Reprogramming of human fibroblasts to induced pluripotent stem cells under xeno-free conditions

The availability of induced pluripotent stem cells (iPSCs)has created extraordinary opportunities for modeling andperhaps treating human disease. However, all reprogrammingprotocols used to date involve the use of products of animal origin. Here, we set out to develop a protocol to generate and maintain human iPSC that would be entirelydevoid of xenobiotics. We first developed a xeno-free cellculture media that supported the long-term propagation of human embryonic stem cells (hESCs) to a similar extent as conventional media containing animal origin products or commercially available xeno-free medium. We also derivedprimary cultures of human dermal fibroblasts under strictxeno-free conditions (XF-HFF), and we show that they can be used as both the cell source for iPSC generation as well as autologous feeder cells to support their growth. We also replaced other reagents of animal origin trypsin, gelatin, matrigel) with their recombinant equivalents. Finally, we used vesicular stomatitis virus G-pseudotyped retroviral particles expressing a polycistronic construct encoding Oct4, Sox2, Klf4, and GFP to reprogram XF-HFF cells under xeno-free conditions. A total of 10 xeno-free humaniPSC lines were generated, which could be continuously passaged in xeno-free conditions and aintained characteristics indistinguishable from hESCs, including colonymorphology and growth behavior, expression of pluripotency-associated markers, and pluripotent differentiationability in vitro and in teratoma assays. Overall, the resultspresented here demonstrate that human iPSCs can be generatedand maintained under strict xeno-free conditions and provide a path to good manufacturing practice (GMP) applicability that should facilitate the clinical translation of iPSC-based therapies.

Is spinal stenosis assessment dependent on slice orientation? A magnetic resonance imaging study.

INTRODUCTION: Lumbar spinal stenosis (LSS) treatment is based primarily on the clinical criteria providing that imaging confirms radiological stenosis. The radiological measurement more commonly used is the dural sac cross-sectional area (DSCA). It has been recently shown that grading stenosis based on the morphology of the dural sac as seen on axial T2 MRI images, better reflects severity of stenosis than DSCA and is of prognostic value. This radiological prospective study investigates the variability of surface measurements and morphological grading of stenosis for varying degrees of angulation of the T2 axial images relative to the disc space as observed in clinical practice. MATERIALS AND METHODS: Lumbar spine TSE T2 three-dimensional (3D) MRI sequences were obtained from 32 consecutive patients presenting with either suspected spinal stenosis or low back pain. Axial reconstructions using the OsiriX software at 0°, 10°, 20° and 30° relative to the disc space orientation were obtained for a total of 97 levels. For each level, DSCA was digitally measured and stenosis was graded according to the 4-point (A-D) morphological grading by two observers. RESULTS: A good interobserver agreement was found in grade evaluation of stenosis (k = 0.71). DSCA varied significantly as the slice orientation increased from 0° to +10°, +20° and +30° at each level examined (P < 0.0001) (-15 to +32% at 10°, -24 to +143% at 20° and -29 to +231% at 30° of slice orientation). Stenosis definition based on the surface measurements changed in 39 out of the 97 levels studied, whereas the morphology grade was modified only in two levels (P < 0.01). DISCUSSION: The need to obtain continuous slices using the classical 2D MRI acquisition technique entails often at least a 10° slice inclination relative to one of the studied discs. Even at this low angulation, we found a significantly statistical difference between surface changes and morphological grading change. In clinical practice, given the above findings, it might therefore not be necessary to align the axial cuts to each individual disc level which could be more time-consuming than obtaining a single series of axial cuts perpendicular to the middle of the lumbar spine or to the most stenotic level. In conclusion, morphological grading seems to offer an alternative means of assessing severity of spinal stenosis that is little affected by image acquisition technique.

Predicting clinical scores from magnetic resonance scans in Alzheimer's disease.

