Parkinson's disease (PD) patients experience cognitive impairment which has been related to reduced quality of life and functional disability. These symptoms usually progress until dementia occurs. Some studies have been published assessing the efficacy of cognitive treatments on improving cognition, functional outcome, and producing changes in brain activity.  A critical review was performed to present up-to-date neurorehabilitation effects of cognitive rehabilitation in PD, with special emphasis on the efficacy on cognition, quality of life aspects, brain changes, and the longitudinal maintenance of these changes.  After exclusions, 13 studies were reviewed, including 6 randomized controlled trials for the efficacy on cognition, 2 randomized controlled trials regarding the brain changes after cognitive training, and 5 studies which evaluated the long-term effects of cognitive treatments.  Cognitive rehabilitation programs have demonstrated to be effective on improving cognitive functions, but more research is needed focusing on the efficacy on improving behavioral aspects and producing brain changes in patients with PD. Moreover, there is a need of randomized controlled trials with long-term follow-up periods. Parkinson's disease (PD) is a common neurodegenerative disease, being most of the cases diagnosed at around 60 years . TraditiThese cognitive impairments in PD have been related to grey matter (GM) atrophy, white matter (WM) alterations, brain functional connectivity (FC), and brain activation alterations. PD patients with Mild Cognitive Impairment (MCI) diagnosis have shown GM volume reduction in the frontal, temporal, and parietal lobes, but also in the hippocampus, amygdala, and putamen , 11. AddWith the progression of the disease, cognitive deficits usually deteriorate until dementia occurs after 10 to 20 years , 17. A sDue to the relevance of cognitive deficits, therapeutic strategies are needed to treat cognitive decline. A common cognitive rehabilitation program could be described as a behavioral treatment for cognitive impairment which focused on cognitive abilities and daily living activities, which is based on the restoration, compensation, and optimization of the cognitive functions , 25. CogThe main objective of the present study is to perform a critical review to present up-to-date neurorehabilitation effects of cognitive rehabilitation in PD. The first objective was to examine the efficacy of cognitive rehabilitation programs on cognition and behavioral aspects. The second objective was to review the evidence of the brain changes found after cognitive treatments. Finally, the present study analyzed the long-term effects of cognitive rehabilitation in PD.Studies were included from inception to December 2017. Focusing on the first objective of this critical review, which was to analyze those randomized controlled trials focused on the efficacy of cognitive rehabilitation programs on cognition, we selected only those studies that fulfilled the following criteria: (1) randomized controlled trials; (2) PD patients underwent a cognitive rehabilitation program; (3) the main objective was to investigate the change in cognition; and (4) studies including a PD control group. Among the studies that fulfilled these specific criteria, we also reported (if included in the studies) the results of the efficacy on behavioral or mood aspects, such as depression, apathy, functional disability, and quality of life aspects.Regarding the second objective of the present review, the efficacy of cognitive rehabilitation programs in producing brain changes in PD was determined based on the following criteria: (1) randomized controlled trials; (2) PD patients underwent a cognitive rehabilitation program; (3) studies including a PD control group; and (4) brain changes were evaluated.Finally, focusing on the last objective of the present study, the review about the long-term effects of cognitive rehabilitation programs was based on the following criteria: (1) PD patients underwent a cognitive rehabilitation program; (2) a longitudinal follow-up evaluation was performed; and (3) the main objective was to investigate the change in cognition at follow-up. In this specific section, we included both randomized controlled trials and nonrandomized trials, due to the scarce number of published studies and to have a wider perspective.Databases included were PubMed, Medline, and Google Scholar. The search terms were specified to be found in the title of the studies. The terms were (1) Parkinson's disease/Parkinson disease; (2) cognitive rehabilitation/cognitive training/cognitive remediation; (3) attention rehabilitation/attention training/attention remediation; (4) executive training/executive rehabilitation/executive remediation; (5) memory training/memory rehabilitation/memory remediation; (6) randomized controlled trial/randomized controlled trial; and (7) cognition. The search term combinations in the databases were (1) + (2); (1) + (3); (1) + (4); (1) + (5); and (1) + (6) + (7).A summary of study selection is shown in A summary of the included cognitive rehabilitation studies in PD is shown in A less studied aspect of cognitive rehabilitation is its efficacy on improving mood symptoms or functional disability related to the disease. Following the review-specific criteria, among the randomized controlled trials in PD, only five studies have evaluated the change in functionality and mood aspects –35 and tMoreover, detecting variables that predict the efficacy of cognitive treatments is an important aspect to take into account in order to understand the cognitive rehabilitation process, which could guide researchers to develop more effective programs and clinicians to personalize treatments for patients . DespiteLittle is known about the neurobiological effects of cognitive rehabilitation programs in PD. To date, literature is scarce about the presence of cerebral changes associated with cognitive rehabilitation programs assessed with structural and functional MRI techniques in PD. One study evaluated the effects of group-based attention rehabilitation on brain functional activity in PD patients . PD patiA later study in PD patients evaluated the changes in brain activity after a 3-month integrative cognitive rehabilitation program . PD patiThese studies suggest that brain activity changes are possible after a cognitive rehabilitation program in PD. Further studies are needed to replicate and complement these findings.Furthermore, the ultimate goal of cognitive treatments is to ensure that benefits are maintained over long periods of time, but little is known about the maintenance of cognitive improvements over time in PD patients, and a few studies have evaluated it –42, 45. The first study to evaluate the long-term effects of cognitive rehabilitation was published in 2004 by Sinforiani et al. and showed that PD patients attending a cognitive training program combined with motor training during one month showed maintenance of the cognitive benefits after 6 months . HoweverAnother study in PD compared three training groups: “group A” which attended cognitive training, “group B” which attended cognitive training and transfer training, and “group C” which attended cognitive, transfer, and motor training  Table 3Table 3. A later study in PD assessed the long-term effects of cognitive rehabilitation for a longer period of time (12 months) . At baseRegarding the maintenance of neuroimaging changes, to date, only one study has been published assessing the longitudinal effects of cognitive rehabilitation . PD patiThese few studies suggest the maintenance of cognitive improvements after attending a cognitive rehabilitation program in PD patients . HoweverThe studies on the efficacy of cognitive rehabilitation programs in PD suggest that cognitive rehabilitation programs are effective in improving cognition but further research is needed in this field to clarify its efficacy on functional disability and brain activity changes. Also, very little is known about the long-term maintenance of cognitive changes after rehabilitation. There are few cognitive rehabilitation studies in PD which followed the Consolidated Standards of Reporting Trials (CONSORT) guidelines for randomized controlled trials. These make more difficult to find conclusive findings. Future studies should implement these guidelines in order to improve the research quality and validity of findings.All randomized controlled trials in PD for cognitive rehabilitation programs point to the efficacy in improving cognition. However, most of them highlighted the small sample size as a limitation, which makes it difficult to generalize the findings. Additionally, all of them used different types of cognitive training programs, with different duration and type of exercises. One of the future steps to be taken towards understanding the efficacy of cognitive rehabilitation is identifying the characteristics that make an integrative cognitive rehabilitation program effective against cognitive impairment. A review of cognitive rehabilitation concluded that better results may be obtained in a group-based format compared to an individual format . HoweverRegarding patients' characteristics, most of the randomized controlled trials in PD have been performed with PD patients at the early Hoehn and Yahr stages of the disease. Future studies should also include PD patients at more advanced stages to evaluate whether cognitive treatments could also benefit these patients. Interestingly, a study protocol was recently published addressing the efficacy of a cognitive rehabilitation in PD patients with dementia, but results are pending .Moreover, transfer effects to clinical aspects have been found in some cognitive rehabilitation studies in PD; however, other studies found no significant changes. The mechanisms that make possible to transfer benefits to clinical variables are unknown. There is an urgent need of studies analyzing this subject. The last goal of cognitive rehabilitation programs is to improve quality of life of patients. Future studies should also include clinical and functionality scales in pre- and post-treatment neuropsychological assessments.On the other hand, promising findings have been found regarding brain changes after treatment in PD, which support the efficacy of cognitive rehabilitation programs in the disease. Results showing brain connectivity and activation increments after a cognitive treatment of less than 3 months in patients with a neurodegenerative disease are relevant in the field of neurorehabilitation. Future studies should include the MRI acquisition as part of the protocol assessment to evaluate brain changes after treatment and replicate the findings.All these changes have been analyzed at follow-up, and some studies found maintenance of these improvements. Future randomized controlled trials should include follow-up periods in order to replicate previous findings and assess whether the improvements after training could be maintained over time. It would be also interesting to examine the maintenance of these changes in PD patients with and without booster sessions.Another aspect to be taken into account during the rehabilitation process of the patient is the role of the caregiver. Some cognitive rehabilitation studies have included an intervention which focused on psychoeducation with the caregivers of the PD patients , 44. TheIn conclusion, cognitive rehabilitation programs have demonstrated to be effective in improving cognitive functions and may also improve functional disability and produce brain changes in patients with PD. In addition, to provide a complete or integrative treatment, the combination of cognitive training with other types of trainings or the intervention with the caregivers should be further analyzed. More research should be performed in the field, with a view to reaching generalized conclusions and including cognitive rehabilitation in the standard of care of PD patients.


