Both the hepatitis B virus (HBV) and cancer stem cells (CSCs) have been independently implicated in the pathogenesis of hepatocellular carcinoma (HCC). To date, there have been no reports describing HBV infection within CSCs. In this report we describe HBV core (HBcAg) and HBx protein expression within CSCs associated with human HCC. HBV markers were also identified in nonmalignant stem cells present in adjacent nontumor tissue. These findings provide new insights into the pathogenesis of HBV-induced HCC and are potentially relevant to the treatment of both HCC and chronic HBV.  Hepatocellular carcinoma (HCC) is the fifth most common cancer and third most common cause of cancer-related death in the world today . HCC is Although HBV is considered an oncogenic virus, the mechanisms whereby it contributes to the development and/or growth of HCC remain to be fully elucidated . PreviouCancer stem cells (CSCs) represent a small subpopulation of malignant embryonic/progenitor cells that contribute to the development, growth, and metastasis of HCC . CSCs caDespite well documented independent associations between HBV and HCC and CSCs and HCC and reports of increased HBx expression in HCCs with a high prevalence of CSCs, there have been no reports describing HBV markers within CSCs associated with human HCC .A 74-year-old Asian male with chronic HBV underwent surgical resection of a 9 cm HCC. The patient had not received antiviral therapy or chemotherapy or undergone prior ablative procedures. Processed sections of the specimen were deparaffinized, washed with buffer, and incubated overnight with commercially available primary monoclonal murine or rabbit antibodies to HBcAg, HBx, and the stem cell markers: EpCAM, Oct-4, Nanog, and Notch. Sections were then washed and incubated with donkey anti-mouse or anti-rabbit secondary antibodies prior to counterstaining the nuclei with DAPI.As shown in The mechanisms whereby HBV contributes to the development of HCC remain to be fully elucidated. During the course of infection, viral genome exists in both episomal and integrated form, resulting in the transcription of viral proteins including HBcAg and HBx . While HA number of studies have implicated CSCs in the pathogenesis and course of HCC , 9. ThusIn two previous studies addressing a possible association between HBV and CSCs, Arzumanyan et al. described transfection of “mature” HepG2 cells with HBx and the subsequent appearance of “stemness” as reflected by EpCAM, Oct-04, Nanog, HIf-4, and B-catenin expression . The autThe present report is the first to describe a direct association between HBV and CSCs as well as HBV and nonmalignant liver stem cells in humans. This association raises interesting questions that warrant further research. For example, does HBV infect CSCs directly or does the virus infect nonmalignant stem cells resulting in their malignant transformation? Alternatively, does HBV infect mature hepatocytes causing them to dedifferentiate into a stem cell lineage as proposed by Arzumanyan et al. and Holczbauer et al. , 11? If In conclusion, to our knowledge, this is the first report that describes HBV marker expression within CSCs associated with human HCC and nonmalignant stem cells associated with adjacent non-tumor tissue. In addition to providing important insights into the pathogenesis of HCC, the natural history of HBV, and new target cell populations for HBV treatment, these findings should encourage the search for other oncogenic viruses in other CSC associated cancers.


In the critically ill patient, early and effective hemodynamic management including fluid therapy and administration of vasoactive drugs to maintain vital organ perfusion and oxygen delivery is mandatory. Understanding the different approaches in the management of critically ill patients during the resuscitation and further management is essential to initiate adequate context- and time-specific interventions. Treatment of hemodynamic variables to achieve a balance between organ oxygen delivery and consumption is the cornerstone. In general, cardiac output is considered a major determinant of oxygen supply and thus its monitoring is regarded helpful. However, indicators of oxygen requirements are equally necessary to assess adequacy of oxygen supply. Currently, more and more less or even totally non-invasive monitoring systems have been developed and clinically introduced, but require validation in this particular patient population. Cardiac output monitors and surrogates of organ oxygenation only enable to adequately guide management, as patient’s outcome is determined by acquisition and interpretation of accurate data, and finally suitable management decisions. This mini-review presents the currently available techniques in the field of hemodynamic monitoring in critically ill patients and briefly summarizes their advantages and limitations. Since its introduction in the early 1970s the pulmonary artery catheter (PAC) was widely used and regarded as the “holy grail” in hemodynamic monitoring  reportedIn general, no specific monitoring device has been found to reduce mortality in critically ill patients , which iDuring the time, when the PAC came under intense debate, more and more alternatives and especially less-invasive techniques were developed and evaluated in experimental and clinical studies. Besides transpulmonary indicator , dilution techniques, less-invasive technologies like esophageal Doppler and uncalibrated pulse contour analysis  were introduced. Furthermore, as a logical step, several manufacturers have promoted and introduced totally non-invasive devices for advanced hemodynamic monitoring: e.g., thoracic bioimpedance, bioreactance, and ultrasound techniques. Although described first in the early 1990s, non-invasive systems for assessment of radial artery pressure curves based on the volume clamp method according to the Peñaz principle and applanation tonometry are currently intensively studied in critically ill patients.per se is only of limited value as its interpretation (Frank-Starling relation) requires simultaneous information on cardiac preload and/or fluid responsiveness (as an a priori estimate of the impact of fluid loading on cardiac output). However, some but not all of the techniques listed below simultaneously provide information on cardiac output and cardiac preload or fluid responsiveness, respectively. Since not intended here and thus without claiming completeness, Table In general, cardiac output as major and flow determinant variable of systemic oxygen delivery is often considered helpful in the management of critically ill patients. However, measurement of cardiac output status quo should be given. Today, as reported in a current survey from Switzerland, 56% of intensivists use transpulmonary thermodilution (1% lithium dilution), 31% a PAC, 9% echocardiography, and 3% an uncalibrated pulse contour system, respectively  as a measure of oxygen transport has been proposed since the trial by Rivers et al. in 2001  arterial cannulation and without need for a reference method, data are conflicting and reliability of measuring absolute cardiac output correctly is still a matter of clinical studies –30. HoweOne clinically increasingly attractive technique is the passive leg raising (PLR) maneuver while using a continuous monitoring system which allows real-time following of its effects . PLR is One step further is the use of totally non-invasive techniques for continuous measurement of blood pressure and cardiac output in critically ill patients. Finger-cuffed systems –37 whichAs a classical Doppler principle-based technology, assessment of cardiac output by esophageal flow probe requires the velocity-time-integral and the vessel cross-sectional area . Thus byin situ for up to 72 h has recently been introduced into the market. Although this method is gaining popularity as a continuous mode of echocardiographic assessment in critical care, it is clearly operator-dependent and needs further evaluation in anesthesia and intensive care , disposable transesophageal echocardiography probe which may be kept ive care .A complete other approach are non-invasive Fick-principle-based techniques. For instance, transcutaneous assessment of soluble indicators  has been suggested for measurement of cardiac output. In this technique,  venous indicator injection and downstream concentration measurement  allow recording concentration-time curves after adequate mathematical analysis cardiac output. However, the indicator concentration is obtained indirectly from optical densities and the relation between both has primarily been derived in healthy volunteers. Since absolute concentrations are mandatory for correct assessment of cardiac output and since physical properties of the skin and other tissues may significantly differ from healthy humans, data in critically ill patients were not convincing . AnotherBoth, bioreactance and bioimpedance are non-invasive methods for estimation of cardiac output. Known for many decades, thoracic bioimpedance allows continuous estimation of cardiac output based on the specific electric resistance of the thorax which changes during the cardiac cycle. A small current is transmitted between electrodes placed on the lower thorax and neck, and the voltage is measured. The electric impedance is derived from the voltage, and is inversely proportional to aortic blood flow. The accuracy of thoracic bioimpedance is affected by alterations in extravascular lung water, respiratory cycle-dependent alterations in venous blood flow, increased aortic stiffness, interference from the mechanical ventilator, and movement of the electrodes. Data on the validity of thoracic bioimpedance in relation to thermodilution estimation of cardiac output have been conflicting and refinements in this technique are required . A similWhile the bioimpedance measures changes in amplitude, bioreactance relies on changes in signal frequency. Thoracic biorectance is more refined than thoracic bioimpedance as the ability to accurately measure phase shifts is less dependent on external noise and electrical interference. First data comparing thoracic biorectance to thermodilution techniques suggests that the methods are comparable , 54. HowLast but not least, pulse oximetry as a fully non-invasive technique may be a helpful tool in assessing hemodynamics . In prinper se is not outcome relevant but the underlying treatment algorithms are more important. Up to date, it seems indicated that with higher patients’ morbidity application of non-invasive techniques should be held restrictive (In conclusion, independently the methodological considerations and requirements to guarantee adequate accuracy, calibrated systems are still most reliable for the measurement of cardiac output in critically ill patients. However, the monitoring system trictive . While wSamir G. Sakka is a member of the Medical Advisory Board of PULSION Medical Systems SE, Feldkirchen, Germany.


