Salinity is a key abiotic property of inland waters; it has a major influence on biotic communities and is affected by many natural and anthropogenic processes. Salinity of inland waters tends to increase with aridity, and biota of inland waters may have evolved greater salt tolerance in more arid regions. Here we compare the sensitivity of stream macroinvertebrate species to salinity from a relatively wet region in France (Lorraine and Brittany) to that in three relatively arid regions eastern Australia , South Africa (south-east of the Eastern Cape Province) and Israel using the identical experimental method in all locations. The species whose salinity tolerance was tested, were somewhat more salt tolerant in eastern Australia and South Africa than France, with those in Israel being intermediate. However, by far the greatest source of variation in species sensitivity was between taxonomic groups (Order and Class) and not between the regions. We used a Bayesian statistical model to estimate the species sensitivity distributions (SSDs) for salinity in eastern Australia and France adjusting for the assemblages of species in these regions. The assemblage in France was slightly more salinity sensitive than that in eastern Australia. We therefore suggest that regional salinity sensitivity is therefore likely to depend most on the taxonomic composition of respective macroinvertebrate assemblages. On this basis it would be possible to screen rivers globally for risk from salinisation. The salinity (the concentration of dissolved major inorganic ions) of inland waters has a large influence on the biotic communities One method of assessing the risk of chemical contaminants to biota is to compile so called species sensitivity distributions (SSDs). SSDs are cumulative distribution functions of the (measured) sensitivity of a group of organisms to a chemical contaminant 2SO4, etc.) and end-points measured There is a general lack of salinity sensitivity data in most parts of the world. Furthermore, the sensitivity data that do exist has been collected using a range of methods differing for example are the durations of exposure, life-stages exposed, salts used  lethal salinity sensitivity of stream macroinvertebrates to salinity, collected from freshwaters in eastern Australia, France, Israel and South Africa. In all localities species were tested using an identical method. Our chief aim was to determine if the salinity tolerances of the macroinvertebrate communities and taxonomic groups differ between the regions. Furthermore we consider whether it is reasonable to combine salinity sensitivity data from one part of the world with those from other parts and whether taxonomic composition of species included in the regional SSDs are representative of the taxonomic composition in respective regions.50 values) over 72 h of salinity exposure was used here because they are relatively easy and quick to measure allowing for their determination across a large number of species 50 values are correlated with the maximum salinity at which species have been recorded in nature 50 values. Additionally the eggs and hatchling of stream macroinvertebrates are more salt sensitive than older aquatic stages 50 values and parameters calculated from their distribution are not meant to be directly predictive of maximum salinity at which species of stream macroinvertebrate can maintain viable populations in nature.The concentration of salt lethal to 50% of a population  and Queensland experiments at 25°C (±2°C). The French experiments were conducted at 18°C (±1°C) and the Israel experiments at 21°C (±1°C). The salinity sensitivities data in the various regions reflect tolerances under typical water temperatures at the sites and times when macroinvertebrates were collected.In all regions a wide range of species was selected for testing in order to cover as many taxonomic groups that were present as practical. We attempted to select species for testing so that we had representative samples of species from each region. Artificial sea water was used as the salt source, with Ocean Nature  used in Australia and South Africa and Instant Ocean  in France and Israel. An experiment was performed in Australia with two species with both of these artificial sea waters. It showed no significant difference in the 72 h LC50 values  SSDs were examined as Kaplan-Meier functions. Interval censored LC50 values  were assumed to be the mid-point of the interval. The Log Rank (Mantel-Cox) test was used to compare for statistical significant differences between SSDs for each of the localities and between SSDs for the different taxa. This method was used as it involves the least assumptions.  and Tarone-Ware tests). Mean LC50 values (as estimated from the Kaplan-Meier functions) of different groups were considered to be statistically different when they had non-overlapping 95% confidence intervals.First we determined whether there are any differences in the distribution of species which happened to be tested in the four regions. Given the presence of right censored LC50 values for particular taxa (mostly Classes or Orders) and regions was conducted using two methods. Firstly excluding right censored LC50 values, a two-factor ANOVA (taxa and regions) was used to compare 72 h LC50 values. However, there were two taxa (Crustaceans and Hydracarina) with less than two non-censored 72 h LC50 values per region. These taxa were excluded from the aforementioned two-factor ANOVA and analysed separately using single factor ANOVA (region). The 72 h LC50 values were square root transformed in order to better meet the assumptions of normality and homogeneity of variance required by ANOVA. These assumptions where checked by examining box-plots and q-q plots.The comparison of differences in LC50 values and 95% confidence intervals were compared. As before, Kaplan-Meier functions compared Log Rank (Mantel-Cox) test.Secondly, using all data, for each taxa Kaplan-Meier functions were calculated from which mean 72 h LC50 values were tabulated and compared non-statistically as there were too few species to allow for statistical tests.The mean and standard deviations of families and genera for each region of non-censored 72 h LCWe estimated the proportion of stream macroinvertebrate species from each order in France and south-east Australia. (Israel and South Africa were not considered here as we were unable to make estimates in these countries.) This was done to determine if the species tested formed representative samples of the richness of orders in these two regions. As our interest was in considering spatial variation  in salinity sensitivity we did not consider any seasonal or other temporal variation in the presence of species within the regions. In France the number of flowing water species in each order was estimated from two sources: Limnofauna Europaea Similar checklists of species in Eastern Australia were not available; however the Victorian and South Australian Environmental Protection Authorities (EPA) have a database of species collected from 2966 samples from two habitats edge/pool and where present riffles across these states 50 values was representative of their region then there should be a regression between these percentages close to the line y = x. That is if q% of the species from an order were tested, then ≈ q% of species in the region should be from this order.For the two regions , we determined if the percentage of species tested in a taxonomic order is similar to the percentage of species estimated to be in the relevant region. If sample of species with LC10 scale that each group of species has its own mean 72-h LC50 but that all groups have the same variance. The model is able to use both right and interval censored data without relying on the mid-point rule We used a Bayesian statistical model, adapted from Hickey et al. a priori. This is equivalent to the statement that we would not change our prior beliefs over the collection of mean log10 LC50s from the individual species-subsets, where in this case the subsets are defined according the aforementioned quasi-grouping. By making this assumption, we can envisage the quasi-taxonomic group means as a random sample from a hyper-population. In other words, on a log10 scale each quasi-taxonomic group SSD mean parameter is assumed to come from a normal distribution with a grand mean parameter  and standard deviation – the hyper-parameters for the model.Here we used the following quasi-taxonomic groups: (1) Coleoptera and Odonata, (2) Crustaceans, (3) Diptera and Hemiptera, (4) Ephemeroptera, (5) Hydracarina, (6) non-arthropods, (7) Trichoptera and Plecoptera. These groups were chosen as analysis showed that most species within them had similar 72-h LC50 values and the variance in LC50 within these groups was broadly similar (see results). The only difference between the analysis used here and Hickey et al. 6). The hyper-population standard deviation was assigned a Uniform  distribution based on the reasoning of Gelman Fitting a Bayesian model requires the specification of prior distributions over parameters. We used standard non-informative prior distributions: over the homogenous per-quasi-taxonomic SSD variance parameter (Inverse-Gamma with shape and rate parameter set to 0.001); over the hyper-population grand mean , eastern Australia and Israel  but not eastern Australia and South Africa . Likewise the Kaplan-Meier functions from France and Israel were not statistically different . The South African Kaplan-Meier function was statistically different from those in France  but not Israel . The mean 72 h LC50 values of species tested in eastern Australia and South Africa were significantly higher than in the other regions  and the mean 72 h LC50 value of rare species was greater than that of common species  was to the right (more tolerant) of the SSD for abundant species . Breaking these groups down further, there were similar Kaplan-Meier functions for annelids (segmented worms), molluscs and turbellaria (flatworms) . The exception was that molluscs were more tolerant than turbellaria . We do note that the number of annelid and turbellaria taxa examined was low  and that the SSDs, as represented by the Kaplan-Meier functions, for these taxa have to be considered as preliminary. Both Hydracarina and insects were more tolerant than all of the aforementioned non-arthropod groups  but there was no statistically significant difference in the Kaplan-Meier functions of Hydracarina and insects . It is, however, noteworthy that there were no salt sensitive Hydracarina observed with the most sensitive mite’s 72 h LC50 value being 19.3 mS/cm .Non-arthropods tended to be more salt sensitive than insects and Hydracarina (water mites), while crustaceans tended to be more tolerant still . There watworms) , Table 1.3 mS/cm . Crustac2 = 4.176 to 77.266, P<0.001 to 0.041). Trichoptera and Plecoptera were the next most sensitive orders. The difference in the Kaplan-Meier functions of these two orders  was not statistically significant. Trichoptera was statistically more sensitive than all remaining orders  except Diptera . Plecoptera was more sensitive of all remaining orders  except Diptera  and Hemiptera . The Kaplan-Meier functions for Diptera and Hemiptera were similar . Both Diptera and Hemiptera were more sensitive than Coleoptera and Odonata . Finally the Kaplan-Meier functions for Coleoptera and Odonata were not statistically significant from each other . Only one species of each of Lepidoptera and Megaloptera  was tested  and to maximise the sample size (species) in each group.Species were split into 7 groups: (1) Coleoptera & Odonata, (2) Crustaceans, (3) Diptera & Hemiptera, (4) Ephemeroptera, (5) Hydracarina, (6) non-arthropods, and (7) Trichoptera and Plecoptera. The groups with multiple taxa were chosen as there was no evidence that these taxa had different distributions of 72 h LC50 values between the regions  but there were highly statistically significant differences between the taxa groups . There was also no significant interaction between these factors . Hydaracarina and crustaceans were not included in the above ANOVA as ≤1 non-censored LC50 value was available from one or more region. Considering crustaceans only, there was a significant difference between the regions , with the mean (square root transformed) 72 h LC50 values of crustaceans being higher in eastern Australia than France but not Israel  72 h LCt Israel . The lact Israel .50 censored status) mean 72 h LC50 values calculated from the Kaplan-Meier functions for Coleoptera & Odonata, Crustaceans, Ephemeroptera, Non-Arthropods and Trichoptera & Plecoptera differed between some of the regions , Israel (for Trichoptera & Plecoptera) or South Africa (for Non-Arthropods). For other pair-wise comparisons between regions there were no significant differences, at the 0.05 level. The same order of relatively sensitivities of the taxa groups between regions, as shown for the mean LC50 values  estimated to be present in eastern Australia and France were positively correlated with the proportion of species tested in these locations , although this correlation was not statistically significant in France. In both locations, the line of best fit was very similar to the one to one line (y = x) . So on aThe quasi-taxonomic groups Diptera & Hemiptera and Hydracarina were relatively richer in France than in Eastern Australia . Non-art1, for example, for France was approximately half that for Victoria and South Australia (50) sensitivity both regions were more similar  of stream macroinvertebrates between the regions. In terms of simply comparing all species tested between the regions the mean 72 h-LC50 value was approximately 10 mS/cm greater in Eastern Australia and South Africa than in France and Israel  collected from Israel.(PDF)Click here for additional data file.Table S2Salinity sensitivity data (mS/cm @ 25°C) collected for common species from France.(PDF)Click here for additional data file.Table S3Salinity sensitivity data (mS/cm @ 25°C) collected for rare species from France.(PDF)Click here for additional data file.