The current view of neuroplasticity depicts the changes in the strength and number of synaptic connections as the main physical substrate for behavioral adaptation to new experiences in a changing environment. Although transcriptional regulation is known to play a role in these synaptic changes, the specific contribution of activity-induced changes to both the structure of the nucleus and the organization of the genome remains insufficiently characterized. Increasing evidence indicates that plasticity-related genes may work in coordination and share architectural and transcriptional machinery within discrete genomic foci. Here we review the molecular and cellular mechanisms through which neuronal nuclei structurally adapt to stimuli and discuss how the perturbation of these mechanisms can trigger behavioral malfunction. In the search for mechanisms that underlie behavioral plasticity, functional and structural changes at synapses are at the core of the theoretical framework. Processes such as long-term potentiation (LTP) or synaptogenesis are thought to be crucial for the adaptation of neuronal circuits to changing environmental conditions . Both stIn eukaryotic nuclei, DNA is wrapped around an octameric histone core comprising of two copies of each of the canonical histones H2A, H2B, H3 and H4. This basic structure, known as a nucleosome, is repeated along the double-stranded DNA, with a fifth type of histone (the linker histone H1) bridging together consecutive nucleosomes. In this fashion, long DNA strands condense with architectural proteins to form chromatin. Based on the level of compaction we can distinguish three main forms of chromatin. These forms differ biochemically with respect to the presence of specific post-translational modifications (PTMs) at the histone tails and to the binding of structural proteins. Euchromatin, a transcriptionally active form is characterized by permissive marks such as the trimethylation of histone 3 at lysine 4 (H3K4me3), and the acetylation of different lysine residues at the histone tails. In contrast, heterochromatin is a transcriptionally silent form, and is decorated by repressive epigenetic marks. It can be found in two different functional states: constitutive heterochromatin that is characterized by DNA methylation at CpGs and histone H3 trimethylation at lysine 9 (H3K9me3), and facultative heterochromatin, which, as suggested by its name, can harbor transcriptional activity and is marked by H3K27me3 .Although the folding of chromatin fibers during cell division is very similar among all cells , the spaApart from chromocenters and peripheral heterochromatin, the interphasic neuronal nucleus is structurally complex  Fig. 1b1b. BasedNuclear architecture and genome organization depend on the integrity of the nuclear envelope, a boundary that separates the cytoplasm from the nucleoplasmic reticulum. This boundary is composed of two phospholipid bilayers spanned at intervals by proteins that act as nuclear pores. The nuclear envelope is not an inert barrier, it participates in different processes including gene regulation and the transport of ions and macromolecular cargos . Its geoInternally attached to the nuclear envelope is the nuclear lamina, whose main components are the lamin proteins A/C, B1 and B2 . These pLamin-associated chromatin domains (LADs) are enriched in transcriptional and epigenetic repressors . AlthougNuclear bodies are subnuclear divisions that lack a membrane. In addition to the afore discussed chromocenters, which are the most prominent type of nuclear body in mature neurons, one can also typically find (i) a single nucleolus where rRNA transcription takes place, (ii) the Cajal bodies (CBs) that are adjacent to the nucleolus and are the site for small nuclear ribonucleic protein (snRNP) assembly, (iii) nuclear speckles that are highly enriched in splicing factors, and (iv) promyelocytic leukemia (PML) bodies that hold unknown functions  Fig. 1b1b. As diThe nucleoplasm is not an inert and homogeneous matrix filled with euchromatin fibers as once thought. Static electron microscopy images have since been challenged by the dynamic scenario revealed by molecular studies that explore short and long-range interactions between DNA sequences that are located thousands of bases apart or even in different chromosomes . The useFine submegabase 3D interactions are essential for neuronal commitment and are also likely to contribute to the regulation of gene expression during neuronal plasticity processes. We will discuss in the next section the novel techniques available and the seminal studies investigating how neuronal activation causes changes in the fine structure of the nucleoplasm.de novo long-range chromatin interactions genome-wide, and DNase I hypersensitive sites sequencing (DNase-seq), Formaldehyde-Assisted Isolation of Regulatory Elements (FAIRE)-seq and Assay for Transposase-Accessible Chromatin (ATAC)-seq allow the assessment of changes in DNA accessibility [Given their small scale, transcription-related and activity-driven dynamic changes in chromatin fibers may escape structural analyses when employing microscopy techniques, but can be tackled by molecular studies investigating long and short-range chromosomal interactions . This issibility . These nsibility , 35. Forsibility , and ultsibility .Bdnf, it has been observed that upon kainate-induced seizures there is both a weakening of its interaction with the lamina as well as the relocation of one allele from the nuclear margin to deeper areas within the nucleus [Bdnf alleles with poised RNAPII. Intriguingly, the detachment from the lamina persisted beyond the transient increase in transcription, which leaves open the possibility that this structural change could contribute to sensitization of affected neurons for ulterior reactivation [Bdnf locus has also been reported to occur in neuronal cultures after depolarization [ASCL1 (encoding for the Mash1 protein), along with other proneural genes, move from the nuclear periphery where they remain transcriptionally silent to the central nucleoplasm where they become transcribed [A key level of genome organization is the movement of genes within the interior of the nucleus. Fundamental contributions in the eighties demonstrated chromosomal movements in seizure foci of the human cortex. These movements were found to affect particularly the X chromosome although they were independent of the patient’s sex . Consist nucleus . This retivation . A similrization . These mrization . These gnscribed , 42.Enhancers are defined as regulatory sequences rich in transcription factor (TF) binding sites that regulate gene activation and are distal to the transcription start site (TSS) . They arFos in the nervous system seem to rely in the action of at least five enhancers that surround the locus and differentially respond to various stimuli  [The expression of cell type-specific and brain region-specific genes often relies on enhancer sequences that act specifically only in those cells, while being methylated and inactive elsewhere . Interesulation) .aka CCCTC-binding factor), mediator and cohesin are important components of the insulator complex that appear in distinct combinations depending on the range of interaction. CTCF and cohesin locate together in active regulatory sequences where they mediate long-range constitutive interactions. They are fundamental building blocks behind insulated chromosomal neighborhoods containing super-enhancers necessary for cell identity [Another type of regulatory sequence that relies on chromatin looping are the insulators. Insulators are described as chromatin regions that protect against the activating influence of distal enhancers associated with other genes . The proidentity . For insidentity . Whereasidentity , CTCF plidentity , 55. In identity , cohesinidentity . The invidentity , while aidentity .The term transcription factory refers to discrete foci in the eukaryotic nucleus where transcription occurs . These mde novo acetylation of SINEs located around their promoter  residues at the C-terminus domain (CTD) of RPB1, the largest subunit of the RNAPII complex . Althougter Fig. . This prter Fig. . Two houAs introduced in previous sections, the neurons in some developmental and degenerative disorders often display gross nuclear aberrations, while psychiatric disorders have been associated with more subtle changes Table . We discMED12 [FMR1 gene, which give rise to fragile X-associated tremor/ataxia syndrome (FXTAS), accumulate more heterochromatin but in smaller foci [Mutations in genes encoding proteins important for nuclear architecture  frequently result in neurodevelopmental disorders . This isMED12  that encMED12 , whereasMED12 . FurtherMED12 , while tMED12  that manMED12 , and an MED12 . Other sler foci .Another type of genetic disorders associated with abnormal nuclear architecture are laminopathies in which the nuclear lamina is prominently disrupted. This group of disorders includes Hutchinson–Gilford progeria syndrome (HGPS) that is caused by mutations in the gene encoding lamin A . IntriguDrosophila models of tauopathy and human samples from Alzheimer’s patients [Large-scale chromatin reorganization is often observed in neurons undergoing degeneration. Thus, irregularities in nuclear shape, particularly mediated by B-type lamins, have been described to precede heterochromatin relaxation, DNA damage and neurodegeneration in both patients . Furtherpatients . Other apatients .Grin2b (encoding for a subunit of the NMDA receptor) and the loss of a loop tethering the promoter to a Setdb1 target site positioned 30 kb downstream of the TSS [GRIN2B loop, is often found in schizophrenic patients and correlates with impaired working memory and schizotypic features. Notably, isogenic deletions of loop-bound sequences in mice impaired cognitive performance and decreased Grin2b expression [GAD1  and an enhancer sequence 50 Kb upstream was found reduced in the prefrontal cortex of schizophrenic patients [FKBP5, which encodes the co-chaperone FK506 binding protein 5, and enhancer sequences located in introns 2 and 7 is associated with an increased risk of developing stress-related psychiatric disorders after childhood trauma [NRG1), a putative schizophrenia susceptibility gene regulating the excitatory-inhibitory balance, are associated with an increase in NRG1 transcripts in the prefrontal cortex, suggesting that this region could function as a transcriptional enhancer. Intriguingly, the presence of these repeats correlated with an earlier age of onset of the symptoms. However, long-range interactions between the intronic sequence and the promoter remain to be experimentally proven [Aberrant chromatin loopings have been recently implicated in psychiatric disorders. For example, Akbarian and colleagues first found that overexpression of the histone methyltransferase Setdb1 caused the heterochromatinization of the promoter of  the TSS . Furtherpression . The sampatients . A simild trauma . Anothery proven . There ay proven  and impuy proven , but molGiven the difficulty of examining the specific contribution of chromatin conformation changes through gain- and loss-of-function experiments, most of the evidence discussed above is correlative. A recent study by our team investigating transgenic mice that express high levels of GFP-tagged H2B in forebrain principal neurons has provided evidence for a causal role of aberrant chromatin organization in the emergence of neuropsychiatric traits . Neuronain vivo of some enhancers carrying CTCF-binding sites relies on their relative orientation and the precise architecture of chromatin domains [Excitingly, the use of engineered transcription factors has recently demonstrated that the local manipulation of epigenetic profiles at a given gene is sufficient to control drug- and stress-evoked transcriptional and behavioral responses, thereby providing seminal evidence for a causative role for those epigenetic marks . Similar domains .As reviewed here, numerous studies have illustrated that nuclear architecture and genome topology are key for understanding neuronal function and dysfunction. Changes in subnuclear structures and chromatin loopings have been found to occur in different neuronal plasticity paradigms. Similarly, the disruption of chromatin structures is a landmark for numerous neurological disorders. Although such a disruption likely contributes to the onset of a disorder, a clear distinction between cause and consequence is still missing, except for some monogenic disorders (often associated with ID) caused by mutations in architectural proteins or regulatory sequences. Although the specific contribution of architectural proteins and the changes in 3D chromatin organization to neuroplasticity and neuropathology largely remain to be determined, new light will soon be shed now that novel techniques such as super-resolution microscopy, NGS-based techniques for the analysis of DNA conformation and CRISPR/Cas9-based epi-editing have emerged. These innovative approaches will facilitate a high resolution determination of the 3D organization of the genome, in parallel to a systems-level interrogation of the consequences of gene expression, the identification of loci associated with aberrant function, and even the manipulation of DNA conformations to promote or correct transcriptional changes.


