Dionex. An ACE5C18 reversed phase column (250×4.6 mm×5 µm) was used. The elution (1 ml/min) was performed using a solvent system comprising solvent A (5% of formic acid) and solvent B (methanol), using a gradient starting with 5% of solvent B until 2 min, linearly increasing to 65% of solvent B until 65 min and decreasing to 5% at 65 min and held constant until 75 min, at 35°C. The injection volume was 50 µl. The PDA detector was set in the range of 200-600 nm. Wavelength of 280 nm was used to identify catechins and caffeine and wavelength of 325 nm was used to detect hydroxycinnamic acids. The compounds were identified based on their retention time when commercial standards were available and by comparison with the UV-spectrum. The composition of GT infusion was analyzed at 0, 24, and 48 hr after its preparation.Animalsad libitum.The design and experimental procedures were performed in accordance with the EU regulations (Directive 2010/63/EU) on protection of animals used for experimental and other scientific purposes. Forty-one, five-week-old ICR mice , were obtained from Harlan-Interfauna (Spain). Animals were placed in polycarbonate cages using wood chips for bedding. During experimental protocol, animals were housed in controlled conditions of temperature (23±2°C), humidity (55±5%), lighting (12/12 hr light/dark cycle), and air system filtration (10–20 ventilations/hr). Standard laboratory diet (Harlan) and GT were provided ChemicalsBBN was purchased from Tokyo Kasei Kogyo (Japan). The GT leaves (Thea Sinensis L.) were purchased from Augusto Coutinho Ervanários . Folin-Ciocalteu reagent, GA, ABTS, trolox (Tx), Qr were purchased from Sigma (USA).Experimental designMales group I: BBN+GT (n=8); group II: BBN (n=7); group III: GT (n=6); Females group IV: BBN+GT (n=7); group V: BBN (n=7); group VI: GT (n=6)]. BBN was prepared in a concentration of 0.05% with bidistilled water and absolute alcohol. It was administered to animals by gavage in mean doses of 7.25 mg/mouse, two times per week, during ten consecutive weeks. The whole GT (0.5%) was daily prepared and given ad libitum to groups I, III, IV and VI for 20 consecutive weeks in dark bottles. The GT was prepared by adding five grams of tea leaves to boiled water and letting it rest for four min. Ponderal homogeneity index (PHI) , was determined to evaluate the homogeneity between groups. Animals’ initial and final body weights were measured to determine weight gain (WG) . The organs’ relative weights were measured as the ratio of the mouse’s organ weight by the mouse’s final weight.After one week of acclimatization period, animals were randomized in six experimental groups each [Sample collection®, France) overdose. Complete necropsies were performed after mice macroscopic evaluation. The spleen, liver, kidneys, lungs, and heart were collected and weighed. The mice urinary bladders were collected and weighed according to the technique described by Oliveira et al . Continuous data were analysed using ANOVA. Pathological examinations were analysed by the chi-square test. Data were expressed as mean±standard deviation (SD). Results were considered statistically significant when This study evaluated the effect of oral administration of whole GT on the development of urothelial lesions chemically-induced by BBN in female and male mice. We observed that the concentrations of total phenols and flavonoids, and antioxidant capacity were higher in GT leaves when compared with GT infusion . In ordeThroughout the course of the experiment two animals died during BBN oral gavage (one from group II and one from group V). We did not observe any changes in animals’ behavior or clinical signs of distress/discomfort. During the experimental protocol, the groups showed a similar food and water intake. PHI, initial and final weights and WG were similar among groups. We did not find any statistically significant differences in the mean values and relative weights of spleen, liver, kidneys, lungs, heart, and urinary bladder (data not shown).Males from group III (GT) and females from group VI (GT) did not show any urothelial lesions. In males, the incidence of urothelial lesions was greater in animals from group I (BBN+GT) than animals from group II (BBN). Nevertheless, we observed a lower incidence of simple hyperplasia, squamous metaplasia, and dysplasia in group IV females (BBN+GT) compared with group V (BBN). However, these results were not