Machine learning and pattern recognition methods have been used to diagnose Alzheimer's disease (AD) and mild cognitive impairment (MCI) from individual MRI scans. Another application of such methods is to predict clinical scores from individual scans. Using relevance vector regression (RVR), we predicted individuals' performances on established tests from their MRI T1 weighted image in two independent data sets. From Mayo Clinic, 73 probable AD patients and 91 cognitively normal (CN) controls completed the Mini-Mental State Examination (MMSE), Dementia Rating Scale (DRS), and Auditory Verbal Learning Test (AVLT) within 3months of their scan. Baseline MRI's from the Alzheimer's disease Neuroimaging Initiative (ADNI) comprised the other data set; 113 AD, 351 MCI, and 122 CN subjects completed the MMSE and Alzheimer's Disease Assessment Scale-Cognitive subtest (ADAS-cog) and 39 AD, 92 MCI, and 32 CN ADNI subjects completed MMSE, ADAS-cog, and AVLT. Predicted and actual clinical scores were highly correlated for the MMSE, DRS, and ADAS-cog tests (P<0.0001). Training with one data set and testing with another demonstrated stability between data sets. DRS, MMSE, and ADAS-Cog correlated better than AVLT with whole brain grey matter changes associated with AD. This result underscores their utility for screening and tracking disease. RVR offers a novel way to measure interactions between structural changes and neuropsychological tests beyond that of univariate methods. In clinical practice, we envision using RVR to aid in diagnosis and predict clinical outcome.

Clinical indicators for the initiation of endotracheal suction in children: An integrative review.

BACKGROUND: Critical decisions and interpretation of observations by the nurse caring for the paediatric intensive care (PIC) patient can have dramatic and potential adverse impact on the clinical stability of the patient. A common PIC procedure is endotracheal tube (ETT) suction, however there is inconsistent evidence regarding the clinical indicators to guide and support nursing action. Justification for performing this procedure is not clearly defined within the literature. Further, a review of the literature has failed to establish clear standards for determining if the procedure is warranted, especially for paediatric patients. OBJECTIVE: The objective of the review is to identify current clinical indicators used in practice to determine why ETT suction should be performed. METHOD: An integrative review using a systematic approach to summarise the empirical and theoretical evidence within the literature as it relates to clinical practice was used. RESULTS: Consensus of opinion indicates that ETT suctioning should only be performed when clinically indicated. There is no general consensus regarding which clinical indicators should be measured and used to guide the decision to perform ETT suctioning. CONCLUSION: Research is required to identify the clinical indicators that could be used to design a valid and clinically appropriate tool to use to assist in the decision making process to perform ETT suction.

EuroCMR (European Cardiovascular Magnetic Resonance) registry: results of the German pilot phase.

OBJECTIVES: During its German pilot phase, the EuroCMR (European Cardiovascular Magnetic Resonance) registry sought to evaluate indications, image quality, safety, and impact on patient management of routine CMR. BACKGROUND: CMR has a broad range of applications and is increasingly used in clinical practice. METHODS: This was a multicenter registry with consecutive enrollment of patients in 20 German centers. RESULTS: A total of 11,040 consecutive patients were enrolled. Eighty-eight percent of patients received gadolinium-based contrast agents. Twenty-one percent underwent adenosine perfusion, and 11% high-dose dobutamine-stress CMR. The most important indications were workup of myocarditis/cardiomyopathies (32%), risk stratification in suspected coronary artery disease/ischemia (31%), as well as assessment of viability (15%). Image quality was good in 90.1%, moderate in 8.1%, and inadequate in 1.8% of cases. Severe complications occurred in 0.05%, and were all associated with stress testing. No patient died during or due to CMR. In nearly two-thirds of patients, CMR findings impacted patient management. Importantly, in 16% of cases the final diagnosis based on CMR was different from the diagnosis before CMR, leading to a complete change in management. In more than 86% of cases, CMR was capable of satisfying all imaging needs so that no further imaging was required. CONCLUSIONS: CMR is frequently performed in clinical practice in many participating centers. The most important indications are workup of myocarditis/cardiomyopathies, risk stratification in suspected coronary artery disease/ischemia, and assessment of viability. CMR imaging as used in the centers of the pilot registry is a safe procedure, has diagnostic image quality in 98% of cases, and its results have strong impact on patient management.