Estrogen plays an important role in the regulation of cardiovascular physiology and the immune system by inducing direct effects on multiple cell types including immune and vascular cells. Sex steroid hormones are implicated in cardiovascular protection, including endothelial healing in case of arterial injury and collateral vessel formation in ischemic tissue. Estrogen can exert potent modulation effects at all levels of the innate and adaptive immune systems. Their action is mediated by interaction with classical estrogen receptors (ERs), ERα and ERβ, as well as the more recently identified G-protein coupled receptor 30/G-protein estrogen receptor 1 (GPER1), via both genomic and non-genomic mechanisms. Emerging data from the literature suggest that estrogen deficiency in menopause is associated with an increased potential for an unresolved inflammatory status. In this review, we provide an overview through the puzzle pieces of how 17β-estradiol can influence the cardiovascular and immune systems. In addition to its essential role in sexual development and reproduction in females, estrogen is involved in a wide range of physiological processes in different tissues , even inIn women, estrogen circulating levels fluctuate during the menstrual cycle and its concentration changes in relation to age . The mosE2 prevents endothelial dysfunction, vascular inflammation and atherosclerosis . In addiThe immune system demonstrates remarkable sex differences: females tend to have a more responsive immune system compared to their male counterparts. The outcome and survival rates from e.g., infections or sepsis are sometimes better in females than in males . FemalesThe aim of this review is to discuss the multifaceted role of estrogen in vascular biology and in the immune response, particularly in the monocyte/macrophage system, and to further integrate available evidence  regarding the estrogenic control of double-edged processes such as angiogenesis and metabolism.The effects induced by estrogen in different tissues are the result of the activation of transcriptional and non-transcriptional signal pathways. Estrogen exerts both rapid and long-term actions through their binding with ERs . SeveralESR1, ESR2 and GPER1, respectively. The two intracellular receptors have different molecular weights; in particular, ERα consists of 595 and ERβ of 530 amino acids, respectively [The genes encoding ERα, ERβ and GPER1 are ectively . Their sectively . Other rectively .ERα is a ligand-dependent transcription factor that exerts its genomic, also called nuclear actions through binding to chromatin and mobilization of cofactors to influence the transcription of its target genes. A fraction of ERα can elicit membrane signaling (non-genomic effects) by association with the plasma membrane ,39. RapiThe estrogenic membrane receptor GPER1 belongs to the family of G protein-coupled receptors and is characterized by the presence of seven transmembrane helices. The organization of the seven helices involves the amino-terminal portion located outside the cell and the carboxy-terminal portion in the cytoplasm. Cytoplasmic loops are involved in the selective binding and activation of various heterotrimeric proteins ,42. E2 bERα is expressed in the vascular tissue ,49; althSeveral of estrogen cardiovascular actions are actually mediated by direct effects on the vessel wall resulting in the control of endothelial function and plasma lipid profile. In particular, estrogen increases the synthesis and release of nitric oxide (NO) and prostacyclin, well-known endothelial-derived vasodilators and anti-platelet agents, and negatively regulates production of several pro-inflammatory mediators in situations of vascular injury . More spEndothelial cells also express GPER1 , which mIn conclusion, estrogen mediates both rapid and longer-term effects on the vessel wall. Novel vascular target genes regulated by ER subtypes are being identified, thereby providing potential opportunities for pharmacological intervention.Additional puzzle pieces that need to fit together include estrogen, angiogenesis and metabolism. Migration and proliferation of endothelial cells are closely involved in re-endothelialization and angiogenesis. Angiogenesis consists of a number of subsequent biological events and is a tightly regulated process. In adult organisms, angiogenesis is virtually absent under normal conditions, except in the female reproductive tract, where it is routinely observed in the uterus in association with E2 fluctuations . E2 stimIn pathological circumstances, such as breast cancer, a clear association has been made between estrogen, ER expression by endothelial cells, angiogenic activity and/or tumor invasiveness . In this+/H+ exchanger-1 (NHE-1) activity as reviewed recently by De Francesco et al. [During angiogenesis, endothelial cells must increase their metabolic activity to generate energy quickly and to facilitate the incorporation of nutrients into biomass. De Bock and colleagues  demonstro et al. .Experimental evidence accumulated over the past decade indicates that the direct effect of E2 on endothelial cells explains some cardiovascular benefits of the ovarian sex steroid hormone , but theNew data are redefining macrophages as diverse, polyfunctional and plastic cells that respond to the needs of the tissue at steady state and during disturbed homeostasis. Inflammation plays a critical role in the onset and progression of degenerative diseases, and is characterized by activation of tissue-resident macrophages as well as monocyte-derived macrophages that originate and renew from adult bone marrow. Under normal conditions, these cells provide immune surveillance and host defense in tissues to maintain homeostasis. However, upon sensing changes in the microenvironment, macrophages become activated, undergoing a morphological and functional switch . ActivatEstrogen has been shown to act as regulator of the immune function of the monocyte-macrophage system, especially regarding the production of cytokines. For instance, estrogen treatment in ovariectomized animals reduces expression of vascular MCP-1 and leukocyte infiltration into injured tissues, such as arteries and lung ,89. EstrMacrophages have long been recognized as crucial regulators of vascularization and healing ,100; in Local and systemic metabolism is integrated at the cellular level to regulate immune cell function. By interacting with ER subtypes as discussed in These findings point to a complex and partially unresolved role of estrogen in immune and inflammatory responses . Here weRecently, ER expression in human monocytes and macrophages has been investigated, increasing the number of pieces of this already complex puzzle. Both cell types express all ERs . Human pAs noted above, E2 is able to modulate the activation of different macrophage immune phenotypes ,109. TheThe regulation of the immune response to infection or tissue damage is a complex interplay of multiple factors, but it has long been recognized that estrogen steers the innate and adaptive immune systems at various levels. Thus, we believe that pharmacological targeting of macrophage estrogen pathways may restore the impaired resolution of inflammation associated with aging and chronic inflammatory disease.It has been reported that young women generally have much lower rates of cardiometabolic disease than men. However, midlife women lose this apparent protection during the menopausal transition, so that cardiometabolic disease is most common in post-menopause than any other stage of a woman’s lifespan. In fact, fundamental aspects of metabolic homeostasis are regulated differently in males and females ,31,111, Sex steroid hormones alter the biology of vessel wall cells and the inflammatory cells that accrue as atherosclerosis progresses differently in the early versus later stages of the disease . Hence, An age relationship of estrogen–monocyte/macrophage number and function has long been identified, which may have several implications for postmenopausal health ,116. StuThe role of E2 in regulating macrophage function is still an evolving topic. In particular, there is interest in understanding how E2 levels in vivo influence the activation of macrophage phenotypes in physiological conditions at different stages of the menstrual cycle as well as in pathological conditions associated with changes in circulating estrogen levels. This piece fits into the broader puzzle of how estrogen pathways impact on macrophage function and, consequently, on immune response, angiogenesis, wound healing and metabolism . FurtherThe role of estrogen and its multiple receptors in health and disease is heterogeneous. This makes trying and putting the numerous puzzle pieces together a rather complex task. The protection against cardiovascular disease in women during reproductive age is related, at least in part, to estrogen since endogenous E2 levels and ER expression differ considerably between sexes. Estrogen prevents endothelial dysfunction and atherosclerosis by promoting endothelial healing and increasing angiogenesis. The number of puzzle pieces and with them our knowledge of the mechanisms of estrogen action is growing . Today,


Macrophages represent key players of the immune system exerting highly effective defense mechanisms against microbial infections and cancer that include phagocytosis and programmed cell removal. Recent findings highlight the relevance of the non-neuronal cholinergic system for the regulation of macrophage function that opens promising new concepts for the treatment of infectious diseases and cancer. This mini review summarizes our present knowledge on this topic and gives an outlook on future developments. Tissue-specific macrophages are bound to a distinct tissue and may differ greatly in their function and morphology .1 As a component of the mononuclear phagocytic system, macrophages have varying functions within the scope of the cause defense, central role with the initiation and regulation of defense reactions (inflammation), with their most important mission being phagocytosis  II molecules on the cell surface. These antigen–MHC-II complexes are then recognized by T-helper cells, resulting in specific immune reactions that may include the release of antibodies by B cells.Macrophages are big, movable, eukaryotic cells, which develop from monocytes (blood cells which belong to the class of leukocytes) that have left the circulating blood, and that belong to the cellular immune system.ocytosis , and beyocytosis  of micro3 However, cancer cells often express CD47 and/or other antiphagocytic ‘don't-eat-me’ signals that protect them from PrCR-mediated recognition and phagocytosis. Interestingly, blocking the interaction of CD47 on cancer cells and its corresponding receptor SIRPα on macrophages results in efficient PrCR of tumor cells but not of normal cells both in