This review summarises the findings and applications from neuroimaging studies in dementia with Lewy bodies (DLB), highlighting key differences between DLB and other subtypes of dementia. We also discuss the increasingly important role of imaging biomarkers in differential diagnosis and outline promising areas for future research in DLB. DLB shares common clinical, neuropsychological and pathological features with Parkinson’s disease dementia and other dementia subtypes, such as Alzheimer’s disease. Despite the development of consensus diagnostic criteria, the sensitivity for differential diagnosis of DLB in clinical practice remains low and many DLB patients will be misdiagnosed. The importance of developing accurate imaging markers in dementia is highlighted by the potential for treatments targeting specific molecular abnormalities as well as the responsiveness to cholinesterase inhibitors and marked neuroleptic sensitivity of DLB. We review various brain imaging techniques that have been applied to investigate DLB, including the characteristic nigrostriatal degeneration in DLB using positron emission tomography (PET) and single-photon emission computed tomography (SPECT) tracers. Dopamine transporter loss has proven to reliably differentiate DLB from other dementias and has been incorporated into the revised clinical diagnostic criteria for DLB. To date, this remains the 'gold standard' for diagnostic imaging of DLB. Regional cerebral blood flow, 18 F-fluorodeoxygluclose-PET and SPECT have also identified marked deficits in the occipital regions with relative sparing of the medial temporal lobe when compared to Alzheimer’s disease. In addition, structural, diffusion, and functional magnetic resonance imaging techniques have shown alterations in structure, white matter integrity, and functional activity in DLB. We argue that the multimodal identification of DLB-specific biomarkers has the potential to improve ante-mortem diagnosis and contribute to our understanding of the pathological background of DLB and its progression. Dementia with Lewy bodies (DLB) is the second most common form of neurodegenerative dementia following Alzheimer’s disease (AD), accounting for approximately 15% of cases at autopsy iodoamphetamine and 99mTc-ethyl cysteinate dimer. The majority of studies have consistently highlighted a distinctive pattern of occipital hypoperfusion in DLB when compared to AD, affecting both primary visual cortex and visual association areas, and including the precuneus [123I]iodoamphetamine also demonstrated relatively preserved medial temporal lobe perfusion in DLB in addition to occipital hypoperfusion in DLB when compared with AD methylpiperidin-4-yl acetate and propionate. Klein and colleagues [Autopsy findings of profound cholinergic deficits in DLB (greater than in AD) have led to the application of PET ligands to evaluate the cholinergic system in DLB lleagues  have demlleagues . This palleagues ,25. On tlleagues . The spalleagues .11C-dihydrotetrabenazine have also examined nigrostriatal projections in DLB and AD, and previous studies have demonstrated its potential to differentiate DLB from AD. Compared to controls, significantly decreased binding of 11C-dihydrotetrabenazine was predominantly found in the posterior and anterior putamen and caudate nucleus in DLB, while differences were found between AD and controls [PET studies using controls .Besides nuclear imaging methods, the clinical utility of structural neuroimaging with magnetic resonance imaging (MRI) for differential diagnosis of dementias is also well established. Structural MRI has been widely used to compare regional structural changes in patients with DLB against AD, PDD and their healthy controls. In this section, we summarise the principal findings using MRI to distinguish DLB from other subtypes of dementia Table .Both cross-sectional and longitudinal studies have shown that DLB is associated with less pronounced global atrophy than AD . The mosIn parallel with the MTL findings, patients with DLB generally demonstrate relative preservation of episodic memory capabilities compared to AD while experiencing impairments in attentional and visuospatial tasks. Hence, Ballmaier and colleagues  hypothesConversely, the midbrain appears to be more affected in DLB than in AD, with the substantia innominata showing greater atrophy ,36. The 123FP-CIT SPECT functional changes involving the loss of DAT in DLB [In light of the T in DLB , associaT in DLB  have obsThere have also been attempts to compare GM loss between DLB and PDD. These results have converged to reveal a pattern of more pronounced GM loss in DLB compared to PDD. This is in agreement with the PET and pathological findings of greater amyloid burden in DLB. It is noteworthy that localisations of GM reductions in DLB relative to PDD appear to be variable amongst studies. While Burton and colleagues  did not Recent advances in image processing allow automatic extraction of whole brain cortical thickness information from structural MRI. Cortical thickness has been shown to demonstrate high precision and sensitivity in detecting alterations in morphology resulting from neuropathological changes. Hence, it has been used in increasing numbers of studies as a marker to separate AD and PDD from healthy controls. Recently, a multivariate classification study of cortical thickness demonstrated 82% sensitivity and 85% specificity for differentiating AD from DLB . SpecifiWhite matter hyperintensities (WMHs) are areas of high signal intensity commonly detected using T2-weighted or fluid inverted recovery sequences in periventricular and deep subcortical white matter regions. In older people they are often a marker of cerebral small vessel disease, accumulating over time. WMHs are commonly attributed with loss of myelin and axons, and mild gliosis , but canActive task and resting state functional MRI (fMRI) are the main neuroscience tools to examine cerebral function related to cognitive tasks or during rest through changes in blood oxygen level-dependent signal. There are still relatively few fMRI studies in DLB, but different patterns of functional connectivity between AD and DLB have been reported Table . A recenGiven the high prevalence of visuoperceptual impairments in DLB patients, task-based fMRI studies have investigated the functional integrity of the visual system in DLB. A task-based fMRI study involving visual presentations of colour, face and motion paradigms found greater activation in the superior temporal sulcus in DLB compared to AD during the motor part of the tasks . Anotherin vivo information about white matter microstructural integrity by utilising the anisotropic nature of diffusion in neuronal white matter tracts [Diffusion tensor imaging (DTI) provides r tracts . White mDTI studies in AD have consistently found elevated MD in the hippocampus and decreased FA in the main limbic pathways. Much less is known about DTI changes in DLB. Previous DTI studies on DLB used ROI-based or voxel-based techniques with considerable variability in their findings. Some studies have reported diffusion abnormalities of the corpus callosum and the frontal, parietal, occipital, and, to a lesser extent, temporal white matter when compared to controls, while other studies have found very little change in DTI parameters compared to controls and AD patients ,54. The 1H magnetic resonance spectroscopy (MRS) measures the peak signals from several different metabolites within a single examination period, and it has increasingly been used in differential diagnosis of dementia through the identification of respective spectroscopic profiles of various dementia subtypes  compared to AD patients (1.1%) .This review summarises the current imaging literature of DLB in the context of its differentiation from other causes of dementia, discusses the increasingly important role of imaging biomarkers in differential diagnosis, and outlines promising areas for future research. Collectively, the imaging findings have yielded important insights into the underlying pathophysiology of this condition while showing potential promise in improving clinical differentiation of DLB from other types of dementia.To date, the marked reduction of dopaminergic activity in the basal ganglia is the most characteristic imaging finding of DLB, and has been considered as a significant step in aiding the clinical diagnosis of DLB. Cerebral perfusion studies have also revealed a distinctive pattern of deficits in parietal and occipital regions. At the structural level, another robust observation concerns the relative preservation of MTL in DLB that is consistent with preserved memory functions. Other techniques such as DTI, MRS, and fMRI coupled with novel analytical approaches have also revealed information for differential diagnosis of DLB.While the field has made substantial progress in delineating the imaging characteristics associated with dementia subtypes, the ability to detect structural patterns that enable accurate prediction of diagnosis for specific individuals ultimately determines the clinical value of MRI and the measurements obtained from it. The reliable application of these methods in routine radiological practice may be facilitated by non-expert-dependent, automated methods of analysis.In terms of practical considerations, SPECT and PET are both generally well tolerated investigations with few contra-indications, though both involve radiation exposure, limiting the number that any one subject should have. In most countries, SPECT is more widely available than PET. MRI is now widely accessible but more unsuitable for those with claustrophobia, pacemakers or metallic implants in or around the head. fMRI requires specialist stimulus presentation, which limits its applicability to research studies. In addition, some computerized paradigms in task-based fMRI might not be suitable for elderly dementia patients. Other modalities, particularly DTI and MRS, require extensive post-processing analysis time, also potentially limiting their routine application for clinical use.It is worth mentioning the limitations of the current research. At present, the overwhelming majority of neuroimaging studies in DLB are cross-sectional, relatively small in size, and in participants in established stages of the disease. Therefore, larger prospective longitudinal studies are warranted to confirm the utility of many imaging techniques and monitor disease progression in early disease stages as well as at risk individuals and patients with mild cognitive impairment. Furthermore, studies involving multimodal neuroimaging data and larger cohorts are likely to make novel contributions in evaluating the utility of combined biomarkers in DLB.Lewy Body Dementia, edited by Ian McKeith and James Galvin. Other articles in this series can be found at http://alzres.com/series/LewyBodyDementia.This article is part of a series on AD: Alzheimer’s disease; DAT: Dopamine transporter; DLB: Dementia with Lewy bodies; DTI: Diffusion tensor imaging; FA: Fractional anisotropy; FDG: 18 F-fluorodeoxygluclose; fMRI: Functional magnetic resonance imaging; GM: Gray matter; MD: Mean diffusivity; MRI: Magnetic resonance imaging; MRS: Magnetic resonance spectroscopy; MTL: Medial temporal lobe; PDD: Parkinson’s disease dementia; PET: Positron emission tomography; ROI: Region of interest; SPECT: Single-photon emission computed tomography; VBM: Voxel-based morphometry; WMH: White matter hyperintensity.EM, LS and GBW have no competing interests. JTO’B has acted as a consultant for GE Healthcare and Lilly.