The risk of influenza infection depends on biological characteristics, individual or collective behaviors and the environmental context. The Cohorts for Pandemic Influenza (CoPanFlu) France study was set up in 2009 after the identification of the novel swine-origin A/H1N1 pandemic influenza virus. This cohort of 601 households (1450 subjects) representative for the general population aims at using an integrative approach to study the risk and characteristics of influenza infection as a complex combination of data collected from questionnaires regarding sociodemographic, medical, behavioral characteristics of subjects and indoor environment, using biological samples or environmental databases.Households were included between December 2009 and July 2010. The design of this study relies on systematic follow-up visits between influenza seasons and additional visits during influenza seasons, when an influenza-like illness is detected in a household via an active surveillance system. During systematic visits, a nurse collects individual and environmental data on questionnaires and obtains blood samples from all members of the household. When an influenza-like-illness is detected, a nurse visits the household three times during the 12 following days, and collects data on questionnaires regarding exposure and symptoms, and biological samples  from all subjects in the household. The end of the follow-up period is expected in fall 2012.The large amount of data collected throughout the follow-up will permit a multidisciplinary study of influenza infections. Additional data is being collected and analyzed in this ongoing cohort. The longitudinal analysis of these households will permit integrative analyses of complex phenomena such as individual, collective and environmental risk factors of infection, routes of transmission, or determinants of the immune response to infection or vaccination. The first human cases of influenza caused by a novel swine-origin A/H1N1 pandemic influenza virus variant (H1N1pdm) were reported in Mexico and the United States in April 2009 , BoliviaSeveral studies already reported risk factors for seasonal or pandemic influenza infection in households. These studies focused on including individual characteristics of index patients and their household contacts -10 or hyThis cohort was designed to assess the relative risk of infection by the H1N1pdm virus. We first intended to include 1000 households (about 2100 subjects) which would have permitted to detect covariates associated to a relative risk ≥ 1.4 with a 80% power and 5% significance, assuming a cumulative incidence of 10% and intra-household correlation of 0.3.2) according to population density  in a st· A “landline” list of 25 households: these households were chosen as those with the phone number immediately following a landline number drawn in this area. Since landline numbers are geographically allocated, this method ensured reaching households who chose not to be listed in the national directory.· A “mobile phone” list of 7 households: these households were directly drawn in the national directory, in order to reach households without a fixed phone.Addresses were iteratively drawn from the 40 lists of 130,000 households each and these households were phoned to present the study and, upon meeting eligibility criteria, were sent a written description of the study. A household was considered as “pre-included” when a referent member sent back a filled form to confirm his agreement. According to this method, 1,280 households were pre-included, i.e. agreed to be visited by a nurse for an inclusion visit involving all household members. We anticipated that 20% of pre-included households would finally decline to participate in the cohort.A household was defined as a person or group of people occupying the same domicile. All households were eligible to participate in the cohort, provided at least one member was over 18 years of age and French-speaking. A household member was defined as a person living at least half his/her time in the household. All household members were eligible, regardless of age. The inclusion of a household required the participation of all members: the refusal of one or more member(s) prevented the inclusion of other members.Five thousand one hundred and two households were contacted by phone in order to achieve our targeted number of 1280 pre-included households . Data was collected on the 601 remaining households (1450 subjects). According to population census data , these hThe main objective of this study was to identify individual and collective determinants of H1N1pdm infection; therefore we tried to collect comprehensive data about subjects and their environment, in addition to biological samples. Several household visits are carried on by nurses for this purpose . As the households’ addresses were geocoded, we were able to get additional information from public databases regarding the immediate surrounding environment of households. An overview of data collected from questionnaires at entry in the cohort is shown in Figure · Systematic yearly visits After the inclusion visit, systematic follow-up visits are carried on between influenza seasons. During a systematic visit, a nurse collects or updates individual and environmental data on questionnaires, completes previously missing data, and obtains blood samples from all members of the household. Two waves of systematic follow-up visits have already occurred . A third wave is expected by the end of the second year of follow-up (summer 2012).· Influenza-like illness (ILI) visits During the influenza season (as defined by the French surveillance network [·  network ), we use network ). A free· During these visits, a detailed questionnaire collects data about the circumstances of possible exposure to influenza viruses and the chronology of symptoms (if any) in all subjects. Nasal swabs are collected from all subjects. A stool sample and a throat swab are also collected from subjects with ILI, as well as a blood sample from those over 10 years of age. Moreover, a self-swab procedure is previously sent to the households in order to collect virological samples when a visit by a nurse within the first 48 h is not possible. Nasal swabs are used to identify various respiratory viruses by PCR and biochips allowing for multiple diagnosis tests.· This series of three visits can occur several times in the same household during an influenza season. There were 23 ILI alerts during the 2009–2010 season (as households were still being included) and 143 during the 2010-2011 season, all of which triggered up to three ILI visits.Vaccination visits In order to update serological information, a blood sample was collected from subjects who had an influenza vaccination, between 2 and 4 weeks following this vaccination. There was one vaccination visit following the inclusion visits; 29 vaccination visits were conducted following the first wave of follow-up visits and 69 following the second wave.· The cohort was initially designed to include households before the 2009 pandemic season and to follow subjects during the two subsequent influenza seasons. We obtained funding in June 2009, the cohort protocol and questionnaires were finalized in July 2009, and the protocol obtained ethical approval on September 8, 2009. Households were pre-included between September 25 and December 17, 2009 and inclusion visits began on December 4, 2009 – as the final administrative authorizations were obtained. The inclusion period was extended until July 31, 2010, in order to get a relevant sample size for the planned analyses, some of which were postponed until the following season. A total of 575 households (96%) were included after the first pandemic season .The protocol of the CoPanFlu-France study was approved by the research ethics committee “Comité de Protection des Personnes Ile-de-France 1” on September 8, 2009. Information was previously given by investigators to participants indirectly through written descriptions of the study and training of the nurses, and directly by e-mail and telephone for any question. Written informed consent was obtained for all subjects.Many analyses have been recently completed or are currently being carried out. Based on inclusion data, we used a data-driven approach to identify factors associated with a high anti-H1N1pdm serological titer. We are conducting several analyses to identify risk factors associated with influenza infections . Nasal swabs are being analyzed to identify various respiratory viruses and the characteristics of infected subjects. Blood samples collected during ILI visits are used to study innate immunity against influenza and the related transcriptome. Determinants of vaccination against influenza have also been identified, and other studies are being conducted in the field of social science and risk perception. Several other analyses are expected soon from different collaboration partners in various biomedical fields.The main strength of this cohort is the large amount of available and expected data and the different biological samples to be collected, which will permit to carry on many studies in various biomedical fields. To our knowledge, this project is the first attempt to study so thoroughly the determinants of infections by respiratory viruses in a large sample of households randomly selected in the community. This approach is likely to provide new insights from the interaction of sparse data usually studied separately, especially with the help of data-driven methods such as those already under development in the field of non-communicable diseases ,16. AddiWe designed the CoPanFlu-France study in order to enable inference to the French general population, yet we cannot exclude a selection bias induced by the proportion of contacted households who refused to participate. However, a comparison between CoPanFlu subjects and population census data  suggestsWe wish we were able to set up this project a few months earlier, in order to include households before and to follow-up subjects during the 2009 pandemic season. Due to organizational impairments, the inclusion process was delayed and data regarding ILIs were collected retrospectively, sometimes up to 6 months after the epidemic. Thus, this timeline of inclusion may have induced recall or reporting biases for the 2009 season, and we were not able to collect enough pre-pandemic blood samples and nasal swabs during this first H1N1pdm season to investigate laboratory-confirmed infections. Another consequence of this delayed inclusion process is that we decided to stop inclusions as only 601 out of the 1000 expected households were included. This limit is the main reason why we decided to postpone the end of the study until 2012 instead of 2011 as initially expected, in order to collect data during an additional influenza season.Pr Carrat reported not having shares or paid employment with pharmaceutical companies; received honoraria from Novartis, GlaxoSmithKline and Boiron and received travel support to attend scientific meetings from Novartis. Other authors have no competing interest to declare.NL, FC and AF drafted the protocol of the study. All authors contributed to conception and design of the study. NL, PF, MSe, RMD and FC participated to the acquisition of data. NL and FC carried out the statistical analyses. NL and FC drafted the manuscript and all authors revised it critically. All authors gave final approval of this version of the manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2458/12/417/prepubDesign and methods of the CoPanFlu-France cohort: representativeness of the population sample.Click here for file