statistically significant between groups . The meaMost research works conducted with GT use only one purified or partially purified component. In this study we evaluated the effect of whole GT infusion in mice experimental urinary bladder cancer induction instead of a particular compound, because this mimics the normal human tea consumption.et al (In order to characterize the composition of GT, we performed a total phenols, flavonoids, and antioxidant capacity analysis. According to our results, GT leaves had a higher total phenols, flavonoids, and antioxidant capacity than GT infusion; however, approximately 50% of total phenols, flavonoids, and antioxidant capacity remained in the infusion. These results differ from the study conducted by Sato and Matsushima  that repet al . ad libitum. In this study we chose BBN oral administration by gavage, because the oral route was selected to administrate GT infusion. The animals’ health states were normal during the experiment. The mortality rate observed in this study was associated with oral gavage, and in accordance with Arantes-Rodrigues et al (Taking into account that frequent mice manipulation can induce severe distress, we decided to provide GT infusion in bottleses et al .et al male mice are more likely than females to develop urinary bladder cancer induced by BBN (The groups that only drank GT infusion did not have any urothelial lesions. Although in humans the incidence of urinary bladder cancer is greater in men than in women, in this work we observed similar results between male and female mice. According to Miyamoto d by BBN . AnimalsOther studies have demonstrated the antitumor capabilities of GT , howeverAnimals from BBN and BBN+GT groups only developed preneoplastic lesions. The number of inflammatory aggregates was lower in animals that drank GT. We can conclude that the whole GT infusion had influence on urothelial inflammation. For future investigations we suggest the use of different GT infusion concentrations.


Fenofibrate offers a number of benefits on the cardiovascular system and it is plausible that its anti-inflammatory, anti-oxidant and anti-fibrotic effects and enhancement of cardiac metabolic performances may account for its direct cardioprotective effects.In this study we aimed to investigate the effect of fenofibrate on endothelial function assesed by vascular studies and levels of soluble E-selectin (sE-selectin) as well as the effect on plasma myeloperoxidase (MPO) in patients with type 2 diabetes mellitus (T2DM) without previous use of lipid-lowering medication.27 patients (14 men and 13 women) with T2DM and good glycemic control  treated with metformin monotherapy, without previous use of lipid-lowering medication were enrolled in this study. Vascular studies included measures of brachial artery diameter before and after release of a suprasystolic ischemia. FMD was calculated as the percent (%) change in arterial diameter following reactive hyperemia. Student’s paired t test and Wilcoxon Signed Ranks Test were used to compare values before and after fenofibrate therapy.Fenofibrate therapy significantly increased post ischemia mean brachial artery diameter at 60 s  and at 90 s . FMD response to hyperaemia at 60 s increased with 4.5 ± 13.7% . After 8 weeks of fenofibrate therapy, plasma MPO levels decreased to 49.5  ng/ml  and mean plasma sE-selectin levels decreased to 67.1  ng/ml, .In patients with T2DM without previous treatment for dyslipidemia, short-term treatment with fenofibrate improved vascular endothelial function as demonstrated by increased post ischemia mean brachial artery diameter, increased FMD and decreased plasma sE-selectin and favorably affected plasma MPO levels. Therefore, fenofibrate may be considered a protective cardiovascular drug in this group of patients.ANZCTR12612000734864)(Australian New Zealand Clinical Trials Registry  Type 2 diabetes mellitus (T2DM) is associated with a high risk of cardiovascular (CV) events, irrespective of the presence of other traditional risk factors . CurrentMyeloperoxidase (MPO) is a predominantly leukocyte-derived enzyme, involved in the initiation, destabilization of atherosclerotic plaque and genesis of acute coronary syndromes . Plasma Therefore, drugs that have the potential to influence both vascular function and MPO have become highly interesting, especially if already used in the treatment of other CV risk factors in persons with