Listeria monocytogenes is a Gram-positive human-pathogen bacterium that served as an experimental model for investigating fundamental processes of adaptive immunity and virulence. Recent novel technologies allowed the identification of several hundred non-coding RNAs (ncRNAs) in the Listeria genome and provided insight into an unexpected complex transcriptional machinery. In this review, we discuss ncRNAs that are encoded on the opposite strand of the target gene and are therefore termed antisense RNAs (asRNAs). We highlight mechanistic and functional concepts of asRNAs in L. monocytogenes and put these in context of asRNAs in other bacteria. Understanding asRNAs will further broaden our knowledge of RNA-mediated gene regulation and may provide targets for diagnostic and antimicrobial development. Listeria monocytogenes is a Gram-positive, facultative foodborne pathogen that causes a severe life-threatening disease (listeriosis) in susceptible humans and animals. Complex regulatory mechanisms allow L. monocytogenes to adapt and survive in a wide range of environmental conditions  and infect a variety of hosts including mammalia and insects  in which the major virulence determinants are organized   elements in various bacterial genomes added a further layer of complexity in our understanding of bacterial gene regulation. In the last decade a myriad of non-coding RNAs (ncRNAs) of different genomic origin, length, function, and mechanisms of gene regulation were identified  the thermosensor forms a complex secondary structure that prevents translation of PrfA by interfering with the Shine-Dalgarno (SD) region . An important example in Trans-encoded small RNA (sRNA) could be considered as the second category. Those transcripts regulate genes located elsewhere on the genome and share only limited complementarity with the target. They often interact with multiple different target transcripts, and therefore function analogous to human microRNA , is located on the opposite DNA strand of their target and therefore share a high degree of complementarity with it. There is growing evidence that asRNAs are present in several Gram-positive and Gram-negative bacterial species and families with a high variability in prevalence and genomic density , distinct media, and organs  and under stress conditions (hypoxia and low temperature). This study demonstrated the influence of regulatory RNAs in response to different microenvironments.The reliable detection of antisense RNA is challenging because of technical difficulties. A major problem using microarrays, for instance, is artificially generated products during cDNA synthesis from RNA -detection they identified 86 additional ncRNAs, including 50 novel asRNAs , differences in origin and mechanisms  short, (ii) long, (iii) 3′UTR, (iv) 5′UTR, and (v) excludon Figure .L. monocytogenes are for example rliE, rli23, rli25, rli29, rli30, and rli35  was 2149 nucleotides in length and spans across neighboring genes partially including lmo2098 . It might represents an effective way to regulate neighboring genes. For example, transcription of 7 Figure .lmo0733 and lmo0743 both encoding putative transcription regulators, interact through 3′UTR . For example, R Figure . The traL. monocytogenes—the excludon  transcription, (ii) transcript stability, or (iii) translation.Although next generation sequencing was instrumental in the identification of several novel asRNAs in Staphylococcus aureus, Streptococcus pyogenes, and Vibrio anguillarum   encodes a transcriptional regulator, which activates transcription of the neighboring pdu and cob genes in the presence of propanediol. Pdu and Cob are essential for the catabolism of 1,2-propanediol catabolism and vitamin B12 biosynthesis. Propanediol is a byproduct of the metabolism of commensal intestinal bacteria. The ability to metabolize propanediol is important for pathogenicity and provides a survival advantage for bacterial during infection. In the process of propanediol catabolism vitamin B12 is required as a cofactor for involved enzymes. The reported asRNA anti1150 (aspocR) overlaps with the pocR gene. Interestingly, aspocR is controlled by a vitamin B12 dependent riboswitch that prematurely terminates transcription of aspocR in presence of vitamin B12 and thereby generates only a small transcript previously known as rli39. Subsequently, PCR-based experiments confirmed that pocR transcription was negatively regulated by aspocR. Additional experiments using ectopically transcribed aspocR showed inhibitory action in trans on pocR expression. These findings emphasize that the utilized mechanism is rather transcription attenuation or inhibition of translation than transcription interference or modulation of transcript stability in this case. Given that pocR is important for vitamin B12 biosynthesis, here antisense regulation seems to be rather a fine-tuning mechanism than an on- off-switch  is most likely involved in regulating central metabolic pathways in L. monocytogenes hundreds of ncRNAs, including even more than hundred asRNAs possibly implicated in the regulation of 102 Listeria genes, were discovered to date.Technological and methodological advances transformed the field of RNA-mediated gene regulation in bacteria and provided insight into an unexpected complexity. In L. monocytogenes show the dependency of some antisense transcripts on transcription factors or even the absence of a metabolite, the importance of experimental conditions is highlighted.This number seems rather low compared to the scope reported from other bacteria and will presumably rise with further studies. Yet, as recent findings in L. monocytogenes has proven to be a valuable model organism for studying asRNA regulation and given rise to novel discoveries like the excludon concept that could then be transferred to other bacteria.Also despite the rather low extent of asRNAs reported to date, L. monocytogenes likely act through different mechanisms and could either function as an on-off switch or fine regulators of a particular network. Thereby, asRNAs might be involved in regulating metabolic processes, virulence and determinants of host inflammatory response. In addition, the impact of asRNA regulation is spread as many targets of antisense regulation then again affect the expression of other genes .It might be speculated that asRNAs in However, our understanding about mechanisms and function remains limited to few individual transcripts. Mechanistic and functional validation of ncRNAs, including asRNAs, will shed further light into the extent of RNA-mediated regulation in bacteria. This understanding may then allow to develop new approaches for therapeutics.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


New NSAIDs may have a growing role in management of women's health. This review summarizes the current use of non-steroidal anti-inflammatory drugs (NSAIDs) in obstetrics, gynecology and infertility. These medications are commonly used in different fields of reproductive medicine, for pain management after operative procedures and to relieve dysmenorrhea. In addition to their analgesic effect, NSAIDs are helpful in the management of menorrhagia by decreasing menstrual blood loss. NSAIDs alleviate pain associated with medical abortion, assist in undertaking natural cycle  At higher concentrations, NSAIDs are also known to reduce production of superoxide radicals, induce apoptosis, inhibit the expression of adhesion molecules, decrease nitric oxide synthase, decrease proinflammatory cytokines , modify lymphocyte activity and alter cellular membrane functions. In obstetrics and gynecology, NSAID’s have long been used to control acute and chronic postoperative pain, menstrual pain, pain related to medical abortions, menorrhagia, intrauterine device, assist in fertility treatment, and administered as tocolytics in preterm labor. The benefits of NSAIDs for specific gynecological diagnoses and the type of methodology used to establish efficacy are summarized in Non-steroidal anti-inflammatory drugs (NSAIDs) are commonly used first line drugs for treatment of different medical situations in the field of obstetrics and gynecology. Their anti-inflammatory properties make them most useful in the management of disorders in which pain is related to the intensity of the inflammatory process. Most currently available traditional NSAIDs act by inhibiting the prostaglandin G/H synthase enzymes, known as the cyclooxygenases. Those enzymes convert arachidonic acid to the unstable intermediates prostaglandin Gversus acetaminophen found that all of the NSAIDs studied  were effective in treating women with dysmenorrhea, and all of the NSAIDs were more effective than acetaminophen [versus placebo for primary dysmenorrhea and found that NSAIDs were significantly more effective for pain relief than placebo , with little evidence of the superiority between any individual NSAIDs [Primary dysmenorrhea is cyclic menstrual pain, usually described as “cramping”, without an identifiable associated pathology. Primary dysmenorrhea more commonly begins within two years after menarche, often accompanied by low backache, nausea and vomiting, headache, or diarrhea. Research has shown that women with dysmenorrhoea have high levels of prostaglandins, hormones known to cause cramping abdominal pain . The mosminophen . A CochrMenorrhagia is defined as prolonged (more than 7 days) or heavy (more than 80 mL) cyclic menstruation. Excessive prostaglandin synthesis has also been implicated in primary menorrhagia [NSAIDs in adequate dosages decrease ovulatory bleeding by approximately 30-40% with less reduction in anovulatory cycles . CochranIntrauterine device (IUD) is the most common method of reversible contraception worldwide . Pain anPerformance of medical abortions with mifepristone, progesterone antagonist, or methotrexate, followed by administration of prostaglandin analogue misoprostol has gained popularity during the last two decades. One of the major side effects of medical abortion is abdominal pain in response to misoprostol. NSAIDs were often avoided to treat this side effect, because of concern over their potential inhibition of prostaglandin-induced uterine contractions. However, recent studies have not shown interference by ibuprofen on the action of methotrexate and misoprostol in medical abortions at up to 56 gestational days . In anotin-vitro fertilization (IVF) treatments. The use of indomethacin to reduce the incidence of spontaneous ovulation in natural IVF cycles have been supported by several studies [The use of NSAIDs can induce reversible infertility in humans, underlining the observation that COX inhibition prevents normal reproductive processes. Young women are receiving NSAIDs for different indications such as inflammatory joint disease, developed luteinized unruptured ovarian follicles ,17. Norm studies ,19. Indo studies . IndometAnother retrospective study evaluated the number of spontaneous ovulations occurring before oocyte retrieval in modified natural cycle IVF  with and without the use of indomethacin . IndometProstaglandins play an important role in endometrial function. Pregnant human myometria express both cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2) . ThroughNSAIDs have proven to be effective agents in attenuating the progression of term labor and preterm labor in human studies . NSAIDs versus COX-2 inhibitor (rofecoxib and nimesulide)  did not demonstrate any differences in maternal or neonatal outcomes. Compared to any other tocolytic, COX inhibitors were again associated with a decrease in the proportion of women delivering at less than 37 weeks' gestation (RR 0.53), and a trend to a reduction in the proportion of women delivering less than 48 hours from initiation of treatment.The development of COX-2 specific inhibitors opened new therapeutic options to obstetricians and gynecologists as these drugs have been hailed as better-tolerated alternatives to conventional NSAIDs. COX-2 inhibitors significantly relax the human myometrium. Nonetheless their use is not very popular today due to association between COX-2 inhibitors and cardiovascular (CV) and thromboembolic events. A review of 13 randomized trials was done to assess the effects of NSAIDs as a tocolytic agent on maternal, fetal and neonatal outcomes . NSAIDs A concern about possible effects of NSAIDs on the fetal ductus arteriosus, antenatal constriction and closure leading to postnatal persistent patent ductus was also addressed in the review mentioned above . In thisAnother meta- analysis of fifteen retrospective studies and six case controlled studies found a statistically significant association between periventriclar leukomalacia and administration of antenatal indomethacin in premature infants . In addiNecrotizing enteroclitis with administration of antenatal indomethacin in premature infants. This adverse effect was attributed to the influence of the indomethacin on cerebral and mesenteric blood flow . In this article we have reviewed the specific implications of NSAIDs in obstetrics, gynecology and infertility. These medications were found to be effective not only in pain management, but also had additional beneficial influence such as decreasing menstrual bleeding, preventing premature ovulation in natural IVF cycles and serving as tocolytics in preterm labor. The newer COX-2 specific inhibitors may have a role in the treatment of primary dysmenorrhea, and may be effective in postoperative pain as well as pain associated with endometriosis.


