Glucocorticoid hormones (GCs) have profound effects on bone metabolism. Via their nuclear hormone receptor – the GR – they act locally within bone cells and modulate their proliferation, differentiation, and cell death. Consequently, high glucocorticoid levels – as present during steroid therapy or stress – impair bone growth and integrity, leading to retarded growth and glucocorticoid-induced osteoporosis, respectively. Because of their profound impact on the immune system and bone cell differentiation, GCs also affect bone regeneration and fracture healing. The use of conditional-mutant mouse strains in recent research provided insights into the cell-type-specific actions of the GR. However, despite recent advances in system biology approaches addressing GR genomics in general, little is still known about the molecular mechanisms of GCs and GR in bone cells. Here, we review the most recent findings on the molecular mechanisms of the GR in general and the known cell-type-specific actions of the GR in mesenchymal cells and their derivatives as well as in osteoclasts during bone homeostasis, GC excess, bone regeneration and fracture healing. Glucocorticoid hormones (GCs) are major stress hormones released by a hierarchical hormonal axis. The circadian rhythm and psychological and physiological stress trigger the hypothalamus to release corticotropin-releasing factor (CRF), which in turn acts on the pituitary, stimulating its release of adrenocorticotropic hormone (ACTH). ACTH acts on the zona fasciculata of the adrenal cortex for the release of GCs, which belong to the steroid class of hormones. Cortisol as the major GC in humans and corticosterone, the major GC in rodents, act on virtually all cells in the body via the GC receptor (GR) . ConsequHere, we review the recent mechanistic evidence of how GCs act via the GR and thus affect skeletal cells, and how this translates to alterations in bone mass and modification of fracture healing.Both endogenous and synthetic GCs exert their effects via nuclear receptors such as the GR (NR3C1) and the closely related mineralocorticoid receptor (MR) (NR3C2); both are ligand-induced transcription factors . Only inThe ligand-bound GR can dimerize and bind directly to specific palindromic sequences in the genome (GC responsive elements (GRE) or glucocorticoid receptor-binding sites (GBS)) and in the vicinity to binding sites of tissue-specific transcription factors  in cortical bone, and thus, effects on cortical bone might differ from those in humans. Second, there is a strong variety in GC effects on bone concerning different mouse strains. Swiss Webster mice described by Weinstein and colleagues  and other soluble and membrane-bound factors. In addition to osteoblasts, osteocytes were recently discovered to be a major source of osteoclast-inducing factors, including RANKL .Figure The disturbance of these processes leads to bone loss because of enhanced bone resorption and/or diminished bone formation. Cell-type-specific genetic modulation of bone cells in mice confirmed the strong cell-autonomous impact of GCs on bone mass.Osteoclast activity is greatly increased at the onset of GC excess, but declines with prolonged GC excess. This dual activity results from complex, in part opposing, mechanisms of GCs on osteoclast function and maturation.In particular, in cell systems, an induction of RANKL by simultaneously reducing the osteoclast differentiation inhibitor osteoprotegrin (OPG) was observed . These cells were originally defined as stromal bone marrow cells with multi- or at least tri-lineage differentiation potential into adipocytes, osteoblasts or chondrocytes. Finally, MSCs should be capable to give rise to new bone when transplanted ectopically into rodents . NumerouSome experimental conditions suggested that GCs contribute to adipocyte differentiation and that this would be at the expense of osteogenic differentiation, providing an attractive model to explain two side effects of GC excess, namely fat redistribution and GIO . Certainin vitro studies varied from bone marrow stromal cells to adipose-tissue stromal cells and ectodermal-derived calvarial pre-osteoblasts. Surprisingly, all of these cultures can be directed into distinct differentiation direction, given that the respective culture medium cocktail is provided. These cultures frequently contain cells of mixed character, and it remained unclear whether they originated from homogenous progenitors or whether these cultures consist of a mixture of distinct, specifically committed cells that expand only under the respective conditions. Furthermore, we observed that impaired GR dimerization in cells of GRdim mice (described earlier) completely abrogates the induction of adipocytes from progenitor cells  activation –insulin-like growth factor 1 (IGF1) axis; whereas GH triggers IGF1secretion in the liver, both act on the cartilaginous growth plate in growing bones. GC excess is accompanied by a decline in GH and IGF1 titres as well as action  turned out to be responsive , are now tested in clinical trials for atopic skin diseases. However, some of these compounds with steroidal scaffold still exert side effects , also GR dimer inducing agents were postulated to improve steroid therapy  on the OPG/RANKL ratio. They concluded that these compounds suppress the OPG/RANKL ratio less than prednisolone in two human osteoblast cell lines -18 and (R)-21 were described to have less effect on bone mass than prednisolone. And this was demonstrated even at concentrations showing a stronger reduction of inflammatory disease score than prednisolone in a model of collagen-induced arthritis , the deletion of GR in osteoblasts leads to reduced bone mass. Ablation of GR in osteoblasts decreases bone volume in the trabecular compartments of particular bones when using GRTaken together, dependent on the model, GCs and the GR are slightly anabolic at very distinct bone sites, either femora, vertebrae or tibia. In contrast, MR seems to be rather catabolic and whether it mediates GC effects remains to be elucidated. The different background strains of the respective mouse lines may also contribute to the differential effects on bone mass.Patients with GIO are at increased risk for fractures, and it can be anticipated that the process of fracture healing is disturbed because of the strong effects of GCs on virtually all participating cell types. However, there are no clinical studies on fracture healing in GIO patients. Furthermore, to date clinical data on bone healing in patients under short-term GC medication are lacking. However, such observations would be extremely important, because GCs are widely prescribed for patients with inflammatory and other disorders. In the following section, we review the literature on the effect of exogenous and endogenous GCs on fracture healing in preclinical models.Fracture healing starts with a fine-tuned inflammatory phase with the orchestrated actions of immune cells and cytokines. Because of tissue and vessel disruption, endogenous danger associated molecular patterns (DAMPs), including histones, mitochondrial DNA, and ATP, are released, which trigger activation of the immune system . ImmediaProper resolution of inflammation is mandatory to enable normal fracture healing. Any disturbance of the inflammatory phase is known to influence fracture healing outcome negatively; examples are prolonged inflammation, increased or altered profile of inflammatory mediators as probably caused by concomitant inflammatory disorders, including severe trauma, rheumatoid arthritis, osteoporosis, and diabetes, and experimental depletion of certain immune cells .GCs could participate in the resolution of inflammation also during fracture healing. However, they have both adverse and beneficial roles on bone cells, depending on the exposure and timing. However, how GCs orchestrate fracture healing remains largely unknown and only recent studies with conditional-knockout mice shed some light into this complicated process, as described below.Interestingly, only a few studies to date have analysed the effects of GCs on fracture healing. These studies demonstrated different effects of short- and long-term administration of synthetic GCs on bone repair. Short-term medication with GCs did not significantly disturb bone regeneration . The reaCol2CreERT2 mice attenuated endochondral bone healing in tibial metaphyseal fractures by transiently increasing the cartilaginous fraction of the callus, but still resulted in a normal healing response -6 levels in the serum, increased IL-1β concentrations in the initial fracture haematoma and a significantly higher number of T cells infiltrating the fracture callus. During callus formation, endochondral ossification was disturbed in the absence of the GR, as confirmed by persisting cartilage in GRCol2CreERT2 mice. In the late healing phase, bony bridging of the fracture gap was reduced, resulting in poor mechanical properties of the healed bones. Therefore, the GR has a protective role in fracture healing by shaping the inflammatory response and by promoting cartilage-to-bone transition . Bone represents a major tissue in appendages; intriguingly it is completely regenerated after amputation of salamander limbs and fish fins. As discussed previously in this review, progenitor cells are thought to be the cellular source of newly forming osteoblasts during mammalian bone repair, whereas mature osteoblasts  do not appear to contribute to bone repair . Advances in The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of this review.This work was funded by the German Research Foundation within the Collaborative Research Centre ‘Danger Response, Disturbance Factors, and Regenerative Potential after Acute Trauma’ .


