The field of neuroprotection generated consistent preclinical findings of mechanisms of cell death but these failed to be translated into clinics. The approaches that combine the modulation of the inhibitory environment together with the promotion of intrinsic axonal outgrowth needs further work before combined therapeutic strategies will be transferable to clinic trials. It is likely that only when some answers have been found to these issues will our therapeutic efforts meet our expectations. Stroke is a clinically heterogeneous disease and combinatorial treatments require much greater work in pharmacological and toxicological testing. Advances in genetics and results of the Whole Human Genome Project (HGP) provided new unknown information in relation to stroke. Genetic factors are not the only determinants of responses to some diseases. It was recognized early on that “epigenetic” factors were major players in the aetiology and progression of many diseases like stroke. The major players are microRNAs that represent the best-characterized subclass of noncoding RNAs. Epigenetic mechanisms convert environmental conditions and physiological stresses into long-term changes in gene expression and translation. Epigenetics in stroke are in their infancy but offer great promise for better understanding of stroke pathology and the potential viability of new strategies for its treatment. The classical molecular targets for stroke include those involved in oedema/inflammation control, axonal regeneration/plasticity, neurogenesis/angiogenesis, and events that support recovery. For decades, old targets for stroke were based on observations of molecular and cellular changes after stroke. Numerous inflammatory markers, growth-associated proteins, cell cycle proteins, NMDA receptors, molecules involved in synaptic plasticity, dendritic branching, neural sprouting or extracellular matrix remodelling were key targets. The field of neuroprotection generated consistent preclinical findings of mechanisms of cell death but these failed to be translated into clinical therapies. Many clinical trials were carried out using doses that were already known to be ineffective in preclinical trials, or employing time delays outside the established therapeutic window. Some trials were based on preclinical data showing relatively weak effects or those that were only established in one limited model. Similar problems may occur in the field of neural repair without careful work on the key points associated with clinical translation . The eff Stroke is a clinically heterogeneous disease, with infarcts commonly occurring in different tissue compartments (white matter and gray matter) and brain regions , and occurs most often in aged individuals. Combinatorial treatments require much greater work in pharmacological and toxicological testing. Further, treatments that promote anatomical rewiring will need to be administered in combination with behavioural activity to help “stamp in” patterns of brain rewiring that are adaptive and to avoid the formation of maladaptive patterns of wiring. A promising experimental treatment will need, at the very least, to be tested in several different rodent stroke models and aged animals. Despite these issues, it is becoming clear that the partial recovery that is commonly seen after stroke is associated with a reorganization of brain circuitry, and those methods that can safely and effectively enhance this reorganization could potentially have great clinical value . It is i In recent years, the field of neural repair in stroke has identified cellular systems of reorganization and possible new molecular mechanisms. However, conceptual barriers now limit the generation of clinically useful agents. First, it is not clear what the causal mechanisms of neural repair are in stroke. Second, adequate delivery systems for neural repair drugs failed and need to be determined for candidate molecules. Third, ad hoc applications of existing pharmacological agents that enhance attention, mood, or arousal to stroke were unsuccessful. New approaches that specifically harness the molecular systems of learning and memory provide a new avenue for stroke repair drugs. Fourth, combinatorial treatments for neural repair need to be considered for clinical therapies. Finally, neural repair therapies have as a goal altering brain connections, that is, rewiring cognitive maps and active neural networks. These actions may also trigger a unique set of “neural repair side effects” that need to be considered in planning clinical trials . Future The Whole Human Genome Project (HGP) early in the XXI century ushered in a wave of optimism and anticipation that new therapies and even cures for many diseases would soon be forthcoming . Aside fRNAs are an integral component of chromosomes and contribute to their structural organization , 38. It The examples above provide proof-of-principle that RNA can regulate gene expression at many levels and by using a wide array of mechanisms. The ENCODE project showed that at least 93% of analyzed human genome nucleotides are transcribed in different cells, with similar findings in mice and other eukaryotes, which indicate that there may be a vast reservoir of biologically meaningful RNAs that could greatly exceed the ~1.2% encoding proteins. A fraction of RNAs with short open reading frames (ORFs) potentially encodes peptides but on the other side of the ledger many currently annotated ORFs are not conserved and may be false, which could reduce the number of protein-coding genes in the human genome. There has been debate about whether these ncRNAs are (in the main) functional or simply noise. In some cases, it may be the transcript or merely the act of transcription, or both, that are relevant. Nevertheless, many observations indicate that substantial numbers of ncRNAs are intrinsically functional. These include the fact that many loci produce spliced  transcripts that are developmentally regulated. A large fraction of ncRNAs are expressed in specific regions of the brain, exhibiting precise cellular locations. Some mark new domains within the cell, which means that ncRNAs are also set to have a major impact in cell biology. Comparative analyses indicate that ncRNA promoters are, on average, more conserved than those of protein-coding genes and that ncRNA sequences, secondary structures, and splice site motifs have been subject to purifying selection. Moreover, many ncRNAs are evolving quickly, and some have undergone recent positive selection, as exemplified by HAR1 RNA expressed in the human brain, which contains the sequence conserved in mammals that most rapidly diverged after the human-chimpanzee separation. Although the need for large-scale approaches to explore the function of ncRNAs is evident, a glance at the genome browser will show noncoding expressed sequence tags associated with most genes of interest that may have regulatory functions. ncRNAs are already being identified as markers for cancer and associated with other complex diseases such as coronary disease, diabetes, and Alzheimer's. The elucidation of their function may significantly contribute to the understanding and treatment of such conditions. It may also transform our understanding of the genetic programming of multicellular organisms, particularly as it appears that regulation dominates the information content of complex systems . The predisposition to and the development of cerebrovascular diseases involves the dynamic interplay between environmental and intrinsic vascular, systemic, and CNS risk factors. Increasing evidence suggests that disruption of these homeostatic and plasticity events involves an array of deregulated epigenetic processes . Appreci≈20–25 nucleotides) RNA molecules identified as micro-RNAs (miRNAs) [The most recently recognized category of epigenetic mechanisms includes the pathways involved in the transcription, processing, and action of a class of short ((miRNAs) . MicroRN(miRNAs) . Differe(miRNAs) . This ob(miRNAs) , suggestMultipotent mesenchymal stromal cells (MSCs) have potential therapeutic benefit for the treatment of neurological diseases and injury. MSCs interact with and alter brain parenchymal cells by direct cell-cell communication and/or by indirect secretion of factors and thereby promote functional recovery. In another study, using Multipotent mesenchymal stromal cells (MSCs) treatment of rats subjected to middle cerebral artery occlusion (MCAo) significantly increased microRNA 133b (miR-133b) level in the ipsilateral hemisphere. In vitro, cultured neurons treated with exosome-enriched fractions from MSCs exposed to MCAo brain extracts significantly increased the neurite branch number and total neurite length. This study provides the first demonstration that MSCs communicate with brain parenchymal cells and may regulate neurite outgrowth by transfer of miR-133b to neural cells via exosomes .Further, microRNAs (miRNAs) regulate formation of myelinating oligodendrocytes. Overexpression of miR-219 and miR-338 as oligodendrocyte-specific miRNAs is sufficient to promote oligodendrocyte differentiation. These findings illustrate that miRNAs are important regulators of oligodendrocyte differentiation, providing new targets for myelin repair . ANRIL (NCBI EntrezGene 100048912) is an lncRNA with an unknown function that is associated with the development of atherosclerosis, diabetes, and aneurysms, possibly through effects on vascular smooth-muscle proliferation and migration. GOMAFU (NCBI Entrez Gene 440823) is another lncRNA that is expressed in the nucleus of developing neural cells. Although the function of GOMAFU is unknown, a case-control association study identified a single nucleotide polymorphism associated with the GOMAFU locus as a susceptibility factor for cardiovascular disease [ Long ncRNAs represent another important and emerging subclass of ncRNAs that may also play a role in stroke. Long ncRNAs have roles in local and long-range chromatin remodelling, transcriptional regulation, and alternative splicing and other forms of post transcriptional RNA processing . They ar disease . In addi disease .  