Atherosclerosis is a chronic inflammatory disease of the vasculature that causes significant morbidity and mortality from myocardial infarction, stroke, and peripheral vascular disease. Landmark clinical trials revealed that mineralocorticoid receptor (MR) antagonists improve outcomes in cardiovascular patients. Conversely, enhanced MR activation by the hormone aldosterone is associated with increased risk of MI, stroke, and cardiovascular death. This review summarizes recent advances in our understanding of the role of aldosterone and the MR in the pathogenesis of vascular inflammation and atherosclerosis as it proceeds from risk factor-induced endothelial dysfunction and inflammation to plaque formation, progression, and ultimately rupture with thrombosis, the cause of acute ischemia. The role of the MR in converting cardiac risk factors into endothelial dysfunction, in enhancing leukocyte adhesion and infiltration into the vasculature, in promoting systemic inflammation and vascular oxidative stress, and in plaque destabilization and thrombosis are discussed. A greater understanding of the mechanisms by which the MR promotes atherosclerosis has substantial potential to identify novel treatment targets to improve cardiovascular health and decrease mortality. Atherosclerosis is a vascular inflammatory condition that is the primary cause of myocardial infarction (MI), stroke, and limb ischemia. Through substantial research efforts, what was originally thought of as simply a buildup of cholesterol in the arteries is now known to be a chronic inflammatory process involving multiple interacting cell types . BriThe mineralocorticoid receptor (MR) is well-known as a regulator of blood pressure in the kidney, where it promotes sodium retention when activated by its hormone ligand aldosterone (Aldo). MR can also be activated by other mechanisms, including glucocorticoids in tissues lacking the cortisol-inactivating enzyme 11beta-hydroxysteroid dehydrogenase-2 and ligand-independent activation by mechanisms still under investigation. MR-independent effects of Aldo have also been reported. These additional mechanisms will not be discussed as they have been reviewed elsewhere [reviewed in Ref. –6] and a−/−) mouse model rapidly develops atherosclerotic plaques that mimic the human pathology. Using this model, Tikellis et al.  contribute to vasodilation by producing nitric oxide via endothelial nitric oxide synthase (eNOS) activity. Nitric oxide production is impaired early in atherogenesis, and the downstream effects of this can be measured olactone . Hyperteies [ROS ]. In anoies . In pri−/− mice, Aldo treatment enhanced aortic leukocyte infiltration -1, and vascular cell adhesion molecule (VCAM)-1, which interact with leukocyte surface proteins to promote leukocyte–endothelial adhesion and facilitate trans-endothelial migration of leukocytes into the vasculature [reviewed in Ref. ]. Aldo altration . In the nary ECs . Recentlnary ECs . ICAM-1 nary ECs . Kirsch nary ECs  later sh−/− mice, eplerenone treatment reduced plaque size, macrophage infiltration, and oxidative stress  development of endothelial dysfunction and systemic inflammation in response to cardiovascular risk factors; 2) EC and SMC production of cytokines and surface expression of leukocyte adhesion molecules, resulting in recruitment, adhesion, and transmigration of leukocytes from the circulation to the vasculature; (3) leukocyte activation, oxidative stress, and MMP expression; (4) development of plaque instability; and (5) vascular thrombosis (Figure  developm EC and SThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


BRAF mutant melanoma have demonstrated impressive efficacy, and combination treatment with these agents may prove to be even more effective. However, these drugs are not curative, mainly because of the relatively rapid development of drug resistance. Furthermore, they can produce undesired, and even unanticipated, side effects, including the emergence of neoplastic lesions harboring activating RAS mutations. Two recent reports reveal new considerations for the optimal approach to targeting this key oncogenic pathway in melanoma, highlighting the importance of combination treatment and therapeutic scheduling.Selective inhibitors of the kinases BRAF and MEK for the treatment of patients with otherwise refractory  The discovery of a highly recurrent mutant allele of the gene encoding the RAF-family kinase BRAF in human melanoma through cancer genome resequencing has set the stage for revolutionary new treatment strategies for this challenging disease . This BRBRAF mutation [BRAF alleles, the current BRAF inhibitors in fact promote ERK signaling through an unintended allosteric activation of dimers of BRAF with the related kinase CRAF, leading to MEK and ERK activation. This appears to be particularly relevant in cells harboring an activated allele of NRAS, which encodes a RAS-family kinase. Indeed, approximately 20% of melanoma patients treated with the BRAF inhibitors vemurafenib or dabrafenib develop keratoacanthomas and/or squamous cell carcinomas of the skin that can harbor activating RAS mutations (mostly HRAS), suggesting that the oncogenic potential of these RAS mutations is realized specifically in the context of drug treatment [Another challenge to the use of these new agents - particularly for the BRAF inhibitors - is the unanticipated, paradoxical activation of ERK signaling in patients' cells that do not harbor the mutation . Thus, ireatment . More rereatment .BRAF mutant melanoma, revealing new therapeutic strategies that can potentially improve outcomes for such patients.Despite the dramatic clinical benefit associated with these new 'personalized' drug treatments for melanoma patients, the issues described above highlight some important remaining challenges, and the many published reports on this topic over the past 3 years point to the intense interest in better understanding this key pathway in melanoma. Here, I highlight two such recent studies ,9 that aBRAF mutant tumor who was diagnosed with chronic myelomonocytic leukemia (CMML) while undergoing treatment with vemurafenib. The leukemic cells in this patient were associated with an activating NRAS G12R mutation, consistent with a potential role for vemurafenib in inducing ERK activation, similar to that seen in the RAS-mutant skin lesions observed in melanoma patients treated with BRAF inhibitors. Thus, vemurafenib treatment may have accelerated the development of a previously unsuspected RAS-mutant leukemia. The patient's melanoma was effectively managed with intermittent vemurafenib treatment, and the authors [NRAS-mutant leukemic cells were still detectable following treatment, the patient had experienced symptomatic improvement over an 85-week treatment period, until the time of the report.The first of these reports, from Paul Chapman and colleagues , describ authors  observedNRAS mutation to demonstrate that BRAF/MEK co-treatment could inhibit proliferation of NRAS mutant leukemic cells, and that, unlike BRAF inhibition alone, which promoted ERK activation, co-treatment resulted in effective suppression of ERK activation. They concluded that the use of intermittent dosing with combined MEK and BRAF inhibitors prevented the paradoxical ERK activation normally seen with sustained BRAF inhibition in cells that do not harbor an activating BRAF mutation. The authors noted that the potential anti-leukemic effects of MEK inhibitor treatment may have been partially mitigated by co-treatment with vemurafenib, which promotes ERK activation in the NRAS mutant leukemic cells. Although this study reflects the experience of a single patient, MEK inhibition appears to be effective in RAS-mutant myeloid malignancies, as evidenced by some early clinical studies [The investigators  also exa studies , and the studies  suggest NRAS and MEK1, amplification or alternative splicing of BRAF itself, and increased expression of the COT1 kinase or various receptor tyrosine kinases [BRAF mutant colorectal cancers to epidermal growth factor receptor (EGFR) activation [BRAF mutant melanoma. Indeed, they found that post-treatment melanoma biopsies revealed evidence of increased EGFR expression.In a second report, from Rene Bernards and colleagues , a novel kinases . Buildintivation , these itivation  exploredSOX10) gene as a regulator of EGFR expression in four different tested melanoma cell lines. Moreover, SOX10 knockdown was sufficient to confer vemurafenib resistance, and this was associated with increased EGFR expression. Additional studies revealed that SOX10-mediated suppression of transforming growth factor β receptor expression was responsible for SOX10's repressive effect on expression of EGFR as well as on other receptor tyrosine kinases that appear to contribute to vemurafenib resistance. The role of SOX10 in regulating EGFR in melanoma was further supported by the discovery of an inverse relationship between SOX10 and EGFR gene expression in a panel of BRAF mutant melanoma cell lines, as well as in a few BRAF mutant melanoma patient samples following treatment-associated resistance [Then, to determine how EGFR expression is regulated in melanocytes, a cell type that does not normally express EGFR, Bernards and colleagues  conductesistance .BRAF mutant melanoma cells was surprisingly detrimental to their proliferative capacity, and was only tolerated in the context of BRAF inhibition. This finding suggests that, in the absence of drug, adaptive mechanisms underlying drug resistance may come at a 'fitness cost', potentially explaining clinical findings of reversible drug resistance in vemurafenib-treated melanoma patients. Thus, in patients whose cancer progresses on treatment and are consequently taken off therapy, residual tumor cells that lack the proliferation-compromising resistance mechanism (EGFR expression in this case) may have a selective growth advantage, leading to their enrichment as a dominant clone within the relapsed tumor. In such a patient, re-treatment with a BRAF inhibitor would be expected to lead to partial tumor regression. This highlights the dynamic nature of tumor cell heterogeneity in the context of treatment sensitivity and resistance, and the potentially important role of treatment scheduling in producing optimal clinical benefit.Interestingly, these investigators  had alsoBRAF mutant melanoma. Although they address distinct aspects of treatment-associated challenges (acquired resistance and oncogenic side effects), they both relate to the less appreciated issue of treatment scheduling. This will undoubtedly become a more prominent topic of study as both laboratory-based and clinical investigators continue to optimize strategies for combination treatments that can manage these tumors more effectively and safely.In summary, these two recent reports ,9 providCMML: Chronic myelomonocytic leukemia; EGFR: Epidermal growth factor receptor; SOX: Sex determining region Y-box.The author is an employee of Genentech, Inc. and a shareholder of Roche Pharmaceuticals.