Multiple sclerosis (MS) is a chronic demyelinating inflammatory disease of the central nervous system (CNS) affecting prevalently young adults , 2. The Innate immunity represents the immediate non-specific defense against infections and dangerous agents acting through its essential arms such as inflammation and blood coagulation –10. The Many evidences reported below support a significant presence of local and systemic thrombotic events in MS confirming the global over-stimulation of innate immunity for both its inflammatory and coagulant components.Several extensive studies have shown the association between venous thromboembolism and MS from its early stages. In a large population-based cohort study evaluating data from a 30-year Danish National Registry, MS was associated with increased long-term risk of venous thromboembolism . AnotherCases of cerebral thrombosis have been also reported in MS, mainly attributed to the effect of lumbar puncture and methylprednisolone therapy, however, in some cases no pre-disposing condition was found –19.Moreover, a collaborative genome-wide association study screen in MS patients, not only implicated a multitude of genes coding for cytokine pathway, co-stimulatory, and signal transduction molecules of immunological relevance, but also for a vascular cell adhesion molecule (VCAM1) mediating leukocyte–endothelial cell adhesion and signal transduction . InteresThere are several studies confirming platelet –24 and cInterestingly, in the review of D’Haeseleer and colleagues, all the vascular risk factors reported to be associated with MS such as reduced physical activity, smoking, endothelial dysfunction, platelet activation, thrombophilia, and hyper-homocysteine are essentially pro-thrombotic conditions. Conversely, other vascular risk factors such as atrial fibrillation, flutter, arterial hypertension, diabetes mellitus, dyslipidemia, and obesity have not been associated with MS .Decreased cerebral blood volume and flow, as well as its prolonged brain mean transit time, were demonstrated in all forms of MS and even in clinically isolated syndrome, both in white and gray matter, by SPECT, PET, and dynamic susceptibility contrast-enhanced MRI . In my oAccording to D’Haeseleer et al., I think that MS could unlikely be caused by the obstructed extracranial venous drainage. The CCSVI theory explains the perivenular pattern of MS lesions by extravasation and degradation of erythrocytes due to blocked venous drainage with consequent iron deposition causing the inflammatory demyelinating lesions. However, the perivenular distribution of MS lesions, named “Dawson’s fingers,” prevalently around the ventricle-based brain veins, has long been thought to be the result of inflammation and coagulation around the major axis of medular veins .Following the first single center study , many grEven the short-lasting improvements reported by many operated patients that were considered placebo effect by the authors of the review , may be Nevertheless, in my opinion, several studies carried out to test this theory have indirectly demonstrated the presence of the widespread endothelial damage likely due to the persistence of inflammatory-thrombotic processes. The emerging evidences on the link of jugular venous reflux with several CNS diseases have not clarified their cause–effect relationship , howeverMore convincingly, Kirk and colleagues much earlier found the microvascular endothelial tight junctions abnormalities in vessels of any size, especially in active lesions as compared to normal appearing white matter, supporting a causative role of diffusible inflammatory mediators . In factMany evidences in literature support the role of the coagulation system as an expression of innate immune activation both in MS and in experimental allergic encephalomyelitis (EAE).In 1935, Putnam already proposed the pivotal role of venule thrombosis in MS . AnotherChapman highlighted the relevance of thrombin in inflammatory brain diseases , 43. ThrThe activated microglia, a cellular component of innate immunity in the brain, along with fibrin deposition, were both found in acute/early MS and in rat EAE lesions, representing a first stage of tissue injury before active demyelination and massive T-cell infiltration . Fibrin All reported here data highlight that the activation of coagulation system, linked to innate immunity, is a mandatory process during the early immune reaction toward the tissue damage of any kind. This reaction is independent of the underlying cause of injury and precedes a specific adaptive immune response in both MS and EAE.Although the histopathological studies have only rarely found cerebral venous thrombosis in MS, there is convincing evidence supporting the presence of inflammatory-thrombotic processes during the overall course of the disease, from its early phases. This might confirm the relevant role of innate immunity in MS not only for its inflammatory component –6, 47, bThe early systemic activation of innate immunity suggests a direct triggering by pathogens rather than by an indirect autoimmune CNS reaction. The innate immunity not only stimulates and modulates adaptive immunity at the beginning of disease but also mediates the neurodegenerative changes in progressive phase of MS , 6. WhilThe physiological innate immune response to infections does not usually determine the thrombotic manifestations due to an effective action of the natural anti-coagulant system. However, thrombotic manifestations can take place when the natural anti-coagulant system fails to counterbalance the prolonged or intense coagulation processes. These conditions may occur occasionally in immune-competent people during particularly severe infections, or may manifest persistently during chronic latent infections occurring in individuals with unregulated immunity and genetic or environmentally induced impairment of anti-coagulant system. MS and other immune-mediated disorders associated with thromboembolism may fall into this last category. However, there is no doubt that if the activation of innate immunity is directly triggered by viral infection, playing a protective role against infection, our therapeutic intervention should be not greater than necessary to compensate the reduced anti-coagulant capacity.Several evidences support persistent inflammatory-thrombotic processes in MS confirming the prolonged activation of innate immunity, likely by chronic latent infections. Experimental evidences coming from the literature here reported may open new therapeutic perspectives for MS patients. In fact, it is already possible to interfere with the coagulation system at various levels of the cascade, and clinical trials trying to transfer the promising results on EAE to humans are needed.No conflict of interest or financial interest is reported. T. Koudriavtseva reports consulting fees from Bayer Schering, and Institutional grant from Merck Serono, Biogen Idec, Novartis, Bayer Schering outside the submitted work.


Chronic kidney disease is strongly linked to neurocognitive deficits in adults and children, but the pathophysiologic processes leading to these deficits remain poorly understood. The NiCK study  seeks to address critical gaps in our understanding of the biological basis for neurologic abnormalities in chronic kidney disease. In this report, we describe the objectives, design, and methods of the NiCK study.The NiCK Study is a cross-sectional cohort study in which neurocognitive and neuroimaging phenotyping is performed in children and young adults, aged 8 to 25 years, with chronic kidney disease compared to healthy controls. Assessments include (1) comprehensive neurocognitive testing ; (2) detailed clinical phenotyping; and (3) multimodal magnetic resonance imaging (MRI) to assess brain structure , functional connectivity , and blood flow . Primary analyses will examine group differences in neurocognitive testing and neuroimaging between subjects with chronic kidney disease and healthy controls. Mechanisms responsible for neurocognitive dysfunction resulting from kidney disease will be explored by examining associations between neurocognitive testing and regional changes in brain structure, functional connectivity, or blood flow. In addition, the neurologic impact of kidney disease comorbidities such as anemia and hypertension will be explored. We highlight aspects of our analytical approach that illustrate the challenges and opportunities posed by data of this scope.The NiCK study provides a unique opportunity to address key questions about the biological basis of neurocognitive deficits in chronic kidney disease. Understanding these mechanisms could have great public health impact by guiding screening strategies, delivery of health information, and targeted treatment strategies for chronic kidney disease and its related comorbidities. Chronic kidney disease (CKD) is strongly linked to neurocognitive dysfunction in adults and children -4, but tAlthough these studies have increased awareness of neuroanatomical abnormalities in CKD, a key question remains: What are the specific physiologic disturbances in CKD that lead to neurocognitive changes? Potential mechanisms include metabolic neuronal injury  and, perThe Neurocognitive Assessment and Magnetic Resonance Imaging Analysis of Children and Young Adults with Chronic Kidney Disease (NiCK) Study was designed to address this critical gap in our understanding of the biological basis for neurologic abnormalities in CKD. With funding from the Pennsylvania Department of Health, this study uses a comprehensive interdisciplinary approach and leverages unique resources at the Children’s Hospital of Philadelphia and the University of Pennsylvania to combine comprehensive neurocognitive assessments and state-of-the-art multimodal magnetic resonance imaging (MRI) in children and young adults with CKD.In this report, we describe the objectives, design, and methods of the NiCK study, and outline how the study provides the opportunity to address key questions in the field. We will then describe our approach to data analysis, focusing on the challenges and opportunities posed by the multitude of variables inherent in clinical, neurocognitive, and neuroimaging data of this scope. Finally, we will outline future directions for this rich dataset, including the potential for novel unbiased scientific discovery, and discuss the potential public health impact of this study.a comprehensive battery of traditional neurocognitive testing, anda computerized neurocognitive batteryDetailed neurocognitive description of children and young adults with CKD, along with concurrent age-matched healthy controls, using:Detailed clinical description to explore the impact of clinical risk factors such as hypertension and anemia; andStructural