European French-speaking study from the GEMO group on bone metastases management: a special focus on external beam radiotherapy practice survey.

This study seeks to perform a survey of patterns of practice among the different physicians involved in the bone metastases management, with special focus on external beam radiotherapy (EBRT).A questionnaire about bone metastases based on clinical cases and supplemented with general questions, including medical therapies, EBRT and metabolic radiotherapy strategies, surgery, and supportive care approaches, was sent to 4,706 French-speaking physicians in Belgium, France, Luxemburg, and Switzerland.Overall, 644 questionnaires were analyzed. Twenty-eight percent concerned the radiotherapy approach and were judged adequate to respond to the part dedicated to EBRT. Sixty-nine percent of physicians used a total dose irradiation of 30 Gy delivered in ten fractions. A large majority (75%) used two opposed fields prescribed at mid-depth (30%), or with non-equally weighted fields (45%). Seventy percent irradiated also above and below the concerned vertebra. A dosimetry planning treatment was done in 85% and high-energy megavoltage photons were used in 42%. Moreover, 54% physicians used short course radiotherapy in routine.Radiotherapy remains the mainstay of treatment of bone metastases, but there is substantial heterogeneity in clinical practice. Guidelines and treatment protocols are required to improve the treatment quality.

Encéphalites virales [Viral encephalitis: update]

Suspicion of viral encephalitis should always be considered as a medical emergency and the prognosis depend on both the immune status of the host and the virulence of the virus. Among them, the herpes simplex virus is by far the most important one since it can be associated with severe encephalitis in immunocompetent host, and because a good response to acyclovir can be expected when rapidly initiated. Nevertheless, confirmation of the diagnosis requires exclusion of both metabolic or toxic encephalopathy and inflammatory encephalitis of non-infectious origin. In addition, other germs than viruses can mimic viral encephalitis and must be taken into consideration. The purpose of this review is to update the investigation that should be performed in clinical practice for any patient with suspicion of acute viral encephalitis.

Postmortem imaging of sudden cardiac death

Postmortem imaging is increasingly used in forensic practice in cases of natural deaths related to cardiovascular diseases, which represent the most common causes of death in developed countries. While radiological examination is generally considered to be a good complement for conventional autopsy, it was thought to have limited application in cardiovascular pathology. At present, multidetector computed tomography (MDCT), CT angiography, and cardiac magnetic resonance imaging (MRI) are used in postmortem radiological investigation of cardiovascular pathologies. This review presents the actual state of postmortem imaging for cardiovascular pathologies in cases of sudden cardiac death (SCD), taking into consideration both the advantages and limitations. The radiological evaluation of ischemic heart disease (IHD), the most frequent cause of SCD in the general population of industrialized countries, includes the examination of the coronary arteries and myocardium. Postmortem CT angiography (PMCTA) is very useful for the detection of stenoses and occlusions of coronary arteries but less so for the identification of ischemic myocardium. MRI is the method of choice for the radiological investigation of the myocardium in clinical practice, but its accessibility and application are still limited in postmortem practice. There are very few reports implicating postmortem radiology in the investigation of other causes of SCD, such as cardiomyopathies, coronary artery abnormalities, and valvular pathologies. Cardiomyopathies representing the most frequent cause of SCD in young athletes cannot be diagnosed by echocardiography, the most widely available technique in clinical practice for the functional evaluation of the heart and the detection of cardiomyopathies. PMCTA and MRI have the potential to detect advanced stages of diseases when morphological substrate is present, but these methods have yet to be sufficiently validated for postmortem cases. Genetically determined channelopathies cannot be detected radiologically. This review underlines the need to establish the role of postmortem radiology in the diagnosis of SCD.

Official Positions for FRAX® clinical regarding international differences from Joint Official Positions Development Conference of the International Society for Clinical Densitometry and International Osteoporosis Foundation on FRAX®.