Heart failure with preserved ejection fraction (HFpEF) is characterised by a high incidence of metabolic comorbidities that share the potential to induce both systemic and coronary microvascular inflammation and oxidative stress. These pathophysiological alterations contribute to increased passive stiffness of the myocardium and to diastolic dysfunction, both hallmarks of HFpEF. Passive myocardial stiffness depends mainly on two components: the extracellular matrix (ECM) and the cardiomyocytes. Quantitative and qualitative changes in collagen metabolism leading to myocardial fibrosis determine the ECM-based stiffness of the myocardium. Different noninvasive diagnostic tools to assess myocardial fibrosis are being developed, some of which have demonstrated to correlate with clinical status and prognosis. Cardiomyocytes mainly alter the passive stiffness through alterations in the giant myofilament titin, which serves as a spring. By modifying its phosphorylation state or by direct oxidative effects, titin determines cardiomyocyte-based passive stiffness. Probably the relative importance of cardiomyocyte-based changes is more important in the beginning of the disease, whereas ECM-based changes become more prominent in the more advanced stages. The present review focuses on these changes in ECM and cardiomyocytes in HFpEF and their potential prognostic and therapeutic implications. In contrast to heart failure with a reduced ejection fraction (HFrEF), patients with heart failure with a preserved ejection fraction (HFpEF) still do not benefit from evidence-based treatment options in the absence of a profound knowledge about its pathophysiology. Therefore, HFpEF therapy is aimed at comorbidities and at reducing signs and symptoms of congestion . Indeed,Although there are many other pathophysiological findings in HFpEF, as recently discussed comprehensively elsewhere , this reThe signs and symptoms of HFpEF are based on increased myocardial stiffness, leading to diastolic left ventricular (LV) dysfunction, which is defined as the inability of the heart to fill to an adequate end-diastolic volume at acceptably low pressures in the absence of endocardial or pericardial disease . AlthougLV diastole can be subdivided into two components: myocardial inactivation and myocardial stiffness. This myocardial inactivation is the consequence of dissociating contractile myofilaments and calcium reuptake into the sarcoplasmic reticulum. Myocardial stiffness can be attributed to the viscoelastic properties of the myocardium  in HFpEF patients compared with controls . These fHowever, quantification of the total collagen content with CVF seems to have less functional implications than the relative amount of the stiffer collagen type I over the more compliant collagen type III, or the amount of cross-linked collagen by lysyl oxidase  risk of complications and sampling error, both inherent to the procurement of an endomyocardial biopsy. Cardiac magnetic resonance (CMR) imaging, for example, allows for quantification of diffuse myocardial fibrosis by measurement of longitudinal relaxation time (T1 mapping). Several T1 mapping methods have been validated with endomyocardial biopsies to assess diffuse myocardial fibrosis. These techniques include post-contrast T1 mapping, calculation of extracellular volume fraction using MOLLI (Modified Look-Locker inversion recovery) sequences, and equilibrium contrast CMR . Indeed,After fibrosis is detected, its clinical or prognostic consequences need to be established. As mentioned above, extracellular volume fraction can be determined with CMR in HFpEF patients as a measure of diffuse myocardial fibrosis . ExtraceThe number of biomarkers that are currently being studied or developed reflecting myocardial fibrosis in HFpEF is growing exponentially and a detailed discussion of this topic is beyond the scope of this review. The most important and promising biomarkers were recently discussed elsewhere . EspeciaAssociations of myocardial CVF with parameters related to diastolic dysfunction such as LV end-diastolic pressure  or the Epassive) of single isolated, membrane-permeabilised cardiomyocytes was shown to be significantly higher during muscle lengthening than in HFrEF, despite increased CVF in HFrEF versus HFpEF [passive. More recently, functional experiments on small myocardial muscle strips allowed the differentiation between cardiomyocyte- and ECM-based Fpassive. In an HFpEF patient population undergoing coronary artery bypass surgery, force measurements were performed on epicardial biopsy samples obtained during surgery [passive account for more than two-thirds of total Fpassive in HFpEF [passive was also shown to be increased and to correlate with left atrial diameter [passive are therefore dependent on sarcomere length, but the actual operating range of sarcomere length in HFpEF patients is unknown. The giant protein titin forms a unique filament network in cardiomyocytes, which engages in both mechanical and signalling functions of the heart and will be discussed in more detail later [In this regard, in HFpEF patients, passive stiffness . However, CVF was not increased in these animals compared with the control group and the increase in Fpassive was attributed to a stiffer cardiomyocyte compartment [Of interest, the functional relevance of myocardial collagen on diastolic dysfunction may depend on the stage of the disease as illustrated in a ZSF1 (Zucker diabetic fatty/Spontaneously hypertensive heart failure F1 hybrid) rat model in which HFpEF is induced by obesity and diabetes mellitus on top of arterial hypertension . Twenty-partment .passive in HFpEF patients [Finally, as previously mentioned, not only the quantity of collagen but also some qualitative aspects such as the degree of collagen cross-linking  or the cpatients . On the patients .passive upon stretch [passive by means of isoform switching, phosphorylation and oxidative modifications [Next to the observed ECM changes, cardiomyocytes also undergo changes in HFpEF. When compared with HFrEF, cardiomyocytes in HFpEF patients are larger and stiffer with higher F stretch . In card stretch . Titin d stretch . Titin iications . In the ications . Howeverications .in vitro administration of PKA decreased Fpassive in isolated cardiomyocytes, suggesting a titin phosphorylation deficit in HFpEF [in vitro administration of PKA or PKG [passivein vitro [Titin can be divided into certain regions, and especially the I-band is known to contain two spring elements: the N2-B unique sequence (N2-Bus) and a region rich in proline, glutamate, valine, and lysine (PEVK) . Many sein HFpEF . Indeed,A or PKG , but alsA or PKG , 46. On in vitro , but in in vitro . It was in vitro . The clipassive and cardiomyocyte hypertrophy [For HFpEF, especially the relative hypophosphorylation of PKG-dependent titin sites, is an interesting finding that offers potential therapeutic targets Fig. . Not onlertrophy . On top ertrophy . Inflammertrophy . Also, dertrophy . The finertrophy . This capassive [Besides indirect effects via decreased NO-sGC-cGMP-PKG signalling, oxidative stress can also have direct effects on titin-based stiffness Fig. . The N2-passive .passive decreased, whereas incubation with reduced glutathione increased Fpassive, suggesting that also the redox state plays a modifying role in titin-based stiffness that needs to be studied in more detail  domains, which make up the majority of elastic titin. Usually parts of these Ig domains are folded into crypts and they can become unfolded and expose cryptic cysteines to disulphide bonding or S-glutathionylation during stretch . A recenail Fig. .in vitro and myocardial inflammation and fibrosis in vivo [Cardiomyocytes and myocardial ECM are not two completely independent compartments and a close interaction can be expected. Both have been shown to contribute to myocardial stiffness  and to b in vivo . Since c in vivo , future  in vivo . The imp in vivo .passive without any change in ECM turnover at 20 weeks of age [This interplay between endothelium, ECM and cardiomyocytes has several implications. Firstly, therapeutic options that target either the ECM (e.g. spironolactone) or the cardiomyocytes (e.g. sildenafil), cannot be expected to ‘cure’ HFpEF. Secondly, changes in cardiomyocyte function due to inflammation or oxidative stress may trigger ECM changes and, vice versa, ECM changes caused by chemical or oxidative endothelial-ECM signalling or by mechanical stress can be expected to disturb normal cardiomyocyte functioning . Thirdlys of age . On the s of age , 28.passive [This hypothesis has important therapeutic implications. If cardiomyoctes predominate the initial phases of HFpEF, it is probably useful to improve cGMP-PKG signalling to reduce cardiomyocyte Fpassive . Howeverpassive . In the passive. However, further mechanistic studies are necessary to elucidate the relative contribution and interplay between the two mechanisms, which may depend on the aetiology and comorbidities as well as on the stage of the disease.HFpEF is a complex disease of which the pathophysiology is gradually becoming unravelled. Both structural and functional alterations in cardiomyocytes and the ECM have been reported, leading to increased myocardial FOn the other hand, it is essential to develop noninvasive biomarkers for the early identification of the alterations in these two components. For instance, noninvasive techniques such as CMR with T1 mapping and circulating ECM biomarkers are promising, although future studies are needed to assess their potential to truly diagnose, stage or predict outcome and response to therapeutic strategies in HFpEF.This work was supported by a grant from the European Commission .