MRI to explore the effects of CKD on brain structureResting state blood oxygenation level dependent functional MRI (BOLD fMRI) to evaluate connectivity within brain networks that modulate specific neurocognitive functionsArterial spin labeled perfusion MRI to evaluate global and regional cerebral blood flow.Multimodal magnetic resonance imaging (MRI) to identify differences in brain structure, functional connectivity, and cerebral blood flow, using:The overarching objectives of the NiCK study are (1) to determine how CKD and its associated comorbidities affect neurocognitive function, and (2) to understand the neurobiological basis for cognitive abnormalities in CKD. The NiCK study seeks to achieve these objectives using the following three-pronged approach:This multifaceted approach will allow us to integrate findings from neurocognitive testing, clinical phenotyping, and multimodal MRI to develop a unique multi-parametric neurocognitive phenotype for CKD. This innovative approach to assessment will provide new knowledge to further our understanding of mechanisms underlying neurological dysfunction in CKD.2, including dialysis and post-transplant), compared to healthy controls matched on age and socioeconomic status (using insurance status as a proxy).The NiCK study consists of a cross-sectional study of 90 pediatric and young adult subjects, aged 8 – 25 years, with Stage 2 to 5 CKD .Table The lower age limit of 8 years ensures improved compliance with imaging procedures. Since the selected neurocognitive measures are standardized largely in English, participants must have English as their primary language. To ensure that study results reflect the effects of kidney disease, patients with a number of comorbidities that independently affect brain function  or the ability to complete test measures  are excluded from participating , past medical history, family history, and current medications. Participants then undergo clinical, imaging, and neurocognitive assessments as described below.Participants’ blood pressure (BP), heart rate, respirations, height, weight, and body mass index are measured. Laboratory data collected includes complete blood count, comprehensive metabolic panel, calcium, phosphate, cystatin C, lipid panel, and urine studies for total protein, albumin, and creatinine. Urine pregnancy testing is performed in post-pubertal girls prior to MRI scanning. All participants undergo 24-hour ambulatory blood pressure monitoring (ABPM).A battery of age-specific neurocognitive assessments Table  is perfoIn addition to the traditional neurocognitive assessments outlined in Table After appropriate MRI safety screening, participants undergo non-sedated, non-contrast MRI of the brain, acquired on a Siemens Verio 3 T scanner equipped with a 32-channel head coil. The FDA- and manufacturer-approved sequences used are shown in Table An automated processing pipeline consisting of extensively validated methods is applied for processing sMRI images. The processing pipeline includes: extraction of the brain parenchymal tissue using multi-atlas skull-stripping ; inhomogBy summarizing the very high dimensional image data with much fewer variables, calculated volumetric measurements allow us to perform quantitative analyses of regional brain volumes and fractional anisotropy, and to evaluate the extent of brain atrophy and cerebrovascular disease such as infarcts and leukoareosis.BOLD fMRI is used to evaluate connectivity within specific cognitive networks in the absence of external stimuli (resting state BOLD fMRI). Following standard registration procedures (implemented via the program FIRST) , fMRI daASL MRI uses spin-labeled blood water as an endogenous tracer to measure regional cerebral blood flow (CBF). This study acquired pseudocontinuous ASL  sampled A sample size of 90 CKD subjects and 90 healthy controls was calculated based on the assumption that the prevalence of MRI abnormalities in CKD subjects will be 12.5% and will be 1% in normal controls. This sample size provides 80% power to detect such a difference with 95% confidence.The comprehensive approach employed in the NiCK study provides the opportunity to address some key issues and challenges in the field.Vascular disease is a well-known independent risk factor for cognitive impairment and dementia in adults . Adults By comparing neurocognitive test results with MRI data, we will be able to evaluate whether specific deficits correlate with changes in structure, connectivity, or CBF in particular regions of the brain. For example, executive function is a domain known to be affected in children with CKD . We woulHypertension has been associated with neurocognitive dysfunction in adults -32 and cBrain maturation involves a complex series of structural and functional changes throughout childhood, adolescence, and young adulthood . By stud2) allows us to explore neurologic changes across the continuum of disease severity. However, this diversity also introduces a greater number of covariates that must be accounted for in our analyses.The data collected in the NiCK study provides great opportunities to test hypotheses regarding differences in brain structure and function between individuals with CKD and healthy controls. However, the heterogeneous nature of CKD participants in this study presents both challenges and opportunities. Their wide range of kidney function  assessment for correlation between ABPM variables – the variables chosen for our primary analyses were those that were not highly correlated with each other, and thus potentially most informative about different aspects of BP physiology.There are a large number of blood pressure (BP) variables generated by 24-hour ABPM. These include measures for systolic and diastolic BP (SBP and DBP) and heart rate (HR), and can be broken down into waking hours, sleeping hours, and the total 24 hours. Potential variables include mean BP, mean BP index , BP load , nocturnal BP dipping (percent decrease in mean BP from waking to sleeping), hyperbaric index (area enclosed by the line of ambulatory BP readings above and the 95th percentile limit line for BP readings below), and BP and HR variability (coefficient of variation of BP readings and HR). Many of these variables are highly correlated with each other (e.g. mean BP and BP load); however, a growing number of studies show that certain ABPM variables, for example abnormal nocturnal dipping or increased BP variability, are independent predictors of adverse cardiovascular and neurocognitive outcomes. Our approach in selecting which ABPM variables to focus our analyses on has been two-pronged: (1) review of the literature to create an This study includes comprehensive neurocognitive phenotyping in youth with CKD. Although traditional neurocognitive batteries (TNB) have been used extensively in individuals with CKD, this is the first study to utilize a computerized testing (the Penn CNB) in this population. Both the TNB and CNB provide comprehensive assessments of multiple neurocognitive domains. However, since the exact tests differ between the two batteries, one challenge we encounter is aligning specific tasks in each battery with neurocognitive functions tested. Table Interpretation of performance across the TNB and CNB presents another challenge. Most TNB tests measure only accuracy, whereas most CNB tests measure both accuracy and speed. This is potentially important, as recent studies have shown that deviations from normal brain developmental trajectories are associated with significant deviations in performance for speed, but not accuracy . For somOur primary approach for analysis of sMRI, fMRI, and ASL MRI data in the NiCK study is to compare group differences in pre-specified ROIs, based on anatomic partitioning into structural/functional brain regions.Using these pre-specified ROIs, we can explore specific hypotheses – for example, ROIs encompassing the prefrontal cortex and frontotemporal areas would be hypothesized to be affected by CKD, given literature describing deficits in executive function in this population . We can Whole brain approaches can also be used to explore the global effects of specific CKD comorbidities – for example, using ASL, we can examine the effects of hypertension and anemia on global cerebral blood flow (CBF), or CBF within gray matter and white matter compartments.a priori correlations with the neurocognitive data will be explored in an effort to identify underlying neurological processes for manifest neurocognitive dysfunction.Additionally, a priori partitioning into anatomical ROIs. This unbiased discovery approach has the potential to yield new, previously unsuspected connections between regional brain abnormalities and CKD comorbidities.Although the primary analyses in NiCK will be hypothesis-driven, the nature of the imaging data collected also provides the opportunity to explore potential new connections and generate new hypotheses. For example, one hypothesis-free approach to analysis of the sMRI data uses voxel-based analysis ,39 – thiA new machine learning methodology, the brain development index (BDI) , summariThe long-term goal of the NiCK study is to develop a multi-parametric neuroimaging biomarker for patients with CKD. Using machine learning and pattern recognition methods, a classification system incorporating all the imaging measurements can be developed for individual patients. Multivariate pattern classification combining signals from sMRI, ASL and fMRI can be performed using the COMPARE   method. The NiCK Study represents the most comprehensive neurocognitive and neuroimaging phenotyping ever performed in children and youth with CKD. Our innovative approach of integrating multimodal MRI with neurocognitive and clinical data provides a unique opportunity to answer key questions in the field. These include understanding the independent neurologic impact of CKD, the neurologic effects of hypertension and other cardiovascular risk factors, and the consequences of CKD on neurodevelopment from childhood through young adulthood. Understanding these mechanisms could have great potential public health impact. First, this study could help to identify improved screening strategies to identify patients at high risk of adverse neurocognitive outcomes . Second, neurocognitive impairment may affect adherence to the complex medical regimens that are routinely prescribed to patients with CKD. By improving our recognition of these deficits in our patients, we may be able to deliver health information in a more individualized and effective manner. Finally, by identifying mechanisms through which modifiable risk factors such as anemia and hypertension affect neurocognition, this study may help to guide targeted treatment strategies to prevent future cognitive decline.