Haematopoietic stem cell transplantation (HSCT) has been tried in the last 15 years as a therapeutic option in patients with poor-prognosis autoimmune disease who do not respond to conventional treatments. Worldwide, more than 600 patients with multiple sclerosis (MS) have been treated with HSCT, most of them having been recruited in small, single-centre, phase 1–2 uncontrolled trials. Clinical and magnetic resonance imaging outcomes from case series reports or Registry-based analyses suggest that a major response is achieved in most patients; quality and duration of response are better in patients transplanted during the relapsing–remitting phase than in those in the secondary progressive stage.An interdisciplinary group of neurologists and haematologists has been formed, following two international meetings supported by the European and American Blood and Marrow Transplantation Societies, for the purpose of discussing a controlled clinical trial, to be designed within the new scenarios of evolving MS treatments.Objectives of the trial, patient selection, transplant technology and outcome assessment were extensively discussed. The outcome of this process is summarized in the present paper, with the goal of establishing the background and advancing the development of a prospective, randomized, controlled multicentre trial to assess the clinical efficacy of HSCT for the treatment of highly active MS. Treatment alternatives are especially needed for this group of patients, i.e. those failing multiple treatments and rapidly accumulating clinical disability.Current treatments of multiple sclerosis (MS) include high-dose corticosteroids for the treatment of exacerbations and long-term immuno-modulation/-suppression to reduce the number and severity of relapses, and slow down the progression of disability.relapses .2 Howeve3 In the last 15 years HSCT has also been explored to treat patients with severe autoimmune diseases (AD) who were deteriorating despite receiving standard treatments.4 The rationale is derived both from experimental models5 and from the observations of positive effects on a coincidental AD of transplants given for a conventional haematological indication.6Haematopoietic stem cell transplantation (HSCT) is a well-established procedure for the treatment of poor-prognosis haematological malignancies.7 Most patients were treated in small phase 1–2 trials, either single-815 or multi-centric;17 currently no data are available from prospective comparative trials. Two single-centre, retrospective analyses of long-term outcome were recently published and both showed a sustained progression-free survival (PFS) beyond 5 years after HSCT.18 The neurological outcome was considerably more favourable in patients transplanted in the relapsing–remitting (RR) phase18 and/or showing an inflammatory pattern at magnetic resonance imaging (MRI) during pre-transplant screening.15 Indeed, case series and reports of excellent outcome in particularly aggressive forms of MS20 support the notion of efficacy of HSCT in MS patients with prominent inflammatory activity. The risk of treatment-related mortality (TRM) in HSCT, conventionally perceived to be unacceptably high, has decreased since 2001 to 1.3%, according to an analysis of the European Blood and Marrow Transplantation Group  Registry,7 likely due the avoidance of aggressive regimens which resulted in frequent toxicity. In particular, the use of busulfan was found to be significantly associated with higher TRM in multivariate analysis,21 and high doses of rabbit anti-T-lymphocyte globulin (ATG), associated with ex-vivo T-cell depletion and an intense conditioning regimen (CR), resulted in unexpected occurrence of EBV-associated lymphomas.22 In 2004, the EBMT launched a prospective, randomized phase 2 trial  comparing autologous HSCT with mitoxantrone, with new MRI lesions as the primary endpoint (number of new T2 lesions at 1 and 2 years after the randomization). In total, 21 patients were randomized to the two trial arms and the follow-up analysis is currently ongoing. Two phase 2, single-arm trials were carried out in the USA  and Canada , respectively; both trials closed accrual in 2010. The Canadian trial has reported results in preliminary form showing complete suppression of relapses and of new MRI lesions in 23 evaluable patients over a median follow-up of 5 years.12 The US HALT-MS trial preliminary results in 25 patients with highly active, treatment-refractory RRMS have reported a 77% event-free survival at 2 years post-HSCT utilizing a stringent composite endpoint that included relapses, new MRI lesions and progression of neurological disability.23Autologous HSCT for MS has been repeatedly and recently reviewed.24 One year later EBMT and NIAID supported an international meeting in Florence, aimed to update knowledge and strategies of HSCT for AD. Panels of specialists were appointed for each major AD (www.adflorence.org) and the MS panel included neurologists, haematologists and statisticians from both Europe and North America.25 There was uniform agreement about the need for a prospective, controlled trial aimed at investigating the role of HSCT in the treatment of aggressive MS. This report describes the progress to date of this interdisciplinary group of specialists in the design of such a trial.The cumulative experience described above is highly suggestive of a strong treatment effect of HSCT on inflammatory manifestations of MS, but the efficacy and safety of HSCT compared with currently available therapies has not yet been established. In November 2008 a meeting of experts in MS and bone marrow transplantation was held in Minneapolis under the auspices of Center for International Blood & Marrow Transplant Research (CIBMTR), the National Institute of Allergy and Infectious Diseases (NIAID), and the EBMT in order to establish shared criteria for the assessment of efficacy of HSCT in MS and evaluate the feasibility of a new prospective, multicentre trial.Two approaches to a prospective trial were considered : a cohor1 with differences in their introduction expected in different countries due to regulatory issues. Furthermore, there is no standard of therapy for patients failing approved treatments for MS who are eligible for HSCT, therefore they are treated differently according to centre policy and local regulations on drugs and reimbursement policies. As a result of these considerations, a consensus view emerged that the trial should be designed with randomization between autologous HSCT and Best Available and Approved Treatment (BAAT). The BAAT is defined as, and will consist of, the treatment available as licensed therapy to the individual patient in his/her country of residence and approved under the applicable healthcare scheme.There was considerable discussion regarding the most appropriate comparator. Although some discussants favoured a single agent , there were prevailing concerns that some agent/s might not be available in all participating countries. Approval of an additional 3–4 new therapies for RRMS is foreseen in the next 2 years,To establish the safety and efficacy of autologous HSCT in comparison with approved therapies in a specific subset of highly active RRMS;To address questions related to the immunopathogenesis of MS by accompanying mechanistic studies;To create a comprehensive repository of biosamples for planned and future mechanistic studies.The definition of inclusion criteria was felt to be a key point. The aim of the panellists was to capture a subset of patients with relatively early MS who are at high risk of suffering early disease progression based on their high inflammatory activity. Prognostic predictions are difficult in MS, and three domains were considered important to formulate criteria that select subjects at greater risk of poor prognosis and potential eligibility for the trial: (1) clinically significant relapses; (2) MRI activity; and (3) treatment failure. The following specific criteria were selected:■ ≥1 severe relapses  of ≥2 in motor, cerebellar or brain stem deficit  and/or incomplete recovery from clinically significant relapses;andor accumulation of ≥ 0.3 T2 lesions/month in two consecutive MRI 6–12 months apart.■ ≥1 gadolinium-positive (Gd+) lesion of diameter ≥ 3 mm ○ The definition of ‘highly active’ RRMS includes:○ Patients are eligible after failure of conventional treatment.Patients aged 18–45 years with Expanded Disability Status Scale (EDSS) score between 2.5 and 5.5, who are within 5 years from initiation of the first therapy with DMAs and who fulfil the criteria for highly active RRMS within the past 2 years.26 and (3) one report of a worse outcome after HSCT in patients aged over 40 years.21The upper limit of 45 years was chosen for the following reasons: (1) the low probability of older patients to fulfil the inclusion criteria; (2) their faster shift to the secondary progressive phase of disease;27 continuing Gd+ or new T2 lesions ≥6 months from starting therapy (same as ‘highly active’).■ Evidence of relapses, increased EDSS .This last item was extensively discussed since some neurologists consider failure of only first-line therapy as ‘too early’ to randomize patients to HSCT. Others consider the above criteria for ‘highly active’ MS as an indicator of poor prognosis, therefore the randomization at this stage being appropriate. Taking all these considerations into account, it was decided to allow, but not to require, the inclusion of patients after a first-line treatment failure only. Patients will be stratified according to the treatments administered before the randomization, should numbers permit see .The informed consent process should include a detailed discussion of the treatment risks and non-trial treatment alternatives. Patients must be adequately screened to exclude coincidental organ failure, to minimize the transplant-associated toxicity.Most of the MS panellists estimated that 8–10 patients fulfilling the inclusion criteria present each year at their respective institutions .A major response should be expected by an aggressive treatment involving significant risk such as HSCT. Such response could be defined as PFS lasting 5 years free from further immunosuppressive treatment. A long-term follow-up, such as the proportion of patients still progression-free at a given interval (e.g. 3 or 5 years), would greatly increase the discrimination power between the two treatment arms; however, there was consensus that a follow-up duration longer than 2 years before assessing the primary outcome would not be feasible for the core trial. In the ASTIMS trial, randomization prior to HSCT hampered recruitment, due to the rapidly evolving clinical course of patients; this concern should be carefully considered in the trial design. For this reason, we favoured a ‘time to event’ endpoint, allowing patients initially randomized to BAAT who continue to deteriorate to cross-over to HSCT once they have reached the ‘time to’ endpoint.