FigureThis process is intimately linked with ncRNA expression and is another epigenetic process, RNA editing, a mechanism for altering nucleotides in RNA molecules that allows the generation of significant diversity of transcripts in a highly environmental-responsive manner. In transient global cerebral ischemia models, the death of hippocampal CA1 pyramidal neurons is mediated by selective downregulation of an RNA-editing enzyme leading to defective editing of the ionotropic glutamatergic -amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid GluR2 receptor subunit, which influences the vulnerability of hippocampalCA1 pyramidal neurons to ischemia-associated cell death . By alteAPOB (NCBI Entrez Gene 338) mRNA, which encodes an important apolipoprotein found in chylomicrons and low-density lipoproteins. Mutations of the APOB gene and its regulatory region cause dyslipidemias , and genetic variants of APOBEC1 and APOBEC2 are associated with high levels of serum low-density lipoproteins and increased atherosclerosis [APOB mRNA editing; however, APOBECs may play additional roles within the brain. APOBEC-mediated DNA recoding protects the stability of the genome and also enhances its diversity and plasticity [ In addition, the apolipoprotein-B-(ApoB-) editing catalytic subunit (APOBEC) family of RNA editing and DNA recoding enzymes may also play a role in stroke. These enzymes are cytidine deaminases that edit (deoxy)-cytidine to (deoxy)-uridine and act on RNA and DNA molecules . One of clerosis . The APOasticity . Althougasticity . Furtherasticity . Also, lasticity . This suggests a possible evolutionary mechanism to account for the multigenerational inheritance of complex cognitive and behavioural traits and risk profiles for stroke in response to both productive and adverse environmental events. Apart from the molecular mechanisms responsible for epigenetic regulation, a broad variety of evidence has implicated epigenetic regulation in long-term environmental influences on gene regulation. One of the best-known such examples is the epidemiological work of Pearce, who identified a cohort of Dutch individuals with a uniquely elevated risk of coronary artery disease . The comTransient global cerebral ischemia (TGCI) following systemic hypoperfusion is associated with selective and delayed death of hippocampal CA1 pyramidal neurons through the mediation of a series of parallel epigenetic processes. Within vulnerable neurons, there is selective downregulation of ADAR2 and defective Q/R site editing of the ionotropic glutamatergic AMPA, GluR2 receptor subunit, resulting in the expression of the death-promoting calcium permeable GluR2 isoform and associated impairment in GluR2 mRNA and protein expression, receptor assembly, membrane trafficking, and synaptic targeting. Heterogeneity in ADAR2- mediated GluR2 Q/R site editing enhances the vulnerability of hippocampal CA1 pyramidal neurons to global ischemia-associated neurodegeneration. In parallel, TGCI induces the selective expression of REST within these vulnerable neurons with associated suppression of GluR2 and the CA1- selective m-opioid receptor 1 (MOR1) in inhibitory interneurons through a series of histone modifications, including MOR1 promoter H3/4 deacetylation, H3K9 dimethylation and associated recruitment of the G9a histone methyltransferase. This has been postulated to represent a failed attempt of inhibitory interneurons to dampen the excitotoxicity of CA1 pyramidal neurons by disinhibiting GABA release. Ischemia-induced alterations in the histone code may be the result of early dephosphorylation and inactivation of components of the neuronal ERK1 and CREB1 signal transduction pathways that simultaneously reduce expression of the antiapoptotic, bcl2 gene and activate expression of the proapoptotic, caspase-3 effector pathway –83. There is also evidence that the more common type of focal stroke syndrome due to occlusion of the middle cerebral artery is associated with aberrant DNA methylation and histone H3 deacetylation, and that systemic administration of a potent HDAC inhibitor reduces the volume of the ischemic infarction whereas concurrent application of an HDAC inhibitor with a DNA demethylating agent confers neuroprotection against mild but not severe ischemic injury . IncreasTransient focal ischemia in adult rat brain regulates the expression of microRNAs predicted to target proteins known to mediate inflammation, transcription, neuroprotection, receptor function, and ionic homeostasis in the brain. The mRNA levels for proteins important to microRNA biogenesis pathways, including Drosha, Dicer, the cofactor Pasha, and the precursor microRNA transporter Exportin 5, were not altered after transient ischemia. However, transient ischemia repressed miR-145 expression, which resulted in increased translation of its mRNA target, superoxide dismutase-2, in post-ischemic adult rat brain. It is interesting to note that in silico studies revealed eight microRNAs induced by transient ischemia with complementarity to 877 gene promoters, suggesting that microRNAs also regulate gene expression . There iWhole genome expression microarrays can be used to study gene expression in blood, which comes in part from leukocytes, immature platelets, and red blood cells. Since these cells are important in the pathogenesis of stroke, RNA provides an index of these cellular responses to stroke. Human studies show gene expression changes following ischemic stroke. These gene profiles predicted the cause of stroke in 58% of cryptogenic patients. New techniques to measure all coding and noncoding RNAs along with alternatively spliced transcripts will markedly advance molecular studies of human stroke .Platelets are crucial for the maintenance of haemostasis and contribute to thrombosis and vessel occlusion that underlies stroke and acute coronary syndromes. Although platelets are anucleate, they do contain mRNAs and are capable of protein synthesis . Human pMutations in mitochondrial DNA are responsible for a spectrum of mitochondrial encephalomyopathies, including mitochondrial encephalopathy with lactic acidosis and stroke like episodes. Although the DNA sequences that harbor these mutations generally do not code for proteins, many of them encode transfer (tRNAs) and ribosomal RNAs (rRNAs). The array of clinical symptoms seen in mitochondrial disorders highlights the functional importance of nonprotein-coding RNAs (ncRNAs) such as tRNAs and rRNAs that are transcribed from nonprotein-coding DNA sequences. In fact, the pathogenesis of a spectrum of neurodevelopmental, neurodegenerative, and neuropsychiatric diseases is increasingly being associated with mutations of ncRNAs .The brain is a conspicuous consumer of energy resources, and a major consequence of cerebral ischemia is the disruption of energy metabolism and exhaustion of adenosine triphosphate. Because RNA can rapidly be activated, modified, transported, and degraded, it serves as a highly flexible, high fidelity, information encoding, and functional molecule. The ability of RNA molecules to dynamically store, transform, and transmit both “digital” and “analogue” information is a key feature of RNA-based systems .Studies support the potential for microRNAs as novel biomarkers for vascular injury and diseases. Expression profiling of microRNAs in ischemic rat brains revealed significant changes in several micro-RNAs, and some of the microRNAs highly expressed in ischemic brain were detected in blood samples . PeripheEvidence also suggests that microRNAs serve as effectors in neointimal lesion formation, and in angiogenesis in normal and injured brain. The miR-17–92 cluster is highly expressed in human endothelial cells and miR-92a, a component of this cluster, targets several mRNAs for proangiogenic proteins. Overexpression of miR-92a in endothelial cells blocked angiogenesis, and systemic administration of an miR-92a antagomir led to enhanced blood vessel growth and functional recovery of damaged tissue in mouse models of limb ischemia and myocardial infarction . In a siFor treatment of stroke, RNA-based therapies and additional epigenetic strategies are extremely promising. Indeed, approaches for gene silencing that use short regulatory ncRNAs, including miRNAs and related short interfering RNAs (RNA interference) have already been used to identify new molecular targets for treating stroke, such as Bcl-2 and 19-kDa interacting protein 3  and carbμ opioid receptor 1 [RNA regulons. In postischemic neurons, vulnerability to cell death is associated with pathological alterations in RNA operon and regulon dynamics and stress responses that lead to translational [In addition, REST and CoREST modulate the expression of several classes of ncRNAs, including miRNAs and lncRNAs. These molecules act as dynamic modular platforms for the recruitment of a broad array of epigenetic factors to neural gene loci in which they orchestrate site-specific and genome-wide chromatin remodelling. One of the molecular mechanisms that underlie cell death after transient global ischemia is REST dependent repression of the GluR2 subunit and ceptor 1 , 105. REceptor 1 . These oceptor 1 . Furtherceptor 1 . Higher-lational . These olational . Specifilational . These mlational . The forlational . Thus, ulational . Studies of epigenetic mechanisms in stroke are in their infancy but offer great promise for better understanding of stroke pathology and the potential viability of new strategies for its treatment. Correspondingly, inhibitors of histone modification have been suggested to be neuroprotective in animal models of cerebral ischemia and intracranial haemorrhage. In turn, miRNAs have been shown to play diverse roles in neuronal, glial, and endothelia responses to stroke. In addition, miRNAs have been suggested to regulate the effects of ischemia on aquaporin expression and function and in some cases may be neuroprotective. miRNAs may also help explain gender-based differences in responses to cerebral ischemia. RNA editing, a related epigenetic mechanism that is partly responsible for generating the exquisite degrees of environmental responsiveness and molecular diversity. In addition, the development of future therapeutic strategies for locus-specific and genome-wide regulation of genes and functional gene networks through the modulation of RNA transcription, posttranscriptional RNA processing , and RNA translation. These novel approaches for neural cell- and tissue-selective reprogramming of epigenetic regulatory mechanisms are likely to promote more effective neuroprotective and neural regenerative responses for safeguarding and even restoring central nervous system function. Data accumulated to date strongly suggest that further studies of these mechanisms are well justified and that future publications resulting from these studies are worthy of careful attention.