vitro and in vivo. Notably many cancer cells, in contrast to normal cells, express pro-phagocytic ‘eat-me’ signals such as CRT, that interact with other factors, including Bruton's tyrosine kinase (Btk), a member of the Tec non-receptor protein tyrosine kinase family, which has a crucial role in the regulation of the innate immune response. Recently it has been shown that macrophages express CRT, and that Btk-mediated toll-like receptor signaling results in trafficking of CRT to the cell surface, where this ‘eat-me’ signal can contribute to PrCR of cancer cells.4Recently, it has been shown that macrophages can eliminate tumor cells via a highly regulated immunosurveillance mechanism called programmed cell removal (PrCR).5 Otto Loewi proved in 1921 in a frog heart that a chemical, which he called first vagus compound, and which Henry H Dale identified later as ACh, was responsible for the transfer of a nervous impulse to the heart.5 Today, ACh is known as one of the most important neurotransmitters in humans and many other organisms.5–7 ACh is regarded as a classical neurotransmitter that exerts its effects at least in part via binding to two different classes of receptor molecules, the nicotinic  and the muscarinic ACh receptors , that are both expressed in different subtypes.7 nAChRs have been characterized as binding and effector proteins to mediate chemical neurotransmission at neurons, ganglia, interneurons and the motor endplate.8 mAChRs have been characterized as binding and effector proteins to mediate chemical neurotransmission at neurons and effector organs such as heart, smooth muscle fibers and glands.8 This historical view of ACh acting exclusively as a neurotransmitter has recently been revised based on observations published both early and late in the last century, identifying the NNCS.8Acetylcholine (ACh), an ester of acetic acid and the monovalent aminoalcohol choline, represents the first neurotransmitter that has been identified.5–8). The ubiquitous synthesis of ACh and the expression of n- and mAChRs in mammalian cells gives an impressive example of the complexity of biological systems.8 All receptor subtypes and signal transduction pathways used by cholinergic neurons are also used by single non-neuronal cells to communicate among each other and to maintain their phenotypic functions and thus organ homoeostasis.8 Up to now, little is known about the relevance of the NNCS for human health, but recent findings highlight its importance for the regulation of macrophage function, opening promising new concepts for the treatment of infectious diseases and cancer10  and M(3) receptor expression was confirmed using immunocytochemistry.11 Interestingly, an important role of nAChRα7 expression and function in macrophage survival and apoptosis using in vitro polarized (M1 and M2) bone marrow-derived macrophages (BMDMs) from wild-type and nAChRα7 knockout mice has been demonstrated  into the airways. Extracellular HMGB1 impairs macrophage phagocytosis and increases the mortality of mice infected with Pseudomonas aeruginosa. Recently, it was demonstrated that GTS-21 [3--anabaseine dihydrochloride], an α7nAChR agonist, inhibits hyperoxia-induced HMGB1 release into the airways, enhances dose-dependently phagocytic activity of macrophages and improves bacterial clearance from the lungs in a mouse model of ventilator-associated pneumonia. Moreover, it was shown that vagus nerve activity augments intestinal macrophage phagocytosis via nAChRα4β2 –M(5)) were demonstrated using real-time PCR .11 In thnstrated .12 TheseAChRα4β2 .14 The vartments .14 This α7 (directed against AChRα7) or nicotine revealed regulation of various signaling pathways that are involved in phagocytosis, including Fc gamma R-mediated phagocytosis, Jak-STAT-, PI3K-Akt-, Calcium-, mTOR-, Notch- and Wnt-signaling pathways.15Recently, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of differentially regulated miRNAs in whole-rat brains (that contain microglia) after exposure of animals with lipopolysaccharide, Ab16–19Macrophages represent key players of the immune system exerting highly effective defense mechanisms against microbial infections and cancer, which include phagocytosis and PrCR. Recent findings highlight the relevance of the NNCS for the regulation of macrophage function that opens promising new concepts for the treatment of infectious diseases and cancer.Until today, many compounds have been identified, which directly or indirectly can increase the effects of ACh on corresponding receptor molecules. The different cholinesterase inhibitors  belong to the latter. Irreversible inhibitors of the cholinesterase include well-known insecticides such as parathion (E 605), or other highly neurotoxic chemical agents such as sarin, tabun and many others, which often even in small quantities cause death by overstimulation of cholinergic synapses. Other chemical compounds such as neostigmin belong to the group of reversible cholinesterase inhibitors. Parathion is also used as an antidote for curare. The antagonistic interplay between parathion and curare gives an impressive example of the complexity of the NNCS, and highlights the diversity of its multiple promising targets for pharmacological modulation that may also open new avenues for the pharmacological regulation of macrophage function. Curare blocks the docking station for ACh, thereby immobilizing the nervous system and causing suffocation death. Because the active substances of curare are competitive tailors, their effect can be antagonized by an excess of ACh. Blocking of acetylcholinesterase by parathion results in such an excess of ACh and in restoring nerve cell function. However, parathion is also a poison, and aftercare with atropin is often necessary. Another interesting chemical that modulates the NNCS is physostigmin (eserin) that prevents the splitting of ACh by cholinesterase in choline and acetate. Moreover, a broad variety of compounds has been identified that block the effects of ACh on its corresponding receptors (most importantly muscarinic receptors), exerting the so-called anticholinergic effect. Certain alkaloids work anticholinergic, for example, atropin, hyoscyamin or scopolamin.20 and the adrenergic system is a direct opponent of the cholinergic system in the nervous system,21 it is tempting to speculate whether these two systems antagonize each other in macrophages as well.It has to be emphasized that the NNCS interacts with a broad variety of other hormonal networks including the adrenergic system. As adrenergic receptors are also expressed in macrophagesIn conclusion, an increasing body of evidence now convincingly demonstrates that pharmacological modulation of the NNCS to regulate phagocytosis and PrCR in macrophages represents a promising new strategy for the prevention and/or therapy of many health disorders including infectious diseases and various types of cancer.


SRY (in humans)/Sry , although there are notable exceptions. Two species of spiny rats: Amami spiny rat (Tokudaia osimensis) and Tokunoshima spiny rat (Tokudaia tokunoshimensis) and two species of mole voles (Ellobius lutescens and Ellobius tancrei), lack a Y chromosome/Sry and possess an XO chromosome system in both sexes. Such rodent species, prototherians , and fish and reptile species that demonstrate temperature sex determination (TSD) seemingly call into question the requirement of Sry for brain sexual differentiation. This review will consider brain regions expressing SRY/Sry in humans and rodents, respectively, and potential roles of SRY/Sry in the brain will be discussed. The evidence from various taxa disputing the requirement of Sry for brain sexual differentiation in mammals (therians and prototherians) and certain fish and reptilian species will be examined. A comparative approach to address this question may elucidate other genes, pathways, and epigenetic modifications stimulating brain sexual differentiation in vertebrate species, including humans.Brain sexual differentiation is orchestrated by precise coordination of sex steroid hormones. In some species, programming of select male brain regions is dependent upon aromatization of testosterone to estrogen. In mammals, these hormones surge during the organizational and activational periods that occur during perinatal development and adulthood, respectively. In various fish and reptiles, incubation temperature during a critical embryonic period results in male or female sexual differentiation, but this can be overridden in males by early exposure to estrogenic chemicals. Testes development in mammals requires a Y chromosome and testis determining gene  SRY/Sry gene. However, brain sexual differentiation is generally orchestrated by a surge in gonadal steroid hormones during embryo development followed by a second surge later in adulthood. This paradigm is considered “organization-activational programming” and occurs in a sex-specific manner . Female guinea pigs prenatally exposed to testosterone propionate during a specific developmental window had permanent rewiring of the neural circuitry and as adults, demonstrated male-pattern behaviors , sexual differentiation of the gonad is dependent upon the Y chromosome and the SRY (in men)/Sry . For consistency, the gene annotation form Sry will be used throughout the manuscript, unless studies within humans or the human form of SRY are being described.For characteristic sex-dependent behaviors, especially those seen in males, a second spike in steroid hormones at adulthood  is required for full elaboration of sex-specific neurobehavioral pathways       might act as a “miRNA sponge” that binds to ncRNAs and in the process mitigates their gene silencing ability -expressing neurons within the substantia nigra (SN) in adult males  in human males but not females , which resulted in a significant upregulation of SRY C cell line  is inserted into single-cell mouse embryos (hSRYON), this early activation of SRY results in impaired brain neurogenesis and several other disorders, including inability of the resulting pups to suckle, development of fatty liver disease, arrested alveologenesis in the lung, sporadic myocardial fibrosis, and thymic hypoplasia  mice at birth, they undergo retarded postnatal growth and development and die with multi-organ failure before 2 weeks of age. The findings suggest that while SRY might regulate various pathways in the brain and other organs, premature activation of this gene can be deleterious to normal development and physiological function of these organs.To understand and uncouple the role of Sry from other Y chromosome genes, has been to delete Sry from the Y chromosome and then insert it as a transgene on an autosomal chromosome in both XX and XY chromosome bearing mice  in the anteroventral perventricular nucleus (AVPV), the medial preoptic area (MPOA), and the ventromedial nucleus compared to mice