○ Definition of treatment failure is the occurrence of a severe relapse (see above) or sustained EDSS worsening , or a new (Gd+) lesion ≥3 mm or accumulation of >0.3 mm T2 lesions/month in two consecutive MRI 6–12 months apart.The primary outcome is the time to treatment failure, compared between treatment arms.Patients reaching the primary endpoint will be given the chance to receive the treatment of the other arm .This primary endpoint optimizes the trial design, in that it requires a shorter follow-up as compared with a 2-year trial based on a relapse rate endpoint, and preserves a similar statistical power, especially in case of early post-treatment treatment failure events. The option to cross-over for patients reaching the endpoint will facilitate the availability of patients for a randomized trial.Secondary objectives will include:○ Comparison of overall survival;○ Treatment-related mortality;○ MRI outcomes including lesion and atrophy metrics, to be defined;○ Rates of disease progression;○ Rate of adverse events, infectious complication;○ Quality of life;○ Improvement of disability;○ Response to post-failure treatment between treatment arms; and○ Need for any MS-related treatment.We calculated that 114 patients (57 in each group) are required to provide a power of 90% (at a confidence level of 5%) to detect a clinically meaningful relative reduction in the actuarial cumulative probability to have a failure over 1 year of 50% (corresponding to a hazard ratio (HR)=0.44) in the HSCT group as compared with the BAAT group.Sample size was calculated by using a log-rank test on the assumption that 40% of failures would occur in the BAAT group at 1 year, with an exponential distribution for time to failure, with an accrual time of 1 year and a follow-up of 2 years.The intensity of the immunosuppression delivered by the transplant treatment is dependent upon several factors:The use of chemotherapy, such as cyclophosphamide (Cy) in the stem cells mobilization regimen;Ex-vivo T-cells depletion (purging);An intense CR;The inclusion of ATG or monoclonal antibodies  in the CR to maximize the immunosuppression .28 With time, toxicity of such regimens became a clinical issue,29 and at the same time evidence emerged that less aggressive treatments have a favourable risk/benefit profile, as recently reviewed.30 Indeed, the importance of the disease phase (RRMS vs. secondary progressive MS) over the intensity of CR in determining the outcome was increasingly perceived by the teams involved in transplant programmes,18 and this is reflected by the gradual increase in RRMS cases treated being reported to the EBMT Registry over the years . The initial experiences of autologous HSCT for MS were dominated by high-intensity regimens as a high rate of relapse was expected, following some initial reports in ADs.he years .21 This regimen is still widely used due to the good safety/efficacy profile in lymphoproliferative diseases. BEAM has more recently been included in MS trials in Asia31 and in the USA . A lower-intensity (non-myeloablative) regimen, based on Cy and ATG or alemtuzumab, was also reported from Burt and colleagues at Northwestern University32 in a group of patients with early RRMS. The analysis of outcomes showed a low toxicity, but a trend to a higher frequency of relapses or recurrence of MRI activity (23%) than in higher-intensity CRs, although patients were reported to respond well to further immunotherapies. A prospective comparative trial would be needed to draw conclusions about its safety and efficacy profile.The most widely used CR in Europe for MS is the intermediate-intensity BEAM/ATG regimen .2 followed by G-CSF, 10 mcg/kg. Peripheral blood stem cells (PBSC) will be collected by continuous flow leukapheresis, targeted to cryopreserve ≥3×106 CD34+ cells/kg.• Patients will be mobilized by the administration of cyclophosphamide, 4 g/m○ Mobilization of stem cellsThe combination of Cy and G-CSF is preferred because2) Cy regimen;17Most patients improve after mobilization that includes a standard  + peri-transplant ATG is preferred by most of the transplanters as an intermediate-intensity regimen.■ ‘BEAM’ regimen  do not seem likely to achieve the goal of 5 years of disease and treatment-free survival, when considering the only prospective trial reported so far.32 A Brazilian prospective study compared BEAM/ATG vs. Cy/ATG, reporting a similar outcome but a lower toxicity in the latter. However, the patients included in both groups all had advanced disease, which limited the usefulness of the comparison; also, a high dose of horse ATG was administered in the BEAM group, and may have caused the high observed toxicity (TRM 7.5%) which exceeds any other report.37 Finally, high-intensity regimens (i.e. busulphan + Cy) can also be considered; it is thought that toxicity might be higher, but with the advent of intravenous formulations of busulphan and monitoring of blood levels, this might be minimized, as reported in the Canadian trial.12 All the participants agree that data from the Canadian trial are highly promising. However, the choice of the CR for a large, prospective, multicentre trial needs to be based on published data either from prospective studies and/or from Registry analysis, and therefore BEAM was considered the best choice at the current time. New published data on the CR might change the current preference.BEAM+ATG is the most commonly used regimen in Europe, and it showed a satisfactory toxicity profile both in the EBMT Registry analysis38 that autologous HSCT indeed leads to extensive renewal of the T-cell repertoire provided crucial evidence to document that autologous HSCT goes beyond a profound and long-lasting immunosuppression, which can be achieved by conventional treatments. However, many questions remain regarding the therapeutic mechanisms of autologous HSCT, and these should be examined during the prospective phase 3 trial. Therefore, a comprehensive repository of biomaterials should be created in order to allow interested investigators to address important questions in parallel with the trial and later. An assessment of immune reconstitution will be integrated in the trial and will be carried out on freshly collected blood specimens. A policy will regulate the utilization of the stored biological samples for qualified research projects, and its implementation will be overseen by a specific study committee.The demonstration by Muraro et al.The target population of the trial constitutes a relatively small subset of all patients with RRMS, and in order to meet patient accrual within a period of 2 years a large number of countries and participating centres need to be involved. Currently a clear sign of interest has been received from groups in Canada, France, Germany, Italy, Switzerland, the USA, and the UK. Other centres in Brazil, Spain, and the Czech Republic might follow. Each centre will need an established team of MS neurologists and bone marrow transplanters, as well as access to facilities including MRI and a laboratory infrastructure for the collection, characterization and storage of the biological samples for research. Criteria will be established to select centres with the required capacity and experience. Nursing care will require interaction between neurological and haematological nursing staff. A comprehensive plan for data collection, management, quality control and analysis for this complex international study will need to be developed.Conducting a phase 3 controlled multi-centre trial comparing a treatment such as autologous HSCT with other treatments will not only be a major organizational effort, but will also be expensive. We do not expect a high level of interest in autologous HSCT for treatment of MS from pharmaceutical companies, which may even consider the treatment as market competition. Therefore, funding will have to be sought from public funding bodies and charitable foundations. A strategy for funding should consider and coordinate public resources at the international , national , and local  level.We believe that the proposed outline protocol represents a solid foundation for the development of a randomized controlled phase 3 trial of HSCT for MS. Interested neurologists, bone marrow transplanters and MS patient associations are invited to engage in the final stages of protocol development and in the trial planning.


Recovery after stroke correlates with non-active (stunned) brain regions, which may persist for years. The current study aimed to evaluate whether increasing the level of dissolved oxygen by Hyperbaric Oxygen Therapy (HBOT) could activate neuroplasticity in patients with chronic neurologic deficiencies due to stroke.A prospective, randomized, controlled trial including 74 patients (15 were excluded). All participants suffered a stroke 6–36 months prior to inclusion and had at least one motor dysfunction. After inclusion, patients were randomly assigned to "treated" or "cross" groups. Brain activity was assessed by SPECT imaging; neurologic functions were evaluated by NIHSS, ADL, and life quality. Patients in the treated group were evaluated twice: at baseline and after 40 HBOT sessions. Patients in the cross group were evaluated three times: at baseline, after a 2-month control period of no treatment, and after subsequent 2-months of 40 HBOT sessions. HBOT protocol: Two months of 40 sessions (5 days/week), 90 minutes each, 100% oxygen at 2 ATA. We found that the neurological functions and life quality of all patients in both groups were significantly improved following the HBOT sessions while no improvement was found during the control period of the patients in the cross group. Results of SPECT imaging were well correlated with clinical improvement. Elevated brain activity was detected mostly in regions of live cells (as confirmed by CT) with low activity (based on SPECT) – regions of noticeable discrepancy between anatomy and physiology.The results indicate that HBOT can lead to significant neurological improvements in post stroke patients even at chronic late stages. The observed clinical improvements imply that neuroplasticity can still be activated long after damage onset in regions where there is a brain SPECT/CT (anatomy/physiology) mismatch.NCT00715897ClinicalTrials.gov  Intensive functional therapy and rehabilitation programs for post stroke patients are considered essential for maximizing the patients' quality of life 3 of normal brain tissue contains about 1 km of blood vessels, high oxygen supply is essential for repair of the stunned regions. Indeed, as has been demonstrated by previous studies, an increase in dissolved oxygen has several beneficial effects in damaged brain tissues 2 partial pressure rises above 1,110 mmHg. Hence, it is reasonable to expect that HBOT can be an efficient  method for increasing tissue/cellular oxygenation and thus effectively evoking neuroplasticity in the chronically non-active areas during the late post-stroke phase.Years of clinical experience revealed that the dramatic spontaneous recovery from stroke occurs mainly within the first 30 days, though moderate and severe stroke survivors continue to improve for at least 90 days Many physiological pathways, each with a different characteristic time, are spontaneously activated following the onset of stroke. Therefore, a challenging question to be addressed considers the optimal time lapse after stroke to start the HBOT procedure. It should also be kept in mind that signals and chemical cues associated with cell death during the acute stage of stroke might, in fact, promote repair during recovery The study was performed as a prospective, randomized, controlled, two-group trial. The population included patients of ages 18 years or older, who had either ischemic or hemorrhagic stroke 6–36 months prior to their inclusion. All patients had to have at least one motor dysfunction. Exclusions were based on chest pathology incompatible with HBOT, inner ear disease, claustrophobia and inability to sign informed consent. Additional exclusions were based on dynamic neurologic improvements during the last month . Smoking was not allowed during the study. All patients signed written informed consent; the protocol was approved by the local Helsinki committee. The study was conducted in the hyperbaric and research units of Assaf-Harofeh Medical Center, Israel.After signing an informed consent form, the patients were invited for baseline evaluations. Included patients were randomized into two groups (1∶1 randomization): a treated group and a cross group. The neurologic functions as evaluated by National Institutes of Health Stroke Scale (NIHSS) The clinical severity