Stress and anxiety play an important role in the development and maintenance of drug and alcohol addiction. The bed nucleus of the stria terminalis (BNST), a brain region involved in the production of long-term stress-related behaviors, plays an important role in animal models of relapse, such as reinstatement to previously extinguished drug-seeking behaviors. While a number of neurotransmitter systems have been suggested to play a role in these behaviors, recent evidence points to the neuropeptide corticotropin releasing factor (CRF) as being critically important in BNST-mediated reinstatement behaviors. Although numerous studies indicate that the BNST is a complex brain region with multiple afferent and efferent systems and a variety of cell types, there has only been limited work to determine how CRF modulates this complex neuronal system at the circuit level. Recent work from our lab and others have begun to unravel these BNST neurocircuits and explore their roles in CRF-related reinstatement behaviors. This review will examine the role of CRF signaling in drug addiction and reinstatement with an emphasis on critical neurocircuitry within the BNST that may offer new insights into treatments for addiction. Alcohol and drug addiction are chronically relapsing disorders in which alcohol/drug use progresses from initial stages of limited, non-dependent intake to later stages of uncontrolled abuse Koob, . One proStressors and negative affective states, such as anxiety and depression, are often cited by recovering addicts as key instigators of drug craving and relapse Sinha, . Drug/alAltogether, these ideas support an important role of stress-related neurocircuitry in the progression of addiction and in relapse. Clinical studies on relapse have been paralleled and now extended in preclinical studies utilizing reinstatement models  appears to contribute to the use of a number of different drugs. Acute and chronic alcohol/drug exposures and withdrawal increase CRF biosynthesis in the CeA -Crhtm1(cre)Zjh/J) line and the ROSA-tomato [strain B6.Cg-t(ROSA)26Sor < tm14(CAG-tdTomato)Hze > /J] line. Crossing these two lines of mice resulted in offspring where a red fluorescent protein (tomato) was targeted to cre containing neurons, which in this case were neurons that produced cre under the control of the endogenous Crf promoter/enhancer elements (CRF-tomato mice). The CRF-tomato mice were found to have high levels of tomato expression in brain areas known to be dense in CRF-producing neurons, like the paraventricular nucleus of the hypothalamus, the CeA, and the BNST, while brain regions that are known to have little CRF-producing neurons, like the cortex and striatum, were shown to have sparse tomato expression.While it has been established that elevated CRF levels in the BNST are important for stress-induced reinstatement, one remaining question is the source of elevated extracellular CRF in the BNST in response to stress exposure. CRF could be released from local neuronal sources, from extrinsic CRF projections from the CeA, or both . This repeated ethanol exposure/withdrawal paradigm has been shown to increase anxiety-like behaviors during withdrawal  are GAD+/VGlut− while other subtypes are VGlut2+/GAD− or VGlut3+/GAD+ (Kudo et al., Some electrophysiological studies indicate that BNST projections to the VTA are likely to be glutamatergic, as they enhance VTA neuron firing (Georges and Aston-Jones, Overall these findings indicate that the BNST sends a mixture of neurotransmitters to the VTA. However, what is less clear is whether distinct types of BNST projection neurons synapse to different VTA neurons. Recent evidence indicates that selective optogenetic stimulation of VTA GABA neurons disrupts reward consumption (van Zessen et al., The precise role of distinct VTA-projecting BNST neurons in reinstatement is not yet fully understood. For instance, although evidence suggests that BNST neurons that project to the VTA can be mainly GABAergic, but also glutamatergic or potentially both (Kudo et al., In addition to its role in stress-induced reinstatement described above, recent evidence may suggest that BNST CRF neurocircuitry could also play a role in cue-induced reinstatement. BLA DA receptor activation is critical for cue-induced reinstatement (See et al., Importantly, behavioral evidence also shows a potential role for the BNST in cue-induced reinstatement models. For instance, recent findings indicate that pharmacological inactivation of the BNST can reduce cue-induced reinstatement (Buffalari and See, Interestingly, while clinical evidence shows that exposing recovering addicts to drug-associated cues results in enhanced feelings of craving, recent findings indicate that these same cues also increase feelings of negative affect (Fox et al., The findings reviewed here suggest that a catecholamine-CRF-glutamatergic signaling pathway in the BNST plays an important role in the reinstatement to drug-seeking behavior, an important animal model of relapse to alcohol/drug addiction. While this pathway is clearly important in stress-related behaviors, especially in stress-induced reinstatement, further studies suggests that this pathway may also be important in cue-induced reinstatement. Therefore, pharmacotherapies targeting this pathway may be useful in the prevention of relapse to both drug-associated cues and stressors. Unfortunately, relapse can be a life-long struggle in recovering addicts, which means that pharmacotherapies to prevent relapse likely need to be taken daily for extended periods of time. Therefore these therapies need to be well-tolerated and devoid of harsh side-effects. As described earlier, agonist therapies targeting the DA aspect of this pathway may be problematic from the side-effect standpoint due to effects on the cardiovascular system and abuse liability. DA antagonist therapies are also problematic for their potential for extra-pyramidal (Peacock et al., Since DA and β-AR activation enhances BNST activity via CRFR1 activation, then CRFR1 antagonists might be a better alternative for the effective long-term prevention of both cue and stress-induced relapse. CRFR1 antagonists have been shown to reduce ethanol intake following withdrawal in a number of preclinical studies (Funk et al., 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.


Dendritic cells (DCs) initiate and control immune responses, participate in the maintenance of immunological tolerance and are pivotal players in the pathogenesis of autoimmunity. In patients with autoimmune disease and in experimental animal models of autoimmunity, DCs show abnormalities in both numbers and activation state, expressing immunogenic levels of costimulatory molecules and pro-inflammatory cytokines. Exogenous and endogenous danger signals activate DCs to stimulate the immune response. Classic endogenous danger signals are released, activated, or secreted by host cells and tissues experiencing stress, damage, and non-physiologic cell death; and are therefore referred to as damage-associated molecular patterns (DAMPs). Some DAMPs are released from cells, where they are normally sequestered, during necrosis . Others are actively secreted, like Type I Interferons. Here we discuss important DAMPs in the context of autoimmunity. For some, there is a clear pathogenic link . For others, there is less evidence. Additionally, we explore emerging danger signals. These include inorganic materials and man-made technologies  developed as novel therapeutic approaches. Some nanomaterials can activate DCs and may trigger unintended inflammatory responses. Finally, we will review “homeostatic danger signals,” danger signals that do not derive directly from pathogens or dying cells but are associated with perturbations of tissue/cell homeostasis and may signal pathological stress. These signals, like acidosis, hypoxia, and changes in osmolarity, also play a role in inflammation and autoimmunity. Box 1).Dendritic cells (DCs) are specialized antigen-presenting cells (APCs) that help maintain peripheral tolerance and orchestrate the adaptive immune response by presenting both endogenous and exogenous antigens and producing immune modulatory factors, such as costimulatory/inhibitory molecules and cytokines  expressed on both immune and non-immune cells. Since PAMPs are also expressed by the normal flora of mammalian mucosas, they are also called Microbial Associate Molecular Patterns (MAMPs).Damage-associated molecular pattern: endogenous molecular structures that are normally contained within the cell interior and hidden from the immune system, and are liberated upon tissue damage. Examples include ATP, HSPs, and HMGB1. These molecules are recognized by a number of receptors, including PRRs, and are capable of inducing inflammation and immune responses in the absence of infection.Danger signal: a danger signal may be any substance or event that is able to activate DCs and therefore initiate immune responses. For the purposes of this review, we have split the danger signals into three categories shown below. They all have in common an association with cellular stress and the ability to activate DCs.Classic danger signals: we use the term “classic” to refer to danger signals that are not emerging or homeostatic (defined below). The classic danger signals are molecular species either associated with pathogens (PAMPs) or directly derived from tissue injury damage-associated molecular patterns (DAMPs) as described above, or secreted by activated immune cells as amplifiers of the immune activation.Emerging danger signals: we use the term “emerging” to turn attention to new fields of research and technology that may pose new challenges to the immune system. Specifically, emerging danger signals include inorganic materials and man-made technologies  often used as novel therapeutic strategies. These materials have the potential to activate DCs directly or indirectly by inducing tissue damage and release of DAMPs.Homeostatic danger signals: with the term “homeostatic” we aim to highlight a less appreciated category of endogenous danger signals. Homeostatic danger signals generally represent perturbations in tissue steady state often as a result of inflammation. These perturbations include (but are not limited to) hypoxia, changes in acidity, or osmolarity, and metabolic stress.The recognition of tissue and cell damage – which may involve several types of innate immune cells beside DCs and cascades such as the complement – has been proposed to initiate multiple processes Bianchi, , includiDendritic cells collaborate with other cells of the innate immune system to initiate what is often called “sterile inflammation and immunity;” that is, the activation of the innate and adaptive immune response in the absence of pathogens , self-antigen-loaded DCs induces, or accelerates autoimmunity . As DCs are central to antigen presentation and the initiation of the immune response, it was first shown that DCs respond to endogenous danger signals released from dying cells . Some cytokines act as both primary and secondary danger signals, when they are secreted by damaged/dying cells (Box 2). Many primary endogenous danger signals are released from the interior of the cell when the cell loses plasma membrane integrity upon necrosis . Nucleic acids are both the primary self-antigens and potent DAMPs, activating DCs through TLR7 and TLR9 and inducing the production of Type I IFNs  murine embryonic fibroblasts accumulate endogenous retroviral DNA in the cytosol which induces excessive Type I IFN production via STING . EVEs are sections of viral DNA that have integrated into the mammalian genome during evolution Stoye, . The bes7  by ectonucleotidases  is a well-established endogenous danger signal , SLE, EAE, and diabetes  are disrupted leading to the production of low molecular weight degradation products such as hyaluronic acid (HA) and heparan sulfate, which have been shown to activate DCs and initiate inflammation , but exogenous particles not derived from microbes, such as silica, crystals, and nanoparticles can also induce DC