lacking the Sry transgene. The investigators ascribed the results to the fact that Sry transgene upregulated gonadal hormones, and that the results were independent of genetic sex for the brain cells. However, the differences in PRir could also be due to direct effects of Sry transgene expression in these brain regions, which was not tested in this study. Co-transfection studies, as detailed above, could be performed to determine if Sry directly upregulates the expression of PR, as has been identified with TH.Another approach that has been used to disentangle the role of Sry+ , XYSry− , and XYSry+ . Behavioral testing with the FCG model that uncouples the potential effects of Sry from other Y chromosome-associated genes and mice gonadectomized at adulthood demonstrates that mice with two XX chromosomes consumed more food during daylight hours and had increased adiposity than XY mice  mice were more active, consumed less food per body weight, and when tested in the elevated plus maze (EPM) and zero maze, these groups demonstrated enhanced anxiogenic behaviors compared to XX and XY gonadally male mice (Sry+)  expression in the suprachiasmatic nucleus (SCN) and suppressed photic phase-induced behavioral responses compared with gonadal female FCG mice  expression in the cerebellum and hippocampus independent of sex chromosome complement or gonadal sex  mice have been created with TALEN via oocyte injection  behavior when paired with WT males. Generation of Sry-green fluorescent protein (GFP) mice confirmed that Sry is expressed in the gonad and brain in day 12 embryos  technology is another method that has been used to generate transgenic knockout mice . Both of these genera possess an XO system in males and females, and the mechanisms underpinning gonadal and brain sex determination have remained elusive. Detailed below are genomic and gonadal gene expression studies with these two genera. To date, no research group has performed a global assessment of the brain transcriptomic profile in Y chromosome-deficient species within these two genera to determine which genes possibly compensate for the absence of Sry expression in the brain of these males.In therian mammals, sex determination is dependent upon the sex chromosome complement and in particular Matthey, . SeparatEllobius lutescens) and Zaisan mole vole (Ellobius tancrei) lack the Y chromosome and Sry, whereas the closely related species, Southern mole vole (Ellobius fuscocapillus) and the three other mole voles within this genus have retained the XY system. This loss of the Y chromosome in the two Ellobius species likely occurred through two independent evolutionary events that occurred ~0.6 and 2–5 million years ago (MYA) for E. lutescens and Ellobius talpinus, respectively  deletion removing a highly conserved SOX/TCF site , dosage-sensitive sex reversal, adrenal hypoplasia critical region, on chromosome X, gene 1 (Dax1), steroidogenic factor 1 (Sf1), forkhead box protein L2 (Foxl2/Pistr1), Sox9, Sox3, and doublesex and mab-3 related transcription factor 1 (Dmrt1)  are confined to three islands in the Nansei Shoto archipelago in Japan. The two species though vary in chromosome number with Tokudaia osmiensis (residing on the Amami-Oshima Island) having 2n = 25 chromosomes and Tokudaia tokunoshimensis (from the Tokunoshima Island) possessing 2n = 45 chromosomes. The Y chromosome and Sry are no longer extant in Amami spiny rat (Tokudaia osimensis) and Tokunoshima spiny rat (Tokudaia tokunoshimensis), and the loss in the two species likely occurred through separate evolutionary events . Er71 is also upregulated by SRY in species containing this gene  and Zfy are two other Y-associated genes that translocated to the distal part of the long arm of the X chromosome in the Amami spiny rat  spiny rats (Ornithorhynchus anatinus) and echidnas . While both monotremes possess five sets of XY chromosomes, they lack Sry  resides on chromosome 6 of platypuses and chromosome 16 in echidnas , Sf1, LIM Homeobox 1 (Lhx1), LIM Homeobox 2 (Lhx2), Fibroblast Growth Factor 9 (Fgf9), Wnt Family Member 4 (Wnt4), R-Spondin 1 (Rspo1), and GATA Binding Protein 4 (Gata4)  that resides on the shortest and oldest platypus Y5 chromosome , which is exclusively expressed in the testis and kidneys of males . While the gene(s) stimulating sexual differentiation in monotremes have remained elusive, these species are further evidence that gonad and brain sexual differentiation can occur in the absence of Sry.Mammals are comprised for two classification groups: therians, which includes placental mammals and marsupials) and prototherians . This divergence traces back to 190 million years ago (MYA). Animals within the monotreme order include the duck-billed platypus (Sry. Many of these species rely on temperature sex determination (TSD) or other mechanisms to guide sexual differentiation of the gonad and possibly the brain. Several other review articles provide an in-depth discussion of the hormonal control of sexual differentiation in fish  demonstrate protandrous sex changes with a monogamous mating strategy such that a single fish can begin life as a male but then undergo sexual differentiation to become a female, which is likely modulated by transcriptomic changes at the brain and gonad level , who demonstrate GSD. In medaka, the DM domain gene on the Y chromomosome/doublesex and mab-3 related transcription factor 1b on the Y chromosome (dmy/dmrt1bY) and dmrt1 appear to take the place of therian Sry in regulating sexual differentiation  and sox9a2 but suppressing the expression of rspo1 , 27 sox genes have been discovered   possess at least five sox genes , along with the brain and spleen  . From 250 days of age, this sox17 form was significantly greater in females, and the mRNA expression pattern correlated with gonadal aromatase activity. The promoter of cyp19a is hypermethylated in juvenile male sea bass but exposure to an elevated temperature increases cyp19a promoter methylation in females, which is associated with decreased expression of this gene  genome revealed genes within the sox11a gene (Lc-sox11a) in large yellow croaker (Larimichthys crocea) also shows temporal—and sex-dependent expression differences in the gonad and brain , but it remains to be determine whether there are sex-dependent differences in this species  followed by Northern blot analyses, soxlz was identified, and the protein encoded by this gene bound strongly to an oligonucleotide sequence also recognized by mouse Sry and Sox5  for 2 weeks to 25 ng/L ethinyl estradiol  results in decreased expression of sox9a and nuclear receptor subfamily 5 group number 1b (nr5a2l) transcripts in the testis that presumably contributes to spawning failure in exposed males  but a decrease in estrogen receptor-β (esr2).While amhy) in Patagonian perjerrey (Odontesthes hatcheri), gsdf in Oryzias luzonensis (related to medaka), anti-Müllerian hormone receptor type II (amhr2) in tiger pufferfish-fugu (Takifugu rubripes), and sexually dimorphic on the Y chromosome (sdY) in rainbow trout  superfamily, Wnt-pathway, sox, dm-domain, and high mobility group-box families  found that the earliest gonadal sexual differentiation markers appeared at 11–12 days post-fry (dpf) and included wnt4b and wt1a , catenin beta 1b (ctnnb1b), dax1a, foxl2, foxl3, nanos1a, nanos1b, rspo1, steroidogenic factor (sf1), sox9a, and sox9b.Other sex determining genes characterized in therian mammals have been discovered in different fish species, such as, anti-Müllerian hormone Y (Epinephelus merra), which demonstrates protogynous sex change, revealed that foxl2 was elevated in the brain, pituitary, ovary, and gill but was downregulated during late transitional stage to male , a marine fish with ZW sex determination. TALEN injection of dmrt1 into ZZ mutant fish stimulates ovary-like testis development with disrupted spermatogenesis, an increase female-associated transcripts (foxl2 and cyp19a1a) in the gonad, but reduced expression of male-related genes (sox9a and amh) . In those with two sexes, sexual differentiation can occur via GSD with male (XX/XY) and female (ZZ/ZW) heterogamety or due to TSD , incubation temperature of the egg during a critical period of embryonic development modulates gonadal sex and sex-differences in body growth, adult morphology, aggressiveness, reproductive physiology, and neurobehavioral organization and programming  also lack sex chromosomes and demonstrate TSD. When we exposed painted turtles eggs that were incubated at the male producing temperature (MPT) to the endocrine disrupting chemical, bisphenol A (BPA), it resulted in partial gonadal sex reversal to female, feminized their behavioral responses, and altered brain gene expression patterns, especially for transcripts associated with mitochondrial and ribosomal function relative to control males  after incubation at the MPT (33.5°C), including wnt11, histone demethylase kdm6b, and transcription factor cebpa  is another gene associated with the MPT and male sexual differentiation  typically demonstrate GSD. However, this can be overridden when eggs are incubated at higher temperatures, which gives rise to sex-reversed female offspring. In females generated through high temperature but not normal chromosomal females, an intron is retained in mature transcripts from two Jumonji family genes, jarid2 and jmjd , and thus it is unlikely to be responsible for sex determination in turtles with TSD , upregulation of dmrt1 and downregulation of sox9 might exert a role in male and female sexual differentiation, respectively , it is not the primary initiator of male sex determination . Such findings would suggest that multiple pathways exist to sculpt brain masculinization even within therian mammals.Surprisingly, there are rodent species within Sry is not a requisite for gonad and brain sexual differentiation as these species lack this gene. Both male and female duck-billed platypuses and echidnas contain five sets of X and Y chromosomes that resemble avian sex chromosomes  sex determination, fish, and reptilian species can demonstrate GSD and/or TSD. Unlike most other taxa, sexualization in fish is exceptionally plastic, which may confer an evolutionary advantage in rapidly changing environmental conditions  driving gonad and brain sexual differentiation across vertebrate species. Future studies should be directed at using transgenic mice and naturally occurring animal models lacking Sry to address this exciting possibility.This latter study introduces the concept that  Sekido, . One exaSRY/Sry is expressed and might affect certain pathways in the brain of men and select rodent models, respectively, it is also clear that brain sexual differentiation in therian and prototherian mammals, fish, reptiles, and likely in other vertebrate species occurs in the absence of this gene. However, it is uncertain which genes that presumably reside on autosomal chromosomes underpin brain sexual differentiation in these animals. Sry has been postulated to induce epigenetic changes in the brain, but it remains to be determined which ones are critical in modulating masculinization. Even less is known about the potential sex-dependent epigenetic cascade of events within the brain of species lacking Sry that might orchestrate masculinization or feminization of the brain. Such sexually dimorphic epigenetic modifications might be universal across taxa and possibly even precede the actions of Sry. In sum, a comparative taxa approach that examines atypical animal models where males are devoid of Sry might hold the key to unlocking other genes, pathways, and epigenetic changes essential in sculpting brain sexual differentiation.The question posed at the outset was whether SRY is required for brain sexual differentiation. While there is evidence The author confirms being the sole contributor of this work and has approved it for publication.