Selenium supplementation reduces the products of oxidative stress in kidney failure patients and can be protective against cisplatin nephrotoxicity.et al entitled, the effects of vitamin E and selenium on cisplatin-induced renal toxicity in cancerous patients treated with cisplatin-based chemotherapy, they found that, selenium can be used to reduce cisplatin-induced nephrotoxicity  have been completely observed by the author.None.


Many a times their benefits are overshadowed by harmful effects on non-target organisms including humans.4 There are no molecular studies to investigate the underlying mechanism of action of ES in resulting cellular damage, genomic instability and ill health. A recent study by Sebastian and Raghavan5 demonstrated the mechanism of ES-mediated testicular toxicity and male infertility using a mice model system.Endosulfan (ES) is an organochlorine pesticide associated with harmful side effects on humans that include speculated infertility and teratogenic outcomes.+ cells too showed variations in treated animals, suggesting impact at the level of the lymphoid system.Comparing the experimentally validated bioavailability in mice and the known concentrations of ES reported in accidental and occupational exposure in humans, we resorted to physiologically relevant sublethal concentrations of ES in the investigation. At the whole-body level, ES induced more weight fluctuations in males as compared with females. RBC, WBC and platelet levels were also affected upon ES exposure, indicating toxicity. CD19Detailed histopathological analyses revealed that among all the organs, liver, lungs and testes were affected, whereas brain, intestine and kidney showed no sign of toxicity. This was also reflected at the functional level.In testes, high degree of atrophy and tubular necrosis was seen, with many seminiferous tubules having fully or partially depleted spermatogonial mother cells and spermatids. Further detailed analyses through TUNEL assay revealed long-term testicular cell death, indicative of persistent damage.n) and 1n (spermatids) cells. These results indicate that ES treatment significantly perturbed a complete cycle of spermatogenesis, causing testicular atrophy and depletion of cell populations.Following exposure to ES, fluorescence activated cell sorting analysis of different spermatogenic cell populations at various time points spanning spermatogenesis showed remarkable reduction in G1 (spermatocytes and quiescent mother cells), S (dividing mother cells), G2/M  levels. Interestingly, a dramatic reduction in the sperm count upon ES exposure was observed. While significant reduction in actively motile sperms was noted, an increase in nonmotile sperms was also evident due to testicular cell damage and death. These results revealed that the epididymal sperm number and quality were severely compromised upon ES exposure.Does the dramatic effect on sperm count and motility affect fertility? It appears to be the case. Extensive experiments on mating using ES-treated male mice revealed that the number of infertile males increased upon exposure to ES. This suggests that the reduced sperm count and motility contributed towards infertility in males. However, we did not find any synergistic effects on fertility levels when both males and females were exposed, indicating that ES-induced infertility could be male specific.In conclusion, using a variety of approaches, the authors demonstrate that, coupled with pathophysiological changes, ES induced maximal effect on testes causing cell death and depleting testicular cell populations. ES-induced changes in testes were spermatogenesis dependent and led to the reduction in sperm quality and quantity, resulting in male infertility .5). This observation in conjunction with the physiological effects exerted by ES gives us new insights into the mechanisms of ES-mediated toxicity.Interestingly, treatment with ES led to ROS-mediated DNA damage and elevated levels of error prone DNA repair leading to genomic instability  antagonist.pothesis . Importapothesis . Therefo11 Besides, the questions addressed here are of global concern due to increasing world trade of farm produce and lack of appropriate quality control measures, pertaining to the usage of pesticides in several countries.11 This study could be further extended to other spectra of pesticides with such speculated adverse effects and would be a first step towards better rationalised usage of pesticides for the benefit of mankind.This study is of special interest considering the growing concerns about health hazards of pesticides and lack of in-depth understanding of it.


In the past two decades, the Hedgehog (HH) family of secreted proteins has been demonstrated to play an essential role in both animal development and various human diseases . Sonic HSHH, we observed that SHH potency was substantially higher when expressed at physiologically relevant levels than when overexpressed. We rationalized that these differences in activity might result from alterations in some post-translational modification of SHH. We therefore developed a modified purification protocol to isolate SHH from cells expressing endogenous-like levels of SHH, and subjected peptides from this purified SHH protein to microcapillary liquid chromatography-tandem mass spectroscopy. This analysis demonstrated that SHH-Np was heterogeneously modified at a highly conserved amino-terminal Cys25 residue, by distinct fatty acids that varied in terms of their length and saturation, as well by as a number of currently unidentified moieties. The spectrum of these modifications was dynamic, as the types of fatty acid and their degree of saturation were modulated upon changes in the growth conditions of the cells or the level of SHH expression. When SHH was expressed at low levels, 30% of SHH-Np was modified with unsaturated fatty acids, and this percentage was substantially increased upon SHH overexpression. Upon serum deprivation, SHH-Np was predominantly modified by unsaturated fatty acids. These results suggest that the fatty acid modification of SHH-Np is modulated in response to changes in the growth/metabolic environment of the cells producing it.Using tissue culture cells expressing different levels of in vitro, and ex vivo in dissected chicken embryonic limb bud tissue, indicated that SHH-Np modified with unsaturated fatty acids undergoes altered intracellular trafficking to lipid rafts and has reduced potency. Lipid rafts are wellcharacterized plasma membrane niches where SHH-Np is known to enrich, and from where it is likely secreted in a long-range acting form [Functionally, lipid incubation studies ing form . Thus, aing form . PerturbDrosophila HH can act in a hormone-like manner to sense nutrient availability and accordingly modulate lipid metabolism [Interestingly, tabolism . Specifitabolism . Furthertabolism . Therefo


The endothelium separates blood and the vascular wall. Endothelial stimulation by agonists or by shear stress has effects on the smooth musculature in the vessel wall by paracrine communication. After the finding that intact endothelium is essential for acetylcholine to produce arterial vasodilation, various agents were discovered that stimulated the endothelium to produce endothelium-derived relaxing or contracting factors (EDRF/EDCF), the major EDRF being nitric oxide (NO). Additionally to vasodilation, NO inhibits platelet and monocyte adhesion and smooth muscle cell proliferation and migration, all key factors in the genesis and progression of atherosclerosis. Thus, dysfunctional endothelium, characterised by limited NO production, has lost part of its vasodilatory function and promotes atherosclerosis.In 1986, Ludmer and colleagues infused acetylcholine and the NO donor nitro-glycerine in the left anterior descending artery (LAD). Nitro-glycerine invariably caused LAD vasodilation but acetylcholine caused only vasodilation in the normal arteries, while arteries with stenosis showed vasoconstriction or even temporal occlusion. This study paved the road for invasive assessment of coronary endothelial integrity and its association with atherosclerosis in the coronary circulation. In 1989, Cox and colleagues measured flow-mediated LAD diameter changes by quantitative angiography, using adenosine infusions to increase blood flow. Dilation was markedly impaired in atherosclerotic vessels. However, all vessels dilated with nitro-glycerine. Hence, absence of flow-mediated dilation in atherosclerosis reflects impaired endothelial vasodilator function. Later, in 2009, Lavi and colleagues demonstrated that coronary segments with a higher degree of endothelial dysfunction were associated with necrotic plaque.Intracoronary endothelial function assessment, while yielding important information, is not feasible for large-scale application because it is expensive and requires catheterisation. Looking for non-invasive alternatives, measurement of flow-mediated dilation (FMD) of the brachial artery was an important step forward. Measuring in a peripheral arterial bed instead of in the coronary circulation is defendable from the viewpoint that atherosclerosis can be regarded as a systemic condition. FMD is non-invasive: increased flow through the artery is induced by reactive hyperaemia after a 5-minute occlusion of the arm, arterial diameter changes are measured by ultrasound. Peripheral arterial tonometry (PAT) was a next step; this non-invasive method also relies on reactive hyperaemia after a 5-minute occlusion of the arm but compares baseline and reactive hyperaemia plethysmograms measured at a fingertip; hence, PAT is done in a microvascular bed instead of in a conduit artery. Because PAT is low risk, easy to perform and highly operator-independent, it is suitable for large-scale measurement. Consequently, there is much interest to validate this technique in all stages of atherosclerotic disease.In the current issue of this Journal, Van den Heuvel and colleagues  publish Earlier, Rubinshtein and colleagues  had demoThe mechanism of reactive hyperaemia is the same as that of autoregulation: with insufficient blood supply, metabolites accumulate in the interstitial space and cause arteriolar vasodilation (smooth muscle cell relaxation) by direct and by endothelium-mediated effects. E.g., smooth muscle cells as well as endothelial cells have adenosine receptors and, consequently, the metabolite adenosine acts directly on smooth muscle but also indirectly, by acting on endothelial cells that in turn produce NO. Hence, microvascular vasodilation does not faithfully represent endothelial function because it is only partly endothelium dependent.Disruption of laminar flow at branching points in the circulation influences the local endothelial layer via mechanotransduction, thus promoting conditions for atherosclerosis; the microvasculature in the fingertips is not representative for such atherosclerosis-prone sites in conduit arteries.Vascular tone in the arteriovenous anastomoses in the finger is sympathetically controlled rather than by NO. This complicates interpretation of PAT measurements in terms of endothelial function.Endothelial function is influenced by daily-life factors such as diet, smoking, hormone level changes during the menstrual cycle, diurnal and seasonal factors, stress, environmental factors like room and skin temperature, exercise, all at the cost of limited reproducibility of PAT measurements.Negative or mixed conclusions require critical analysis. What can be expected of PAT for the assessment of coronary atherosclerosis? There are several concerns: (1) reactive hyperaemia is not uniquely endothelium dependent; (2) the site of measurement is not representative for the sites where atherosclerotic lesions emerge; (3) the microvasculature in the fingertip abundantly includes anastomoses; (4) endothelial function is subject to many daily-life stimuli/events:All mentioned factors weaken the link between PAT measurements and endothelial function, thus hampering reliable atherosclerosis assessment and risk stratification by this technique. Nevertheless, a recent review considering a variety of measures of peripheral artery function  gives hoNone.None declared.