activation and inflammatory disease. Indeed, it has long been known that inhalation of fine particulate material can cause inflammatory and fibrotic lung disease . The inflammatory nature of non-living particulate matter, like crystals and nanoparticles, may stem in part from their hydrophobic character  or inorganic materials  and engineered to display a molecule of interest or contain a therapeutic payload. Using nanoparticles and synthetic material systems, it may be possible to modulate the immune response through direct manipulation of DCs  to form what is termed a bio-corona . During localized inflammation  or tissue ischemia  extracellular pH can reach pH values as low as 5.5  (Garber and Cahalan, + and Cl− ions and RVD (Compan et al., Earlier studies demonstrated that hypotonicity influence immune cell function, for example in neutrophil shedding of L-selectin (Kaba and Knauf, in vivo in the context of autoimmunity.In contrast to hypotonicity, high salt concentrations do not seem to activate inflammasome activation, as hypertonic solutions are not able to induce caspase-1-dependent release of cytokines IL-18 and IL-1β (Compan et al., Decreased oxygen tension is another characteristic disturbance of inflamed or damaged tissue (Nizet and Johnson, A number of lines of evidence suggest that hypoxia acts as a homeostatic danger signal. Low oxygen levels leads to the generation of ROS, affecting both metabolism and mediators of inflammation. Furthermore, it is well established that HIF-1α signaling is closely tied to NF-kB signaling. In neutrophils, survival in hypoxic microenvironments, such as those found in infected tissue, is mediated by HIF-1α-dependent NF-kB activation (Walmsley et al., Studies of DC responses to hypoxia are at times conflicting. It is unclear if hypoxia in itself activates DCs (Jantsch et al., HIF-1α expression is associated with a number of inflammatory and autoimmune diseases, including within the synovium of RA patients (Hollander et al., N-formyl peptides which mimic bacterial peptides, and ATP as described above (Arnoult et al., As a number of excellent reviews on mitochondria-derived danger signals have recently been published (Arnoult et al., Mitochondrial-derived danger signals are associated with the inflammatory response upon tissue damage and have been implicated in the inflammatory response to trauma. Severe traumatic injury may result in SIRS, a life threatening condition in which extensive tissue injury leads to systemic inflammation and shock reminiscent of sepsis (Stoecklein et al., Mitochondrial-derived CpGs are also an important danger signal in autoimmune disease. For example, injecting mtDNA into the joints of mice induces myeloid cell-mediated arthritis, but injection of nuclear DNA does not (Collins et al., Furthermore, as mitochondria are central to metabolism and cellular energy production, they represent a key sensor of homeostatic perturbations (Desai et al., Perturbations in homeostasis or mitochondrial function also lead to the generation of ROS, which are an important danger signals in their own right. ROS are normal byproducts of respiration, but may also function as signaling molecules and antimicrobial agents under controlled conditions (Valko et al., In addition to being a source and sensor of danger signals, mitochondrial dysfunction may directly influence the course of autoimmune and autoinflammatory diseases. This seems to be due to the participation of mitochondria in regulating the inflammasome and cell death. Indeed, inflammasome activation in the context of monogenic autoinflammatory diseases has been suggested to cause mitochondrial dysfunction (Escames et al., The immune response is often described as a double-edge sword. Too little immunity leads to uncontrolled infections, while too much immunity results in inflammatory damage, autoinflammatory disease, and some forms of autoimmunity. The delicate balance needed to keep immunity within this narrow range is the result of complex biochemical interactions. A threshold likely exists whereby tonic factors suppress noise in the system and other factors amplify or regulate immunologic signals in order to have a quick and efficient immune response Germain, . Danger Danger signals are therefore diverse in form and function. Indeed, there are a number of conceptual frameworks in which danger signals are characterized, all of which have been extremely useful Janeway, . Here weThe 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.


Current treatment of bacterial and fungal infections heavily relies on strategies which aim to inhibit and kill pathogens with high specificity. These strategies are very successful and antibiotics have contributed to increasing human life expectancy more than any other class of therapeutic drugs. However, antibiotics are losing efficacy as a result of high selection pressure and rapid resistance development. Thus, strategies that rely on boosting natural host defenses are gaining more attention, since compounds targeting host mechanisms should control infections regardless of the antibiotic resistance levels of pathogens. Antimicrobial peptides (AMPs) are considered as ideal candidates for such novel anti-infective strategies since they combine direct antibiotic activities with modulation of immune responses Figure . Howeversensu stricto belong to the latter class and were first isolated from mammals, and subsequently also found in invertebrates and plants.All multicellular organisms produce AMPs to protect surfaces and tissues from invading pathogens. These peptides have been referred to as AMPs and more recently as host defense peptides (HDPs). HDPs are ancient effector molecules of innate immunity with multiple functions. They do not share specific sequence similarities, but can be generally defined as amphiphatic cationic peptides consisting of 12–50 amino acids. They are either linear  or have tertiary structures stabilized by disulfide bonds  is also involved in the biosynthesis of other major cell envelope polymers . Synthesis of C55P-anchored molecules always starts with the transfer of a sugar moiety to the lipid carrier, forming a pyrophosphate linkage. This structural motif is highly conserved, as it is part of several essential building blocks and therefore cannot be easily modified to confer resistance.Cell wall biosynthesis is a prominent target of clinically used antibiotics. For example, the glycopeptide vancomycin, a last-resort antibiotic for treatment of infections with multi-resistant Gram-positive bacteria, binds to the Dahlia merckii) bind specifically to inositol phosphoryl-containing sphingolipids leading to membrane permeabilization and ion efflux  with fungal glucosylceramides causes the production of radical oxygen species and apoptosis as well as cell wall stress, septin delocalization, and ceramide accumulation , lipoteichoic acid, CpG oligonuclecotides; via Toll-like receptors (TLRs)] or inflammatory effectors such as cytokines  and iseganan (from porcine protegrin-1) have been shown effective in the prevention of diabetic food ulcer and irradiation-induced oral mucositis, respectively , and formulation (e.g., in liposomes; Oren et al., in vivo half-life (Andes et al., The cationic amphiphilic and peptidic nature of AMPs is often considered unfavorable for development of systemic drugs. However, protease lability, contributing to low serum half-life, may be overcome by different approaches, including the use of peptidomimetics, peptides composed of unusual or Antimicrobial mechanisms based on defined target molecules such as lipid II reduce the risk of unspecific membrane disruption and cytotoxic activities, although HDPs clearly have some specificity for microbial membranes; eukaryotic membranes may be less susceptible due to the absence of anionic lipids on the lipid bilayer surface, the lack of a strong membrane potential gradient and the presence of cholesterol (Hancock and Sahl, S. aureus and vancomycin-resistant enterococci. Notably, these IDRs combine anti-infective and anti-inflammatory properties. IDR-1 and IDR-1002 contribute to bacterial clearance by inducing chemokines secretion and enhancing leukocyte recruitment. Moreover, the peptides suppress the induction of several proinflammatory cytokines, thereby dampening immune-mediated inflammation and preventing tissue damage (Scott et al., The increasing knowledge of the importance of immunomodulatory HDP functions, has led to the synthesis of so called innate defense regulator peptides (IDRs; Easton et al., Taken together, it appears a most promising approach to design future anti-infective drugs that target host defenses and may combine this with targeted antibiotic activities, even more since classic antibiotics such as macrolides also appear to have immune modulatory properties (Tauber and Nau,


O-methyl modification at their 3´ termini, and this is suspected to complicate their accurate quantification. miRNA precursors have been detected in cell lines and other tissues, where their amount does not necessarily correlate with the amount of their mature miRNA due to different regulation processes controlling their biogenesis. Methods currently used to identify and quantify sRNAs face unique challenges due to their short length, as well as the high sequence similarities between them. The correct identification, discrimination and profiling of the different types of sRNAs, including their available isoforms, are crucial for understanding of the regulatory networks they are involved in.Small RNAs (sRNAs) are known to play an important regulatory role in a wide range of organisms and biological processes through expression regulation of a diverse array of genes. Several classes of sRNA including plant microRNAs (miRNAs), small interfering RNAs (siRNAs) and Piwi-interacting RNAs (piRNAs) carry an O-methyl 3'modification.To compare between three high-throughput commercial platforms (Agilent and Affymetrix microarrays and Illumina next-generation sequencing (NGS)), two different approaches were applied: (a) endogenous human placenta miRNA quantification and ranking were assessed across the platforms, and (b) 12 synthetic transcripts were artificially spiked into human placenta total RNA at known input amounts and further analyzed. These transcripts included six mature miRNAs, three precursor miRNAs and three miRNAs carrying an O-methyl modified miRNAs in comparison with their matching mature miRNAs; nevertheless modified miRNAs are biologically active. In contrast to the expected, precursor miRNAs were detected, although mostly at low levels. Such signals can be erroneously interpreted as mature miRNA in the Agilent and NGS platforms.Clear differences in the endogenous miRNA expression ranking were observed between the three platforms, yet Agilent and NGS showed the highest fit. When ranking miRNA expression values, none of the miRNAs were similar across all platforms. Furthermore, all the miRNAs diverged in expression ranking between at least two platforms. To understand these differences, a detailed exploration of miRNA attributes related to sequence structure and composition, including the number of observed isoforms different from the canonical miRNA, was performed. We demonstrate that the percentage of G, T, A, GG, TT, GC and TA in the sequence, RNA folding energy, number of isoforms, mismatches and 5' insertions per miRNA were significantly different between different ranking levels. For the three platforms used, the amounts of spiked-in miRNAs introduced and detected demonstrated linear correlations. However, there was a large variation in the intensity values observed for different spiked-in miRNAs at a certain concentration: up to 500 in Affymetrix, 10 in NGS, and 5 in Agilent. All platforms have a reduced capability (as low as 6%) of detecting the spiked-in All tested platforms do not capture the absolute miRNA quantity and therefore are applicable for relative but not for absolute abundance studies. The power of miRNA detection strongly depends on the platform used and on miRNA sequence attributes and modifications.