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Understanding mechanisms that control immunity is central in the quest to gain insights into the etiopathology of many of the diseases that afflict modern societies. New results implicate the nervous system as a central player in controlling many aspects of both the innate and adaptive arms of the immune response. Furthermore it is now well appreciated that a novel group of autacoids termed as specialized proresolving mediators, which are enzymatically produced from essential fatty acids, orchestrate the immune response promoting the termination of inflammation as well as tissue repair and regeneration. The present brief review discusses evidence for the crosstalk between the nervous system and leukocytes in regulating SPM production. We will also discuss the impact that this has on controlling tissue resolution tone and the resolution of both infectious and sterile inflammation. Current Opinion in Immunology 2018, 50:48–54Innate immunityThis review comes from a themed issue on Gwendalyn J RandolphEdited by Issue and the EditorialFor a complete overview see the Available online 16th November 2017https://doi.org/10.1016/j.coi.2017.10.007http://creativecommons.org/licenses/by/4.0/).0952-7915/© 2017 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license  and docosahexaenoic acid (DHA)-derived resolvins (Rv) and the n−3 DPA and DHA-derived protectins (PD) and maresins (MaR)   (see . These maR) . The observation that ILC-3 numbers were significantly decreased in the peritoneum of vagotomised mice demonstrates that the biological actions of these cells are not solely restricted to the gastro-intestinal epithelium. Our studies demonstrated that these cells are also found in milky spots within the greater omenutm in proximity to macrophages as well as neuronal bodies arising from the gastroepiploic nerve; a nerve that is linked to the right vagus nerve via the gastroepiploic plexus . This hibacteria , 32 as wbacteria , 34 in cIn the lamina propria ILC-3 are central in regulating tissue macrophage responses by producing regulatory factors such as GM-CSF, a mechanism that can be either protective  or pathoE. coli inoculation, significantly reducing the number of leukocytes recruited to the peritoneal cavity, shortening the resolution interval (i.e. the time it takes for recruited neutrophils to traffic out of the peritoneum) and upregulated the ability of peritoneal leukocytes to phagocytose and kill bacteria [Given that leukocytes are central in SPM biosynthesis, loss of PCTRs in mice following the disruption of peritoneal neuronal signaling indicates that the neuro-immune plexus is essential for controlling PCTR biosynthesis in peritoneal leukocytes. In this context production of SPM, in particular PCTR1, by ILC-3 is central in regulating peritoneal macrophage phenotype and responses whereby co-incubations of peritoneal macrophages with either ILC-3 or PCTR1 restores their lipid mediator profiles and their ability to uptake bacteria . This wabacteria  for a de4. Netrin 1 also increases SPM production in vivo during acute-self limited inflammation, were this protein upregulates exudate RvD5 and PD1 concentrations during zymosan initiated peritonitis. Loss of netrin 1 expression in both vagotomized mice and netrin 1 heterozygote mice lead to dysregulated LM production following sterile challenged, including a reduction in exudate RvD1 and an increase in the inflammation initiating eicosanoids LTB4 and PGE2 [The regulation of lipid mediators by neuronal circuits is also central in the control of inflammation to sterile injury. The vagus nerve regulates the expression of the axonal guidance molecule netrin 1, a protein that in addition to regulating neuronal development also controls immune responses , 40, 41.remotio infectiones agens (clearance of infective agents), remotio obruta (clearance of debris), avaritiam ex munere (gain of function) and indolentia  [The biological actions of SPM are now appreciated to be relevant to many experimental as well as human settings where they regulate the cardinal signs of resolution that include algesia) , 38. Dysalgesia) , 38, 42 The authors declare that they have no competing interests.


Neuroblastoma is a tumor arising in the peripheral sympathetic nervous system and is the most common cancer in childhood. Since most of the cellular and molecular mechanisms underlying neuroblastoma onset and progression remain unknown, the generation of new in vivo models might be appropriate to better dissect the peripheral sympathetic nervous system development in both physiological and disease states. This review is focused on the use of zebrafish as a suitable and innovative model to study neuroblastoma development. Here, we briefly summarize the current knowledge about zebrafish peripheral sympathetic nervous system formation, focusing on key genes and cellular pathways that play a crucial role in the differentiation of sympathetic neurons during embryonic development. In addition, we include examples of how genetic changes known to be associated with aggressive neuroblastoma can mimic this malignancy in zebrafish. Thus, we note the value of the zebrafish model in the field of neuroblastoma research, showing how it can improve our current knowledge about genes and biological pathways that contribute to malignant transformation and progression during embryonic life. Neuroblastoma (NB) is the most common extracranial solid tumor arising in the peripheral sympathetic nervous system (PSNS), including spinal sympathetic ganglia and the adrenal medulla . This deOver the past years, zebrafish has become an attractive alternative tool to the classical mouse model for studying many human diseases, spanning from pediatric to adult tumors and from blood to neurodegenerative disorders . NotIt is important to highlight that a complete understanding of the biological processes underlying zebrafish PSNS development will be useful for defining the molecular pathways contributing to malignant transformation and NB progression. The biological information obtained with the zebrafish in vivo model might contribute to the discovery of genes and molecular pathways that are dysfunctional during the early stages of NB development.NB presents as either a localized or metastatic disease, and patients with NB have varying survival rates. Patients with a localized tumor have a good outcome, with an overall survival (OS) of more than 99% 5 years after diagnosis. In contrast, the OS of children older than one year with a metastatic tumor is less than 40% 5 years after diagnosis , 8.MYCN oncogene amplification, which is observed in 20% of patients with NB, plays a role in tumor progression and aggressiveness [MYCN is frequently observed in patients who are resistant to any therapy and is currently a prognostic marker of risk stratification in patients with NB [ALK (anaplastic lymphoma kinase) was the first gene to be identified as a familial NB predisposition gene [ALK tyrosine kinase domain, although mutations F1174L and R1275Q are the most frequently observed and are associated with a more negative prognosis [LMO1 [BARD1 [LINC00340 [MYCN gene amplification plays a major role in driving NB tumorigenesis, while ALK gene mutation cooperates with MYCN to enforce tumor aggressiveness [A large assortment of copy number variations (CNVs), ranging from numerical CNVs in localized tumors to structural CNVs in metastatic tumors, has been observed in NB. The most frequently observed structural CNVs are loss of chromosomes 1p, 3p, 4p, 9p, 11q and 14q and gain of chromosomes 2p and 17q [siveness . The gen with NB . Other grognosis , 16. Mor1 [BARD1  and LINCINC00340  have beeINC00340  and Molesiveness , 22.MYCN oncogene in neural crest-derived cells driven by the rat tyrosine hydroxylase promoter. These mice spontaneously develop NB exclusively in the sympathoadrenal system with high resemblance to human tumors, demonstrating the involvement of MYCN in NB genesis [Previous studies have established different in vivo models of NB. Mouse models of NB are clinically relevant tools for studying the growth and metastasis of this aggressive malignancy. Orthotopic and subcutaneous xenograft mouse models have been extensively generated for the preclinical testing of new therapeutic strategies against NB –30. Nota genesis –37.MYCN gene amplification, the in vivo forced expression of genes that are normally involved in sympathoadrenal development during embryonic life (such as ALK and LIN28B) drives NB formation [Other transgenic mouse models have helped to elucidate the cellular mechanisms of NB pathogenesis. In addition to ormation , 39. ThuIncreasing evidence indicates that NB arises from the NCC lineage during embryonic life ; thus, The cells of the sympathetic ganglia and mature adrenal medulla originate from a transient pool of NCCs. These cells pass through four critical developmental phases. First, NCCs undergo an epithelial to mesenchymal transition (EMT) from the most dorsal region of the neural tube and differentiate into sympathoadrenal precursors. Then, sympathoadrenal precursors migrate toward the ventral portion of the notochord near the dorsal aorta. Finally, these cells activate the neuronal differentiation program, giving rise to the mature sympathetic ganglia and chromaffin cells of the adrenal medulla.After neurulation, NCCs undergo an EMT from the roof plate of the neural tube, delaminating from the neuroepithelium and then migrating through the periphery, where they differentiate into several cell types, including cells of the peripheral nervous system and enteric nervous system, pigmented cells, Schwann cells, adrenal medullary cells, and cartilage cells of the craniofacial skeleton –43. The KRT19, ERBB3, TWIST1 and TCF3 EMT-related genes are differentially expressed in a cohort of patients with NB. Thus, in addition to MYCN gene amplification, the