of the stroke was blindly assessed by a trained physician according to the NIHSS μ = 0.12/cm) attenuation correction Brain single photon emission computed tomography (SPECT) was conducted with 925–1,110 MBq (25–30 mCi) of technetium-99methyl-cysteinate-dimmer(Tc-99m-ECD)at 40–60 min post injection using a dual detector gamma camera  equipped with high resolution collimators. Data was acquired in 3-degree steps and reconstructed iteratively with Chang method . “No change” was assigned to no visual difference in the number or size of perfusion deficits; “mild change” to a reduction in number or size of perfusion defects; “significant change” to a global perfusion increment in addition to diminution of defect numbers or size. Differences in evaluation were resolved after mutual reviewing. A comparison of the SPECT results with anatomical imaging CT was conducted in order to evaluate the extent of perfusion deficit in relation to the anatomical lesion. All SPECT analysis were done while blinded to the laboratory and clinical data.Quality of life was evaluated by the EQ-5D questionnaire The statistical analysis considerations are detailed in Appendix A. SAS software  was used. Continuous data is expressed as means ± STD (standard deviation) and compared by unpaired t-test for inter-group comparison and by paired t-test for intra-group comparison. Categorical data is expressed in numbers and percentages and compared by chi-square test. P values<0.05 were considered statistically significant. All randomly allocated patients were included in the safety analysis and those who had post-baseline assessment were included in efficacy analyses.j, defined as:The analysis aims to better quantify and compare changes in the clinical scores, while taking into consideration the high patient-to-patient variability. The idea was to inspect, for each patient at each time stage, the scaled relative differences in each of the clinical scores. More specifically, we calculated for a specific patient (j) the scaled relative difference SRDj is the value of a clinical score at the end of the time stage (either treatment or control), and SIj is the score at the beginning of the time stage. We note that the symbol<> indicates average over the values of the patients in the group. For example, <SFj> means the average of SFj over all patients (j) that belong to the group. The abbreviation STD means the standard deviation between the values of the patients in the group. This analysis enables quantitative inspection of the changes in the clinical scores as is further explained and illustrated in j–SIj>) is not divided by STD(SFj–SIj).Where SFThe study included 74 patients (August 2008-October 2010). 7 patients from the treated group and 8 patients from the cross group were excluded: 8 refused the SPECT, 3 had no measurable paresis, 1 had a medical problem, 1 had a stroke during the control period, and 2 refused to quit smoking .Twenty four patients (80%) from the treated group had a history of ischemic stroke; of those, 17(71%), 3(13%), 2(8%) and 2(8%) patients were classified as TOAST 1, 2, 3 and 4, respectively. Twenty five patients (86%) from the cross group had ischemic stroke; of those, 18(72%), 3(12%), 2(8%) and 2(8%) patients were classified as TOAST 1, 2, 3 and 4, respectively; p = 0.8 for comparison of the TOAST classification between the groups. Of the 6 patients (20%) in the treated group that had hemorrhagic stroke, 5(83%) had anterior circulation stroke; and of the 4 patients with hemorrhagic stroke in the cross group, 3(75%) had anterior stroke. Baseline patients’ characteristics are summarized in The results of the neurological evaluations, including the NIHSS and the ADL and the quality of life estimates EQ-5D and EQ-VAS, are summarized in Clinical evaluations revealed statistically significant improvements in the NIHSS measures following treatment both in the HBOT-treated group b: p = 0Clinical evaluations revealed statistically significant improvements in the ADL score following treatment both in the HBOT-treated group  period 36% had mild improvement and only 6.2% had significant improvement (p<0.001). After HBOT, the cross group demonstrated 43% significant improvement and 29% mild improvement (p<0.001) (data not shown in tables).The improvements in the SPECT were mostly in regions showing noticeable discrepancy between the CT and SPECT–the earlier mentioned stunned regions of low activity living cells. The following examples of three typical patients illustrate the associations between the improvements in the patients’ clinical conditions and evaluations and the changes in their brain activity (indication of the activation of neuroplasticity) as reflected by changes in their corresponding SPECT images:Baseline brain SPECT images demonstrating hypoperfusion in the right fronto-parietal region, right postero-medial frontal and posterior-parietal perfusion lesions with no significant changes after the control period . In compThese SPECT images are of a 61y old woman from the cross group, suffering from left hemiparesis due to ischemic stroke that occurred 1 year prior to inclusion. Baseline NIHSS showed minor facial paresis, no ability to hold her left hand against gravity, some ability to hold her left leg against gravity for less than 5 seconds and mild-to-moderate sensory loss. In ADL, she needed help in bathing, dressing and climbing stairs. She was unable to do any housework. After HBOT, she was able to hold her hand and leg against gravity without significant sensory loss. She could move her fingers, was independent in bathing, dressing, shopping and cooking.SPECT images at the end of HBOT demonstrating significant improvement of perfusion deficit in the left hemisphere  involvinSPECT images demonstrating improvement in the peri-infarct region following HBOT. The images are of a 64y old woman from the treated group suffering from right hemiparesis due to ischemic stroke that occurred 26 months prior to inclusion. After treatment, the leg hemiparesis was resolved, her hand function improved significantly but she did not regain all fine motor skills. Six patients had mild-moderate barotrauma of the middle ear. After several days of rest they returned and completed the protocol. Two patients with a history of epileptic seizures prior to their inclusion in the study had mild episodes of convulsion (consciousness was fully maintained) during the study. Both patients were already treated with anti-epileptic drugs prior to their inclusion.In the current study, the effect of HBOT on chronic neurological deficiency due to stroke was evaluated in a prospective, randomized controlled study. Statistically significant improvements were obtained following treatment for almost all treated patients from both the HBOT-treated group and the HBOT-treated cross group , as was evaluated by NIHSS, ADL, brain SPECT and life quality. The significance of the improvements in this chronically debilitated population of patients is further noticeable when compared to the lack of improvement during the control (no-treatment) period of the cross group .This is the first prospective, randomized clinical study evaluating the effect of HBOT in the late post-stroke period (6 months to 3 years after the acute event). There are two major reasons for selecting this study population. First, by carefully selecting patients with chronic stable neurological deficiency we were able to avoid unexpected changes in their condition. In this regard, the selection proved very useful since the control group demonstrated neurological stability with no outliers. The second reason was, as discussed in the introduction, to test our hypothesis that the optimal time for the HBOT procedure should be during the regenerative and not during the degenerative stage. While it is not possible to mark a clear line between the regenerative and the degenerative phases The issue of “how to handle the control group” was discussed by a multidisciplinary team including physicians specializing in hyperbaric medicine, physicists specializing in neuronal-glia interactions and the ethics committee. The patients can tell if pressure is increased or not, so the pressure must be increased also in the control group. The only way to administer “placebo” of HBOT is to bring the patients to the hyperbaric chamber and to increase the environmental pressure to an extent that the patients will feel it in their ears. The minimal pressure needed to gain such a feeling should be 1.3 ATM. Henry’s law states: “the amount of a given gas dissolved in a given type and volume of liquid is directly proportional to the pressure of that gas in equilibrium with that liquid”. Thus, hyperbaric environment significantly increases the dissolved oxygen pressure even if a person holding his breath Since increasing the pressure even without adding oxygen can also increases the dissolved oxygen partial pressure, the only way to maintain normal (placebo) levels of dissolved oxygen is to supply air with lower than normal level of oxygen, which we deemed unethical. To partially compensate for this inherent limitation, the patients in the cross group started with a two-month control period of no treatment, at the end of which they were crossed to two months of HBOT sessions. To gain better validity of the results, we used the scatter plot analysis of the changes in the combined neurological evaluations. The scatter plots  show chath century, there was an ongoing debate about the time window available for induction of neuroplasticity. The improvements in the chronic late stage reported here support the view that neuroplasticity can be activated months to years after the acute event when a proper brain stimulation (such as HBOT) is applied. More specifically, the current study included patients that underwent stroke more than 6 months prior to treatment and after their condition reached a steady state (no improvements were monitored for at least a month). These important and unexpected findings arein agreement with other recent findings revealing that many aspects of the brain remain plastic even at adulthood During most of the 20Current imaging technologies reveal that the stunned brain areas (regions of high anatomy-physiology mismatch) may persist for months and years after an acute brain event In any case, the observed reactivation of neuronal activity in the stunned areas imply that increasing the plasma oxygen concentration with hyperbaric oxygenation is a potent means of delivering to the brain sufficient oxygen for tissue repair: HBOT might initiate a cellular and vascular repair mechanism and improve cerebral vascular flow To conclude, in this study we provide, for the first time, convincing results demonstrating that HBOT can induce significant neurological improvement in post stroke patients. The neurological improvements in a chronic late stage demonstrate that neuroplasticity can be operative and activated by HBOT even long after acute brain insult. Thus, the findings have important implications that can be of general relevance and interest in neurobiology. Although this study focused on stroke patients, the findings bear the promise that HBOT may serve as a valuable therapeutic practice in other neurological disorders exhibiting discrepancy between the anatomical and functional evaluation of the brain.Protocol S1Clinical Study Protocol.(DOCX)Click here for additional data file.Checklist S1CONSORT 2010 checklist.(DOCX)Click here for additional data file.Form S1Informed consent form (English translation).(PDF)Click here for additional data file.Text S1Statistical, Randomization and Placebo Considerations.(DOCX)Click here for additional data file.Text S2Scatter plot analysis of the clinical scores.(DOCX)Click here for additional data file.