Post operative atrial fibrillation (POAF) is more common than before due to increased numbers of cardiac surgeries. This in turn is associated with increased incidence of post operative complication, length of hospital stay and subsequent increase the cost of hospitalization. Therefore preventing and/or minimizing atrial fibrillation by pharmacological or nonpharmacological means is a reasonable goal. POAF has also been associated with postoperative delirium and neurocognitive decline. The precise pathophysiology of POAF is unknown, however most of the evidence suggests it is multifactorial. Different risk factors have been reported, and many studies have evaluated the prophylactic effects of different interventions. This review article highlights the incidence, risk factors, and pathogenesis, prevention, and treatment strategies of POAF. Postoperative Atrial Fibrillation (POAF) is more common than before due to the increased number of cardiac surgeries. This in turn is associated with an increased incidence of postoperative complications, length of hospital stay, and subsequent increase in the cost of hospitalization. Therefore, preventing and / or minimizing atrial fibrillation by pharmacological or non-pharmacological means is a reasonable goal.2Postoperative atrial fibrillation has also been associated with postoperative delirium and neurocognitive decline.4 The preThe true incidence of postoperative atrial fibrillation (POAF) following cardiac surgery is unclear. Reported incidences range from 10 - 65%. This range is wide, because studies that examined Atrial Fibrillation (AF) following coronary artery bypass graft (CABG) differ in baseline patient characteristics, type of surgery, methods of detection, and definitions of AF. j27 j2627]Beta-blockers have been the most studied drugs to date, for the prevention of POAF. These drugs are primary in AF prevention and should be used routinely in every patient.29 Severa34P < 0.0001); Furthermore, there was also a significant reduction in ventricular tachyarrhythmia, strokes, and hospital stay. The authors concluded that in the absence of a contraindication, prophylactic amiodarone should be implemented as a routine therapy for high-risk patients undergoing cardiac surgery.[Amiodarone is a class III antiarrhythimic drug that also has beta and alpha adrenergic-blocking properties. It plays a role in controlling the sympathetic overstimulation seen in patients undergoing cardiac surgery. A short perioperative course of oral amiodarone added to a routine beta-blockade has proved to be a very promising approach of management. This combination therapy was associated with a 50% lower incidence of postoperative atrial tachyarrhythmia in patients undergoing valve replacement / repair and /or CABG. In a large prospective trial, the Prophylactic Oral Amiodarone for the Prevention of Arrhythmias that Begin Early After Revascularization, Valve Replacement, or Repair (PAPABEAR), the number needed to treat was only 7.5, to prevent one patient from developing POAF. The resu surgery.There are three mechanisms by which atrial pacing decreases and / or prevents AF:Avoiding the trigger for AF by suppressing atrial premature beatsReduction of bradycardia-induced dispersion of atrial depolarization that contributes to the electrophysiological substrate for AFUsing dual atrial pacing, such mechanisms minimize and / or prevent the development of intra- atrial entry, and hence, AF. Meta-analyses,–40 demonNumerous studies have evaluated the nondihydropyridine calcium-channel blocker. The finding shows that calcium-channel blockers reduce the risk of supraventricular tachyarrhythmia. However, some studies that suggested using this drug preoperatively found an increased incidence of atrioventricular (AV) block and low-output syndrome, which was attributed to the negative inotropic and chronotropic effects of this medication. Therefore, the use of these agents should be considered with caution until more information on their safety profile become available.Several studies point out that statins reduce inflammation in patients with coronary artery disease. Observational studies have previously suggested that patients on statin therapy have a lower incidence of POAF after CABG. The prosExperimental studies in rats and dogs showed that polyunsaturated fatty acids (PUFAs) have significant antiarrhythmic effects on the atrial muscle.44 Furtheet al,[A randomized trial by Calo et al, showed tCorticosteroids have anti-inflammatory properties; hence it has been studied for the treatment of POAF. Clinical trials in the past have shown significant reduction in the incidence of POAF in those patients who received steroid compared to controls. There is an increase in inflammatory mediators postoperatively, which predisposes the development of POAF in susceptible patients. A multicenter trial, consistiet al,[A meta-analysis by Miller et al, suggesteEuropean Heart Journal two years ago.[N-acetylcysteine (NAC) is an antioxidant agent that minimizes cellular oxidative damage.50 It hasAlthough POAF is transient and self-limiting, treatment is indicated for patients who are hemodynamically unstable, and who develop cardiac ischemia or heart failure. The current treatment includes control of ventricular rate, and restoring / maintaining sinus rhythm in addition to prevention of thromboembolism.Most of the recent evidence suggests that rhythm control is better than rate control.The reason being that rhythm control maintains the patients in sinus rhythm, with decreasedlength of hospital stay. DifferenIf POAF results in acute heart failure, myocardial ischemia or hemodynamic instability urgent electrical cardioversion should be performed. It should also be considered electively to restore sinus rhythm after the first onset of AF, if the pharmacologic trial has failed to resume a sinus rhythm.The postoperative recovery period is characterized by increased autonomic nervous system activity and adrenergic stress, and this in turn makes it difficult to control the ventricular rate in patients with POAF. Beta blockers are the therapy of choice, particularly in patients with ischemic heart disease; however, beta blockers are relatively contraindicated or poorly tolerated in patients known to have asthma or Bronchospasms, acute decompensated heart failure, or high-grade atrioventricular (AV) conduction block. Alternatively, other AV nodal blocking agents can be tried instead. Their dosages and side effects are presented in .Postoperative Atrial Fibrillation is associated with an increased risk of perioperative strokes,59 but thPostoperative Atrial Fibrillation is the most common arrhythmia after cardiac surgery. The frequency of this arrhythmia is increasing, most likely due to the increasing number of elderly patients undergoing cardiac surgery.Currently, there are significant variations in the prevention strategies for POAF, with varied supportive evidence. The most recent evidence suggests that beta-blockers are effective, safe, and can be used in most patients. Therefore, unless contraindicated, beta-blockers should be continued perioperatively or initiated in all patients. In addition, amiodarone, statins, N-3 PUFAs, or NAC, may be used with beta-blocker as an adjunctive therapy. Such a combination therapy has been shown to be beneficial in reducing POAF.


Persistent patterns of neural activity that last long after the offset of a stimulus are thought to be the neural substrate for short-term memory. Because the observed decay of persistent activity in memory circuits is much slower than the typical decay time constants associated with synaptic or intrinsic neuronal dynamics, it has been suggested that network interactions must be used to prolong the duration of persistent activity. Most often, these network interactions have been assumed to mediate positive feedback between neurons that supports a long-lasting reverberation of activity. However, most positive feedback models do not naturally fit the architecture of working memory-storing structures in neocortex that have been suggested to exhibit a close balance between excitation and inhibition. Furthermore, positive feedback models of analog memory storage are highly non-robust against commonly studied perturbations in network connectivity.Here, we suggest a complementary mechanism for generating persistent activity based on the principle of corrective negative-feedback: an error-correcting signal of the form of a time-derivative of activity reduces memory slip when it occurs. Using analytic calculations, we show that neocortical circuit models with the observed balance in strength, but with different kinetics, between excitatory and inhibitory synaptic inputs, produce a negative-derivative feedback signal that counteracts drifts in persistent activity. The networks maintain a continuum of stable firing rates even in the presence of intrinsic input-output nonlinearity, while still remaining responsive to external memory inputs. More generally, the networks act as temporal integrators of their inputs, for example converting step-like input into linearly ramping activity.Memory networks operating in this balanced regime are robust against many commonly studied perturbations to synaptic weights that grossly disrupt the performance of persistent activity circuits based on positive feedback. Specifically, in response to uniform changes in synaptic excitation, synaptic inhibition, intrinsic neuronal gains or loss of a fraction of excitatory or inhibitory neurons, there is minimal decay or instability in persistent firing. Furthermore, spiking network models implementing derivative feedback generate persistent firing with Poisson-like statistics, as has been observed experimentally. This observed highly irregular activity occurs across a graded range of firing rates, and arises because the close balance between excitation and inhibition results in spikes being triggered primarily by fluctuations in, rather than means levels of, synaptic inputs.To generate experimental predictions that distinguish among different mechanisms for short-term memory, we compared the correlation structure of excitatory and inhibitory inputs in the negative-derivative feedback models to that of typical analog memory models based on positive feedback. Negative-derivative feedback models exhibit a strong positive correlation between inhibitory and excitatory synaptic inputs, as suggested by recent experiments. By contrast, similarly responding neurons in positive feedback models either exhibited anti-correlations or weak correlations between their excitatory and inhibitory inputs.Altogether, this work suggests a new paradigm for short-term memory storage based upon a balanced network with cortical-like architecture. Stabilization of responses through negative feedback is a common principle in engineering control systems. Our work suggests that a similar principle might be inherent to the circuitry of working memory systems.