expression level of EMT-related genes might become an additional prognostic marker for patients with NB. Despite these recent discoveries, there is no solid evidence from studies using in vivo models that supports the relationship between EMT and NB pathogenesis. Therefore, additional studies will be necessary to elucidate whether EMT plays a role in tumor development and eventual metastasis in NB. Consequently, the window of time in which EMT impairment might lead to the modulation of tumor-associated signals should be defined.Recently, in vitro studies have suggested that an EMT might also be involved in NB progression, and Nozato and colleagues  found thAfter an EMT begins, NCCs acquire a polarized morphology and follow two migratory pathways away from the neural tube: the dorsolateral and the ventromedial Fig. . NCC-derMYCN appears to be required for the migration, survival and/or differentiation of NCCs that migrate to the dorsal aorta. Accordingly, homozygous mutant mouse embryos display embryonic lethality (at approximately 11.5 days of gestation), and several developing organs are affected, including the dorsal root ganglia and sympathetic ganglia of the PSNS [The development of the PSNS in zebrafish has been well examined , 55. In the PSNS –67.Fig. N-Cadherin, CXCL12, and Ephrins, play a critical role in the initiation of the segmental organization of sympathetic ganglia in a discrete and metameric pattern [When sympathoadrenal precursors reach the dorsal aorta, other molecules, such as  pattern . Moreove pattern . The sym pattern . In 2002 pattern  demonstr pattern . Thus, i pattern . This phphox2b and zash1a, which in turn activate the expression of hand2 and later markers, such as phox2a, GATA2 and AP-2 alpha (tfap2a) [hands off) fails to reach the complete differentiation of sympathoadrenal cells, even though some early PSNS markers are normally expressed [Other transcription factors are required for sympathoadrenal cell development and maintenance, such as (tfap2a) –75. For xpressed . An analxpressed , 77.TH) and dopamine B-hydroxylase (DBH) genes, which are involved in the conversion of the amino acid l-tyrosine to noradrenalin [TH, and the complete maturation of sympathetic ganglia and chromaffin cells is reached at 28 dpf [Finally, sympathoadrenal cells undergo catecholaminergic terminal differentiation for the formation of the mature sympathetic chain and the adrenal gland. This last differentiation step requires the expression of tyrosine hydroxylase  but do not express markers of catecholaminergic differentiation, such as TH or DBH, which indicates that these malignant cells originate from a different neural lineage than human NB. Despite these differences, Hagoromo-derived tumors seem to arise through a similar mechanism as in humans [fgf8 appears to function as an oncogene not only in numerous malignancies, including prostate and breast cancer in mammals [The histological screening of a collection of zebrafish insertional mutants revealed that four independent lines carrying mutations in the netrance . These fn humans . Thus, f mammals , 94, butMany cellular and molecular features of NB have been identified over the past decades, and some of these genetic aberrations are now considered strong and powerful predictors of patient outcome. Nevertheless, there is still more complexity behind this pediatric neoplasm, and continued advances are needed to translate this information into more efficient clinical therapy. Although murine and avian models have been used to dissect PSNS development and NB pathogenesis , zebrafi


Chronic kidney disease (CKD) can be a life-threatening condition, which eventually requires renal replacement therapy through dialysis or transplantation. A lot of effort and resources have been invested the last years in the identification of novel markers of progression and targets for therapy, in order to achieve a more efficient prognosis, diagnosis, and treatment of renal diseases. Using experimental models of renal disease, we identified and studied two promising candidates: periostin, a matricellular protein with high expression in bone and dental tissues, and discoidin domain receptor 1 (DDR1), a transmembrane collagen receptor of the tyrosine kinase family. Both proteins are inactive in physiological conditions, while they are highly upregulated during development of renal disease and are primarily expressed at the sites of injury. Further studies demonstrated that both periostin and DDR1 are involved in the regulation of inflammation and fibrosis, two major processes implicated in the development of renal disease. Targeting of either protein by genetic deletion or pharmacogenetic inhibition via antisense oligonucleotides highly attenuates renal damage and preserves renal structure and function in several animal models. The scope of this review is to summarize the existing evidence supporting the role of periostin and DDR1 as novel biomarkers and therapeutic targets in CKD. Chronic kidney disease (CKD) represents a major burden for modern societies affecting almost 10% of the population worldwide, while its growing incidence accounts for the doubling of the global number of deaths caused by CKD over the past 25 years , transforming growth factor-β (TGF-β), platelet-derived growth factors (PDGFs), connective tissue growth factor (CTGF), endothelin-1 (ET-1), and key pro-inflammatory cytokines . Inhibition of these factors in animal models was shown to efficiently ameliorate the progression of renal disease by limiting renal inflammation and fibrosis. However, only renin-angiotensin system (RAS) antagonists are presently applied in clinical practice with limited potential in cases of human CKD, while drugs targeting some of the above mediators have come up to clinical trials but there is no certainty that they will be finally approved for use in patients , 7.Our team has recently identified two novel mediators of renal disease which may serve as promising biomarkers and/or targets for therapy of CKD: periostin and discoidin domain receptor 1 (DDR1). This review will summarize the latest literature on these proteins focusing on renal diseases and discuss their potential of constituting the basis of future treatment against CKD.Periostin is a 90-kDa secreted matricellular protein originally identified because of its high expression in periosteum and periodontal ligament . AlthougThe protein is composed of three distinct regions serving different functions: an N-terminal cysteine-rich EMI domain reported to bind to collagen I, fibronectin and Notch 1, a tandem repeat of four fasciclin-I domains containing binding sites for BMP-1, tenascin-C, as well as several integrins, and a C-terminal domain which is a site of proteolytic cleavage and splice variant formation –13. The Periostin has been shown to play important roles in the pathophysiology of several organs. In the heart, periostin production by fibroblasts was demonstrated to be crucial for collagen fibrilogenesis and cardiac healing short-term after myocardial infarction, whereas in chronic cardiac disease models periostin null mice were protected from hypertrophy and fibrosis , 15. Stude novo expressed in biopsies from patients with various renal diseases, including diabetes, lupus nephritis, IgA nephropathy, and focal segmental glomerulosclerosis. The protein was over-expressed in areas with periglomerular or interstitial fibrosis and its expression levels were associated with the degree of histological damage and the decline of glomerular filtration rate  and nephrotoxic serum (NTS)-induced glomerulonephritis , 34. Mosin vitro or in vivo in different disease contexts. The pro-fibrotic growth factor TGF-β1 is a known potent inducer of periostin expression  signaling through activation of either the ERK1/2 or JNK pathways in different cell lines. It can also induce the expression of matrix metalloproteinases in vascular smooth muscle, bronchial epithelial, and cancer cells. Moreover, DDR1 activation was shown to induce the PI3 kinase/Akt and NFκB pathways in cancer cells . In addiDeregulated function of DDR1 has been described in various human diseases, including several types of cancer, atherosclerosis, osteoarthritis, and fibrotic diseases. DDR1 was shown to be pro- or anti-tumorigenic in different types of cancer. More specifically in renal cancer, phospho-DDR1 (pY792/6) was found to be preferentially enriched in papillary RCC compared to clear cell RCC samples . On the de novo expressed in damaged podocytes, while it was also found increased in the glomeruli of patients with lupus nephritis and good pasture syndrome  and its expression levels correlate very well with the stage of the pathology and the decline of renal function , 33–35. In terms of therapy, a promising target should be easily accessible by potential drugs, while its silencing or inhibition in animal models of CKD should preserve renal structure and function. In this context, both periostin and DDR1 may represent potential targets for therapy of renal disease. The expression of periostin is primarily localized at the sites of injury both in animal models and human biopsies of CKD, which demonstrates that periostin is highly associated with renal damage. For example, periostin was found to be produced by vascular cells in hypertensive nephropathy, by tubular epithelial cells in ureteral obstruction and PKD, and by glomerular podocytes and parietal epithelial cells in glomerulonephritis , 33–35. Discoidin domain receptor 1 is also over-expressed and activated in several animal or human renal pathologies, while its inhibition even after the establishment of renal damage attenuates renal inflammation and fibrosis and preserves kidney function [–54 and TWe identified periostin and DDR1 as novel promising targets for therapy which could be applied in the future for the prevention and/or treatment of patients with CKD. Our observations in biopsies and animal models of CKD demonstrated that both periostin and DDR1 are expressed in low levels under physiological conditions, are highly activated after renal damage and expressed primarily by injured cells, are well correlated with CKD progression, while their deletion or pharmacogenetic inhibition largely protects from development of renal disease. Despite the recent advances, several aspects of the mechanisms of action of periostin and DDR1 are still elusive. The most crucial limitation for pursuing the targeting of these new candidates in human CKD is the current lack of validated tools available for use in humans ; however, we are confident that this limitation will be surpassed in the near future as the field is rapidly expanding, which will hopefully allow a most efficient prognosis and treatment of CKD.NP reviewed the literature and prepared the manuscript. CC supervised and reviewed the manuscript.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.