Intensive breeding and selection on desired traits have produced high rates of inherited diseases in dogs. Hereditary retinal degeneration, often called progressive retinal atrophy (PRA), is prevalent in dogs with disease entities comparable to human retinitis pigmentosa (RP) and Leber's congenital amaurosis (LCA). Recent molecular studies in the English Springer Spaniel (ESS) dog have shown that PRA cases are often homozygous for a mutation in the RPGRIP1 gene, the defect also causing human RP, LCA, and cone rod dystrophies. The present study characterizes the disease in a group of affected ESS in USA, using clinical, functional, and morphological studies. An objective evaluation of retinal function using electroretinography (ERG) is further performed in a masked fashion in a group of American ESS dogs, with the examiner masked to the genetic status of the dogs. Only 4 of 6 homozygous animals showed clinical signs of disease, emphasizing the need and importance for more precise studies on the clinical expression of molecular defects before utilizing animal models for translational research, such as when using stem cells for therapeutic intervention. The domestic dog has a unique population history with bottlenecks that has shaped the diversity and structure of the canine genome. The first bottleneck can be traced back about 7,000–50,000 generations  and reflHereditary retinal degeneration, often called progressive retinal atrophy (PRA) in dogs and also in cats, is a group of diseases of the photoreceptors that exists in various forms. Rods and cones are usually primarily affected, with time leading to bilateral blindness. More than 100 dog breeds are on the list for those that may be affected, in which at least 15 mutations are prevalent in 34 specific dog breeds . For cathttp://www.genome.gov/12511476) has simplified the task of identifying genes responsible for diseases and traits in dogs. Similarly, a full genome sequence (10X) of the cat has recently been completed . A number of the PRAs have been designated gene symbols reflecting either the specific cells involved in the hereditary retinal dystrophy or the protein involved in the retinal degenerative condition.Through advancement in the understanding of hereditary disease processes affecting the outermost portion of the retina, PRA has been further characterized biochemically, electrophysiologically, morphologically, and genetically. Using molecular methods, including the elucidation of causative mutant genes for several hereditary retinal disorders, much knowledge has been gained especially in regards to disease mechanisms . The avaprcd mutation [http://omim.org/entry/268000].Although PRA is usually manifested with a breed specific phenotype, the same allelic mutation may be shared by several different breeds, such as for the mutation . In othemutation . Importamutation . The premutation . Due to mutation , 13. ThiClassical PRA has been described as a generalized disease of the fundus, affecting the rod photoreceptors primarily and with later involvement of cones. Clinically night blindness is an early sign but with progression of disease, affection of both night and day vision occurs. Ophthalmoscopically early changes have been noted as a generalized diffuse change in coloration and reflectivity of the tapetal fundus with vascular attenuation in later stages, the changes being most severe in the peripheral parts but with later involvement of the entire fundus .PRA in the English Springer Spaniel dog was originally described in USA . There wMutations in the RPGRIP1 gene have been identified as causative to human RP, LCA, and cone-rod dystrophies –19. The Mellersh and collaborators recently mapped the cone rod locus (cord1) to a 14.5 Mb region on dog chromosome 15 (16.54–30.68 Mb: coordinates as in CanFam2.0), which contained the RPGRIP1 gene. A 44 bp insertion in exon 2 was further identified in the RPGRIP1 gene, truncating the protein. This defect segregated completely with the cord1 phenotype of cone rod dystrophy in the Animal Health Trust (United Kingdom) research colony of miniature dachshunds. It was concluded that the mutation was responsible for cord1 disease, due to a mutation in the RPGRIP1 gene. It was further emphasized that dogs with this disease are valuable animal models for exploring disease mechanisms and potential therapies for the human counterpart, LCA .RPGRIP1 mutation in cone rod dystrophy (cord1) was further evaluated as a candidate gene for PRA in ESS dogs using DNA collected at the University of Missouri , from a large number of dogs, unaffected and affected by bilateral, generalized retinal degeneration. The mutation was observed in exon 2 of RPGRIP1 in all of the affected dogs .The RPGRIP1 mutation.This paper describes the results of a research project that was thereafter initiated, in order to characterize the clinical signs of retinal degeneration in the ESS breed and to evaluate the genotype-phenotype correlation in USA in family-owned ESS, in regards to the mutation in the RPGRIP1 gene. Recently, the study was further expanded to also include blood samples from a group of Swedish ESS dogs, with and without clinical signs of PRA and the correlation in regards to the RPGRIP1 mutation.ESS dogs from USA were included in the characterization of disease. They were privately owned dogs for which owners or breeders had requested eye examinations, according to eye scheme routines, since their dogs or their close relatives were used for breeding. Twelve cases with clinical signs of retinal degeneration in 1.5- to 12-year-old ESS dogs were discovered during the two-year study period. All of the affected dogs were homozygous for the Informed consent was obtained from the owners of participating dogs. The clinical study included evaluation of retinal and vision based responses and reflexes: menace, dazzle, and examination of the pupillary light reflexes as well as visual testing by behavior and visual reactions to falling cotton balls . Pupils cord1 test or for clinical biochemistry under the condition of anonymity of the individuals and their owners. DNA was extracted from blood samples and genotyping for the cord1 disease causing allele of the mutated gene RPGRIP1 gene was performed as described by Mellersh et al., 2006 [DNA samples from ESS dogs previously collected by Dr. Gary Johnson's laboratory, University of Missouri, Columbia, USA, were utilized. All samples were collected as surplus from blood specimens submitted for routine l., 2006 .In addition, to investigate whether the cord1 genotype was prevalent among ESS dogs in Sweden, and especially in ESS dogs diagnosed with PRA, blood samples from a total of 14 normal and affected dogs were tested for the 44 bp insertion in exon 2 of the RPGRIP1 gene . The blo2, 0.2 mM of each dNTP and 0.5 units of Taq polymerase . A total of 35 PCR cycles was performed, each with denaturation at 94°C for 1 minute, annealing at 60°C for 40 s and a primer extension at 72°C for 40 s. The fragment length polymorphism was then determined using an ABI 3100 DNA Analyzer and GeneMapper Software , Foster City, CA).Primers for genotyping the 44 bp insertion in exon2 of RPGRIP1 gene were designed using the software Primer3 . PCR ampRPGRIP1 mutation was unknown to the investigator (K. Narfström) at the time of the ERG recordings: 6 dogs were homozygous (affected); RPGRIP−/−, 7 were heterozygous ; RPGRIP−/+, and 1 was homozygous ; RPGRIP1+/+ (Fourteen American dogs were included in a masked electroretinography (ERG) study for objective evaluation of retinal function (for ERG see details below). Blood from these dogs, age between 7 y 9 m and 13 y 10 m, had previously been genotyped by Dr. Johnson's laboratory, as described above. Dogs were chosen for retinal functional evaluation in accordance with the owner's consent and availability. The genetic status of each dog in regards to the GRIP1+/+ . Before Unilateral electroretinographic (ERG) evaluations were performed using a portable ERG unit , with a mini-Ganzfeld dome positioned approximately 1 cm from the right tested eye . For praDogs were deeply sedated by using medetomidine IV , up to 150 micrograms/kg, equivalent to 0.15 mL/kg, and prepared for the ERG session in ordinary room light. Heart and respiratory rates were closely monitored before and throughout the procedure and the dogs were temperature controlled. The dog's head was positioned on a cushion for stabilization. Maximal pupillary dilation was provided for by the use of short-acting mydriatics (see above) and the eye was further topically anesthetized using 0.5% proparacaine hydrochloride . A lid speculum was inserted to ensure that the nictitating membrane as well as the upper and lower eyelids did not interfere with light exposure to the maximally dilated pupils. Platinum subdermal needle electrodes  were used for the ground electrode, positioned on the occipital crest, and for the reference electrode, positioned 3 cm from the lateral canthus, close to the base of the right ear. An active contact lens electrode  was placed on the cornea after instillation of one drop of 2% methylcellulose . The electrodes were connected to a preamplifier and the signals were amplified with a bandpass filter between 0.3 and 300 Hz.2; responses were averaged after 10 flashes given at 2 seconds interval and rod responses were recorded at each time point (test #1–5). The light stimulus intensity was then increased to 3 cd·s/m2 for scotopic standard intensity stimulation and responses averaged and recorded after 4 flashes at 10-second intervals (test #6). Thereafter scotopic high-intensity responses were elicited using 10 cd·s/m2; responses were averaged to 4 flashes administered at 20-second intervals (test #7). The latter two recordings depict a mixture of responses from both rods and cones. After 10 minutes of light adaptation with a background luminance of 30 cd/m2, photopic single flash responses were recorded, using 3 cd·s/m2 of flash stimulus, averaging 32 flashes at an interval of 0.5 seconds (test #8), followed by evaluation of 30 Hz photopic flicker at the same light intensity stimulation (test #9). The latter two recordings were performed in order to evaluate cone and cone pathways, respectively. Data were collected automatically on the compact flash card of the ERG unit, transferred to a computer, printed, and stored for further analysis. ERGs were evaluated and the amplitudes and implicit times for the a- and b-waves were measured as previously described [Each ERG session consisted of scotopic and photopic ERGs in accordance with the “Dog Diagnostic Protocol,” recommended by the European College of Veterinary Ophthalmologists, primarily for evaluation and separation of rod and cone function . This prAfter termination of the ERG session an injection of atipamazole hydrochloride  was administered intramuscularly to reverse the deep sedation .2, pH 7.40. Eyecups were incubated with gentle agitation for at least 2 hours at room temperature. The eyecups were then gross sectioned to obtain 2 × 3 mm pieces from the following regions: the central part of the fundus, temporal to the optic nerve head , superior midperiphery and periphery, and inferior midperiphery and periphery. Samples from these regions were postfixed in 1% osmium tetroxide and embedded in epoxy resin. They were washed with 0.17 M sodium cacodylate, pH 7.4, followed by secondary fixation in 1% osmium tetroxide. Subsequently, the samples were dehydrated via sequential incubation in increasing concentrations of acetone and embedded in epoxy resin. Sections of the embedded samples were cut for both LM and EM examinations. For LM, 1-micron-thick sections were mounted on glass slides and stained with toluidine blue. For EM, sections were mounted on copper grids and were stained with uranyl acetate and lead citrate. LM was performed using a Zeiss Axiophot microscope and EM was