diabetes. Fenofibrate offers a number of benefits on the CV system -14 and iThe main purpose of this study was to investigate the role of fenofibrate on endothelial function, assessed by vascular studies and soluble E-selectin (sE-selectin), and on plasma MPO in patients with T2DM without previous use of lipid-lowering medication.This was an interventional, open label study enrolling patients with T2DM treated with metformin alone, and without previous use of lipid-lowering medication. Participants were recruited from a T2DM population in an outpatient clinic from Romania. According to treatment protocols in place in Romania, each patient with diabetes treated with non-insulinic drugs must attend a specialist visit twice a year.Participants were enrolled between August 1st and September 15, 2012 and followed for 8 weeks.Each participant attended 3 study visits: screening, visits 1 and 2 (8 weeks apart). Starting from visit 1 participants fulfilling inclusion criteria and without any exclusion criteria, received 160 mg fenofibrate/day for 8 weeks. The pills were counted at the end of study; compliance was considered satisfactory if >90% of them were taken.Before and at the end of the study, data were collected on medical history (visit 1), anthropometric variables, blood pressure (BP), FMD of the brachial artery, as well as adverse events (visit 2). Blood samples were collected at screening, visit 1 and 2 in fasting conditions as described below. At visit 1 each participant received instructions to maintain his/her usual nutritional habits and not to modify any drug treatment throughout the study.The research was conducted in accordance with the guidelines in The Declaration of Helsinki and Good Clinical Practice. Study protocol was approved by the Iuliu Hatieganu University of Medicine and Pharmacy Ethics Committee. Written informed consent was obtained from each participant prior to any study procedures.1. The variation of brachial artery diameters and of FMD response to hyperaemia, after 8 weeks of treatment with fenofibrate compared to pre-treatment values2. The variation of plasma MPO and soluble sE-selectin after 8 weeks of treatment with fenofibrate as compared with pre-treatment values.We enrolled men and women ≥18 years of age, with T2DM defined according to World Health Organization criteria (WHO) http://www.nephron.com/MDRD_GFR.cgi). sE-selectin and plasma MPO were measured in plasma (EDTA) using commercially available enzyme-linked immunosorbent assay kits for quantitative detection  on an automated ELISA reader (Tecan).Fasting blood samples were collected in the morning after an 8 h overnight fasting period. Fasting plasma glucose, HbA1c, total cholesterol, high-density lipoprotein (HDL) cholesterol, triglycerides, creatinine, hepatic enzymes were determined by automated chemistry analyzer Konelab 30, Thermo Fisher Scientific Inc, Finland. Low-density lipoprotein (LDL) cholesterol was calculated using Friedewald formula . eGFR waAdverse events were recorded throughout of study. Safety parameters included serum creatinine, eGFR, and hepatic enzymes.Imaging studies of the right brachial artery were performed in fasting condition before and after 8 weeks of fenofibrate therapy with a high-resolution, ultrasound imaging system  using M-mode, and electrocardiogram-triggered ultrasound images obtained with a 10 MHz linear-array transducer. All vascular studies were performed according to a protocol previously described by Celermajer and co-workers , by measStatistical analysis was performed using SPSS-PC 15.0 . Data were considered to be normally distributed if the ratio of skewness to its standard deviation did not exceed the value of 2. Data were expressed as a mean [95% Confidence Interval] for variables with normal distribution, median  for variables with abnormal distribution or frequency for dichotomial variables. Student’s paired t test  and Wilcoxon Signed Ranks Test  were used to compare values before and after fenofibrate therapy. Correlations were assessed with Pearson’s and Spearman’s correlation coefficient analysis. The level of significance was set at 0.05, and all tests were performed two- sided.Twenty seven patients (14 men and 13 women) fulfilling the inclusion criteria and without any exclusion criteria were enrolled in this study. Two participants  did not complete the study: one was excluded from the study due to poor compliance with fenofibrate treatment