Cancers remain a major public health problem worldwide, which still require profound research in both the basic and preclinical fields. Patient-derived xenograft (PDX) models are created when cancerous cells or tissues from patients’ primary tumors are implanted into immunodeficient mice to simulate human tumor biology in vivo, which have been extensively used in cancer research. The routes of implantation appeared to affect the outcome of PDX research, and there has been increasing applications of patient-derived orthotopic xenograft (PDOX) models. In this review, we firstly summarize the methodology to establish PDX models and then go over recent application and function of PDX models in basic cancer research on the areas of cancer characterization, initiation, proliferation, metastasis, and tumor microenvironment and in preclinical explorations of anti-cancer targets, drugs, and therapeutic strategies and finally give our perspectives on the future prospects of PDX models. Cancers are among the leading causes of death worldwide. The Cancer Moonshot 2020 program has been launched in 2016 to transform the cancer research and care ecosystem and double the rate of progress in cancer prevention, diagnosis, and treatment . Novel tPatient-derived xenograft (PDX) models are immunodeficient mice engrafted with patients’ cancerous cells or tissues. The development of PDX models for cancer research, based on the assumption that these models faithfully resemble the original tumors, especially for the patient-derived orthotopic xenograft (PDOX) models , has sigPrkdcscidIl2rgtm1Wjl (NSG) [PrkdcscidIl2rgtm1Sug (NOG) mice [−/− (NSI) mice, which exhibit severe immunodeficiency, lacking T, B, and NK cells, and used these mice in studies of both leukemia and solid tumors [−/− mice, called NSIN, by deleting foxn1 with CRISPR/Cas9 system. The nude NSIN mice showed even higher immunodeficiency than NSI mice by TEI and can be more suitable for studies of tumors with poor engraftment efficiency (data unpublished).Immunodeficient mice engrafted with human immune systems provide powerful models for the study of human immunobiology in vivo, and PDX models with these humanized mice are critical tools for studying the interaction between human immunity and various cancers. In order to establish a PDX model, we need a highly immunodeficient mouse strain. Several types of immunodeficient mice can be used to establish xenograft models: athymic nude mice, SCID, NOD-SCID, and recombination-activating gene 2 (Rag2)-knockout mice . Howeverjl (NSG)  and NODSOG) mice , are witOG) mice –20. We gd tumors –25. As td tumors . RecentlFor the first implantation, patient-derived tumors may be implanted into immunodeficient mice in the form of small tumor fragments or cell suspensions derived from blood of patients or from digestion of tumors into single-cell suspensions. Principal determinants of successful tumor engraftment into immunodeficient mice are the viability and sterility of the human tumor . Cancer Cancerous cells or tissues can be implanted heterotopically or orthotopically and monitored for tumor formation Fig. . In contSince many patients’ primary tumors cannot engraft directly in immunodeficient mice, other methods are needed to establish PDX models for these patients. Primary tumor cells can be reprogrammed to iPSC and then differentiated into the cell type of origin, which then can be used to establish PDX models. PDX models derived through an intermediate iPSC stage could be useful in approximately one third of patients whose primary cells cannot undergo PDXs . An advaRecent advances in immunotherapies highlight the importance of the immune system in tumor progression and treatment, which require PDX models with human immune system to facilitate the study of immunity-cancer interactions and preclinical assessment of cancer immune therapies. To establish human immune system-conditioned PDX models, we first need to generate humanized mice . One method for the generation of humanized mice involves the transplantation of total peripheral blood or tumor-infiltrating lymphocytes (TILs) into immunodeficient mice. These procedures are known to cause severe graft versus host disease (GVHD) 2–5 weeks after injection  and limiBasic cancer research is to characterize cancer biology and explore mechanisms involved for improved understanding or prediction of cancer. PDX models essentially provide important in vivo and ex vivo evidence to aid basic studies of cancer, including tumor characterization, tumorigenesis, and metastasis.Provided that PDX models faithfully mimic human cancers, they can be used to delineate the per se molecular, cellular, and sub-clonal characterizations of various types of cancers. In the PDX model of acute lymphoblastic leukemia (ALL), a rare unfavorable ALL subpopulation has been defined which is dormant and treatment resistant and mimics patients’ primary cells at minimal residual disease . PDX mod+ human brain tumor initiating cells (TICs) that initiate tumors in vivo, providing insights into human brain tumor pathogenesis, giving strong support for the CSC hypothesis as the basis for many solid tumors [PDX models are frequently used to study the cellular components involved in cancer cell initiation and proliferation. The cancer stem cell (CSC) hypothesis suggests that neoplastic clones are maintained exclusively by a rare fraction of cells with stem cell properties. Xenograft assay identified CD133d tumors . The intd tumors ; knockdod tumors ; Notch- d tumors ; short hd tumors . Since id tumors . The inid tumors . MiRNA-1d tumors . Milliond tumors . The prod tumors . A method tumors .Metastasis is the basis of cancer lethality, of which the mechanisms are not fully understood and interventional strategies not well defined. PDX models are useful in defining cell populations and molecules associated with metastasis. Metastasis-initiating cells (MICs) have been proven critical for cancer metastasis. But it is difficult to identify and isolate adequate numbers of MICs from patients for research. PDX models are depositories of MICs. PDX model of human breast cancer was used to identify and isolate MICs through a highly sensitive fluorescence-activated cell sorting (FACS)-based assay . CirculaAnti-cancer therapies exert selective pressure on tumor cells that leads to the preferential growth of resistant subpopulations, necessitating the development of novel generations of therapies to treat the evolving cancers. A critical role for PDX models in preclinical research is to identify therapeutic targets, including specific molecules and molecular interactions. Another major role for PDX models is as a guide for the clinical treatment of cancer patients Fig. . The choFirst, PDX models can help to discriminate the most suitable therapy for cancer patients Fig. . PDX modCCLX models are not adequate for preclinical development of anti-cancer agents because most human cancer cell lines do not accurately reflect human malignant tumors . In contSAMMSON can be targeted to sensitize melanoma to MAPK-targeting therapeutics