Acute renal failure is defined as a rapid decrease in the glomerular filtration rate, occurring over a period of hours to days and by the inability of the kidney to regulate fluid and electrolyte homeostasis appropriately. AKI is a catastrophic, life-threatening event in critically ill patients. AKI can be divided into pre-renal injury, intrinsic kidney disease (including vascular insults) and obstructive uropathies. The prognosis of AKI is highly dependent on the underlying cause of the injury. Children who have AKI as a component of multisystem failure have a much higher mortality rate than children with intrinsic renal disease. Treatment of AKI is subjected to risk stratification and ongoing damage control measures, such as patients with sepsis, exposure to nephrotoxic agents, ischemia, bloody diarrhea, or volume loss, could be helped by optimizing the fluid administrations, antibiotics possessing least nephrotoxic potential, blood transfusion where hemoglobin is dangerously low, limiting the use of nephrotoxic agents including radio contrast use, while maximize the nutrition. Acute kidney injury remains a complex disorder with an apparent differentiation in pathology between septic and nonseptic forms of the disease. Although more studies are still required, progress in this area has been steady over the last decade with purposeful international collaboration. Furthermore, AKI affects about 7% of all hospitalized patients and around 35% of overall intensive care patients. , occurring over a period of hours to days. This leads to an increase in serum concentration of nitrogenous excretory products and creatinine leading to the inability of the kidney to regulate electrolyte and fluid homeostasis appropriately. The term Our knowledge about AKI in the recent years has increased significantly. However, despite the advances and development of efficient techniques for its treatment, mortality and morbidity rates are still very high, especially in critically ill children . In a laAKI can be divided into pre-renal injury, intrinsic kidney disease and obstructive uropathies. Some causes of Acute kidney injury, such as renal vein thrombosis and cortical necrosis, occur more commonly in neonates, whereas Heamolytic Uremic Syndrome is more common in young children, and Rapidly Progressive Glomerulonephritis (RPGN) generally occurs in older children and adolescents. Exposure to teratogenic drugs during pregnancy is one of the main causes leading to AKI in newborns interfering with embryogenic processes like nephrogenesis. These include drugs such as angiotensin receptor blockers, angiotensin-converting enzyme inhibitors and nonsteroidal anti-inflammatory drugs , hypoxic schemic injury and sepsis were leading causes of AKI . In KolkOther studies have demonstrated that very low birth weight, a patent ductus arteriosus, nonsteroidal anti-inflammatory drugs and maternal receipt of antibiotics and was associated with the development of AKI . MoreoveSome causes of AKI, such as Rapidly Progressive glomerulonephritis (RPGN), may present as AKI but rapidly evolve into chronic renal disease. Several renal diseases, such as the Henoch– Schönlein purpura, hemolytic–uremic syndrome (HUS), and obstructive uropathy with associated renal dysplasia, may present as AKI with significant improvement of renal function to near-normal levels, but the kidney function of the child may slowly deteriorate, leading to renal failure several months or years later. Children with AKI due to hypoxic insults, acute glomerulonephritis, HUS and other causes are more likely to demonstrate oliguria (urine output less than 500 ml/24 h in older children or less than 1 ml/kg per hour in younger children and infants). Children with nephrotoxicity, acute interstitial nephritis, contrast nephropathy, and nephrotoxic renal insults including drugs such as aminoglycoside are likely to have AKI with normal urine output.The prognosis of AKI is highly dependent on the underlying cause of the injury. Children who have AKI as a component of multisystem failure have a much higher mortality rate than children with intrinsic renal disease such as HUS and RPGN Recovery from intrinsic renal disease is also highly dependent on the underlying etiology of the AKI. Children with aminoglycoside nephrotoxicity and ATN typically recover normal renal function; however, despite in the past it being thought that such patients are at low risk for late complications, it has been shown that chronic kidney disease can evolve from AKI .It is accepted that the concentration of serum creatinine is a delayed and insensitive measure of decreased kidney function following AKI. Other markers under biological investigation include changes in cystatin C and plasma neutrophil gelatinase associated lipocalin (NGAL) levels, and urinary changes in interleukin- 18 (IL-18), NGAL, and kidney injury molecule-1 (KIM-1) .The definition and staging of AKI has been recently standardized using the RIFLE classification proposed by the Acute Dialysis Quality Initiative Group . The RIFVolume status management in critically ill patients with acute kidney injury is difficult as it is often accompanied by anuria or oligouria as well as total body fluid overload leading to tissue edema. As such, AKI increases the risk of mortality and often occurs in the setting of sepsis or other forms of shock . While the early goal-directed therapy study showed the benefit of adequate volume repletion in critically ill patients with septic shock , there are detrimental effects associated with salt and water overload which can result from resuscitation with colloids and crystalloids. These include difficult wound healing and worsening of lung function .Treatment of AKI is subjected to risk stratification and ongoing damage control measures, such as patients with sepsis, exposure to nephrotoxic agents, ischemia, bloody diarrhea, or volume loss, could be helped by optimizing the fluid administrations, antibiotics possessing least nephrotoxic potential, blood transfusion where hemoglobin is dangerously low, limiting the use of nephrotoxic agents including radio contrast use, while maximize the nutrition.Good nutritional support has been advocated in AKI, since these patients are generally in a developmental growth phase requiring adequate nutrition than adults. Recent data suggests that protein supplementation in critically ill-children of the order of 2-3 g/kg/day for children aged 0-2 years and 1.5-2.0 g/kg/day for children aged 2-13 years .By using low  dose dopamine in children, AKI outcome remained same . AnotherIn adults SOAP and SAFE studies did not show any superiority of crystalloid over colloid administration or vice-versa. However,In a retrospective study of 116 children by (the Prospective Pediatric CRRT Registry Group (ppCRRT), observed, fluid administration to be independently associated with mortality in children started on CRRT,  necessitConverting oliguric AKI into nonoliguric AKI by use of diuretics did not improve the outcome . There hTo conclude, acute kidney injury remains a complex disorder with an apparent differentiation in pathology between septic and nonseptic forms of the disease. Having common definitions and diagnostic tools aids in the research and, consequently, the management of AKI. With the advent of technology and modern techniques, more research into using these instruments with caution should be carried out . Although more studies are still required, progress in this area has been steady over the last decade, with purposeful international collaboration.