Osteoporosis is a serious worldwide epidemic. Increased risk of fractures is the hallmark of the disease and is associated with increased morbidity, mortality and economic burden. FRAX® is a web-based tool developed by the Sheffield WHO Collaborating Center team, that integrates clinical risk factors, femoral neck BMD, country specific mortality and fracture data and calculates the 10 year fracture probability in order to help health care professionals identify patients who need treatment. However, only 31 countries have a FRAX® calculator at the time paper was accepted for publication. In the absence of a FRAX® model for a particular country, it has been suggested to use a surrogate country for which the epidemiology of osteoporosis most closely approximates the index country. More specific recommendations for clinicians in these countries are not available. In North America, concerns have also been raised regarding the assumptions used to construct the US ethnic specific FRAX® calculators with respect to the correction factors applied to derive fracture probabilities in Blacks, Asians and Hispanics in comparison to Whites. In addition, questions were raised about calculating fracture risk in other ethnic groups e.g., Native Americans and First Canadians. In order to provide additional guidance to clinicians, a FRAX® International Task Force was formed to address specific questions raised by physicians in countries without FRAX® calculators and seeking to integrate FRAX® into their clinical practice. The main questions that the task force tried to answer were the following: The Task Force members conducted appropriate literature reviews and developed preliminary statements that were discussed and graded by a panel of experts at the ISCD-IOF joint conference. The statements approved by the panel of experts are discussed in the current paper.

Validation of the Mini Nutritional Assessment short-form (MNA-SF): a practical tool for identification of nutritional status.

OBJECTIVE: To validate a revision of the Mini Nutritional Assessment short-form (MNA(R)-SF) against the full MNA, a standard tool for nutritional evaluation. METHODS: A literature search identified studies that used the MNA for nutritional screening in geriatric patients. The contacted authors submitted original datasets that were merged into a single database. Various combinations of the questions on the current MNA-SF were tested using this database through combination analysis and ROC based derivation of classification thresholds. RESULTS: Twenty-seven datasets (n=6257 participants) were initially processed from which twelve were used in the current analysis on a sample of 2032 study participants (mean age 82.3y) with complete information on all MNA items. The original MNA-SF was a combination of six questions from the full MNA. A revised MNA-SF included calf circumference (CC) substituted for BMI performed equally well. A revised three-category scoring classification for this revised MNA-SF, using BMI and/or CC, had good sensitivity compared to the full MNA. CONCLUSION: The newly revised MNA-SF is a valid nutritional screening tool applicable to geriatric health care professionals with the option of using CC when BMI cannot be calculated. This revised MNA-SF increases the applicability of this rapid screening tool in clinical practice through the inclusion of a "malnourished" category.

Expertises croisées dans la dégénérescence maculaire liée à l'âge. Focus sur la physiopathologie, l'angiogenèse, les données pharmacologiques et cliniques [Review and expert opinion in age related macular degeneration. Focus on the pathophysiology, angiogenesis and pharmacological and clinical data].

Age related macular degeneration (AMD) is a pathological aging of the macula, brought about by the interaction of genetic and environmental factors. It induces geographic atrophy of the retina and/or choroidal neovascularization. In the latter, abnormal vessels develop from the choriocapillaris, with the involvement of VEGF (vascular endothelial growth factor). The VEGF family includes several factors, including VEGF-A, B, C, D, F and PlGF (placental growth factor). Their biological properties and their affinities to the VEGFR1, VEGFR2 and VEGFR3 receptors found on endothelial cells differ. Exudative AMD involves mainly VEGF-A and VEGF-R2. Anti-VEGF agents used in ophthalmology (ranibizumab, bevacizumab and aflibercept) are designed to primarily target this pathway. In vitro, all have sufficient affinity to their ligands. Their therapeutic efficacy must therefore be judged based on clinical criteria. In clinical practice, the minimum number of injections required for a satisfactory result appears to be comparable with all the three. The few available studies on therapeutic substitutions of anti-VEGF compounds suggest that some patients may benefit from substituting the anti-VEGF in cases of an unsatisfactory response to an initial molecule. Although local side effects, including increased risk of geographic atrophy, and systemic effects, including vascular accidents, have been suggested, these risks remain low, specially compared to the benefits of the treatment. Differences in safety between anti-VEGF are theoretically possible but unproven.