both in vitro and in PDX models [PDX models in preclinical cancer research is to aid the identification of cancer-specific biomarkers that can be used for diagnosis, prognosis, and therapeutically targeted. Whole-transcriptome profiling of PDX models to identify both tumor- and stromal-specific biomarkers supports drug efficacy studies and compartment-specific biomarker discovery . PDX modX models . The IGFX models .Conventional chemotherapy is still the mainstay treatment modality for various cancers, and PDX models are valuable tools for the evaluation of chemical drugs in vivo. PDX models have been used to evaluate dozens of small-molecule compounds, mainly kinase inhibitors, in various cancers. Kinase inhibitors have been tested in PDX models for cholangiocarcinoma , chordomMoreover, PDX models are valuable tools for the tests of novel antibodies before clinical application. Antibody-based therapies have been widely used in the clinical treatment of cancer patients, and PDX models have been used to test the use of antibodies for the treatment of various cancers , 99. EspSalmonella typhimurium A1-R, a facultative anaerobe that can grow in the oxic viable region of tumors and in necrotic regions, has shown efficacy against osteosarcoma [PDX models are valuable tools for the careful assessment of attenuated microorganisms in cancer treatment. osarcoma , soft-tiosarcoma , and melosarcoma  in orthoosarcoma . Oncolytosarcoma  and measosarcoma  have beeTargeted cancer therapies often lead to resistance, which can be suppressed through combination drug therapies. Combinatory targeting of two or more onco-signaling pathways is a promising strategy for cancer therapy. We recently used B-ALL PDX models to evaluate the anti-B-ALL efficacy of the combination of disulfiram and copper . PDX modG12D [A major issue in cancer drug development is the low success rate of new agents. Many compounds advance to large phase III studies, which consume considerable resources, but eventually fail because of low efficacy. These poor results arise partly because conventional preclinical models to screen new agents for clinical development have poor predictive value . FurtherG12D . AnotherG12D .TRAC locus with CRISPR/Cas9 enhances tumor rejection, a strategy averting antigen-stimulated differentiation and exhaustion [Adoptive transfer of chimeric antigen receptor (CAR) T cells has shown great promise in treating cancers, especially in B cell leukemia with CAR T cells targeting CD19. PDX models are frequently used for preclinical studies of chimeric antigen receptor (CAR) T cells –123. Novhaustion . The “Onhaustion . Loss ofhaustion . The CARIn summary, PDX models facilitate the discovery and testing of various therapeutic regimens including small-molecule compounds, antibodies, microorganisms, and cytotoxic cells.PDX models can provide in vivo evidences to support in vitro findings, and data from PDX models may lead to new discoveries or hypotheses which can be further investigated by research in vitro. The use of these xenograft models to study human tumor biology and drug screening is, however, limited by several factors, including the replacement of human stromal components  by murine elements, the lack of a functional immune system, and the lack of interactions between human stromal cells and the immune system. The development of PDX models that account for interactions between tumor, stromal, vascular, and immune cells is essential to produce a tumor microenvironment more representative of the human host. PDX models in humanized xenochimeric mice, or XactMice, engrafted with human HSPCs before tumor engraftment expressed the chemical stimuli necessary to give rise to stromal and immune cells that recreated the original tumor microenvironment observed clinically ; nonetheRecent studies using single-cell sequencing suggest that in some PDX models, only a limited number of clones propagate in mice, indicating a selection process . The idePDX models are increasingly used in translational cancer research. These models are useful for the study of cancer biology, biomarker development, drug screening, and the preclinical evaluation of personalized medicine strategies. This review provides a timely overview of the key roles of PDX models in both basic and preclinical cancer research and a detailed discussion of major hurdles in the field.


Presbycusis (age-related hearing loss) is a potential risk factor for tinnitus and cognitive deterioration, which result in poor life quality. Presbycusis-related tinnitus with cognitive impairment is a common phenotype in the elderly population. In these individuals, the central auditory system shows similar pathophysiological alterations as those observed in Alzheimer’s disease (AD), including cholinergic hypofunction, epileptiform-like network synchronization, chronic inflammation, and reduced GABAergic inhibition and neural plasticity. Observations from experimental rodent models indicate that recovery of cholinergic function can improve memory and other cognitive functions via acetylcholine-mediated GABAergic inhibition enhancement, nicotinic acetylcholine receptor (nAChR)-mediated anti-inflammation, glial activation inhibition and neurovascular protection. The loss of cholinergic innervation of various brain structures may provide a common link between tinnitus seen in presbycusis-related tinnitus and age-related cognitive impairment. We hypothesize a key component of the condition is the withdrawal of cholinergic input to a subtype of GABAergic inhibitory interneuron, neuropeptide Y (NPY) neurogliaform cells. Cholinergic denervation might not only cause the degeneration of NPY neurogliaform cells, but may also result in decreased AChR activation in GABAergic inhibitory interneurons. This, in turn, would lead to reduced GABA release and inhibitory regulation of neural networks. Reduced nAChR-mediated anti-inflammation due to the loss of nicotinic innervation might lead to the transformation of glial cells and release of inflammatory mediators, lowering the buffering of extracellular potassium and glutamate metabolism. Further research will provide evidence for the recovery of cholinergic function with the use of cholinergic input enhancement alone or in combination with other rehabilitative interventions to reestablish inhibitory regulation mechanisms of involved neural networks for presbycusis-related tinnitus with cognitive impairment. Subjective tinnitus, mainly induced by hearing loss and emotional states, is heterogeneous, affecting the development of effective intervention strategies. Presbycusis, commonly referred to as age-related hearing impairment, is a potential risk factor for tinnitus  followed up over a mean of 7.3 ± 4.4 years , more than 10% experience dementia, and more than 90% of individuals with dementia have hearing abnormalities  may represent a common neurophysiological basis of presbycusis-related tinnitus and age-related cognitive impairment. We hypothesize that age-related loss of cholinergic innervation of various brain structures may be a common link between