Shh+), which have been shown to mediate many pro-angiogenic effects. In addition to offer a potential source of cardiovascular markers, therapeutical potential of EVs reveal exciting opportunities to deliver specific agents by non-immunogenic means to cardiovascular system.Extracellular vesicles (EVs), including microvesicles (MVs) and exosomes, are small vesicles secreted from a wide variety of cells. Whereas MVs are particles released by the outward budding of the plasma membrane, exosomes are derived from endocytic compartments. Secretion of EVs can be enhanced by specific stimuli, and increased plasma circulating levels of EVs have been correlated with pathophysiological situations. MVs, already present in the blood of healthy individuals, are considerably elevated in several cardiovascular diseases associated with inflammation, suggesting that they can mediate deleterious effects such as endothelial dysfunction or thrombosis. Nonetheless, very recent studies also demonstrate that MVs may act as biological information vectors transferring proteins or genetic material to maintain cell homeostasis, favor cell repair, or even promote angiogenesis. Additionally, exosomes have also been shown to have pro-angiogenic and cardio-protective properties. These beneficial effects, therefore, reveal the potential therapeutical use of EVs in the field of cardiovascular medicine and regenerative therapy. In this review, we will provide an update of cellular processes modulated by EVs of specific interest in the treatment of cardiovascular pathologies. A special focus will be made on the morphogen sonic hedgehog (Shh) associated with EVs (EVs Extracellular vesicles (EVs) comprising exosomes (<100 nm of size), microparticles, and apoptotic bodies (100–1200 nm in size) are physiologically released by almost all cell types and represent endogenous cargos that are able to participate in cell-to-cell communication. The composition of EVs determines the type of message, which they can convey. Indeed, EVs are rich in proteins, mRNAs, and miRNAs as well as lipids, which they can transfer to target cells by ligand/receptor interaction, fusion, and/or internalization. Manipulation of cells generating EVs by pharmacological treatment or transfection allow to produce EVs rich in specific components that can affect selectively the functions of target cells. In this article, we will review the current knowledge of the function and impact of EVs used as therapeutic tools against cardiovascular diseases.In addition to their different size, the criteria that are used to distinguish between the various types of EVs are based on the cellular compartment that they originate from and on their composition . While eExtracellular vesicles have been isolated from the main fluids of the organism and from several solid tissues . However, it is difficult to establish the exact nature of EVs from solid tissues, as the recovered materials can also comprise intracellular vesicles released during the tissue dissociation process . In addiAlternatively, EVs constitute promising therapeutic delivery tools in cardiovascular diseases. Their general capacity to act as bioactive cargoes, particularly through their ability to carry secretory molecules such as cytokines, chemokines, or growth factors as well as exogenous nucleic acids, highlight them as attractive vehicles . In addiAlthough largely identified as deleterious carriers, EVs might also have beneficial effects and deliver protective messages to preserve endothelial function and/or vascular integrity. Indeed, circulating EVs from sepsis patients are able to restore vascular hyporeactivity in vessels treated by lipopolysaccharide . In agre+ stem cells or endothelial progenitor cells (EPCs) have been shown to promote angiogenesis . Indeed, CPC-derived EVs have been shown to stimulate migration of endothelial cells  and to pAltogether, the use of stem cell-derived EVs instead of stem cells engraftment might open new perspectives for cardiac regenerative therapies. This would first resolve safety concerns related to uncontrolled dissemination of transplanted cells or aberrant stem cell differentiation. In addition, immunosuppressive effects reported for certain EVs could favor efficient cardiac tissue regeneration . MoreoveAs described above, EV composition and cargoes transported  vary with cell origin. In addition to use them as delivery vector for specific proteins, their ability to transport and deliver genetic material to non-adjacent cells have highlighted their potential as new delivery vectors in gene-therapy , which revealed higher stability  protein family. Hedgehog signaling is essential in vertebrates for embryo patterning, as it participates in central nervous system development, lateral asymmetry, and anterior–posterior limb axis . In adulin vivo , interleukin-1β, MMP-1 and decrease anti-angiogenic factors such as transforming growth factor β (TGF β)  . EVsShh+t manner , likely t manner .Shh+ have been illustrated following their in vivo intravenous injection in mouse models  release and reversing endothelial dysfunction after ischemia/reperfusion  production  isoforms and catalase] and by inducing cellular Mn-SOD protein expression ] offer a better protection against ventricular dilation and cardiac functional decline than CD34+ cells alone when injected into mice after acute myocardial infarction  further revealed the functional transfer and activation of Shh signaling in recipient cells, which might explain the observed preservation of cardiac function in mice treated with CD34+ (Shh) EVs.Therefore, EVsactivity , 58. Finfarction . In vitrThus, EVs harboring Shh represent an exciting tool for therapy regarding angiogenesis defects related diseases and cardioprotection.Extracellular vesicles might be further used as delivery devices for any molecule of interest. In particular, using naturally occurring secreted vesicles might allow overcoming toxicity or immunogenicity associated with other developed carrying agents like liposomes or nanoparticles. Moreover, this new drug-delivery system might be combined with other existing therapeutic strategies to optimize drug-delivery. Indeed, a recent report demonstrates that adeno-associated viruses (AAVs) are more efficient when encapsulated in EVs (termed vexosomes) than free AAVs for the delivery of cargo into recipient cells. Such a unique entity offers a promising strategy to improve gene delivery . AlternaFurthermore, the use of biological material as carriers might also allow getting molecules in their native conformation, due to natural interaction or folding with membranous environment. Different strategies might be used to specifically target molecules in EVs. For example, addition of different types of membrane anchors can target highly oligomeric cytoplasmic proteins to sites of vesicle secretion and EVs . AnotherDespite interesting perspectives for cardiovascular treatment, EV-based therapies still need more investigations to translate into clinical studies. Lessons from the ongoing exosome-based preliminary clinical trials in the field of cancer would certainly boost such therapeutic perspectives. Particularly, one challenge would be to control the fragile balance between the harmful effects and beneficial ones reported for EVs in the context of cardiovascular diseases. However, cardiovascular field would undoubtedly benefit from optimized developed methods for large-scale production of clinical grade EVs derived from MSC  or dendr+ represent interesting tools to treat diseases related to failed NO pathway and impaired angiogenesis.By inhibiting their deleterious effects, by taking advantage of their regenerative properties or by developing EV-based drug-delivery tools, targeting EVs reveal all the exciting opportunities to treat cardiovascular alterations by non-immunogenic means. In particular, EVs carrying ShhThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The success of systemic adoptive T cell transfer lies in the capacity of the antigen-experienced cytotoxic T lymphocytes to access and persist within the tumour microenvironment. The mimicking of tertiary lymphoid structures that promote a protective immune response against cancer can be achieved using an injectable biocompatible matrix that releases anti-tumour, proliferating T lymphocytes. Prime candidates for this application are liquid, chitosan-based, biocompatible thermogels which rapidly gelify at physiological temperatures. Therefore, we aimed to fine-tune an injectable chitosan-based thermogel formulation that would provide an environment permitting the three-dimensional proliferation and release of T lymphocytes whose activation state can be influenced by the surrounding conditions. We have developed a novel chitosan formulation that is both cytocompatible and injectable, and which has ideal mechanical properties and porosity for T cell encapsulation and growth. With such promising characteristics, we hypothesize that the injection of these T lymphocytes loaded hydrogels into the tumour microenvironment will provide a means for a continuous delivery of T lymphocytes towards the reprogramming of inflammation mechanisms and the reduction of tumour burden.Novel T cell cytocompatible chitosan thermogels were prepared using combinations of gelling agents. Their rheological properties, mechanical strengths, pH, osmolality, and morphology were evaluated. Three formulations were selected for human T cell encapsulation. Biocompatibility was assessed using live/dead staining and fluorescent microscopy. Thermogel- and supernatant-derived T cells and tumour infiltrating lymphocytes were immunophenotyped over time using flow cytometry.in vitro. Use of flow cytometry and microscopy demonstrates which novel thermogel formulation is best suited for cell viability, proliferation, and escape over time, along with the maintenance of T lymphocytes cellular phenotype and an activation status that can be influenced by surrounding conditions. Our injectable three-dimensional T lymphocyte cultures may serve to complement current adoptive cell transfer immunotherapies.We have optimized thermogel formulations supporting the encapsulation of T lymphocytes


The glycosaminoglycan hyaluronan (HA), a component of the extracellular matrix, has been implicated in regulating neural differentiation, survival, proliferation, migration, and cell signaling in the mammalian central nervous system (CNS). HA is found throughout the CNS as a constituent of proteoglycans, especially within perineuronal nets that have been implicated in regulating neuronal activity. HA is also found in the white matter where it is diffusely distributed around astrocytes and oligodendrocytes. Insults to the CNS lead to long-term elevation of HA within damaged tissues, which is linked at least in part to increased transcription of HA synthases. HA accumulation is often accompanied by elevated expression of at least some transmembrane HA receptors including CD44. Hyaluronidases that digest high molecular weight HA into smaller fragments are also elevated following CNS insults and can generate HA digestion products that have unique biological activities. A number of studies, for example, suggest that both the removal of high molecular weight HA and the accumulation of hyaluronidase-generated HA digestion products can impact CNS injuries through mechanisms that include the regulation of progenitor cell differentiation and proliferation. These studies, reviewed here, suggest that targeting HA synthesis, catabolism, and signaling are all potential strategies to promote CNS repair. Up to 20% of the volume of the mammalian central nervous system (CNS) is composed of an extracellular matrix (ECM) that includes proteins, proteoglycans, and glycosaminoglycans . EvolvinThe composition and function of the CNS ECM can differ within distinct areas of the CNS . For exa7 Da. HA can have distinct physiological functions depending on its size, concentration, and localization. These functions include altering tissue hydration and elasticity and creating cell-free spaces that are crucial for cell migration. In addition, HA can induce cell signaling through transmembrane HA receptors.Although numerous studies have implicated proteoglycans in the response to CNS damage and in mediating inflammatory responses and recovery (for excellent recent