Estudi sobre la qualitat de vida de les persones amb demència ateses a la Unitat d'avaluació de deteriorament cognitiu de l'Hospital de la Santa Creu

OBJECTIU: determinar la qualitat de vida de les persones amb demència ateses en una unitat avaluadora de deteriorament cognitiu. MÈTODE: estudi descriptiu transversal amb una mostra consecutiva no probabilística, formada per 42 persones amb demència tipus Alzheimer lleu o moderada i els seus cuidadors. La Qualitat de Vida (QV) es va avaluar amb el qüestionari QoL-AD (Quality of Life Alzheimer’s Disease) en les versions per al pacient (QoL-ADp) i per al cuidador (QoL-ADc). RESULTATS: la mitjana de puntuació del QoL-ADp va ser de 35,38 punts (DE = 5,24) i del QoL-Adc, de 30,60 (DE 5,33). La diferència entre aquests resultats és significativa (p&0,001). Els pacients amb simptomatologia depressiva i els seus cuidadors van puntuar significativament més baix el QoL-AD (p&0,001). En les freqüències per ítems del QoL-ADp s’observa que: més del 75% van valorar com a bona/excel·lent les condicions de vida, família, matrimoni/relació estreta, vida social, situació financera i vida en general; el 61% valoraren bona/excel·lent la capacitat per realitzar tasques a casa; prop del 50% pensava que l’estat d’ànim, l’energia, la salut física, la capacitat per fer coses per diversió i la visió de si mateixos era dolenta/regular, i el 85,7% opinava que la seva memòria era dolenta/regular. CONCLUSIONS: els resultats obtinguts en el QoL-AD no difereixen dels obtinguts en altres investigacions. Suggereixen que les intervencions que genera l’avaluació de la QV en la pràctica clínica inclouen aspectes centrats pròpiament en la malaltia i aspectes vinculats amb les relacions socials.

Imagerie cardiaque non invasive: apport spécifique en clinique des nouvelles modalités (II). Evaluation fonctionnelle [Cardiac imaging: specific clinical role of newly developed non invasive techniques. Part II: functional evaluation].

The non-invasive evaluation of myocardial ischemia is a priority in cardiology. The preferred initial non-invasive test is exercise ECG, because of its high accessibility and its low cost. Stress radionuclide myocardial perfusion imaging or stress echocardiography are now routinely performed, and new non-invasive techniques such as perfusion-MRI, dobutamine stress-MRI or 82rubidium perfusion PET have recently gained acceptance in clinical practice. In the same time, an increasing attention has been accorded to the concept of myocardial viability in the decisional processes in case of ischemic heart failure. In this indication, MRI with late enhancement after intravenous injection of gadolinium and 18F-FDG PET showed an excellent diagnostic accuracy. This article will present these new imaging modalities and their accepted indications.

Therapeutic strategies to improve control of hypertension.

Blood pressure is poorly controlled in most European countries and the control rate is even lower in high-risk patients such as patients with chronic kidney disease, diabetic patients or previous coronary heart disease. Several factors have been associated with poor control, some of which involve the characteristic of the patients themselves, such as socioeconomic factors, or unsuitable life-styles, other factors related to hypertension or to associated comorbidity, but there are also factors directly associated with antihypertensive therapy, mainly involving adherence problems, therapeutic inertia and therapeutic strategies unsuited to difficult-to-control hypertensive patients.It is common knowledge that only 30% of hypertensive patients can be controlled using monotherapy; all the rest require a combination of two or more antihypertensive drugs, and this can be a barrier to good adherence and log-term persistence in patients who also often need to use other drugs, such as antidiabetic agents, statins or antiplatelet agents. The fixed combinations of three antihypertensive agents currently available can facilitate long-term control of these patients in clinical practice. If well tolerated, a long-term therapeutic regimen that includes a diuretic, an ACE inhibitor or an angiotensin receptor blocker, and a calcium channel blocker is the recommended optimal triple therapy.