tinnitus seen in presbycusis-related tinnitus and age-related cognitive impairment. Recovery of cholinergic function may be useful to treat presbycusis-related tinnitus with cognitive impairment by affecting multiple shared pathophysiological targets.Aging and neurodegenerative diseases are the major causes of declining cholinergic function. Aging leads to cholinergic hypofunction of the basal forebrain cholinergic complex, which is the main cholinergic projection to the cerebral cortex and hippocampus. Gradual age-related loss of cholinergic function results from decreased trophic support from nerve growth factor (NGF) and degeneration of dendritic, axonal and synaptic structures, which cause brain function decline, including cognitive impairment  expression. In patients with AD compared with age-matched healthy controls, there is a 50%–90% decline in activity of presynaptic ChAT , which has been associated with cognitive disturbances and age-related sensory loss  showed that NFTs appear earlier than amyloid plaques during normal aging. All cases from people over 48 years old displayed at least a few NFTs , which was preceded by tau pathology in these areas rather than in the brainstem  can impair learning and memory in mice  activity and inhibiting GSK3β activity via the activation of nAChRs, which reduces tau-induced neuronal toxicity and neurodegeneration -based targeted plasticity therapies are effective in patients with neurological diseases Hays, . VNS in Animals studies have shown that GABAergic interneuron deficits result in aberrant excitatory neuronal activity in mouse AD models  nAChR and α4β2 nAChR subtypes could enhance GABA release in hippocampal synaptosomes  vulnerability and brain hypoperfusion. Increased release of neurotoxic and inflammatory mediators has been observed in the brain microvessels of patients with AD Grammas, . FurtherThese results suggest that chronic inflammation and hypoperfusion play important roles in the pathogenesis of presbucusis-related tinnitus with cognitive impairment. Recovery of cholinergic function with AChE inhibitors, including donepezil, tacrine, pyridostigmine, galantamine, rivastigmine and Huperzine A shows potential disease-modifying benefits in the treatment of neuropsychiatric symptoms in patients with AD Linton,  and demeObservations from experimental rodent models indicate that anticholinergic activity might initiate and/or accelerate AD pathology in the tauopathy mouse model by enhancing neuroinflammation, including microglial activation. The recovery of lost cholinergic innervation or function by the cholinesterase inhibitor donepezil or Huperzine A could alleviate tau pathology as well as age- and AD-related chronic neuroinflammation (Yoshiyama et al., The mechanisms underlying cholinergic anti-inflammation were first observed in human immune cells (Wang et al., Subsequently, a similar anti-inflammatory mechanism was also observed in rat CNS. Increased brain ACh induced by Huperzine A activates cholinergic-mediated suppression of nuclear translocation of NF-κB, as well as inducing oxidative stress, glial cell activation, and neuroinflammation in rats with ischemia (Wang et al., in vitro (Shimohama et al., in vitro (Xie et al., in vitro (Mencel et al., The activation of α7nAChRs in neural cells suppresses central inflammatory responses in mice with Parkinson disease (Stuckenholz et al., Animal research suggests that auditory cortical cholinergic inputs from the basal forebrain in adult ferrets contribute to cognitive functions related to the processing of auditory stimuli, including normal auditory perception and adaption to changes in spatial cues (Leach et al., Chronic inflammation leads to BBB dysfunction and increased vascular permeability during aging, as well as in AD and other neurodegenerative disorders (Zlokovic, Loss of cholinergic input during aging and neurodegenerative diseases causes decreased ACh release and brain hypoperfusion. Reduced sensory input can also lead to decreased ACh release in the neocortex and hippocampus (Penschuck et al., Based on animal research, we hypothesize that the cholinergic anti-inflammation mediated by α7nAChR may be one potential mechanism by which hearing loss occurs with tinnitus or cognitive impairment. AChE inhibitors might suppress presbycusis accompanied by tinnitus and may indirectly protect auditory and cognitive function by activating α7nAChR-mediated anti-inflammatory effects in various cells of the brain’s neural vascular unit. This might include the suppression of glial and endothelial activation, neuroinflammation, tau-induced neurotoxicity and decreased gap junctions, as well as improved glutamate and extracellular potassium reuptake by astrocytes. These effects inhibit network hyperexcitability and excitotoxicity in the auditory pathway Figure .Presbycusis is a risk factor for tinnitus and cognitive decline. Cholinergic hypofunction might be a major contributor to presbycusis-related tinnitus and age-related cognitive impairment. Cholinergic denervation in the CNS, might lead to the reduction of both inhibition by NPY neurogliaform cells and cholinergic anti-inflammatory effects on the neural vascular unit mediated by nAChRs, as well as suppression of GSK3β activity and tau-induced neurodegeneration.Implementing VNS and AChE inhibitors alone or in combination with other hearing rehabilitative interventions during the optimal time window may lead to greater disease-modifying benefits in the treatment of presbycusis-related tinnitus with cognitive impairment. However, in the evidence reviewed here, data have mainly been obtained from animal experiments. Age-related hearing loss and AD in humans become apparent very slowly, and are associated with a long preclinical period. Therefore, animal models with a life expectancy of approximately 3 years are not really comparable to humans with these disorders. Further studies are required to elucidate the roles played by M or N cholinergic neuromodulation and distinct GABAergic cell types in the pathophysiological process. Furthermore, it must be investigated whether mechanisms underlying peripheral and central cholinergic regulation are the same.The potential relationship between tinnitus and depressive systems or affective disorders, and the mechanisms underlying this, should also be investigated in rodents. In addition, dynamic changes in CNS-derived biomarkers of cholinergic hypofunction and neuronal impairment in peripheral body fluids should be investigated as possible screening tools for preclinical or early stage disease, predictors of diagnosis, predictors of intervention outcomes. Finally, innovative, specific and selective neuromodulatory methods and multi-center longitudinal cohort studies are also urgently needed.QR and ZY designed the study and analyzed the data. QR, ZY, WZ, JR, CL and RZ provided a consensus agreement on the final hypotheses and drafted the initial version of the manuscript. WZ, JR and QR collected the data. All authors contributed to the final version 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.