reviews see –13) therHA is synthesized at the inner face of the plasma membrane and secreted into the extracellular space as a linear undecorated molecule. In mammals, HA synthesis is achieved by a family of transmembrane proteins known as HA synthases (HASs). The mammalian genome codes for three such synthases, HAS1, HAS2, and HAS3. HA catabolism is carried out by hyaluronidases (HYALs) that differ in their cellular localization and pH optima. Mammals possess multiple hyaluronidase genes, including HYAL-1 through HYAL-5, PH20/SPAM1, and, in humans, a pseudogene designated PHYAL-1. The balance between HA synthesis and catabolism has long been recognized to play roles in CNS development e.g., –17). Her. Her17])In the uninjured CNS, HA is diffusely distributed throughout the white matter but is densely packed in gray matter, including perineuronal nets . Damage In addition to the findings in adult patients and in adult models of stroke, HA is also markedly elevated in perinatal hypoxic-ischemic cerebral white matter injuries. Lesions resulting from these insults are the leading cause of cerebral palsy in survivors of premature birth and contribute to life-long neurobehavioral disabilities. HA accumulates in the ECM in human preterm chronic cerebral white matter lesions coincident with extensive reactive astrogliosis . Similar Has3-null mice have reduced hippocampal volumes and develop epileptic seizures [Seizures also alter HA synthesis in the CNS but whether seizure activity directly increases or decreases HA is not entirely clear. Epileptic seizures can influence both the behaviors of existing neurons and adult neurogenesis (the generation of new neurons). One consequence of these alterations is abnormal sprouting by granule cell axons  in the dentate gyrus of the hippocampus, an area of the brain involved in learning and memory. Degradation of HA in organotypic hippocampal slice cultures with a hyaluronidase significantly reduced kainic acid-induced mossy fiber sprouting, a synaptic rearrangement associated with temporal lobe epilepsy . Interesseizures . These dseizures . It is pFor up to 5 months after dorsal rhizotomy proximal to dorsal root ganglia, Mansour and coworkers  found inA number of studies have demonstrated increased HA in CNS lesions associated with neurodegenerative diseases that influence white matter. For example, HA is elevated in demyelinating white matter lesions from mice with experimental autoimmune encephalomyelitis and patients with multiple sclerosis, both of which are autoimmune diseases where myelin-reactive immune cells induce demyelination –33. HA i HAS1 expressed by reactive astrocytes [HA accumulation in the CNS also occurs during the course of normal aging. Moderate increases in HA were observed in 30-month-old rat brain tissue compared to tissues from younger animals . HA levetrocytes . HA is strocytes , 38 and trocytes . In agretrocytes  observedtrocytes , who obstrocytes . There wAll together, the findings described above demonstrate that HA accumulates, likely due to increased HAS transcription, in a wide variety of insults to the CNS. HA accumulation is usually associated with astrogliosis, and indeed reactive astrocytes demonstrate elevated HAS. This likely accounts for the chronically high levels of HA within glial scars. As indicated by studies in models of spinal cord injury and seizures, HA may initially become degraded due to the possible induction of specific hyaluronidases or other factors in the early injury environment but, at least in chronic lesions, quickly accumulates to higher than normal levels that are sustained for long periods after the initial insult. At least in some insults, this elevated HA can be detected in CSF. Future studies focused on examining when HA concentrations become detectably elevated in the CSF following different insults to the CNS will demonstrate the potential of HA as a diagnostic biomarker for CNS injury.6 Da) to rodent OPCs in vitro, to white matter slice cultures, or to lysolecithin-induced demyelinating corpus callosum lesions blocks OPC differentiation and remyelination [What are the consequences of prolonged HA accumulation in CNS lesions? In addition to its roles in nervous system development and homeostasis, HA can have an impact on nervous system repair by altering the proliferation, migration, and differentiation of progenitor cell populations in the CNS. In demyelinating lesions, including those of multiple sclerosis patients, some recovery of function is associated with the recruitment of oligodendrocyte progenitor cells (OPCs) that differentiate into oligodendrocytes (OLs) that remyelinate spared axons . Howeverlination , 32, 48.lination  and inhilination . Thus, a in vitro and following transplantation into inflammatory demyelinating lesions. Although the authors reported elevated HA in these lesions, it remains to be determined if HA is directly required for the migratory behavior of these cells. Nonetheless, these data suggest that while HA can block OPC maturation when found in excess in demyelinating lesions, HA might also be required to facilitate OPC recruitment to lesions in a CD44-dependent manner.HA may also play a role in OPC recruitment to demyelinating lesions. Following demyelinating insults, OPCs migrate towards damaged axons before differentiating into myelinating OLs. Using an OPC line, Piao and coworkers  found th in vitro in conditions of stress and in vivo following transplantation into the infarct cavity in a stroke model [While HA is mostly observed at low levels in the uninjured adult brain, it remains at high levels in the adult subventricular zone, the subgranular zone of the dentate gyrus of the hippocampus, and the rostral migratory stream, areas where neural stem/progenitor cells (NSPCs) reside throughout life , 51. HA ke model . DegradaCollectively, these studies suggest that HA within NSPC niches plays a critical role in regulating NSPC proliferation and differentiation. This function of HA is recapitulated in demyelinating and possibly other lesions, where OPCs encounter a niche-like environment rich in HA that can influence OPC migration, astrocyte proliferation, and glial cell differentiation. Presumably, the levels and distribution of HA within NSPC niches must be tightly controlled to regulate NSPC differentiation. The challenge will be to identify a means of controlling HA synthesis or catabolism within NSPC niches or areas of damaged CNS tissues in a way that promotes nervous system repair. The use of HA biomaterials, as described above, has the potential to regulate HA functions in both stem cell niches and CNS lesions, offering a potential approach to stem/progenitor cell-based therapies (reviewed in ).As discussed above, there are several mammalian hyaluronidases that catabolize HA in tissues. While digestion of high molecular weight HA can have its own biological consequences, the accumulation of HA digestion products generated by hyaluronidases can also have distinct, size-dependent activities.The first indication that hyaluroidases play a role in the CNS came from the observation that there is a high level of hyaluronidase activity in the developing brain and spinal cord that declines during the early perinatal period . Hyaluro in vitro and in demyelinating mouse and human lesions by immunohistochemistry and, in mice, by RT-PCR [ in vitro with a broad-spectrum hyaluronidase inhibitor (ascorbate 6-hexadecanoate) promoted OPC maturation [Hyal1 mRNA was also elevated in rats following a craniotomy or a controlled cortical impact injury . Other hy RT-PCR . This amy RT-PCR . Treatmeturation , 55 whilFurther evidence that PH20 is elevated in the CNS following injury comes from a sheep model of prenatal white matter injury described above, where elevated ovine PH20 expression was confirmed through multiple approaches . A singlα-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor trafficking into and out of the synapse [It is unclear if endogenous hyaluronidases influence neuronal function. However, a number of studies have suggested that HA within perineuronal nets can influence neuron activity that can be altered by hyaluronidase treatment. For example, the removal of perineuronal nets using hyaluronidase in hippocampal slices from young rats reduced the width of the synaptic cleft and increased the amplitude of excitatory postsynaptic potentials in CA1 axodendritic connections . From th synapse  suggesti synapse . How thiWhile the digestion of HA by hyaluronidases may relieve signaling induced by high molecular weight HA, HA digestion products generated by hyaluronidases may also have their own distinct biological activities in the CNS. For example, digestion products of HA generated by bovine testicular hyaluronidase (whose activity is mostly PH20), but not those of another hyaluronidase directly blocked OPC differentiation and remyelination . This fiAdditional evidence supporting the possibility that hyaluronidase-generated HA products could influence nervous system repair comes from studies utilizing HA oligosaccharides. For example, an HA tetrasaccharide was reported to improve functional recovery following spinal cord injury, possibly by inducing the expression of brain-derived neurotrophic factor (BDNF) and vascular endothelial growth factor (VEGF) in astrocytes within the injury environment . HA tetrThese studies indicate that high molecular weight and digested forms of HA each have distinct functions following nervous system injury and during recovery. Certain sizes of HA products that accumulate in the damaged CNS may induce cellular signals that prevent differentiation and inhibit repair, while others may induce distinct signals that support these activities. Identifying how these products are generated, the precise sizes of HA fragments that influence cell signaling in neural progenitor populations, and the receptors and downstream effectors that regulate these signals will likely lead to novel strategies to promote nervous system repair.There are a number of receptors that signal in response to HA . Among tHA binds to CD44 via an extracellular domain that shares homology with cartilage link protein. Multiple forms of CD44 are generated as a result of extensive RNA splicing and posttranslational modifications , both ofCD44 is expressed throughout both the central and peripheral nervous systems predominantly by glial cells, although some neuronal populations are at least transiently CD44-positive . In para shiverer mice that lack CNS myelin. Furthermore, chronic elevation of CD44 on OPCs leads to pericellular HA accumulation [Demyelinating lesions demonstrate highly elevated CD44 expression on reactive astrocytes and OPCs in conjunction with elevated HA , 63, 70.mulation . Thus, cmulation  suggestiCD44 is also elevated in the hippocampus following seizures , 72. AltLike CD44, RHAMM also exists in multiple isoforms and is expressed by at least subsets of neurons and glial cells, especially oligodendrocytes . Unlike Like CD44, RHAMM is observed in astrocytes in peri-infarct areas following ischemia  and has A number of studies have suggested that HA is a ligand for Toll-Like Receptors (TLRs). Both TLR2 and TLR4 have been reported to bind HA  and theiAltogether, studies to date support the notion that multiple HA receptors are involved in nervous system responses to injury, regulating cell migration, axon outgrowth, and differentiation. What remains to be determined is how these receptors interact to influence each of these cellular behaviors, how receptor activation influences specific intracellular signaling cascades, and which receptors respond to high molecular weight HA versus HA digestion products generated by hyaluronidases. HAS genes by astrocytes and other reactive glia. HA accumulation is accompanied by transcriptional upregulation of CD44 and possibly other transmembrane HA receptors leading to both enhanced HA signaling and anchoring of cell surface HA, contributing to further HA accumulation in the injury microenvironment. Such anchoring of HA to the cell surface by CD44 has been described in activated brain endothelial cells [ HAS genes and HA receptors, the expression of one or more hyaluronidases is also induced, leading to the digestion and clearance of the accumulated HA in the lesions. The balance between HA accumulation and HA degradation by hyaluronidases can influence a number of cell behaviors, such as proliferation and differentiation in NSPC niches  and to