Left ventricular noncompaction (LVNC) is a myocardial disorder probably due to the arrest of normal embryogenesis of the left ventricle. It could be isolated or associated with other extracardiac and cardiac abnormalities, including coronary artery anomalies. Despite the continuous improvement of imaging resolution quality, this cardiomyopathy still remains frequently misdiagnosed, especially if associated with other heart diseases. We report a case of LVNC association with both malposition of the great arteries and a very original coronary artery pattern. Left ventricular noncompaction (LVNC) is a myocardial disorder, which is thought to occur due to the arrest of normal embryogenesis of the left ventricle (LV), leading to distinct morphological characteristics in the ventricular chamber . The affWe present herein a case of LVNC association with both malposition of the great arteries and a very original coronary pattern. This report has been approved by the Institutional Review Board, with informed consent given by the patient.A 30-year-old male, smoker, without family history for congenital or acquired cardiovascular diseases, was referred to our department because of chest/epigastric pain and syncope due to ventricular fibrillation after cocaine overdose. After the usual resuscitation maneuvers, including 3 early direct current shocks, orotracheal intubation, and pharmacological support, his clinical conditions were stabilized. The first echocardiographic evaluation highlighted hypokinesia of the inferior LV wall, with mild systolic dysfunction and mild-to-moderate mitral valve regurgitation. During hospitalization he progressively developed lesion, ischemia, and then necrosis waves in diaphragmatic leads at the electrocardiogram (ECG); moreover the toxicological test results confirmed the recent cocaine abuse, while laboratory ones showed increased troponin I (peak 23.9 ng/mL) and N-Terminal pro-Brain Natriuretic Peptide (peak 2356 pg/mL) levels.Coronary angiography showed a short main stem, originating from the right sinus of Valsalva, splitted into four branches, from right to left: an artery providing the right ventricle (RV) free wall (RCA), a dual left anterior descending (LAD) artery (type I variant of Spindola-Franco ), and fiBecause of frequent but not repetitive ventricular extra beats at the last in-hospital Holter dynamic ECG monitoring, he was discharged soon after loop recorder implantation. This device has never stored relevant arrhythmic events after 2-year follow-up.The embryonic myocardium is composed of a loose meshwork of interwoven fibers separated by deep recesses, which communicate with the LV cavity, allowing an increase in the myocardial surface area and the exchange diffusion from the cavity. From the 5th–8th week of embryogenesis, LV trabecular compaction occurs simultaneously with the invasion of the myocardium by the developing coronary vasculature coming from the epicardium: at the same time in fact the coronary circulation develops and intertrabecular recesses are transformed in capillaries . The traα-dystrobrevin gene was identified subsequently in patients with LVNC and associated with congenital heart diseases [LVNC coexists with another cardiac anomaly approximately in 20% of cases ; sometimdiseases . The clidiseases  as well Diagnostic sensitivity has been enhanced by the introduction of specific morphologic criteria by high resolution echocardiography, CT angiography, and cardiac MRI. Echocardiography could pose inherent problems in assessing the LV apex  and lateThe coincidence of LVNC, malposition of the great arteries, and such a complex coronary anomaly has never been described before. As this case illustrates, accurate diagnosis of a grown-up congenital heart disease could be very tricky, so much so that the role of multimodal imaging assessment is fundamental.


Scaphoid stress fractures are rare injuries that have been described in young, high-level athletes who exhibit repetitive loading with the wrist in extension. We present a case of an occult scaphoid stress fracture in a 22-year-old female Division I collegiate shot-putter. She was successfully treated with immobilization in a thumb spica splint for 6 weeks. Loaded wrist extension activities can predispose certain high-level athletes to sustain scaphoid stress fractures, and a high index of suspicion in this patient population may aid prompt diagnosis and management of this rare injury. Scaphoid fractures are common in young adults and athletes and can lead to significant morbidity even with early diagnosis and appropriate treatment. While scaphoid fractures are most commonly associated with acute wrist trauma, it is notable that chronic repetitive loaded wrist extension can lead to scaphoid stress fractures .Although rare, scaphoid stress fractures have been described in young, high-level athletes who exhibit repetitive loading with the wrist in extension, most commonly in gymnasts –7. With A 22-year-old female Division I collegiate right-hand dominant shot-putter initially presented with a two-month history of progressive, activity-related right wrist pain, with no report of prior trauma. She noted worsening pain with wrist extension during throwing the shot-put but had no complaints of pain or dysfunction with the discus nor with activities of daily living. The patient had been training for three to four hours per day, five times per week, alternating between the shot-put and discus as well as doing Olympic-style weight lifting for seven years prior to presentation.Physical exam revealed tenderness to palpation at the anatomic snuffbox and the scaphoid tuberosity. Wrist flexion, extension, and supination were symmetric, but painful with loaded terminal extension in the dominant wrist. Radiographs of the affected wrist at the time of presentation, demonstrated −1 mm ulnar negative variance, with no evidence of abnormality Figures , and 3. The patient was placed in a removable thumb spica wrist splint and was instructed to avoid any loaded extension of the wrist, including throwing the shot-put and weight-training. She was allowed to throw discus as it did not cause any pain. After three weeks, the patient reported no symptoms or pain with wrist extension. Radiographs obtained at 6 weeks after thumb spica immobilization were negative for any evidence of scaphoid fracture Figures . On physScaphoid stress fractures are very rare injuries, with only case reports available for analysis in the peer-reviewed literature. In each reported case, the patients were competitive, high-level athletes training for multiple hours per day for several years prior to their presentation . While gCommon to all of these athletic activities is the act of repetitive loaded wrist extension. Although the exact factors that lead to stress fractures of the scaphoid remain unclear, it has been suggested that repetitive stress and microtrauma to the bone can exceed native osseous repair mechanisms . In eachLoaded wrist extension creates stresses that are typically centered at the scaphoid waist. In a cadaver study, Weber and Chao reported that 460 to 960 pounds of force applied to an extended wrist was required to acutely fracture the scaphoid, at the waist . A more Interestingly, the majority of scaphoid stress fractures have been reported in young male athletes, with 14 male and only 2 female patients reported in the available literature. The exact mechanism for this apparent gender discrepancy remains unclear but may be related to males reaching skeletal maturity at a later age than females, as adolescence appears to be a risk factor for suffering a scaphoid stress fracture. Similarly, while a direct link to age, sex, and athletic participation remains speculative, scaphoid stress fractures may be in part due to more intense participation in higher-level, longer-duration athletic training during adolescence.Importantly, many of the published case reports regarding scaphoid stress fractures have noted a delay in diagnosis with this injury, with the majority of cases being recognized only after the fracture became apparent on plain radiographs , 3, 5. SThere are no current guidelines specific to the treatment of scaphoid stress fractures. However, for displaced or chronic scaphoid fractures or nonunions, surgical intervention is typically recommended. For nondisplaced or incomplete fractures, as in this case, nonsurgical treatment with immobilization is usually appropriate. Of the cases described in the literature, nine of the cases were treated nonoperatively , 15, 16,In summary, stress fractures of the scaphoid are exceedingly rare but potentially devastating if not recognized and treated promptly. Clinicians should have a high index of suspicion when evaluating an athlete or patient who presents with an insidious onset of activity-related wrist pain and snuffbox tenderness and who is involved in a sport that requires repetitive loaded wrist extension. While the majority of cases described have involved male athletes, scaphoid stress fractures also occur in females. A low-threshold to obtain advanced imaging when radiographs appear negative for scaphoid pathology may aid in the early diagnosis of this rare entity, avoid fracture nonunion, and reduce the need for complex surgical intervention.