Structural abnormalities in first episode psychosis (FEP) tend to be subtle and widespread. Most studies investigating structural abnormalities in this clinical population have used small samples, and therefore may be under-powered. In addition, most studies have examined participants at a single research site, and therefore the results may be specific to the local sample investigated. Consequently, findings from existing studies have often been heterogeneous. This study aimed to overcome these issues by testing for neuroanatomical abnormalities in individuals with FEP relative to healthy controls that are expressed consistently across five independent datasets.Structural Magnetic Resonance Imaging data were acquired from a total of 572 patients with FEP and 502 age and gender comparable healthy controls (HC) at five sites - London (UK), Utrecht (Netherlands), Chengdu (China) and two independent sites at Santander (Spain). Voxel-based morphometry (VBM) as implemented in Statistical Parametric Mapping software (SPM12) was used to investigate differences in gray matter volume (GMV) between the two groups. The statistical analysis was carried out using an analysis of variance (ANCOVA), with diagnostic group and scanning site as factors, and age and gender as covariates of no interest. Neuroanatomical alterations in patients with FEP relative to HC common to the five datasets were identified by comparing the total FEP group against the total HC group, and then using the inclusive masking option (at p<0.05 uncorrected) to identify those regions that survived the comparison between FEP and HC within each dataset. Individual clinical scores from each site were normalised and then used to examine their association with GMV in the total sample. Statistical inferences were made at p<0.05 after family-wise error correction for multiple comparisons.Relative to HC, FEP showed a widespread pattern GMV reduction in fronto-temporal regions bilaterally, including the gyrus rectus, orbitofrontal, temporal, fusiform, precentral and lingual gyri, anterior cingulate and insula as well as in the parietal lobe in the precuneus gyrus. The largest GMV reduction was found in the left gyrus rectus which is part of the inferior frontal lobe. Negative correlations were found between this region and positive symptoms severity  and duration of illness , but not with negative symptoms . Patients also showed GMV increases in the temporal gyrus bilaterally, left inferior frontal gyrus and right cerebellum relative to HC.This study identified a common pattern of fronto-temporal-parietal reductions in five independent FEP samples; in addition, some of these reductions were more pronounced in patients with more severe positive symptoms and longer duration of illness. This pattern of results suggests the presence of symptom- and stage-dependent neuroanatomical alternations in FEP that are expressed above and beyond site-related differences in recruitment criteria and scanning parameters.


Inter-organ crosstalk plays an essential role in the physiological homeostasis of the heart and other organs, and requires a complex interaction between a host of cellular, molecular, and neural factors. Derangements in these interactions can initiate multi-organ dysfunction. This is the case, for instance, in the heart or kidneys where a pathological alteration in one organ can unfavorably affect function in another distant organ; attention is currently being paid to understanding the physiopathological consequences of kidney dysfunction on cardiac performance that lead to cardiorenal syndrome. Different cardiorenal connectors  and non-traditional risk factors potentially contribute to multi-organ failure. Of these, inflammation may be crucial as inflammatory cells contribute to over-production of eicosanoids and lipid second messengers that activate intracellular signaling pathways involved in pathogenesis. Indeed, inflammation biomarkers are often elevated in patients with cardiac or renal dysfunction. Epigenetics, a dynamic process that regulates gene expression and function, is also recognized as an important player in single-organ disease. Principal epigenetic modifications occur at the level of DNA  and histone proteins; aberrant DNA methylation is associated with pathogenesis of organ dysfunction through a number of mechanisms . Herein, we focus on the potential contribution of inflammation in pathogenesis of cardiorenal syndrome. Heart and kidney function are strongly connected. Organ crosstalk, or biological communication between organs, involves cellular, molecular and neural factors; it plays an essential role in physiological homeostasis and in the event of malfunction can induce organ dysfunction ,2,3,4,5.The causal association between CKD and cardiovascular risk was initially deliberated by Bright in 1836 ; his obsFirstly, patients with CKD are amongst the highest risk groups for adverse cardiovascular events and cardiovascular-related mortality and therefore require particular clinical attention . AdditioRecent findings document that detrimental bi-directional interaction between different renocardiac connectors influence vascular endothelium  and contribute to progression of multi-organ failure ,27. UndeInflammation is a potentially important stressor for acute and chronic cardiorenal dysfunction. Altered endothelial regulation , which mIn this review, we discuss clinical and experimental findings related to the role of inflammation in the pathogenesis of cardiorenal syndrome as well as heart and kidney disease. Herein we attempt to establish a role for inflammation in the physiopathology of cardiorenal syndrome per se without taking into account the multifaceted presentations of CKD from early kidney involvement through renal replacement therapy and organ transplantation. Clinical and basic science reports were searched using MEDLINE, PubMed, and Google Scholar with the keywords cardiorenal syndrome, heart–kidney interactions, ischemia, heart and kidney function, inflammation, and various combinations thereof. Finally, experimental data from our own studies in this field were also consulted.The pathogenesis of kidney disease is complicated by the cellular composition and anatomy of nephrons; injured tubular epithelial cells contribute to functional impairment of water and electrolyte homeostasis along with reduced disposal of metabolic waste products. Enhanced inflammatory status also contributes to renal injury and reduced kidney function ,33,34. FVarious triggers  produce pathologically distinct lesions in the kidneys . Renal vStimulation of inflammation mechanisms during AKI results in release of soluble mediators into the bloodstream that exert harmful effects on distant organs. Knowledge on physiopathological mechanisms is limited; however, insights into cellular and molecular mechanisms are currently being addressed in pre-clinical studies. For instance, after organ injury, monocytes (involved in both tissue damage and repair) release cytokines into the peripheral circulation that trigger pathogenic mechanisms in distant organs . MonocytA sophisticated connection or crosstalk exists between the sympathetic nervous and immune systems; activation of the sympathetic nervous system amplifies the release of inflammatory cells  into the bloodstream, resulting in low-grade systemic inflammation  and can Neuroimmunomodulation interventions that are based on bidirectional control by the brain of physiological functions of various organs  are also being tested . Recent Coronary artery disease most often results in an acute coronary occlusive event due to rupture of atherosclerotic plaque; various inflammatory cells contribute to atherogenic pathogenesis and its complications  . Ischemia, infection, and uremic milieu have the potential to stimulate diverse inflammatory components in both kidney and heart disease . LeukocyUnder adverse conditions such as myocardial ischemia, different inflammatory cells, platelets, and fibroblasts contribute to chronic production of eicosanoids and other lipid second messengers; this is potentially deleterious and can result in dysfunctional excitation–contraction coupling, bioenergetic inefficiency, and accelerated necrosis, all of which contribute to myocardial pathology. We refer the reader to the review paper by Jenkins et al. that examined the role of myocardial eicosanoid signaling pathways in cardiac function . Arachidsn-2 position; these plasmalogens markedly affect membrane dynamics [2 catalyzes hydrolysis of the sn-2 ester linkage of glycerophospholipids to generate free fatty acids such as AA and other lysolipid products. The important role of myocardial phospholipase A2-activated release of AA from cellular phospholipids is well established [Myocardial membranes are highly enriched with choline and ethanolamine plasmalogens that comprise esterified AA at the dynamics ,81,82. Pablished ,84,85. D2; COX-1 is expressed constitutively and is involved in cardiac homeostasis [COXs catalyze AA to form prostaglandin Geostasis . On the eostasis ,88 but ieostasis ,90. FurtCatalysis of AA by LOXs to hydroperoxyeicosatetraenoic acids results in production of hydroxyeicosatrienoic (HETE) derivatives. In normal myocardium HETEs contribute to regulation of cardiac function; post-ischemic activation of lipoxygenases may be important in the development of cardiac fibrosis and hypertrophy . UpregulIn cardiac tissues, several CYP450-generated eicosanoids have been identified and linked to the pathogenesis of hypertension, myocardial infarction, AKI and stroke ,95,96,97CYP450-derived AA metabolites have been evaluated in animal models of cardiac pathology ,107. TheIn clinical studies, inflammation and its impact on cardiovascular renal pathogenesis remain to be established. Weiner et al. performed a study with two patient cohorts  ; they doEpigenetic inheritance is responsible for phenotypic differences between cell types in multicellular organisms; epigenetics is considered a dynamic process that regulates gene expression patterns, accessibility to the genome through gene expression and ultimately gene function . In mammedn1, an ET-1 encoding gene [Endothelin (ET-1) also contributes to cardiac and renal physiopathology; elevated ET-1 levels are found in arterial hypertension and chronic renal failure ,126. DNAing gene . Anothering gene ,129. A better understanding of underlying mechanisms involved in organ crosstalk in relation to heart and kidney disease is required to improve clinical outcomes; the same is true for co-morbidities including cardiorenal syndrome. It may not be that important which organ is initially affected, but how pathogenesis in one organ affects other distant organs merits investigation. Inflammation may be a key underlying mechanism in the progression of multi-organ disease; however, epigenetics and their influence on inflammation and disease progression also warrant consideration. Controlled clinical studies with drugs investigating these factors in cardiorenal syndrome patients remain at a preliminary stage, however, results from ongoing studies will undoubtedly improve future therapeutic approaches for patients.