Despite advances in the treatment of patients with early and metastatic breast cancer, mortality remains high due to intrinsic or acquired resistance to therapy. Increased understanding of the genomic landscape through massively parallel sequencing has revealed somatic mutations common to specific subtypes of breast cancer, provided new prognostic and predictive markers, and highlighted potential therapeutic targets. Evaluating new targets using established cell lines is limited by the inexact correlation between responsiveness observed in cell lines versus that elicited in the patient. Patient-derived xenografts (PDXs) generated from fresh tumor specimens recapitulate the diversity of breast cancer and reflect histopathology, tumor behavior, and the metastatic properties of the original tumor. The high degree of genomic preservation evident across primary tumors and their matching PDXs over serial passaging validate them as important preclinical tools. Indeed, there is accumulating evidence that PDXs can recapitulate treatment responses of the parental tumor. The finding that tumor engraftment is an independent and poor prognostic indicator of patient outcome represents the first step towards personalized medicine. Here we review the utility of breast cancer PDX models to study the clonal evolution of tumors and to evaluate novel therapies and drug resistance. Breast cancer is not a single disease but a diverse set of diseases characterized by heterogeneity in histology, genomic aberrations, and protein expression that influence treatment response and patient outcome. Importantly, this heterogeneity cannot be precisely defined through the traditional parameters of histopathology, tumor size, grade, nodal involvement, and biomarker expression that are currently used to guide treatment decisions. Although survival rates following diagnosis have improved in recent years, patients with recurrent disease are almost invariably treatment resistant, highlighting the need for identifying new therapeutic strategies.The heterogeneity of breast cancer is a significant stumbling block for the application of personalized medicine approaches. For this strategy to be successful, a complete set of clinically relevant and validated biomarkers is required, along with the development of companion diagnostic tests to evaluate treatment responses . To dateOver recent years, patient-derived xenograft (PDX) models have emerged as important tools for translational research, with the promise of enabling a more personalized approach to patient care. In this review, we discuss the importance of these models for assessing novel therapies and understanding molecular and cellular mechanisms that contribute to tumor evolution.The traditional histopathological markers used in the clinic do not always reflect the intrinsic subtype . For exacis-acting alterations. In other large cohorts, recurrent mutations in genes not previously associated with breast cancer  and novel translocations were identified [All cancers carry somatic mutations due to imprecise repair of DNA damage. Mutations may be single base-pair substitutions or structural variants including translocations, large deletions, and intra-chromosomal inversions (reviewed in ). Only aentified ,17.With respect to clonal heterogeneity in breast cancer, genomic analysis of triple-negative breast cancers has indicated that some are characterized by a few dominant clones, whereas others may comprise more than 15 . Whole gin vitro model for drug discovery. Whilst they serve as useful tools, there are significant limitations, because continual passage of these cell lines is accompanied by extensive clonal selection and consequent loss of heterogeneity [in vitro and in vivo data obtained with cell lines [The use of preclinical models to test hypotheses is central to cancer research. Unfortunately, long-established human cell lines, and many transgenic mouse models, often fail to recapitulate the key aspects of human malignancies and thus do not adequately predict drug effects in the clinic. Cancer cell lines have been used since the 1970s as an ogeneity . Moreoveogeneity . Their lll lines , in partin vitro culture. Multiple groups have now established cohorts of breast cancer PDXs [In contrast to long-established cell lines, PDXs are propagated through successive generations in murine hosts, thus circumventing the high selection pressure imposed by cer PDXs -32. ImpoThe methodology underpinning the generation of human breast cancer xenograft models has been comprehensively described ,34. BrieHistorically, breast tumors were often implanted subcutaneously, but orthotopic implantation into the inguinal mammary fat pad is optimal because this more faithfully recapitulates the breast tumor stromal microenvironment ,31,38. TTable Given the importance of the tumor microenvironment, different groups have attempted to humanize the mouse mammary fat pad. Based on increased engraftment of human mammary epithelial cells in nonobese diabetic (NOD)/ severe combined immunodeficiency (SCID) mice by co-introduction of an immortalized human fibroblast cell line , Zhang a−/− (NSG) mice are more immunosuppressed and demonstrate higher engraftment rates. In breast cancer, however, NSG and SCID/Beige mice appear to have similar take rates [Multiple mouse strains are available, with slight differences in immunosuppression conferring both advantages and disadvantages. For melanoma, the level of immunosuppression in recipient mice is a key determinant of xenograft establishment . NOD/SCIke rates , while tAll clinically defined subtypes of breast cancer have been established as PDX models ,28,32,37Early passage breast PDX models have been shown to retain the principal molecular characteristics of the corresponding patient tumor at both the genomic and gene expression levels ,32. ThesGenome-wide analyses have revealed that structural and copy number variations are faithfully retained in a large bank of PDXs and their originating tumors ,49. VariBecause multiple passages may be required to expand the model for therapeutic use, Zhang and colleagues performed gene expression analysis on every fifth generation, and demonstrated that generations clustered together across at least five generations and up to 15 generations . Moreovede novo mutations and deletions not present within the primary tumor. While the xenograft retained the primary tumor mutations, it most closely mimicked the mutation spectrum of the metastasis; that is, the PDX was genetically closer to the metastatic lesion than the primary tumor [Tumors are recognized to comprise multiple subclones, based on the analysis of samples from an individual patient at different times , and hypry tumor . In anotry tumor .PDX models have been demonstrated to undergo metastasis in the mouse but it is unclear whether this accurately recapitulates metastases observed in patients. In the case of three primary tumors capable of metastasis in mice, although these correlated with the clinical metastases, additional sites were apparent . For exaOne of the major issues in drug development is the absence of correlation between preclinical data and trial results leading to failure of multiple phase III studies, in part due to the poor predictive value of cell line studies ,20. PDX Basal-like tumors represent ~20% of patients with breast cancer, are typically triple-negative, and carry a worse prognosis due to high rates of local and systemic relapse. The absence of ER and HER2 expression precludes the use of endocrine therapy or anti-HER2 treatment. Systemic treatment is limited to cytotoxic chemotherapy, thus highlighting the need for biomarker development and new treatment strategies . Human-iImpairment of apoptosis is a hallmark of cancer , thus geMost ER-positive PDX models remain dependent on estrogen for tumor growth ,34,37, aVery few therapeutic studies have thus far been carried out with this type of PDX model. However, Marangoni and colleagues tested two HER2/ERBB2-amplified PDX lines, one of which responded to trastuzumab while the other did not . Synergiin vivo endocrine resistance have been recently generated and identified significant deregulation of ER-mediated gene transcription, suggesting that endocrine resistance is both tumor specific and treatment specific [de novo endocrine-therapy resistance in ER-positive breast cancer and discovered point mutations or rearrangements affecting the ESR1 ligand-binding domain, which were unique to the different tumors and were not previously identified by cell line studies [ESR1 variants may be selected in a subset of endocrine-resistant luminal tumors and have immense potential to inform the design of effective therapies to target mutant forms of ESR1. Other mechanisms of endocrine resistance, largely based on cell lines and cell line xenografts, include the utilization of alternate signaling pathways , increased AP1 expression, deregulation of ER-associated co-regulators, or deregulation of the cell cycle and apoptosis [Although the majority of ER-positive breast cancer diagnoses respond to anti-estrogens such as tamoxifen and aromatase inhibitors, their efficacy is limited by intrinsic and acquired resistance. Luminal PDX models with acquired specific . Tamoxif studies . These fpoptosis .Indeed, aberrant PI3K signaling has been implicated as a potential mechanism of endocrine resistance, with upregulation of AKT signaling and acquired sensitivity to mTOR inhibition. The mTOR inhibitor everolimus has been beneficial in the treatment of advanced breast cancers resistant to endocrine therapies . In tamoPDX models are showing increasing utility for the identification of mechanisms of resistance and potential targetable pathways. For example, reverse phase protein assay analysis in combination with an integrated bioinformatic model established upregulation of the PI3K/Akt/mTOR signaling pathway as a candidate driver of resistance to anti-angiogenic agents . MoreoveThe most relevant data on the power of PDXs will ultimately derive from direct comparison of a patient’s clinical response with that of the corresponding PDX to the same drug Figure . Althoug+CD24−, ALDH1+), but these do not universally mark breast cancer stem cells, with variation evident between individual tumors. In cases where tumor samples are too small for cell fractionation, early passage xenografts may be a useful tool for evaluating the existence of cancer stem cells and determining their intrinsic resistance to therapy [+ breast tumor cells enriched for cancer stem cells spontaneously metastasize to the lungs and lymph nodes, thus suggesting a role for cancer stem cells in primary tumor growth as well as metastatic spread [The cancer stem cell hypothesis provides a cellular mechanism to account for phenotypic and functional intra-tumoral heterogeneity. Furthermore, it provides an explanation for resistance to radiation and chemotherapy, as well as eventual tumor relapse. A number of potential markers of breast cancer stem cells have been identified (CD44 therapy . It has  therapy . PDX modc spread . FurtherDespite the ability of PDX models to recapitulate the primary tumor, there are several limitations of which researchers need to be cognizant. To date, the majority of PDX cohorts are biased towards more aggressive tumors, characterized by low ER-positivity, high Ki67, and node positivity. Indeed, the rate of engraftment may serve as an independent predictor of poor patient outcome . SamplesSeveral parameters require optimization to more closely mimic the genesis of human tumors. Given that the stromal compartment and immune system play an important role in breast cancer progression , the losThe wealth of data derived from massive parallel sequencing has now primed the genomic phase of clinical trials with the potential integration of predictive and prognostic biomarkers Figure . Throughin vivo to be studied and the eventual identification of therapies to target metastases.Given the current clinical challenge of eradicating metastatic disease, there is a pressing need for models that better predict metastatic behavior. Xenograft lines with metastatic capacity to the lung, node, and pleura, but not to the liver or bone as yet, have been described ,32. PDX