Asthma represents the most common chronic childhood disease worldwide. Whereas preschool children present with wheezing triggered by different factors , clinical asthma manifestation in school children has previously been classified as allergic and non-allergic asthma. For both, the underlying immunological mechanisms are not yet understood in depth in children. Treatment is still prescribed regardless of underlying mechanisms, and children are not always treated successfully. This review summarizes recent key findings on the complex mechanisms of the development and manifestation of childhood asthma. Whereas traditional classification of childhood asthma is primarily based on clinical symptoms like wheezing and atopy, novel approaches to specify asthma phenotypes are under way and face challenges such as including the stability of phenotypes over time and transition into adulthood. Epidemiological studies enclose more information on the patient’s disease history and environmental influences. Latest studies define endotypes based on molecular and cellular mechanisms, for example defining risk and protective single nucleotide polymorphisms (SNPs) and new immune phenotypes, showing promising results. Also, regulatory T cells and recently discovered T helper cell subtypes such as Th9 and Th17 cells were shown to be important for the development of asthma. Innate lymphoid cells (ILC) could play a critical role in asthma patients as they produce different cytokines associated with asthma. Epigenetic findings showed different acetylation and methylation patterns for children with allergic and non-allergic asthma. On a posttranscriptional level, miRNAs are regulating factors identified to differ between asthma patients and healthy controls and also indicate differences within asthma phenotypes. Metabolomics is another exciting chapter important for endotyping asthmatic children. Despite the development of new biomarkers and the discovery of new immunological molecules, the complex puzzle of childhood asthma is still far from being completed. Addressing the current challenges of distinct clinical asthma and wheeze phenotypes, including their stability and underlying endotypes, involves addressing the interplay of innate and adaptive immune regulatory mechanisms in large, interdisciplinary cohorts. Asthma is a complex chronic pulmonary disease with reversible airflow obstruction, which affects adults and children. The prevalence of asthma symptoms in 13- to 14-year-old children ranges between 5 and 20 % in different parts of the world with partly increasing trend , 7. ThisThere are several tools to diagnose asthma early in life, such as the International Study of Asthma and Allergy in Childhood (ISAAC) criteria or the modified asthma predictive index (mAPI) , 10. DefOne of the most common parameters for classification at school-age is specific IgE, an immunoglobulin used to assess allergic sensitization as a mean of differentiation between allergic and non-allergic asthmatics. Positive specific IgE with characteristic clinical symptoms in addition to pulmonary symptoms is distinctive for allergic asthmatics while a low level and/or negative specific IgE without clinical symptoms is defined as non-allergic asthma phenotype . HoweverAnother way is a more objective, epidemiologic approach driven by data from birth cohorts. Mainly latent class analysis (LCA) is used to identify groups with similar features for a large, heterogeneous group of subjects , 4. EpidOne facet of defining precise and stable phenotypes is the question of predicting outcomes, i.e., progression of wheeze to childhood asthma and persistence of asthma into adulthood. Longitudinal studies following a cohort from infancy to adulthood are limited; however, it is commonly accepted that the origins of adult asthma at least partially lie in childhood events. For instance, experiencing EVW in childhood appears to be a risk for rapid loss of lung function in adult asthmatics. On the other hand, there are many differences in phenotypes of childhood compared to adult asthma, for example, aspirin-sensitive asthma is a subset rather found in adults, and allergic or eosinophilic and non-allergic or non-eosinophilic asthma are not identical in children and adults. Regarding stability of phenotypes, frequent switching seems to be a phenomenon more common in childhood wheeze and asthma, as the phenotypes of adult asthma appear to be more stable, rather showing variations within one phenotype .Over the past three decades, many birth cohorts have tried to elucidate some of the influences contributing to the development of a certain phenotype. Ongoing efforts to harmonize data from different cohorts will allow comparisons and joint analyses . EffortsIntensive efforts to further classify such endotypes especially in children seem to be the most promising approach to further understand the pathogenesis of early-in-life wheeze disorders. This will help to determine whether they are or are not developing into childhood asthma in order to prevent or modify this progression . HoweverThe complex puzzle of the development and manifestation of childhood asthma consists of different parts which are illustrated in Figs. Asthma is a complex disease, and different childhood asthma phenotypes have already been defined by different immunological mechanisms . SeveralImmunological mechanisms in asthma concerning the adaptive immune system have been reduced to the imbalance between Th1 and Th2 CD4+ T cells for many years. However, the shift in favor of Th2 cells cannot explain all features of asthma. In the last years, different T cell subpopulations have emerged with a role in asthma, such as regulatory T cells (Treg) and the recently described Th9 and Th17 cells. Tregs are ascribed immuno-suppressive functions as they induce self-tolerance and suppress allergy and asthma . Even thIL-17 producing Th cells are accepted as a distinct T cell lineage, as Th17 cells do not develop from Th1 or Th2 cells. They were associated with asthma as increased levels of their pro-inflammatory cytokine IL-17 were found in plasma samples and airway tissue of asthma patients. IL-17 induces the production of pro-inflammatory cytokines and chemokines in epithelial cells, fibroblasts, and neutrophilic granulocytes leading to airway inflammation. IL-17 was also found to play a role in the exacerbation of asthma, which is commonly triggered by viral infections mediated mainly via Toll-like receptor 3 , 27. HowMost innate immune cell types like monocytes, lung-specific macrophages, dendritic cells (DC), natural killer cells (NK), mast cells, and especially eosinophils are enriched within asthma patients . This diFurthermore, the recently discovered innate lymphoid cells (ILC) type 2 seem to play a major role in allergic eosinophilic asthma as they secrete asthma-associated cytokines such as IL-13 and IL-5 in response to the epithelia derived cytokines IL-25, IL-33, and thymic stromal lymphopoietin (TSLP) .Transcription and translation of different genes are regulated in a very complex manner by different meshing regulation strategies and are additionally influenced by gene variants between individuals and populations. Cellular regulatory mechanisms are, among others, epigenetic histone acetylation, DNA methylation, and posttranscriptional regulation via miRNA. Together, they are likely mechanisms and currently a primary focus of research studies, which may influence the different asthma phenotypes in children and in adults.In GWAS, several new loci were identified, which are associated with childhood asthma. This involves for instance the ORMDL , 41, TSLBesides genetics, the very complex field of epigenetic mechanisms seems to play an important role in the development of asthma. Especially, prenatal and early childhood DNA methylation and histone modification may be useful targets as new biomarkers for the development and/or manifestation of asthma. Various lifestyle factors were shown to influence epigenetic regulation of individuals such as smoking, obesity, fish oil consumption, and microbe exposure during pregnancy as recently summarized , 53. MatIn the field of posttranscriptional regulation in gene expression, one mechanism is mediated by small non-coding RNA sequences. These miRNAs can bind complementary to messenger RNA (mRNA) and lead to a repression of protein synthesis through degradation of mRNA or transcription blockade. They are gaining in importance as both potential biomarkers and therapeutic targets. Specific miRNA sequences have already been shown to be differentially expressed in various diseases. The role of miRNA in asthma and other respiratory diseases were summarized in the recently published mini review of Maltby et al. . CurrentFor future studies, specific miRNA patterns could be used to identify children at risk for asthma or for discrimination of asthma phenotypes, as the required plasma samples are easily accessible. Yet, several facets of differences and similarities in miRNA expression in children and adults require further studies: miRNA stability over time needs to be investigated. Further, the prerequisite for a screening is a clear discrimination between healthy controls and patients. Also, the specificity of miRNA for asthma needs to be validated as the same miRNA might be differentially regulated in different lung diseases. In the review of Deshpande et al., the preclinical achievements of targeting miRNA were discussed. However, the problem of the delivery mechanisms of miRNA still has to be solved . In summTaken together, epigenetic modifications, combined with functional transcriptional mechanisms, are promising candidates contributing to close the existing gap of knowledge of the molecular mechanisms of asthma and also allergy, in particular for different phenotypes. Yet, the practical application for clinics is still theory, and ongoing and future studies would profit from implementation of direct use for asthma patients to truly impact on asthma diagnosis or treatment.Verification of cellular metabolic products in different body fluids represents a novel approach to identify patients with asthma. The detection and specification of such metabolic molecules have been studied with mass spectrometry (MS) and nuclear magnetic resonance (NMR) spectroscopy in terms of lipids and glucose metabolites. Research focused on airway biofluids of the lung . Yet, toIn summary, multiple studies indicate that the use of endotyping may help to