Ever since they were postulated in 1978, stem cell niches have been identified and characterized in many germline and adult tissues. Comprehensive studies over the last decades helped to clarify the critical components of stem cell niches that include cellular, extracellular, biochemical, molecular, and physical regulators. This knowledge has direct impact on their inherent regenerative potential. Clinical applications demand readily available cell sources that, under controlled conditions, provide a specific therapeutic function. Thus, translational medicine aims at optimizing in vitro or in vivo the various components and complex architecture of the niche to exploit its therapeutic potential. Accordingly, the objective is to recreate the natural niche microenvironment during cell therapy process development and closely comply with the requests of regulatory authorities. In this paper, we review the most recent advances of translational medicine approaches that target the adult stem cell natural niche microenvironment for regenerative medicine applications.The interactions between stem cells and their surrounding microenvironment are pivotal to determine tissue homeostasis and stem cell renewal or differentiation and regeneration  Multipotent stem cells are critical biotherapeutics for regenerative medicine because of their innate ability to restore the structure and function of adult damaged tissues or organs. As a matter of fact, self-renewal, clonogenicity, and multipotentiality are the main common features of adult stem cells. In the transition from preclinical studies to clinical application, however, we should consider a number of hurdles in manipulating stem cells and implement clinically oriented approaches to control stem cell fate and function.The niche is a highly dynamic microenvironment that can adapt to physiological or diseased conditions , 2. The To target the stem cell niche, it might be necessary to regulate its various components such as cell-to-cell contact, cell to extracellular matrix interactions, and mechanical and electrical stimuli in a temporally and spatially regulated manner , 9. ContManufacturing of cell therapy products (CTPs) for clinical application typically requires challenging steps such as the specific definition of identity, potency, and purity of each CTP. These definitions are largely therapy dependent. Towards this purpose, the US Food and Drug Administration (FDA) releases the current Good Manufacturing Practice (cGMP) guidelines and the International Conference on Harmonisation (ICH) introduces a systematic approach to process manufacturing and product management based on scientific knowledge and risk assessment , 11. OveIn the following sections, we discuss the major challenges to limit adult stem cell product variability, and we describe, to the best of our knowledge, the most recent advances for their clinical translation. In general, we highlight the fact that “Clearly, fundamental scientific and medical questions reside within the niche”  to develin vitro through an artificial microenvironment may efficiently avoid the need for direct genetic manipulation, which is more problematic for clinical application. Employing an artificial ECM aims at recreating the in vivo three-dimensional (3D) microenvironment.Contact with the extracellular matrix (ECM) and with other cells represents an important mechanism by which adult stem cells sense the microenvironment and make decisions about their fate . The preNoncellular niches represent the first attempt for the development of defined physical culture conditions. The most recent advances towards therapeutic application include the development of synthetic bioinformative substrates designed at the micro- and nanoscale level . MicrotoThe mechanisms by which topographic information of the biomimetic niche influence stem cell behavior are not completely understood; they appear to involve changes in cytoskeletal organization and structure, mainly at the level of integrins in the cellular membrane as a response to the geometry and size of the ECM. This interaction activates concomitant intracellular signaling cascades and guides stem cell behavior , 31. AddIn general, synthetic peptides and surfaces offer the advantage of being animal component free (ACF) and are potentially scalable. Matrigel, a poorly defined complex ECM isolated from the murine Engelbreth–Holm–Swarm tumor, would not be the ideal choice for clinical applications , 36. Recin vitro. Thus, we further analyze the additional important components of the stem cell niche to proceed to clinical application.Biocompatible hydrogel-based ECMs are employed for the culture of stem cells. Hydrogels are 3D macromolecule platforms obtained by the crosslinking of hydrophilic polymers. Collagen, fibrin, hyaluronic acid, alginate, dextran, chitosan, and agarose are used as components for hydrogels , 38. HowIn vitro, cultured cells are subjected to an environment whose main components are, together with the substrate, the culture medium, the atmosphere, and cell-to-cell interactions. Each of these components participate to the complex network of signaling pathways that eventually determine stem cell fate [in vivo.ell fate , 23. Steell fate , 32. CulA universally optimal culture condition does not exist because stem cells are all different. Stem cell culture parameters are defined for each stem cell type and designed on their intended therapeutic use , 42. FeeGood Cell Culture Practice (GCCP) and Good Manufacturing Practice (GMP) represent the leading guidelines to establish standardized protocols for cell-based therapy and regenerative medicine . As a maThe advantage of defined media, aside from the desirable ethical reduction or complete absence of fetal bovine serum (FBS), is the precise chemical composition which thus facilitates a controlled culture environment for the selective growth of cells. Defined culture conditions allow the establishment and the maintenance of phenotypically well-defined and karyotipycally stable cells.Cell culture conditions are further optimized by the implementation of specific stem cell supplements, that is, recombinant growth factors or cytokines. The selection of the medium additives and their concentrations, especially the growth factors, is critical since it could variably affect the cultured cells. Adult stem cells require ex vivo-specific growth factors that mimic their native microenvironment.β, vascular endothelial growth factor, and platelet-derived growth factor [Growth factors act as mitogens that stimulate cell proliferation and in some cases are crucial to maintain cell characteristics. The most commonly used growth factors in ACF or xenofree (XF) media for human adult stem cells include basic fibroblast growth factor, epidermal growth factor, transforming growth factor-h factor , 47. MosThe specificity of growth factors, their concentration, and synergistic effect play a crucial role in achieving an optimized, cell-specific, defined culture medium. Notably, growth factor requirements can be not only cell-type specific but also species specific: LIF supports the expansion of a mouse but not human ESCs. Secreted molecules, such as colony-stimulating factor and stem cell factor (Kit ligand), play important roles in cell survival.Cell-to-cell interactions involving other classes of molecules are also important: interactions between Eph tyrosine kinase receptors and their Ephrin transmembrane ligands regulate adult stem cell proliferation and migration .in vitro is crucial to guarantee a long-term therapeutic effect in vivo. This critical issue increases our knowledge on the fine regulation of stem cell microenvironment and moves translational research into effective and more reproducible clinical trials.Efficient stem cell manufacturing in vivo. Cell culture incubators normally preserve atmospheric partial oxygen pressure (pO2) which is around 150 mmHg (21% O2). In vivo, physiological pO2 ranges between 50 and 5 mmHg (7–0.7%). Thus, the term “normoxia” referred to standard cell culture systems does not refer to physiological conditions. Lowering the pO2 is beneficial for various adult stem cell types [For decades, cells are cultured under an atmospheric oxygen pressure that is much higher than the one experienced in their niches ll types : Wion etThe stem cell culture medium is dynamic and changes rapidly due to the release and/or consumption of numerous metabolites. For this reason, continuous perfusion of cell cultures with fresh medium through controlled bioreactors is considered a valuable option to standardize cell-manufacturing processes. Bioreactors utilize mechanical forces to influence biological processes under closely controlled conditions. They provide spatially homogeneous cell distribution; deliver physiological relevant concentrations of oxygen, carbon dioxide, and nutrients in the culture medium; and provide physical stimuli to regulate stem cell differentiation and proliferation. In bioreactors, stem cells are expanded in stirred vessels or on perfused scaffolds, and their culture pH and oxygen values are monitored. This controlled process is beneficial in terms of stem cell expansion and differentiation compared to conventional static culture conditions, although autocrine and paracrine loops might be disturbed . The impSerum is a mixture of a large number of components, and its composition is partly uncharacterized. Slight variations in its composition influence key properties of cells because they are highly sensitive to culture conditions. Thus, serum introduces an unknown variable into the culture system, and this represents a challenge to generate consistent and quality-assured cells in clinical-scale production , 53.in vivo condition: hormonal factors for cell growth and proliferation transport proteins that carry hormones, minerals, trace elements , and lipids . Additionally, it stabilizes pH with factors inhibiting proteases (such as α-antitrypsin or α2-macroglobulin), supplies adhesion molecules of the extracellular matrix, and contains factors that protect against shear forces [In cell culture, the use of fetal bovine serum (FBS) as a medium supplement is most widespread. The major function of serum in stem cell culture media is to provide multiple elements that correspond to the r forces , 55.The critical problems related to the presence of FBS in stem cell culture are batch-to-batch variability, fluctuating availability, unexpected cell characteristics, and potential cytotoxicity of uncharacterized factors –58. GstrMost regulatory agencies tolerate the use of xenogenic components in culture media in phase I clinical trials. However, later phase trials are required to employ serum-free or at least xenofree media. Mendicino et al. reported recently that FBS is employed during manufacturing in over 80% of the investigational new drug (IND) applications for mesenchymal stem cell (MSC) products submitted to the FDA . The conThe major benefits of establishing serum-free cell culture systems are in the direction of standardization, that is, limitation of the cell therapy product variability, and elimination of a potential source of contamination , 66. Of in vivo, growth factors such as platelet-derived growth factor (PDGF), transforming growth factor-β (TGF-β), fibroblast growth factor (FGF), insulin-like growth factor-1 (IGF-1), platelet-derived epidermal growth factor (EGF), vascular endothelial growth factor (VEGF), together with attachment factors (fibronectin and vitronectin), and protease inhibitors are exploited for their use [Adult stem cells cannot survive in the absence of serum-specific growth factors as well as other unidentified factors in the serum. In serum-free culture, a separate attachment substrate is required. Human plasma fibronectin is a common adhesion substrate used in serum-free systems . Human pheir use . HoweverPooled human AB serum (HABS) is an additional alternative: it supports proliferation of human mesenchymal stromal cells (hMSCs) and maintains their characteristics throughout ex vivo expansion . FurtherTo overcome the issue of limited collection availability, recombinant forms of human serum albumin are commercially available. Recombinant human serum albumin (r-HSA) is used instead of purified human serum albumin (HSA) . r-HSA iA few serum-free media are also commercially available. Unfortunately, the composition of commercially available proprietary serum-free media is generally unknown. Manufacturers usually do not disclose this information that is often requested by regulatory authorities in clinical settings.The process of developing serum-free media or adapting stem cells to serum-free culture media is complex and time consuming. However, the development of these defined media should be encouraged in view of their intended clinical application. As stem cell therapy industry advances and clinical trials reach their later phases, culture process validation, scale-up, and quality assurance of critical raw material are highly requested.Addressing this need results in significant changes to current culturing technologies for a beneficial shift towards more qualified and compelling therapies. Extracellular matrix (ECM) composition is precisely regulated during normal heart development and its dysregulation results in structural and functional heart diseases.The heart is a biomechanical organ in which the mechanical stress on cardiac cells mainly arises from the hemodynamic load. Dysregulation of either preload in diastole or afterload in systole contributes to the pathogenesis of congenital or adult heart disease. The microenvironment of stem cells in the adult myocardium includes cardiomyocytes, vasculature, interstitial cells, and extracellular matrix, each of them representing a potential target to enhance the regenerative potential of the heart after injury.Cardiovascular diseases represent a major public health priority. Specifically, patients who suffer from myocardial infarction may encounter adverse remodeling that can ultimately lead to heart failure. Prognosis of patients affected by heart failure is very poor with 5-year mortality close to 50%. Despite the impressive progress in the clinical treatment of heart failure in recent years, heart transplantation is still required to avoid death as the result of the inexorable decline in cardiac function. Nonetheless, the morbidities associated with heart transplantation and the limited organ supply demand the development of new stem cell-based approaches for regenerative medicine –84.The human heart is one of the organs which regenerates less in the body, or at least, its regenerative potential is clearly lower than the intestine, liver, bone, or skin . Howeverin vitro with growth factors, hypoxic treatment, or antiaging reagents enhances cellular engraftment, survival, and differentiation before administration. An example of this valuable approach involves the “cardiopoietic” guidance of multipotent adult stem cells: Behfar et al. employed a specific cardiogenic cocktail for human mesenchymal stem cells through manipulation of their culture environment [ClinicalTrials.gov Indentifier: NCT00810238) and in the larger CHART-1  clinical trial to treat ischemic heart failure. So far, preliminary results indicate a positive although not statistically significant trend in the treated group.Current investigation deals with combinatory approaches that employ multiple stem cell types. Preconditioning stem cells ironment . The autEngineered scaffolds represent 3D myocardial tissue for adult stem cell culture; this approach includes synthetic porous scaffolds or scaffold-free cell sheets to increase cardiac contractility and output. In an effort to use physicochemically defined components, recombinant human laminin and