Osteoporosis is characterised by low bone mass and structural deterioration of bone tissue, resulting in increased fragility and susceptibility to fracture. Osteoporotic fractures are a significant cause of morbidity and mortality. Direct medical costs from such fractures in the UK are currently estimated at over two billion pounds per year, resulting in a substantial healthcare burden that is expected to rise exponentially due to increasing life expectancy. Currently bone mineral density is the WHO standard for diagnosis of osteoporosis, but poor sensitivity means that potential fractures will be missed if it is used alone. During the past decade considerable progress has been made in the identification and characterisation of specific biomarkers to aid the management of metabolic bone disease. Technological developments have greatly enhanced assay performance producing reliable, rapid, non-invasive cost effective assays with improved sensitivity and specificity. We now have a greater understanding of the need to regulate pre-analytical sample collection to minimise the effects of biological variation. However, bone turnover markers (BTMs) still have limited clinical utility. It is not routinely recommended to use BTMs to select those at risk of fractures, but baseline measurements of resorption markers are useful before commencement of anti-resorptive treatment and can be checked 3–6 months later to monitor response and adherence to treatment. Similarly, formation markers can be used to monitor bone forming agents. BTMs may also be useful when monitoring patients during treatment holidays and aid in the decision as to when therapy should be recommenced. Recent recommendations by the Bone Marker Standards Working Group propose to standardise research and include a specific marker of bone resorption (CTX) and bone formation (P1NP) in all future studies. It is hoped that improved research in turn will lead to optimised markers for the clinical management of osteoporosis and other bone diseases. In healthy adults, SCL levels correlate positively with age, BMI, and bone mineral content and negatively with osteocalcin and calcium . SCL is An understanding of the source and magnitude of the absolute inter and intra-person variability, including biological, pre-analytic and analytical variation, of each marker is necessary to interpret serial measurements and individualise treatment.Bone turnover shows a circadian rhythm, this is more obvious in the serum and urinary markers of bone resorption. βCTX for example is highest between 01:30 and 04:30 hours and may be more than twice that at the nadir between 11:00 and 15:00 hours , this maThe existence of intra-individual low-frequency biological rhythms, imply that biomarkers can also vary between consecutive days, this is more noticeable in the urinary resorption markers . There iBone turnover varies with the menstrual cycle, research suggests that osteoblastic activity is higher during the luteal period  and boneA comprehensive drug history should also be taken into account when interpreting bone marker results. Anti-resorptive drugs such as bisphosphonates  and hormInflammatory conditions are major precipitators for bone loss, especially rheumatoid arthritis (RA) which is further aggravated by decreased functional activity and the use of glucocorticoids . In a pr12+CVA2); where CV1 is the within-subject coefficient of variation and CVA is the total analytical imprecision. LSC identifies the true physiological change in the marker. In general a change of more than twenty percent is considered significant for formation markers [In light of the above evidence it can be seen that to use bone turnover to monitor change can be quite difficult. In order to minimise problems it is best to measure the BTMs in as similar a set of circumstances as possible. Particular attention should be paid to the time of day and hence research studies tend to use early morning fasted samples. One way in which to help overcome within person variability in serial measurements and to monitor therapy is to use the ‘least significant change’ (LSC) model . LSC at  markers , similar markers .Between person variability is much harder to control, e.g. age, gender, and menopausal status, but is equally important to validate results. Bone metabolism rates are higher in infants up to three years of age, they are relatively stable throughout adolescence but sex-specific increases in bone marker levels are evident during the pubertal growth spurt and are reportedly influenced by pubertal stage rather than age . BTMs arOver the last decade many of the traditional BTM immunoassays have been automated, improving technical performance and increasing their availability. Nevertheless, analytical aspects such as within and between batch precision, accuracy and standardisation, remain problematic. Inter-laboratory variation is also crucial; a European study in 2001, measuring pooled samples of serum and urine in seventy-three laboratories concluded that even with identical assays results for the majority of the markers were significantly different . SimilarAppropriate control of sample collection and preparation is vital for successful BTM measurement. Several BTMs, especially osteocalcin and TRAP5b, are sensitive to thermo degradation and levels can be significantly reduced after only a few hours at room temperature ,23. TRAPBTMs are frequently used in clinical trials and provide valuable information on the efficacy of osteoporotic treatments, but their predictive value is limited by their large biological variation and diagnostically they are less often used for individualized patient care. Other routine laboratory investigations are frequently used to identify or exclude secondary causes of osteoporosis such as hyperparathyroidism, vitamin D status, thyrotoxicosis and hypogonadism .Currently the WHO recommends the use of BMD of the spine and proximal femur measured by DXA as the gold standard to diagnose osteoporosis and its severity . AlthougWomen generally lose about one to two percent of their bone per year after the menopause, however thirty percent lose bone at a faster rate . LongituBMD is widely used to predict osteoporotic fractures but approximately thirty to fifty percent of patients with fragility fractures have T-scores above the osteoporotic threshold . There iBMD and BTMs are independent predictors of fracture risk, recent evidence does not support the use of BTMs to select the optimal treatment, but BTMs can be used to monitor treatment efficacy before BMD changes can be evaluated. Additionally early changes in BTMs can be used to measure the clinical efficacy of an anti-resorptive treatment and to reinforce patient compliance . The effMeta-regression analysis has found no evidence of a relationship between BMD changes and reduction in risk of fractures among patients receiving calcium with or without vitamin D supplementation. Calcium and/or Vitamin D may reduce fracture rates through a mechanism independent of bone density . BTMs onThere has been considerable discussion about how long to treat with bisphosphonates, because these drugs accumulate in the skeleton, leading to a reservoir that continues to be released for months or years after treatment has stopped. These medications also result in a low bone turnover state over time with both resorption and formation reduced. This combined with concerns over microfracture, the possibility that they may prevent bone healing and the association with atypical femoral shaft fractures has led to the belief that it may not be wise to continue these medications indefinitely. It is generally accepted that the need to continue bisphosphonates be reviewed after 5 years and kept under review until ten years of treatment. Depending on the individual circumstances a decision to stop treatment, give a drug holiday or change treatment may be made. If a drug holiday is decided upon then BTMs could be checked at regular intervals, e.g. annually. Once these are rising again and especially on return to pre-treatment levels therapy could be restarted. Such an approach may be particularly useful with longer acting agents such as zoledronic acid . The BTMMore recently anabolic agents such as PTH, e.g. teriparatide, have become available which stimulate osteoblastic activity. Markers of bone formation increase early after the initiation of teriparatide therapy with a delayed, but significant, increase in resorption markers . It has During the last decade significant advances have been made in the identification and characterisation of specific BTMs for use in clinical drug trials and to aid in the therapeutic management of osteoporosis. Technological developments have greatly enhanced assay performance producing reliable, rapid, non-invasive cost effective assays with improved sensitivity and specificity. We now have a greater understanding of the need to regulate pre-analytical sample collection to minimise the effects of biological variation. The use of BTMs to select those at risk of fractures is not routinely recommended partly due to their degree of variability. However, baseline measurements of resorption markers are useful before commencement of anti-resorptive treatment e.g. bisphosphonates or denosumab and can be checked 3–6 months later to check response and adherence to treatment. Similarly a formation marker such as P1NP can be used to monitor bone forming agents such as PTH analogues. BTMs may also be useful when monitoring patients during treatment holidays and aid in the decision as to when therapy should be recommenced. The recent recommendations by the Bone Marker Standards Working Group aim to standardise research and include a marker of bone resorption (CTX) and formation (P1NP) in all future studies. They anticipate that manufacturers will calibrate their assays in future using an international reference standard to establish robust reference ranges. It is hoped that improved research in turn will lead to optimise markers for the clinical management of osteoporosis and other bone diseases. The biochemical assessment, utilizing BSAP, is now the mainstay of the diagnosis and management of metabolic bone disease in chronic kidney disease.AGEs: Advanced glycation end products; ALP: Alkaline phosphatase; BSAP: Bone specific alkaline phosphatase; βCTX: Beta-isomerised carboxy terminal telopeptide of type I collagen; BMD: Bone mineral density; BTM: Bone turnover marker; CTX: Carboxy-terminal cross-linked telopeptides of type 1 collagen; CVA: Total analytical imprecision; CV1: Within-subject coefficient of variation; DKK-1: Dickkopf-related protein 1; DMP1: Dentin matrix protein 1; DPD: Deoxypyridinoline; DXA: Dual-energy X-ray absorptiometry; EDTA: Ethylenediaminetetraacetic acid; Fz: Frizzled protein; GFR: Glomerular filtration rate; GH: Growth hormone; HELP: Type 1 collagen alpha 1 helicoidal peptide; HRT: Hormone replacement therapy; ICTP: Carboxy-terminal cross-linked telopeptide of type 1 collagen; IDS: Immuno-Diagnostic Systems Ltd; IFCC: International Federation of Clinical Chemistry and Laboratory Medicine; IGF1: Insulin-like growth factor 1; IOF: International Osteoporosis Foundation; LRP: Low-density lipoprotein receptor-related protein; LSC: Least significant change; MEPE: Matrix extracellular phosphoglycoprotein; MMP: Matrix metalloproteinases; MoM: Multiple of the median; MoMF: Multiple of the median formation marker; MoMR: Multiple of the median resorption marker; NBHA: National Bone Health Alliance; NTX: Amino-terminal cross-linked telopeptide of type 1 collagen; OB: osteoblast; OC: osteocalcin; OCY: osteocyte; OPG: osteoprotegerin; OPN: osteopontin; PICP: Procollagen type 1 carboxy-terminal propeptide; PINP: Procollagen type 1 amino-terminal propeptide; PPR: PTH/PTHrP receptor; PTH: Parathyroid hormone; PTHrP: Parathyroid hormone related peptide; PYD: pyridinoline; RA: Rheumatoid arthritis; RANK: Receptor activator of nuclear factor kappa B; RANKL: Receptor activator of nuclear factor kappa B ligand; sRANKL: Soluble receptor activator of nuclear factor kappa B ligand; SCL: sclerostin; SD: Standard deviation; SERMs: Selective estrogen receptor moderators; Sost: SclerOSTeosis gene; TRAP: Tartrate resistant acid phosphatase; TSH: Thyroid stimulating hormone; UK: United Kingdom; WHO: World Health Organisation; Wnt: Wingless and Integration-1.JMvL has received a research grant, consultancy and speaker fees from Roche. SPT has received speaker fees from Ely Lilly.GW drafted the manuscript. ME revised the manuscript. SPT and HKD revised and critically appraised the manuscript. JMvL revised, critically appraised and provided overall supervision for the project. All authors read and approved the final manuscript.