and one dropped out soon after fenofibrate treatment initiation for personal reasons and refused to present for reevaluation. Mean duration of diabetes was 3.8 years. All participants had good glycemic control (HbA1c range: 5.9%-7.1%). Doses of metformin ranged from 850 to 3000 mg/day. 76.0% of study participants had a history of hypertension and 20.0% had cardiovascular diseases. Characteristics of study participants are presented in Table Compared with baseline, at visit 3 participants presented significantly lower levels of total cholesterol , triglycerides levels , systolic and diastolic blood pressure . No statistically significant effect was observed on HDL-cholesterol and on LDL-cholesterol (p > 0.05). In addition, no change was observed in fasting plasma glucose, HbA1c, BMI and waist after fenofibrate treatment  , triglycerides  or changes in systolic BP  or diastolic BP .At baseline, MPO values ranged from 16.5 to 236.5 ng/ml, with a median level of 55.0  ng/ml and values of sE- selectin ranged from 15.0 to 142.0 ng/ml, with a mean level of 75.2  ng/ml. After 8 weeks of fenofibrate therapy, plasma MPO levels decreased to 49.5  ng/ml   or between % change in MPO levels and % change in E selectin .2, p = 0.007)  trial and other studies showing that both long-term and short-term fenofibrate treatment have no effect on this marker of arterial damage ,34.Observational studies have suggested that there is a strategic accumulation of MPO at the interface between endothelium and media. This observation led to the hypothesis that MPO could affect vascular tone by depleting endothelium derived NO ,36. SubeA possible explanation for the favorable effects of fenofibrate on endothelial function in T2DM, independent of its antihiperlipidemic action, is that this drug has direct anti-inflammatory and anti-oxidant effects on vascular wall, a hypothesis that was partially confirmed in healthy normolipidemic subjects  but not To our knowledge, no prior human study has examined the effect of fenofibrate on plasma MPO concentration. Given that MPO adversely influences LDL atherogenic properties and HDL functionality, it may be hypothesized that drugs reducing this enzyme level could have certain benefits in reducing the risk for atherosclerotic events.Experimental and clinical studies provide support for a potential impact of statin therapy on tissue and plasma MPO levels both in animal models and in humans -43. In cIf our results regarding the beneficial effect of fenofibrate on plasma MPO and on endothelial function will be confirmed in larger populations of patients with T2DM, this drug could become a valuable option for reducing atherosclerotic process in this group of high cardiovascular risk patients as part of an early and comprehensive preventive strategy.Our results regarding the decline in renal function are concordant with those in the FIELD and Action to Control Cardiovascular Risk in Diabetes (ACCORD) studies showing that fenofibrate caused an acute, sustained plasma creatinine increase ,44. In aThe main limitations of the present study are the relatively small number of patients included and the short duration of the fenofibrate treatment.In patients with T2DM without previous treatment for dyslipidemia, short-term treatment with fenofibrate improved vascular endothelial function as demonstrated by increased post ischemia mean brachial artery diameter, increased FMD and decreased plasma sE-selectin and favorably affected plasma MPO levels. Therefore, fenofibrate may be considered a protective cardiovascular drug in this group of patients.BMI: Body mass index; BP: Blood pressure; CAD: Coronary artery disease; CV: Cardiovascular; eGFR: Estimated glomerular filtration rate; FMD: Flow mediated dilation; HbA1c: glycated hemoglobin; HDL: High density lipoprotein; LDL: Low density lipoprotein; MPO: Myeloperoxidase; NO: Nitric oxide; sEselectin: Soluble eselectin; T2DM: Type 2 diabetes mellitus; WHO: World Health Organization.The authors declare that they have no competing interests.CN and CB were study coordinators, participated in the design of the study, were responsible for individual selection and characterization, carried out the statistical analysis and drafted the manuscript. MP carried out vascular studies and participated to manuscript preparation. NH participated in the design of the study and to manuscript preparation. All authors read and approved the final manuscript.