performed using a JEOL 1200 EX transmission electron microscope.Upon the owner's request due to unrelated medical problems, three American ESS dogs were euthanized and the eye tissue made available for the present study. Advanced PRA had been diagnosed in two of the dogs , while the third dog, 3 years old, had a normal fundus appearance. Euthanasia was performed by intravenous infusion of Beuthanasia-D-Special . The eyes of each dog were enucleated immediately after death and the posterior segment of each eye placed in fixative solution for examination using light and electron microscopy (LM and EM). The fixative included 2.0% glutaraldehyde, 1.12% paraformaldehyde, 0.13 M sodium cacodylate, 0.13 mM CaClRPGRIP1 mutation. Due to the small sample size in the affected group and the fact that the data did not show any extreme outliers, two-sample t-tests with the Satterthwaite approximation for degrees of freedom allowing for unequal variances between the groups were utilized. Due to the marked differences between the groups, results were considered significant at the 0.01 significance level.Descriptive statistics were performed in relation to the masked ERG study using the SAS v9  of the dogs classified as normal  and affected dogs by DNA analysis for the A 10-year-old dog was examined due to severe visual problems reported by the owner, first noted at the age of 8 years. Advanced signs of retinal degeneration were observed by ophthalmoscopy in this dog. None of the other 11 dogs examined with clinical signs of retinal disease  had shown visual problems until the age of 6–9 years, according to the owners.The earliest ophthalmoscopic signs of disease were observed in a 1.5- and a 2-year-old ESS dog, respectively. Both showed increased granularity in the far peripheral tapetal fundus, with minute hyporeflective brown to gray spots in the far periphery of the tapetal fundus . With inBlood from 14 dogs was included in the study in which ten of the dogs had been diagnosed with PRA. Two cases of PRA were found to be homozygous for the disease causing RPGRIP allele (−/−), four were genotyped as heterozygous (+/−) and four of the cases had the homozygous genotype for the normal allele (+/+) .RPGRIP1 mutation through blood testing . For the b-wave these differences were also significant at the same level for 4 of the 5 scotopic low-intensity responses evaluated (#2–5)  and for the photopic flicker response (P < 0.0032). For a- and b-wave implicit times there were no significant differences for the former while for the latter a significant longer implicit time was found for the affected group of dogs in comparison to the normal group for scotopic standard intensity stimulation (P < 0.0066). For ERG amplitude and implicit time details see Results of ERG recordings in a normal 8-year-old ESS and in two affected dogs, 9 and 12 year olds, respectively, are shown in RPGRIP1 mutation by blood testing, had a- and b-wave amplitude and implicit time values that were in complete accordance with the normal group of dogs.Two dogs in the group, found to be homozygous for the In order to evaluate if the cone system was more affected than the rod system by the disease, the percentage reduction for each was calculated. This was performed through comparison of the median response from affected and normal dogs, respectively, using photopic standard intensity (test #8) and photopic flicker responses (test #9) for the cone system and scotopic low-intensity stimulation (test #5) for the rod system. It was found that the cone system was more affected than the rod system: 46% and 58% reduction, respectively, for the cones and 44% reduction for the rods in the affected group when compared with the normal ESS group of dogs.RPGRIP1 mutation.Light and electron microscopies were performed using retinal sections from 3 dogs. The two older dogs had ophthalmoscopic signs of severe bilateral generalized retinal degeneration while the younger, a 3-year-old ESS dog, exhibited a granular but otherwise normal fundus appearance. DNA from the youngest dog only was obtained, and showed homozygocity for the In the 9-year-old dog, bilaterally atrophic retinas with no residual photoreceptor cells and sporadic remnants of cells from inner nuclear and ganglion cell layers were observed, with marked retinal gliosis Figures  and 5.The retinas of the second dog, 6-years-old, showed signs of bilateral generalized retinal degeneration, with complete atrophy of the inferior retina, and a lack of photoreceptor cells in this area, while in the superior retina 1-2 layers of photoreceptor nuclei could be observed.The 3-year-old dog, however, demonstrated mainly normal retinal morphology except for changes in the photoreceptor cell layer at all retinal locations examined: cone cell nuclei appeared slightly abnormal, with dense chromatin and with photoreceptor inner segments condensed and shrunken . PhotoreRPGRIP1 gene. However, four of the 6 dogs homozygous for the RPGRIP1 mutation had clinical signs of disease while 2 were completely normal appearing both by ophthalmoscopic examination and by ERG. Thus, a clear genotype-phenotype discordance was observed in regards to this group of ESS dogs.The present study indicates that a majority of the American ESS dogs with hereditary retinal degeneration can be associated with homozygosity for the disease causing allele of the RPGRIP1 insertion by itself is not sufficient to cause retinal degeneration. It appears likely that additional factors are warranted for initiation of photoreceptor cell death such as additional loci involved as modifiers of the disease, as have been described for various forms of PRA, for example, prcd and X-linked PRA [A large proportion of the genetically and/or clinically affected dogs did not show signs of visual impairment until comparably late in life and 2 were clinically completely normal. The reason for the latter finding is unclear. It could be that the nked PRA , 31. It nked PRA .RPGRIP1 mutation, on a variable genetic background, such as is the case in the human population. In purebred dogs, however, a more uniform phenotype is usually expressed, due to a more homogenous background. This fact is true for most forms of PRA; however, for the cone-rod dystrophies observed in the longhaired and shorthaired Dachshund breeds, with the RPGRIP1 and NPHP4 mutations, respectively, severe heterogeneity has been described for both [Similar findings have been observed in comparable human clinical studies in cone rod dystrophies and degenerations. A phenotypic variation between clinical signs in affected individuals and in the onset of hereditary retinal dystrophies has been observed, also by functional testing in human cone rod dystrophies . The varfor both , 27, 34.RPGRIP1 mutation. Another dog showing clinical signs of retinal degeneration had been diagnosed as ophthalmoscopically normal by a veterinary ophthalmologist one year previously, at the age of 7 years. ERGs showed, however, severely reduced retinal function in accordance with a cone rod dystrophy.In retinal degeneration of the ESS dog clinical signs most often appear comparably late in life and are often difficult to evaluate by the owner. For most of the affected dogs observed in the present study the owners had not noted any visual impairment. The owner of an 11-year-old dog from the present study described that the dog could still play with a transparent frisbee and could easily walk down an indoor stairway in low-lighting conditions. This dog had moderately advanced retinal degeneration with ophthalmoscopically visible changes and was homozygous for the RPGRIP1 mutation, showed ultrastructural changes specifically in cones, while rod photoreceptors were still normal appearing. It is possible that the visual problems become apparent clinically later in life when also the rods start to degenerate. Further, this second phase appears to occur at a variable time point, but most often not until late in life, then leading to a rather fast generalized severe retinal degeneration .It is likely that the cone affection in the disease is early onset, and a defect the dog learns to live with as long as its rod function is normal. Morphology of one 3-year-old ESS dog, homozygous for the RPGRIP1 mutation. In two cases, however, function in genetically affected individuals was found to be within normal limits. These discordant clinical results could be consistent with incomplete penetrance for the mutation, but other factors previously outlined may also be involved.ERG recordings proved to be useful in the masked study of the present investigation for objectively detecting reduced photoreceptor function in accordance with cone rod dystrophy due to the RPGRIP1 may also be present in the ESS breed. One mutated gene, prevalent in at least 32 canine breeds, is prcd, . This could also be a candidate gene since it is known to affect the English and American Cocker Spaniel dog breeds, distant relatives to the ESS dog . Some of the other mutations causing primary photoreceptor degenerations in dogs are ADAM9 [Other affected genes or other mutations in re ADAM9 , CCDC66 re ADAM9 , CNGB3 [re ADAM9 , RD3 [38re ADAM9 , RPE65 [re ADAM9 , VMD2 [4re ADAM9 , PDE6betre ADAM9 , PDE6A [re ADAM9 , and PDCre ADAM9 .RPGRIP1 locus. Four were found to be heterozygous and four were homozygous for the normal wild-type allele (Among the ten PRA cases in ESS dogs previously diagnosed in Sweden, from which DNA was available for the current study, only two individuals were homozygous for the disease causing allele at the e allele . The twoRPGRIP1 and PRA observed in the ESS thus remains to be fully elucidated. There are strong indications that the RPGRIP1 gene is involved in the cone rod dystrophy described herein, but the genotype-phenotype discordance observed shows that the genetic background most probably is more complex than previously suspected. In conclusion, there are strong indications that other mutations or modulating genes may be involved in cone rod dystrophy of ESS dogs and could also be causative to other types of hereditary retinal degenerations in the breed. Further investigations in regards to additional loci or genes required for development of cord1 are therefore warranted.The complete association between An important goal for vision research is to provide effective treatments for the millions of people affected with retinal blinding disorders. Therapeutic intervention using large animal models such as dogs and cats are effective and necessary methods to utilize before proceeding with human treatment trials. Proof of principle was obtained through therapeutic studies using gene therapy in a dog model of LCA , resulti