predict a better response to asthma treatment and potentially novel therapies. Also, there is increasing evidence that selecting patients based on biomarkers can identify subgroups that are likely to respond to biological treatments , 68. ForAdditionally, recent studies about anti-IL-5-treatments have successfully used eosinophil count to select patients. In children, non-invasive transcriptome analysis in sputum gene expression analysis have allowed to form three clusters of near-fatal, severe, and mild asthma in children (and adults) . HoweverCurrent differentiation and classification of phenotypes in particular in young asthma patients will profit from future interdisciplinary research networks which, if successful, will result in identification of subgroups with distinct endotypes subsequently profiting from individualized medicine with decreased side effects. Although novel findings will lead to an increase of parts of the puzzle, these parts have to be combined leading to a more distinct definition of few clearly defined phenotypes.There may be several combinations of molecular analysis imaginable to differentiate distinct phenotypes Fig. . One posFor children and their families, a better prediction of the course of the disease will also contribute to an increased quality of life. Reduction of asthma-related personal and societal burden is achievable within the next decade through the development of novel preventive and possible therapeutic regimens. Figure In summary, the definition of exact asthma phenotypes especially in children is complex. Clinical classification depending on current symptoms does not reflect the heterogeneity and course or stability of the disease and might lead to insufficient treatment. Therefore, the definition of asthma and its phenotypes depending on endotypes including immunological and genetic mechanisms are needed for potentially more effective and tailored therapy in the long term. Developing an “asthma phenotype index” including clinical and molecular criteria of several facets relevant and stable over time would facilitate practical use in clinics, if positive and negative predictive values are superior to current management. In order to truly have an impact on a more specific asthma diagnosis, modulated treatment and patient’s recommendation, these challenges have to be approached in the next decade of asthma research.BALF, bronchial alveolar lavage fluid; CNPs, copy number polymorphisms; COPD, chronic obstructive pulmonary disease; DC, dendritic cell; EVW, episodic viral wheeze; GWAS, genome-wide association study; HDM, house dust mite; IL, interleukin; ILC, innate lymphoid cells; LCA, latent class analysis; miRNA, micro RNA; mRNA, messenger RNA; MTW, multiple-trigger wheeze; NK, natural killer cell; SNPs, single nucleotide polymorphisms; Th, T helper cell; Tregs, regulatory T cells; VOC, volatile organic compounds


Langerhans cells (LCs) have been scrutinized many times in studies of the pathogenesis of graft-versus-host disease (GVHD). As migratory dendritic cells, LCs are capable of direct antigen presentation to cytotoxic T cells. Their self-renewal capacity has led to speculation that persistent recipient LCs could provide a continuous source of host antigen to donor T cells infused during hematopoietic stem cell transplantation (HSCT). In this issue of Experimental Dermatology, a new study examines at the relationship between recipient LCs and chronic GVHD. The role of Langerhans cells (LCs) in the pathogenesis of graft-versus-host disease (GVHD) remains enigmatic. LCs are the paradigmatic migratory dendritic cell (DC) and are potent at priming CD8+ T cells . In haemHuman studies had previously shown that LCs were in cell cycle  and undeExperimental Dermatology examines a new aspect, the relationship between persistent recipient LCs and the occurrence of chronic GVHD (A recent paper in nic GVHD . Perhapsin situ analysis and studies showing high donor chimerism in LCs isolated from epidermal sheets by migration (in vitro culture might somehow favour the detection of donor-derived LCs. However, there is no a priori reason to justify this, and early after transplantation, it is possible to recover high numbers of recipient LCs by migration.The surprising finding is not the lack of an association with chronic GVHD, which is postulated to depend more on indirect antigen presentation by donor-derived DCs –12, but igration ,8. Migrain situ methods are liable to overestimate the proportion of recipient LC nuclei because the intricate branching of LC membrane can lead to the inadvertent inclusion of recipient keratinocyte nuclei. This problem may be minimized using the more intense peri-nuclear distribution of Langerin to detect LC nuclei; Langerin also has the distinct advantage of protease resistance that permits immunofluorescence to be performed after FISH. In addition, Z-stack reconstructions ensure that scored nuclei are completely encapsulated in LC membrane, and post-GVHD skin may be used to provide controls in which a very high proportion of donor LCs is expected. Using these approaches, high donor LC engraftment was also recently observed in a study incorporating a large cohort of minimally conditioned patients (No technique is perfect, and patients .These considerations aside, Andani and colleagues have analysed a homogenous population of patients without GVHD, and their conclusion is that there is significant persistence of recipient LCs in the absence of skin inflammation. This is certainly in keeping with the reports of human limb transplantation, in which donor LCs survive long-term , and higGATA2 mutation lacking DCs but retaining LCs and macrophages also still experience GVHD (The role of LCs in murine transplantation models is still debated. While it is clear that LCs may trigger acute GVHD if they are the only recipient APCs remaining ,15, theynce GVHD –21.in vitro (A new facet of GVHD research is the potential of stable recipient macrophages to promote GVHD. In human transplantation, macrophages outlive LCs and can stimulate allogeneic memory T-cell responses in vitro . In micein vitro –25.In conclusion, we know that many recipient LCs are intact at the moment that donor T cells are infused into humans, and it remains difficult to rule out a contribution of recipient LC-mediated antigen presentation to acute GVHD. However, despite the persistence of many investigators, it remains increasingly hard to envisage that measurements of post-transplant LC chimerism will lead to useful clinical decisions.


For many years, ovarian biology has been based on the dogma that oocytes reserve in female mammals included a finite number, established before or at birth and it is determined by the number and quality of primordial follicles developed during the neonatal period. The restricted supply of oocytes in adult female mammals has been disputed in recent years by supporters of postnatal neo-oogenesis. Recent experimental data showed that ovarian surface epithelium and cortical tissue from both mouse and human were proved to contain very low proportion of cells able to propagate themselves, but also to generate immature oocytes in vitro or in vivo, when transplanted into immunodeficient mice ovaries. By mentioning several landmarks of ovarian stem cell reserve and addressing the exciting perspective of translation into clinical practice as treatment for infertility pathologies, the purpose of this article is to review the knowledge about adult mammalian ovarian stem cells, a topic that, since the first approach quickly attracted the attention of both the scientific media and patients. The human ovary is responsible for providing mature and competent oocytes for reproduction. In addition, it is responsible for the secretion of various hormones and growth factors and cytokines that are involved in signaling pathways of folliculogenesis and oogenesis.Many years, ovarian biology has been based on the principle (dogma) that oocytes reserve in female mammals included a finite number established before or at birth and it is determined by the number and quality of primordial follicles developed during the neonatal period. The restricted supply of oocytes in adult female mammals has been disputed in recent years by supporters of neo-oogenesis. This new threatening-dogma perspective states that renewable germline stem cells (GSCs) are present in the postnatal mammalian ovary.The supporters of neo-oogenesis claim the existence of GSCs in the ovarian surface epithelium (OSE) or the bone marrow (BM) and peripheral blood, which can differentiate in the ovary into oocyte, granulosa phenotype, fibroblast-like cells and in vitro, under appropriate stimulation, in neural and on mesenchymal type cells. Endpoints ranging from oocyte counts to genetic lineage tracing and transplantation experiments support a paradigm shift in reproductive biology involving active renewal of oocyte-containing follicles during postnatal life . AlthougThe reports about existence and potency of adult mammalian ovarian stem cells yielded controversies in the scientific media, like most new and groundbreaking reports. The purpose of this article is to review the knowledge about adult mammalian ovarian stem cells, both pro and con opinions on a topic that, since the first approach, quickly attracted the attention of both the media and patients.When assessing a cell population, one should first refer to the histological organization of the studied structure, in an attempt to identify the different pools of putative stem cells.The female gonad is organized as a parenchymatous organ, with an outer, cortical region and an inner medullar one Figure . It is cPrimordial germline stem cells (PGSs) are of extraovarian origin, migrated in the ovaries during embryonic development. In humans, PGSs originate in the posterior epiblast cells, under the signaling influences of BMP-family morphogens . By weekThe differentiating potential of PGS in vitro is well characterized so far and involves several directions: i) differentiation in mature gametocytes; ii) undifferentiation into embryonic stem cells ; iii) trIn the last few years, some authors reported PGSs originating in the OSE of fetal gonads and give rise to secondary germ cells and primitive granulosa cells . The aduDuring the last few years, several groups reported the presence of very small, embryonic like pluripotent stem cells in various tissues ,27, inclAccepting the idea of an adult germline stem cell, one must