Childhood cancer is the leading cause of death by disease among U.S. children between infancy and age 15. Despite successes in treating solid tumors such as Wilms tumor, disappointments in the outcomes of high-risk solid tumors like neuroblastoma have precipitated efforts towards the early and accurate detection of these malignancies. This review summarizes available solid tumor serum biomarkers with a special focus on mediastinal and abdominal cancers in children. Survival rates for children with cancer have improved over the past decades ,2. RecenSerum tumor markers offer great promise in the management of children with solid malignancies and are integral to disease diagnosis and prediction of treatment response. These cancer biomarkers are biologically measured substances that are expressed by malignant tissues, circulating tumor components, or generated by the host in response to the tumor, and constitutively serve as essential tools to aid clinicians in making diagnosis, staging, and risk assessments. The nonspecific property of some markers to be differentially expressed in other tissues limits their clinical use and hampers accurate diagnosis of disease information. Although there is a wide spectrum of pediatric solid tumors, this review discusses only current trends in the use of serum biomarkers for detection and evaluation of solid tumors in the chest and abdomen of children.Mediastinal tumors are relatively common in infants and children and represent a heterogeneous group of asymptomatic or potentially life-threatening neoplastic lesions that may carry complex diagnostic and therapeutic dilemmas. In general, imaging with a chest radiograph  and a computed tomography (CT) scan gives anatomic guidance for primary tumor location and information on mass morphology, extent, and relation to surrounding structures. Depending on tumor location within 3 different mediastinal compartments , a differential diagnosis may be made along with information on age and clinical history. Anterior mediastinal tumors include germ cell tumors (GCTs), thymic-related, cystic hygromas, and lymphomas. Common masses in the middle mediastinum are lymphomas, bronchogenic cysts, and granulomatous disease. Posterior mediastinal masses are mainly neurogenic tumors and represent the most common tumors in the mediastinum [Pediatric solid abdominal neoplasms encompass a wide spectrum of diseases ranging from lesions causing significant morbidity and mortality [advanced stage neuroblastoma (NB)] to conditions that can be corrected by surgery alone [favorable-stage Wilms tumor (WT)]. The physician needs to determine the nature of the mass in a timely, safe, and cost-effective manner. Likely abdominal malignancies diagnosed in children include NB, WT, hepatoblastoma (HB), lymphoma, soft tissue sarcomas, and GCTs. As for thoracic masses, diagnostic imaging plays a major role in the diagnosis and management of pediatric abdominal masses, local disease staging, identification of distant metastases, and monitoring response to therapy. However, imaging techniques cannot always accurately distinguish between benign and malignant masses and may be discrepant from histologic staging . Table 1The exact incidence of pediatric GCTs is not precisely known as approximately 50% of GCTs are benign [pure mature teratoma which do not secrete α-fetoprotein (AFP) and β-human chorionic gonadotropin (β-hCG)] and go unreported. Malignant GCTs account for 3% of all childhood cancers. GCTs may develop from variations in normal differentiation , aberrant migration , or their combination. Extragonadal GCTs occur at various midline locations , but most commonly affect the mediastinum and sacrococcygeal region. Morphologically, they are divided into seminomas and nonseminomatous GCTs (NSGCTs). Most NSGCTs display mixed histologies, such as embryonal, choriocarcinomas, teratomas, and yolk sac tumors (YSTs). Although predicting the biological behavior in GCTs can be confusing owing to other factors such as patient age, histologic subtype, anatomic site, and clinical stage, serum levels of alpha-fetoprotein (AFP), beta-human chorionic gonadotropin (β-hCG), and lactate dehydrogenase (LDH) are well validated in their utility as noninvasive diagnostic indicators of GCTs . These mThe complete characterization of AFP, β-hCG, and LDH as biologic disease markers have been addressed by numerous reports over the past several decades –47. As aLiver malignancies account for slightly more than 1% of all pediatric tumors, with approximately 100–150 new cases of liver tumors being diagnosed annually in the US . Pediatrl g/L, preterm infants: 158,125 l g/L)  can cause discordance in hCG immunoassay results. Cao and Rej identified the following factors that contribute to the incorrect reporting of hCG results by laboratories: (a) complexity of the hCG molecule and confusion of nomenclature among various forms of hCG; (b) laboratory personnel’s lack of awareness of the distinctions in the forms of hCG and failure to recognize the specificity of assays for measurement of different forms; (c) lack of clarity and uniformity in manufacturers’ reagent labeling; and (d) lack of information on the specificity of each method for the various forms of hCG in most product inserts .It is vital to accurately detect the specific hCG-related molecule in patients with malignant disease. Immunochemical characterization has shown that though intact hCG is abundantly produced by the placenta and germ cell tumors, it is the serum free β-hCG subunit (independent of the common glycoprotein hormone α subunit [GPHα]) that is also a tumor marker in many non-trophoblastic tumors, which include common epithelial cancers. Therefore, it is critical to know which hCG isoforms are measured in order to avoid false-positive results, as most automated commercial laboratory tests, point-of-care tests, and over-the-counter tests are limited in what they detect, as they focus only on regular hCG.etc.), and differential quantifying protein methods such as isobaric tags for relative and absolute quantification (iTRAQ).Despite considerable potential limitations to current pediatric tumor markers, continued progress in tumor marker discovery will likely come from rigorous translational investigations that will integrate multidimensional analyses from various emerging technologies to deliver more specific predictive and prognostic markers. Undoubtedly, next-generation biomarkers will likely stem from the growing omics technologies, which will amalgamate large-scale genomics, transcriptomics, and proteomics to promote personalized cancer care. Consequently, proteomics based biomarker discovery is one of the omics platforms that has garnered much interest in many diseases, including cancer. The enthusiasm towards cancer-related proteomics applications is due to the role proteomics can provide in bridging the gap between genetic alterations underlying cancer and cellular physiology, such that a cell’s genome is relatively stable and gives information of the organism’s gene expression while the proteome is dynamic, complex and variable. Moreover, there is a growing opinion in favor of the role of targeting proteomic measurements for improving blood-based human diagnostics ,155. BloIn addition, other novel work in biomarker discovery involves proximal fluid proteomics. Proximal fluid, the fluid derived from the extracellular milieu of tissues, contains a large repertoire of shed and secreted proteins that are likely to be present at higher concentrations than in the plasma or serum. It has been hypothesized that many, if not all, proximal fluid proteins exchange with peripheral circulation, which has triggered the use of proximal fluids as a primary sample source for protein biomarker discovery .et al. [Lastly, exciting opportunities in nanoscale exploration open new strategies for serum protein analysis. For example, Pujia and colleagues recently described a tool based on biodegradable nanoporous nanoparticles (NPNPs) that allows the harvesting of low-molecular-weight fractions of crude human serum or plasma . NPNPs wet al.  performeIn the continued search for ideal tumor markers in children, it is essential to pursue study designs that are appropriately powered and lend themselves to independent replication. There are several criteria for any biomarker to be considered clinically relevant. Sturgeon and colleagues recently updated the National Academy of Clinical Biochemistry laboratory medicine practice guidelines and quality requirements for the use of tumor markers. The most important feature with regard to these biomarkers is the ability to affect patient management . Savage The progress toward early diagnosis, curative therapy, and favorable outcomes in childhood solid cancer patients and survivors depends not only on developing continued strategies to identify biomarkers for more precise diagnosis, targeted therapy, and possibly prevention, but also designating additional funding or redistributing existing resources for furthering the study of childhood malignancies that continue to have static or poor survival trends. For example, 5-year survival rates for pediatric and adolescent sarcomas  have not drastically changed over the past 2 decades as compared with those for other solid tumors such as non–central nervous system GCTs and Wilms tumor [


Systemic hypotension in preterm infants has been related to increased mortality, cerebrovascular lesions, and neurodevelopmental morbidity. Treatment of hypotension with inotropic medications aims at preservation of end organ perfusion and oxygen delivery, especially the brain. The common inotropic medications in preterm infants include dopamine, dobutamine, adrenaline, with adjunctive use of corticosteroids in cases of refractory hypotension. Whether maintenance of mean arterial blood pressure (MAP) by use of inotropic medication is neuroprotective or not remains unclear. This review explores the different inotropic agents and their effects on perfusion and oxygenation in the preterm brain, in clinical studies as well as in animal models. Dopamine and adrenalin, because of their α-adrenergic vasoconstrictor actions, have raised concerns of reduction in cerebral blood flow (CBF). Several studies in hypotensive preterm infants have shown that dopamine elevates CBF together with increased MAP, in keeping with limited cerebro-autoregulation. Adrenaline is also effective in raising cerebral perfusion together with MAP in preterm infants. Experimental studies in immature animals show no cerebro-vasoconstrictive effects of dopamine or adrenaline, but demonstrate the consistent findings of increased cerebral perfusion and oxygenation with the use of dopamine, dobutamine, and adrenaline, alongside with raised MAP. Both clinical and animal studies report the transitory effects of adrenaline in increasing plasma lactate, and blood glucose, which might render its use as a 2nd line therapy. To investigate the cerebral effects of inotropic agents in long-term outcome in hypotensive preterm infants, carefully designed prospective research possibly including preterm infants with permissive hypotension is required. Preterm animal models would be useful in investigating the relationship between the physiological effects of inotropes and histopathology outcomes in the developing brain. Systemic hypotension is a relatively common complication of preterm birth, affecting approximately one-third of very low birth weight infants. Systemic hypotension has been associated with increased mortality, intraventricular hemorrhage, periventricular leukomalacia, and neurodevelopmental morbidity in the preterm infant , and cerebral oxygen delivery, which are presumably the mechanism for the adverse neurodevelopmental outcome associated with hypotension  is usually avoided because of concerns regarding excessive α-receptor-mediated peripheral vasoconstriction, with a corresponding reduction in cardiac output  is often used as a second or third line of treatment in systemic hypotension, usually in addition to dopamine and/or dobutamine infusion. Adrenaline acts upon α1-, β1- and β2-adrenergic receptors. In a randomized control trial in very low-birthweight infants with early postnatal hypotension, in whom either adrenaline or dopamine infusion was administered , and oxygenation  on cerebral hemodynamic and oxygenation responses and identified significant differences in these responses to specific drugs and doses  do not cross the blood–brain barrier (BBB)  (Penny et al., There is a distinct lack of prospective research addressing cerebral effects of inotropic treatments in preterm infants and their long-term outcome (Shuhaiber et al., 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.