Advantages and disadvantages of combination treatment with antipsychotics ECNP Consensus Meeting, March 2008, Nice.

TERMINOLOGY AND PRINCIPLES OF COMBINING ANTIPSYCHOTICS WITH A SECOND MEDICATION: The term "combination" includes virtually all the ways in which one medication may be added to another. The other commonly used terms are "augmentation" which implies an additive effect from adding a second medicine to that obtained from prescribing a first, an "add on" which implies adding on to existing, possibly effective treatment which, for one reason or another, cannot or should not be stopped. The issues that arise in all potential indications are: a) how long it is reasonable to wait to prove insufficiency of response to monotherapy; b) by what criteria that response should be defined; c) how optimal is the dose of the first monotherapy and, therefore, how confident can one be that its lack of effect is due to a truly inadequate response? Before one considers combination treatment, one or more of the following criteria should be met; a) monotherapy has been only partially effective on core symptoms; b) monotherapy has been effective on some concurrent symptoms but not others, for which a further medicine is believed to be required; c) a particular combination might be indicated de novo in some indications; d) The combination could improve tolerability because two compounds may be employed below their individual dose thresholds for side effects. Regulators have been concerned primarily with a and, in principle at least, c above. In clinical practice, the use of combination treatment reflects the often unsatisfactory outcome of treatment with single agents. ANTIPSYCHOTICS IN MANIA: There is good evidence that most antipsychotics tested show efficacy in acute mania when added to lithium or valproate for patients showing no or a partial response to lithium or valproate alone. Conventional 2-armed trial designs could benefit from a third antipsychotic monotherapy arm. In the long term treatment of bipolar disorder, in patients responding acutely to the addition of quetiapine to lithium or valproate, this combination reduces the subsequent risk of relapse to depression, mania or mixed states compared to monotherapy with lithium or valproate. Comparable data is not available for combination with other antipsychotics. ANTIPSYCHOTICS IN MAJOR DEPRESSION: Some atypical antipsychotics have been shown to induce remission when added to an antidepressant (usually a SSRI or SNRI) in unipolar patients in a major depressive episode unresponsive to the antidepressant monotherapy. Refractoriness is defined as at least 6 weeks without meeting an adequate pre-defined treatment response. Long term data is not yet available to support continuing efficacy. SCHIZOPHRENIA: There is only limited evidence to support the combination of two or more antipsychotics in schizophrenia. Any monotherapy should be given at the maximal tolerated dose and at least two antipsychotics of different action/tolerability and clozapine should be given as a monotherapy before a combination is considered. The addition of a high potency D2/3 antagonist to a low potency antagonist like clozapine or quetiapine is the logical combination to treat positive symptoms, although further evidence from well conducted clinical trials is needed. Other mechanisms of action than D2/3 blockade, and hence other combinations might be more relevant for negative, cognitive or affective symptoms. OBSESSIVE-COMPULSIVE DISORDER: SSRI monotherapy has moderate overall average benefit in OCD and can take as long as 3 months for benefit to be decided. Antipsychotic addition may be considered in OCD with tic disorder and in refractory OCD. For OCD with poor insight (OCD with "psychotic features"), treatment of choice should be medium to high dose of SSRI, and only in refractory cases, augmentation with antipsychotics might be considered. Augmentation with haloperidol and risperidone was found to be effective (symptom reduction of more than 35%) for patients with tics. For refractory OCD, there is data suggesting a specific role for haloperidol and risperidone as well, and some data with regard to potential therapeutic benefit with olanzapine and quetiapine. ANTIPSYCHOTICS AND ADVERSE EFFECTS IN SEVERE MENTAL ILLNESS: Cardio-metabolic risk in patients with severe mental illness and especially when treated with antipsychotic agents are now much better recognized and efforts to ensure improved physical health screening and prevention are becoming established.