Pemphigus vulgaris (PV) is an autoimmune skin blistering disease effecting both cutaneous and mucosal epithelia. Blister formation in PV is known to result from the binding of autoantibodies (autoAbs) to keratinocyte antigens. The primary antigenic targets of pathogenic autoAbs are known to be desmoglein 3, and to a lesser extent, desmoglein 1, cadherin family proteins that partially comprise the desmosome, a protein structure responsible for maintaining cell adhesion, although additional autoAbs, whose role in blister formation is still unclear, are also known to be present in PV patients. Nevertheless, there remain large gaps in knowledge concerning the precise mechanisms through which autoAb binding induces blister formation. Consequently, the primary therapeutic interventions for PV focus on systemic immunosuppression, whose side effects represent a significant health risk to patients. In an effort to identify novel, disease-specific therapeutic targets, a multitude of studies attempting to elucidate the pathogenic mechanisms downstream of autoAb binding, have led to significant advancements in the understanding of autoAb-mediated blister formation. Despite this enhanced characterization of disease processes, a satisfactory explanation of autoAb-induced acantholysis still does not exist. Here, we carefully review the literature investigating the pathogenic disease mechanisms in PV and, taking into account the full scope of results from these studies, provide a novel, comprehensive theory of blister formation in PV. Pemphigus vulgaris (PV) is an autoimmune skin blistering disease characterized by the presence of autoantibodies (autoAbs) directed against keratinocyte surface antigens. It has been well established that autoAbs alone are capable of driving blister formation in PV. Early studies identified the primary target of pathogenic autoAbs as desmoglein 3 (Dsg3) and to a lesser extent, desmoglein 1 (Dsg1). More recently, additional autoAbs specificities have been identified in PV patients that could potentially also contribute to disease pathogenicity . DespiteSince the primary target of PV autoAbs was shown to be a desmosomal protein, most of the earliest theories of acantholysis suggested that the loss of cell adhesion was simply the result of autoAbs sterically hindering the homo- and heterophillic binding of desmosomal proteins between neighboring cells , 4. StudThe observation by multiple groups that PVIgG was seen only to bind at desmosomal areas also supported the idea that autoAb interference of desmosomal interaction drives blister formation –10. In aFurther support for the steric hindrance theory came from later studies attempting to characterize the fine-epitope specificity of anti-Dsg3 autoAbs. It was shown that autoAbs primarily target the amino terminal of Dsg3 which, based on the crystal structure of other classical cadherins, was predicted to facilitate trans-interaction –17. Furt2) fragments, as well as single-chain variable fragments which lack the Fc domain, were able to induce blister formation in vitro proved that Fc-dependent mechanisms were not necessary for blister formation -deficient mice were protected from PVIgG-induced blister formation . Pkg, anIdentification and characterization of the precise signaling pathways driving autoAb-induced acantholysis has been a significant focus for PV research. As a result, large amounts of (often conflicting) information concerning the signaling alterations downstream of anti-Dsg and PVIgG binding have been characterized. Moreover, studies showing that autoAbs in PV sera directed at non-desmoglein antigens can also elicit intracellular signaling have further complicated efforts to elicit the precise mechanisms driving disease , 50. Thep38 is one of the three major families of mitogen-activated protein kinases (MAPK), which are known to play a prominent role in a wide range of cellular pathways . In geneThe significance of p38MAPK signaling in PV pathogenesis was first suggested by a study which observed that PVIgG induced significant increases in the phosphorylation of p38MAPK, MAPKAP2 (MK2), and heat-shock protein (Hsp)27 . The degOther studies assessing the role of PVIgG on p38MAPK activation identified that PVIgG causes keratin retraction and p38MAPK activation within 30 min, and another peak at 6–10 h in cultured human keratinocytes. Only inhibition at the earlier time point was associated with prevention of blister formation and keratin retraction . Anotherin vivo, as well as p38MAPK activation, suggesting auto-phosphorylation of p38MAPK  signaling , RhoA ac2+ concentration in keratinocyte culture medium increases intracellular calcium which in turn induces cell–cell contact (mainly adherens junctions) within 5 min, and formation of desmosomes within 2 h (2) into inositol 1,4,5-triphosphate (IP3) and diacylglycerol (DAG), plays a significant role in calcium-induced keratinocyte differentiation  via EGFR and nitric oxide synthase. The end result is the activation of caspases 3 and 9, which have been proposed to contribute to bister formation  then induce the retraction of the KIFs as well as the turnover of the actin cytoskeleton; (3) the retraction of KIFs from the desmosomal plaques, as well as cytoskeletal rearrangements, then cause a destabilization of desmosomes, and a weakening of cell adhesion; (4) finally, the weakening of cell adhesion, coupled with the mechanical stress induced by cytoskeletal rearrangements, induces a cellular stress response, resulting in the activation of Src, EGFR, and Rac1 (as well as other pathways), and re-initiation/perpetuation of the pathological cycle  binding of autoAbs to target desmosomal antigens  induces the activation of PLC, leading to the activation of PKC  as Ask1 , 156];  to PV –161, butAlthough this model lays a comprehensive framework for the mechanism of blister formation incorporating all of the major signaling pathways implicated in pathogenesis by the literature, future experiments are required to test the validity of this model and more precisely define the series of signaling events which occur downstream of autoAb binding. Determining the degree to which both desmosomal and extra-desmosomal Dsg proteins associate with signaling molecules and whether or not the binding of autoAbs to these proteins is sufficient to elicit intracellular signaling is a high priority to ascertain if these molecules could function as signal transducers and if so, through which pathways. Immunofluorescent studies of keratinocyte monolayers before and after PVIgG treatment may be one way to determine which molecules are physically associated with desmosomal proteins and determine if autoAb binding leads to their activation/translocation. Another important step would be to more precisely define the upstream MAPK cascade that leads to p38MAPK activation. There are multiple upstream signaling molecules which are known to activate p38MAPK and the characterization of the molecules involved in this process could allow for a better understanding of how autoAbs, or additional factors such as hormones or cytokines (which are known to utilize specific cascades) could activate p38MAPK –175. ProDesmosomal proteins as a mechanosensing signaling complex would provide a logical intersection between the two primary theories of autoAb-induced blister formation in PV. Although future studies are still needed, there exists a great potential to significantly and directly affect the development of future treatments in PV. Identification of the signals transduced by autoAb binding could lead to the identification of potentially novel drug targets, or, at the very least allow researchers to focus on disease-specific signaling pathologies, resulting in small molecule inhibitors with a lower chance of harmful side effects and the ability to minimize systemic suppression of the immune system. In addition, large scale profiling of the signal transduction pathways upstream of MAPK may represent an especially beneficial endeavor as such studies could identify cytokines or hormones which may be contributing to disease process. In addition to facilitating the use of disease specific anti-cytokine or hormonal therapies which may already be in use for other disease modalities, such information could be individualized to the specific pathologies driving disease in a given patient, informing more tailored and effective treatment strategies and greatly enhancing clinical management of disease.AS and TS contributed to the conceptualization and writing of the manuscript.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.


The most recent systematic literature reviews and meta-analyses have indicated significant and nutritionally-relevant composition differences between organic and conventional foods. This included higher antioxidant, but lower cadmium and pesticide levels in organic crops, and higher omega-3 fatty acids concentrations in organic meat and dairy products. Also, results from a small number of human cohort studies indicate that there are positive associations between organic food consumption and reduced risk/incidence of certain acute diseases  and obesity. Concerns about potential negative health impacts of organic food consumption (e.g. risks linked to lower iodine levels in organic milk) have also been raised, but are not currently supported by evidence from human cohort studies. However, there is virtually no published data from (1) long-term cohort studies focusing on chronic diseases  and (2) controlled human dietary intervention studies comparing effects of organic and conventional diets. It is therefore currently not possible to quantify to what extent organic food consumption may affect human health. The demand for organic food has increased rapidly over the last 25 years in many developed countries in Europe, North America, and Asia/Oceania . Demand While there is increasing scientific evidence for biodiversity and environmental sustainability-related benefits of organic farming –6, thereorganic crops have higher antioxidant activity and between 18 and 69% higher concentrations of a range of individual antioxidants; increased intakes of polyphenolics and antioxidants has been linked to a reduced risk of certain chronic diseases such as cardiovascular and neurodegenerative diseases and certain cancers ;conventional crops have higher levels of the toxic metal cadmium, and are four-times more likely to contain detectable pesticide residues; there are general recommendations to minimise the intake of pesticides and cadmium to avoid potential negative health impacts ;conventional crops also have higher concentrations of protein, nitrogen, nitrate, nitrite, respectively; increased intakes of these compounds have been linked to both positive and negative health impacts ;organic meat, milk, and dairy products have approximately higher concentrations of nutritionally-desirable omega-3 fatty acids; intakes of very long chain omega-3 fatty acids in Western diets and there are EFSA (European Food Safety Authority) recommendation to at least double their intake ;organic milk was reported to contain higher levels of total conjugated linoleic acid (CLA), higher iron and α-tocopherol concentrations, which are all considered to be nutritionally desirable, although the evidence for health benefits of CLA is mainly from in vitro and animal studies ;conventional milk was estimated to have and higher concentrations of iodine and selenium, respectively; milk is not a major source for selenium, but may be the main source of iodine in countries were iodised salt in not widely available or used; there is concern that the lower iodine content in organic milk may cause iodine deficiency  and associated negative health impacts  ;conventional meat has slightly, but significantly higher concentrations of the saturated fatty acids myristic- and palmitic acid, which were linked to an increased risk of cardiovascular disease .A series of recent systematic reviews and meta-analyses of published data have shown that there are significant differences in the concentrations of nutritionally relevant compounds between organically and conventionally produced foods –14. SpecGRADE  assessment showed that the overall strength of evidence was good or moderate for many of the parameters listed above , but low for others  –14.Also, for a range of parameters  for which significant differences were identified by meta-analyses GRADE assessment showed high inconsistency, poor precision and/or publication bias –14. ThisMeta-analyses carried out prior to 2014 were all based on a smaller evidence base (number of publications/data), but produced broadly similar results, when they analysed the same parameters –11. BranHowever, it is virtually impossible to accurately estimate change in dietary intakes, since there are still substantial gaps of knowledge with respect to composition differences between organically and conventionally produced foods. For example, there is (1) a need to systematically review mycotoxin levels in crops  and/or composition differences in eggs from organic and conventional farms, (2) insufficient data to accurately estimate the magnitude of differences for individual crops and meat products from different livestock species, which means it is currently not possibly to accurately estimate differences in dietary intakes of the most desirable and undesirable compounds from organic and conventional food based diets, and (3) not enough published information to compare concentrations of a wide range of nutritionally relevant nutrients  and undesirable compounds  in a meta-analysis –14. AlsoA small number of human cohort studies and animal dietary intervention studies have identified associations between organic food consumption and specific health, and health-related physiological parameters. Most human cohort studies were mother-and-child dyad cohorts and reported positive associations between organic vegetable and/or dairy consumption and risks of (1) pre-eclampsia in mothers , (2) hypA sub-study (of about 54 000 adults) of the French-Belgium Nutrienet-Sainté cohort reported that regular consumers of organic food had a substantially lower risk of being overweight or obese . The assAlso, a subgroup of a large UK cohort study focused on cancer incidence in middle-aged women showed that there is a weak association between organic food consumption and a reduced incidence of non-Hodgkin’s lymphoma, although the study was based on an observation period of only seven years .However, there are a range of confounding factors that may have influenced the outcome of all cohort studies since organic and conventional consumers are known to differ in a range of other lifestyle factors  which are often difficult to properly factor out in cohort studies ,23,24.There are also a small number of published animal dietary intervention studies in which the effects of organic vs. conventional food consumption were compared  address a range of methodological issues in both the available meta-analyses of composition data and dietary cohort studies ,23,24, (