also try to define the stem cell niche, which, for other defined adult stem cells comprises several compartments: i) a slow-dividing adult stem cell population; ii) a rapid proliferating compartment of precursors cells – transit amplifying cells (TA cells); iii) supportive cells, usually of mesenchimal type. A part from the cellular partners, extracellular matrix  and gradThere are several arguments that both fetal ovary and postnatal ovary are functionally compartmentalized to ensure proper oocyte development. The correct spatial organization and proper neighboring relations between ovarian fetal cells are required during in utero gonad development to generate follicles, as proven by Nicholas et al. In their experiment, they attempt to “reassemble” mouse ovarian tissue from single cell suspensions, in order to further obtain folliculogenesis upon transplantation into depleted ovaries. Until mid-gestation (d13.5), their attempts were unsuccessful. However, once a certain developmental stage has been reached, isolation of cells and in vitro re-arrangement was sufficient to further generate ovarian follicles . BukovskFurthermore, the adult ovarian cortex provides the inhibitory stimuli needed to maintain the oocyte from completing its cell cycle and there are several maturation triggers known so far to induce the completion of meiosis I: i) low cyclic adenosine monophosphate (cAMP); ii) members of Epidermal Growth Factor (EGF) family, such as amphiregulin and ephiregulin ; iii) poThere are reports, however, of in vitro derived GSC from embryonic stem cells under appropriate stimuli by enriched growth media and cultivation on fibroblast feeder layer . Such a Taking into consideration the hypothesis of GCS being harbored in the OSE, implicitly a niche must exist, but it has not yet been described. Very recently, Flesken-Nikitin et al. advanced the idea that hilum region of the mouse ovary harbors a putative niche for the OSE .Along with the postnatal oogenesis hypothesis, also arises the idea that menopause may actually be the result of a compromised somatic niche ,44. ExpeThe multipotency of a stem cell may be demonstrated through several types of experiments: i) specific surface receptors expression analysis that changes along differentiation pathway; ii) generation of lineage-specific cells in vitro; iii) repopulation of a depleted organ in vivo . Such experimental approaches were used for putative adult ovarian germline stem cells, in order to prove their presence and their regenerative abilities. To identify and further characterize OGS, OSE or cortical tissue from mouse and human were used as putative pools. Indeed, both sources were proved to contain in very low proportion cells able to propagate themselves but also to generate immature oocytes in vitro or in vivo- when transplanted into mouse ovaries .Furthermore, recent data argued for their multipotency, when putative ovarian stem cells were successfully differentiated into cells of the three primary germ layers .If the faith of these cells is, by now, proved and accepted to be towards differentiation downstream female germline, their origin is still a controversy. Among the first groups to promote postnatal folliculogenesis, Tilly’s group also proposed at first a bone marrow origin for the putative stem cells ,21. BukoThe last four years were very fruitful in terms of establishing cell culturing conditions for germline precursor cells. Several independent laboratories have reported successful protocols of female germline stem cells culture initiation and subsequent yielding of immature oocytes , as discMaybe not first, but perhaps most acknowledged landmark in OGS is 2004 Tilly’s research group report on postnatal follicular renewal in mouse ovary, from a population of BrdU/mouse VASA homologue (MVH) positive cells, located in the OSE . They alIn 2006, Kerr et al. demonstrated by unbiased stereological methods quantifying both healthy and atretic follicles, that mean primordial follicle numbers per ovary did not decline significantly in postnatal mouse ovaries .During the next few years, “ovarian stem cells” were mostly in a gray zone, the literature providing mostly interesting debates on their existence -55.In 2009, Zou et al. isolated double positive BrdU/MVH cells from the ovarian surface epithelium of new born and adult mice that were further maintained in culture for six months of more. The cells expressed a protein phenotype of stemness  and no markers of meiotic activity or further differentiation down germline lineage . One yeaIn 2004 Bukovsky et al. proposed that adult human ovaries contain germline putative stem cells in the OSE and ovarian cortex, as proven by imagistic studies and immunohistochemistry for zona pellucida proteins (ZPPs) such as PS1 . CytokerThe same group continued their work in 2009, isolated putative stem cells from OSE scrapings in 21 postmenopausal women with no naturally present follicles and oocytes, that further maintained in cell culture. OSE cell culture spontaneously developed oocyte-like cells . Virant-In 2012, based on accumulated knowledge, White et al. optimized the protocol of OSE isolation from patients and established a cell culture that spontaneously generated oocytes. When injected in NOD-SCID mice ovaries, they generated primordial and primary follicles .At the same time, using a lineage-tracing experiment based on DDX4 (DEAD box polypeptide 4), also known as Ddx4 or Mvh (mouse VASA homolog) in mice, Zhang et al. showed that the Ddx4-expressing cells from postnatal mouse ovaries did not enter mitosis, nor did they contribute to oocytes during de novo folliculogenesis , arguingFrom the beginning it should be mentioned that, as there is a lack of ovarian tissue available for research, human reports are scarce.In vivo results are intricately related to in vitro research, especially when transplantation studies involve cell suspensions. In order to correctly interpret the results, one must properly isolate the cell population to be transplanted. More accurate, perhaps, are transplantation models involving the entire postnatal ovary, that would allow tracing of newly generated germ cells. Some experimental models followed the lessons from hematopoietic stem cells research, attempting repopulation of a depleted organ by putative multipotent cells. For gonads, experimental depletion was achieved chemically, through busulphan treatment. Such an approach was used by Johnson et al., who reported postnatal folliculogenesis after elimination of primordial follicles with busulphan treatment. They transplanted wild-type ovaries into transgenic mice expressing green fluorescent protein and reported preantral follicles with GFP negative granulosa cells and GFP positive oocytes . Also foNicholas et al. transplanted mouse fetal ovarian tissue (dissociated to single cell suspension and re-aggregated) into ovarectomized, immunodeficient mice and reported posttransplant folliculogenesis, but only with around and after midgestation - prelevated tissue, with increased efficiency closer to term. Their experiment also included postnatal gonads that were amongst the most efficient in generating new follicles upon transplantation. Interestingly, the failure of pre e12.5 tissue sample to generate follicles was restricted to oocytes only, whereas granulosa cells exhibited reorganization .Niikura et al. described STRA8 positive cells in the surface epithelium of ovaries of aged (20 mo old) mice, that they labeled as “quiescent germ cells”. In order to see if they are still functional, Niikura et al. transplanted senescent ovaries into young hosts and obtained GFP + immature follicles, proving there is a “reserve” of GSC that can be reactivated under appropriate circumstances. To further prove that the senescent ovary cannot sustain the folliculogenesis anymore, in spite of the existent stem cells pool, they reversed the experiment, by grafting young ovaries into senescent female mice and found a significant loss of immature follicles by 50% in 3 weeks .In spite of accumulating data in favor of OSG, the controversy still exists – in 2012 Kerr et al. found no replenish of primordial follicular reserve after chemical ablation of follicles or sterilizing doses of γ-irradiation  and in 2The abnormalities of ovarian function might lead to infertility or manifestation of aggressive cancer . Germ ceFurthermore, other disorders, such as autoimmune diseases  and myelodysplastic syndromes, require medical treatment that can also impair reproductive cells and tissues.Moreover, social and individual factors  have imposed delaying childbearing in women of reproductive age, affecting these women by age-related infertility. Even more so, the continuous delay of childbearing observed in developed countries will result in an increased proportion of women diagnosed with cancer before their first pregnancy . MaternaFertility preservation is an emerging field in medicine that enables women to maintain reproductive health and therefore, stem cells in adult human ovaries are of great interest to reproductive medicine.Thus, oocytes and their precursor cells might be altered metabolically to sustain or increase ovarian function and fertility in women .So far, orthotropic autotransplantation of cryopreserved ovarian tissue has been reported as a successful method to ensure post-chemotherapy fertility in women suffering from different malignancies . The abiTranslation into clinical practice and use of newly identified ovarian stem cells in infertility pathologies is a desiderate that falls in trends with the regenerative medicine concept. However, experience from other fields, such as neurodegenerative diseases, teaches us tha t is not an easily attainable goal. Closer to home, studies on spermatogonial cell line demonstrated that in vitro generation and isolation of spermatogonia is indeed possible -75, but The trend of the last years seems to indicate that there is oogenesis after birth; however, the origin of the new oocytes is still unclear. Discovery of VSELs opens exciting new perspectives on tissue regeneration and regenerative medicine, but the relationship between them and new oocytes is still under debate.The authors declare that they have no competing interests.AA.GG: manuscript writing, conception and final design. AM.E.: collection and assembly of data, manuscript writing. ME.H: design of the study, revising the study critically for intellectual content. All authors read and approved the final manuscript.