There is growing evidence that heterozygosity–fitness correlations (HFCs) are more pronounced under harsh conditions. Empirical evidence suggests a mediating effect of parasite infestation on the occurrence of HFCs. Parasites have the potential to mediate HFCs not only by generally causing high stress levels but also by inducing resource allocation tradeoffs between the necessary investments in immunity and other costly functions. To investigate the relative importance of these two mechanisms, we manipulated growth conditions of great tit nestlings by brood size manipulation, which modifies nestling competition, and simultaneously infested broods with ectoparasites. We investigated under which treatment conditions HFCs arise and, second, whether heterozygosity is linked to tradeoff decisions between immunity and growth. We classified microsatellites as neutral or presumed functional and analyzed these effects separately. Neutral heterozygosity was positively related to the immune response to a novel antigen in parasite-free nests, but not in infested nests. For nestlings with lower heterozygosity levels, the investments in immunity under parasite pressure came at the expenses of reduced feather growth, survival, and female body condition. Functional heterozygosity was negatively related to nestling immune response regardless of the growth conditions. These contrasting effects of functional and neutral markers might indicate different underlying mechanisms causing the HFCs. Our results confirm the importance of considering marker functionality in HFC studies and indicate that parasites mediate HFCs by influencing the costs of immune defense rather than by a general increase in environmental harshness levels. Associations between individual genetic diversity and fitness-related traits are commonly known as heterozygosity–fitness correlations (HFCs) and have been intensively studied in the last decades  Szulkin et al. Many studies investigated HFCs and reported associations with important life-history traits , a naturally occurring nest-based ectoparasite, for which we detected a mediating effect on HFCs in a previous study  were cross-fostered on day 4 by swapping all chicks. We combined the cross-fostering with the brood size manipulation treatment by pairing either broods with the same number of nestlings (unchanged brood size) or broods that differed by two nestlings to create enlarged and reduced broods. We heat-treated nesting material for 3 min using a microwave oven and brushed the nestboxes to remove all remaining parasites . We amplified 47 autosomal microsatellite markers by polymerase chain reaction using QIAGEN Multiplex PCR kit  as described in Saladin and Richner .http://blast.ncbi.nlm.nih.gov) against nucleotide collection of zebra finch and chicken. Markers showing homology to avian expressed sequence tags (ESTs) were considered as presumed functional, otherwise as neutral. According to this method, 13 of the microsatellite markers used were identified as presumed functional  as explained in Szulkin et al.  and linkage disequilibrium (LD) were calculated using FSTAT  starting with the highest order interactions and retained all main effects. The 4-factorial interactions were never significant and thus removed from all models. We started instead with the three-way interactions, which included heterozygosity. To interpret significant interactions, we split the models according to factor levels. The fit of the models was verified by checking residuals for normality and homoscedasticity and by plotting residuals against fitted values. Nestling survival was analyzed using a generalized linear mixed model (GLMM) with a binomial error structure and logit link with the add-on package lme4 . Explanatory variables in the starting models were heterozygosity levels of nestlings, nestling sex, flea infestation treatment, and brood size manipulation. We included heterozygosity levels of foster fathers and foster mothers and original brood size as covariates. We controlled for hatching rank by including it as a two-level factor, indicating whether a nestling hatched on the first day (level 1) or later (level 2). When modeling the swelling response to LPS, we additionally included nestling weight on day 15. The random structure consisted of nest identity, in case of the swelling response nested within observer identity, to correct for the nonindependence of siblings. For all models, we eliminated nonsignificant interactions . Presumed functional markers were less diverse compared with neutral markers in terms of allele number  as well as mean observed heterozygosity .Heterozygosity levels calculated based on the set of neutral microsatellites and presumed functional markers did not correlate . For neutral markers, there was a weak trend for positive HHCs , but g2 did again not differ from zero .Heterozygosity–heterozygosity correlations and F1, 285 = 3.76, P = 0.05, rb = 0.092), while no significant relationship could be detected in parasitized nests  and a positive relationship in parasitized nests . In parasite-free nests, there was no relationship between neutral heterozygosity and body condition of female nestlings , while we found a trend for a positive relationship in parasitized nests .We found a significant interaction between neutral heterozygosity, nestling sex, and flea infestation treatment on nestling body condition Table C. Investz = −0.91, P = 0.36), while in flea-infested nests, the chance of survival was significantly higher for more heterozygous nestlings . Testing for single-locus effects following the procedure described in (Szulkin et al. The relationships between heterozygosity levels and the nestling swelling response, feather length, and body condition could be attributed to effects of heterozygosity within broods, as indicated by the significant within-nest variable in the centered models Table . The relIn this study, we manipulated growth conditions of nestling great tits by infesting broods with hen fleas and simultaneously manipulating the natural brood size and investigated the mediating effect of both treatments on the occurrence of HFCs. We found that nestlings in experimentally enlarged broods had shorter feathers than control nestlings, indicating that brood enlargement deteriorated growth conditions. However, there was no evidence for a mediating effect of brood size manipulation on the occurrence of HFCs, given that the detected effect did not depend on nestling heterozygosity levels. In contrast, the effects of the flea infestation treatment on the investigated traits strongly depended on nestling heterozygosity levels, joining the evidence that parasites are important mediators of HFCs (Coltman et al. In parasite-free control broods, we found no relationship between nestling heterozygosity and growth or survival, but a positive relationship with the swelling response after LPS injection. These results indicate that while all nestlings invested similar amounts of resources into growth and survival, more heterozygous nestlings could additionally invest also into the development of the immune system. Stronger swelling responses to LPS are thought to reflect higher immunocompetence, and therefore, this positive relationship in parasite-free nests is in line with a number of previous studies reporting positive correlations between individual heterozygosity and immunocompetence or parasite resistance (Acevedo-Whitehouse et al. In parasitized nests, no relationship between heterozygosity and immune response could be detected. Ectoparasites have been found to take smaller blood meals from hosts with increased immunocompetence (Bize et al. The detected positive relationships between neutral heterozygosity and survival, feather length, and female body condition in parasitized nests may alternatively be explained not as a cost of increased investments in immunity, but as a result of a generally increased harshness level of the environment. The observation that HFCs are stronger in harsh environments, that is, the parasitized nests, are in line with a number of previous studies (Lesbarreres et al. The correlation between neutral heterozygosity and nestling fitness traits could be attributed to within-brood effects. Removal of extra-pair offspring from the analysis did not qualitatively change these results. Therefore, we can conclude that these within-brood effects were caused by full-siblings. As full-siblings share their ancestry and inbreeding history, HFCs within full-sibling designs are commonly interpreted as evidence for local effects (Hansson et al. g2 values (David et al. Heterozygosity measured across a set of presumed functional genetic markers was negatively linked to nestling immune response in all treatments. Thus, functional heterozygosity seemed to have a detrimental effect on nestlings and contrasts with the positive relationships found with neutral markers. The interpretation of negative correlations between heterozygosity and fitness depends on the underlying biological mechanism causing the HFCs. Under the assumption that the detected HFCs are caused by genome-wide effects of heterozygosity David , negativThe negative effects of presumed functional markers and positive effects of neutral markers have previously been explained as resulting from local effects for functional markers and genome-wide effects for neutral ones (Olano-Marin et al. To conclude, in the present study, we found strong evidence for a mediating effect of parasites, but not of brood size manipulation on the occurrence of HFCs. This suggests that parasites mediate HFCs not only by a general increase in the harshness level of the environment, but also by forcing individuals to increase investments into immune defense. Neutral heterozygosity was found to reveal heterozygosity-dependent costs of these increased investments in terms of reduced survival, feather length, and female body condition for less heterozygous nestlings.