Although the benefit of sirtuin activation in age-related diseases is well-characterized, the benefit of sirtuin activation in acute diseases has been elusive. Here we discuss that, at least in yeast, Sir2 activation prevents programmed cell death induced by the sustained activation of the stress activated protein kinase (SAPK) Hog1, the yeast homologue of the p38 SAPK. Sir2 prevents ROS formation and maximize cell survival upon SAPK activation. The conserved function of Sir2 in age-related diseases and the conserved role of SAPKs open the possibility of a novel role for sirtuins in cell fate determination in eukaryotic cells. Activation of sirtuins has been proved to be positive in age-related diseases -5. HowevThe study by Vendrell et al shows that inhibition of cell growth by sustained activation of Hog1 is caused by the induction of apoptosis-like cell death in yeast. Specifically, activation of the Hog1 SAPK inhibits mitochondrial respiration that leads to an increase in ROS levels  or, to a lesser extent, the YCA1 caspase prevented Hog1-induced cell death. Taken together, Hog1-induced cell death is likely to be caused by apoptosis.Unexpectedly, mutations in the E3 ubiquitin ligase CDC4, as well as the core components of the SCF complex (SKP1 and CDC53) partially suppressed Hog1-induced cell death. SCF mutations resulted in a reduced Msn2/Msn4 degradation that led to an increase in Msn2/Msn4 dependent gene expression (Figure Interestingly, PNC1, an activator of the Sir2 histone deacetylase, is under the control of Msn2/Msn4 transcription factors . The mutThe mechanism by which Sir2 prevents apoptosis in yeast has not been defined. However, whereas Sir2 activation does not alter cellular respiration upon stress, it strongly reduces ROS production and hence, it is likely that this reduction in ROS permits cell survival upon Hog1 activation.Activation of SAPK signalling is essential for cell adaptation to stress. Strikingly, sustained activation of the pathway unravels a more-complex scenario. Therefore, although a decrease in mitochondrial respiration might be important for adaptation to stress, an extended reduction of respiration leads to excessive ROS formation. To counteract cell damage, Hog1 seems to engage a protective program by inducing PNC1 gene expression, which concomitantly activates Sir2 to balance excessive ROS accumulation. Therefore, cell fate is dictated by the balance between ROS induced by Hog1 SAPK and the protective effects of Sir2.Apoptosis is caused by a variety of factors, such as oxidative stress . InductiThe mechanism by which Hog1 regulates mitochondrial respiration and ROS levels remains unknown. It cannot be excluded that regulation of metabolism could lead to an altered TCA cycle and/or mitochondrial respiration. Therefore, the characterization of this phenomenon will allow identifying at which level the MAPKs can interact with the mitochondria: regulating mitochondria biogenesis, respiratory chain formation or respiratory chain activity. This would help to know whether the effect of Hog1 MAPK could be conserved in higher eukaryotes.The effect of sirtuins in apoptotic cell death is not clear. Depending on expression of a specific sirtuin, the cell type and the stimuli, sirtuins prevent or induce apoptosis -29. MoreRecent data on the overexpression of mammalian Sir2 seems to show that Sir2 might not be responsible of lifespan extension albeit important for health in ageing processes . RemarkaApoptosis in yeast is a controversial issue. The benefit that could generate programmed cell death in a unicellular organism has been deeply discussed . It is tThe determination of cell fate upon stress could result of the balance between cell survival and cell death. Once cells are exposed to a stress, Hog1 SAPK is activated and cells enter into an alarm state. Upon a mild stress, Hog1 SAPK is active for a short period of time. Then, Hog1-induced sirtuin activation might be sufficient to prevent ROS accumulation, and the whole population survives. However, upon severe stress or sustained activation of the SAPK, longer activation of the MAPK Hog1 induces an increase on ROS production beyond a certain threshold. It is likely then that the preventive effect of sirtuin activation might not be sufficient to compensate ROS levels and certain cells of the population would undergo apoptosis and provide more resources to the younger cells to adapt and survive to a severe stress. The fact that both pro and anti-apoptotic pathways are controlled by the same protein, Hog1, emphasize the importance of the balance between the two responses that will determine cell fate.Vendrell and collaborators have unraveled the ability of Sir2 to prevent ROS formation and cell death upon MAPK activation in yeast. However, the mechanism by which Sir2 prevents ROS formation remains unclear. It would be interesting in the field of sirtuins to define how Sir2 is able to reduce ROS levels and decipher whether Sir2 is modulating mitochondrial activity per se or it is inducing antioxidant defence mechanisms.It is likely that, as occurs in yeast, the balance in sirtuin activation determines cell fate in higher eukaryotes cells. Recent data show that two-fold overexpression of Sir2 in flies and worms did not extend lifespan at least as much as it was reported before -45. In f


Multiple renal arteries in kidney grafts have been associated with an increased rate of vascular and urologic complications. Our objective is to compare the outcome of transplanted patients who receive a single pedicle kidney with those who receive a multiple arterial graft.To find our the differences in the outcome and complications in patients undergoing kidney transplantation with one single artery or with multiple arteries.We analyzed 147 kidney transplantations, . population divided into two groups: group A for those who presented with only one renal artery, or group B for those with more than one renal artery. Homogeneous vascular reconstructions and implantation rules were applied in all patients. We compared the rates of renal failure between the two groups, urinary and vascular complications, patient and graft survivals and the levels of creatinine clearance during the first year of post-transplantation.No significant differences were found between the two groups regarding to the values analyzed.As many other authors, we do believe that the presence of multiple renal arteries in kidney grafts should not be considered as a predictive factor of transplantation failure. The rate of patients with end-stage renal disease is increasing everyday . Kidney Our goal in this study is to find differences in the outcome and complications of patients with only one artery or with multiple arteries that led us to think in worst, better or equal results in kidney transplantation.Between January 2006 and December 2008 147 cadaveric-donor kidney transplantations were performed in university hospital Virgen de la Arrixaca by the authors of this paper. We evaluated all of cases, including those grafts that were rejected for some reason at the time of the implantation . After cadaveric extraction (most of which were multiorganic) bench surgery was carried out and implantation of the graft performed, preferably in the right iliac fossa, with end-to-side anastomosis of the graft main vessels to the external iliac artery and vein avoiding atheromatous plaques. Both anastomoses were performed with two halves of running non-absorbable monofil 6x0 or 5x0 sutures. Extravesical ureteroneocystostomy (Lich-Gregoir technique) with double-J stent was used for urinary reconstruction. All patients received the standard triple drug immunossuppression.Mean patient age was 50.9 ± 12.24 years (range 18-75 years) and 46.66 ± 16.42 years (range 14-75 years) for the donors; 96 (65.3%) were males and 51 (34.7%) females.Thirty-three grafts (22.4%) had multiple arteries (group B) and the rest, 114 (77.6%), had one single artery (Group A). Regarding bench reconstructions, when there were polar arteries of less than 2 mm in diameter they were ligated (4 patients). If the distance between the two main arteries (more than 2 mm in diameter) was less than 2 cm, a common aortic patch had been used (in 21 patients). However, if it was greater than 2 cm, we carried out side-to-side  anastomosis (4 patients) when the caliber was of equal size or sequential anastomosis in recipient (5 patients) when the calibers were very different. Only two grafts had three arteries, one of them was ligated because of its small caliber, and bench surgery was performed as in double artery grafts. One graft had 4 arteries, so one polar artery was ligated, two were converted to a single arterial vessel after bench reconstruction and 2 end-to-side anastomosis were performed in the external iliac artery. This patient suffered extensive external iliac vein thrombosis when we removed the vessel clamp and transplantectomy was performed.To evaluate the outcome of transplantation we recorded the following factors in the first year after surgery: (a) patient and (b) graft survival; (c) the incidence of vascular complications ; (c) urinary complications ; (d) delayed graft function (DGF), that was defined as when serum creatinine levels increased or remained unchanged after 5 five days post-transplantation, and the levels of creatinine clearance at one, two, three, six, nine and twelve months.The differences between the groups of patients (A and B) were analyzed using SPSS 10 software: t-student test for the means of levels of creatinine clearance, Chi-Square test for the complications rates and Kaplan-Meier plots for the graft and patient survival.The one year graft survival rate was 87.5%; 15 patients lost the graft function . Patient survival was 95.5%; six patients died in this period. No statistically significant differences were found between groups A and B .The incidence of DGF in the 147 patients was 25.9%; 27% in the group A and 21% in the group B and was not significantly different.We only found one case (0.7%) of renal artery thrombosis, one case (0.7%) of renal vein thrombosis and three cases (2%) of renal artery stenosis. The case of artery thrombosis occurred in the patient who was transplanted using the graft with four arteries. Transplantectomy was performed in both cases of vascular thrombosis and the cases of stenosis were successfully treated by balloon angioplasty. No statistically significant differences were found between groups A and B.Eight cases (5.4%) of ureteral obstruction that were treated by balloon ureteroplasty, with only one case of restenosis that required surgery (reimplantation of the ureter into the bladder) had been found. Fourteen patients (9.5%) suffered anastomotic or bladder leakage and urgent reimplantation was performed on all of them. There were no statistically significant differences between both groups.No significant differences between groups A and B were found with respect to creatinine clearance at 1, 2, 3, 6, 9 and 12 months. It could be concluded that in the first year renal function was similar in patients with a single-artery graft compared to those with a multiple-artery graft .Throughout the cadaveric extraction the surgeon could find anatomic anomalies that are not uncommon. In fact, Harrison et al. revised arteriographies of future living-donors and concluded that 25% of people had multiples arteries and less than 50% of them had the simplest form of renal vascular pedicle . Prior tFortunately, after those discouraging studies, due to the long hospital waiting list for deceased donor grafts and the peak in the number of living donors, a larger series of patients were analyzed with very different statistical results. Authors of these studies did not find differences in the outcomes when comparing the patients who received a multiple artery graft with those who received a single pedicle kidney , 10-16. As a matter of fact, we cannot deny that the existence of anatomic anomalies in renal grafts is an added difficulty. They need to be dealt with by an experienced team in vascular reconstruction and involve a higher cold and warm ischemic time that can be improved by using the common aortic patch (Carrel patch). Unfortunately, this technique is not possible in living donation. However this increase in ischemic time is not enough to damage the graft if it is adequately perfused and cooled .One limitation of our study could be the monitoring duration (one year). We do believe that the classic complications associated with vascular anomalies in kidney transplantation  are usually occur early; only arterial stenosis tends to happen later and is usually diagnosed between 6 months and 3 years post-transplantation . The manLike many other authors we cannot conclude that the use of multiple artery grafts is associated with a poorer outcome. Consequently, it is currently unacceptable to reject organs because of this anomaly and as a result more patients can benefit from the best treatment for the end-stage kidney disease. More studies are necessary in order to shed the light on the subject.