recombinant human fibronectin in our hands  and long noncoding RNAs (lncRNAs) act as critical regulators of cardiac development and disease: they necessitate the implementation into future efforts at mimicking the cardiac microenvironment Future models may expand into gene therapies: the analysis of mononuclear polyploid cells naturally occurring in regenerative tissues represents a more recent approach .Several studies performed in the last decades highlight the importance of the microenvironment in which human stem cells grow and maintain their peculiar characteristics. Various components of the human stem cell niche are clarified, and the objective of recreating an appropriate native microenvironment is the current objective of regenerative medicine.Manufacturing human adult stem cells as therapeutics should preferably be performed in animal component-free or reduced animal component systems to avoid the risk of zoonoses. Ideally, the cell culture systems that are engineered for this purpose will minimize exposure to animal cells and proteins by using primarily human or recombinant human components. Furthermore, it is highly desirable to employ physicochemically defined culture media, possibly devoiding complex mixtures such as animal or human serum.We are moving closer to producing stem cell therapy products that have very limited contact with animal products and thus are better candidates for use in regenerative therapies. Although many challenges lie ahead in the industrialization of CTP manufacturing, we find much reason for optimism. Decades of experience with industrial cell line culture processes lay the foundation of engineering CTPs such as bioreactor scale-up, analysis of cellular metabolism, medium design, optimization of expansion strategies, and process control. Meanwhile, our understanding of how cells interact with their environment is improving, and controlled systems that mimic the cellular microenvironment are generating important data sets which are increasingly focused on molecular and cellular information. In parallel, our general understanding of the molecular basis of stem cell states, including adhesion properties, metabolic needs, clonogenicity, and proliferation control, is progressing. Such findings emphasize the importance of a multidisciplinary approach for the development of engineered products, involving the connection of many disciplines such as cell and molecular biology, materials science, biomedical engineering, and medicine. The global perspective is the implementation of a comprehensive cell therapy product including a defined serum-free culture medium, a perfusion system, biosensors, and micro- or nanoscale-designed scaffolds mimicking as far as possible the niche microenvironment that is known to modulate stem cell function. The future lies probably in the development of 3D modular biomimetic systems assembled according to the final purpose of the stem cell culture, for example, stemness maintenance or control of cell differentiation towards clinically relevant cell phenotypes.This massive development requires time and resources and may also involve remarkable changes to be implemented into the original manufacturing process. Additionally, full characterization of the final stem cell product after process development changes is crucial to verify comparability to the original product. It is also critical to carefully examine the quality, safety, and availability of the specific components implemented into the system to ensure that the selection meets the needs for further scale-up of the process and resulting therapeutic product.The future of CTPs relies on the development of cost-effective technologies for cell manufacturing. Given the inherent complexity of CTPs and their production processes, appropriately designed approaches will be essential in transforming today's experimental CTPs into available therapeutics.The advancement of the knowledge and optimization of the integral components of the human stem cell niche are instrumental in this ambitious goal.


Macrophages are present in mammals from midgestation, contributing to physiologic homeostasis throughout life. Macrophages arise from yolk sac and foetal liver progenitors during embryonic development in the mouse and persist in different organs as heterogeneous, self-renewing tissue-resident populations. Bone marrow-derived blood monocytes are recruited after birth to replenish tissue-resident populations and to meet further demands during inflammation, infection and metabolic perturbations. Macrophages of mixed origin and different locations vary in replication and turnover, but are all active in mRNA and protein synthesis, fulfilling organ-specific and systemic trophic functions, in addition to host defence. In this review, we emphasise selected properties and non-immune functions of tissue macrophages which contribute to physiologic homeostasis. Elie Metchnikoff (1845–1916), a comparative zoologist who initially was intrigued by intracellular digestion in invertebrates, became an experimental immunologist when he appreciated the role of phagocytes in tissue orthobiosis and inflammation . Hans Se871–1945 Blood monocytes and tissue macrophages display many properties in common, but they adapt readily upon entering different tissue microenvironments, where they contribute to the general needs as well as specialised functions of individual organs. Reciprocal interactions between neighbouring cells and tissue macrophages are mediated by close contacts as well as secretory products which act at close range or distally via the circulation. Although primarily known as professional phagocytes , which eTo set the stage for subsequent discussion, we briefly summarise some general properties of macrophages relevant to their functions in vivo. The cells originate from progenitors in the embryo and postnatally, as discussed further below, and are seeded throughout the body as relatively long lived, terminally differentiated haematopoietic cells. They are motile and display a range of membrane receptors to recognise and respond to a large repertoire of host-derived and foreign ligands . These iThe uptake and killing of microbes and opsonised cellular targets by oxygen and nitrogen-derived radicals are mediated by assembly and activation of an NADPH oxidase and inducible nitric oxide synthetase, respectively , 43. TheMacrophage gene expression has been intensively studied in recent years and modules of genes regulated by a range of cytokines and other stimuli characterised in tissue-resident and inflammatory macrophages. Both pro- and anti-inflammatory pathways can be induced in macrophage populations, mediated by plasma membrane or cytoplasmic receptors. IFN-γ, for example, induces a characteristic profile of genes and proteins, often termed “classical or M1 activation” ; prototyThe immune system comprises a complex network of cellular and soluble components that collaborate to respond to challenges, both infectious and non-infectious, identified as potential threats to the organism. Accordingly, immune constituents are widely distributed throughout tissues with specialised lymphoid organs, such as splenic white pulp and lymph nodes, mostly providing unique sites for the induction of specific acquired immunity.To date, newcomers to the field are still surprised to learn that healthy tissues incorporate immune cells, in particular, macrophages which are cells commonly associated with protection against infection and as mediators of tissue damage. Macrophages, unlike neutrophils, are integral components of tissues; they contribute to organ development and maintenance of homeostasis through mechanisms similar to those exploited during infection to detect pathogens and trigger inflammation. Widespread distribution of macrophages is accompanied by substantial tissue-specific phenotypic and gene expression profile differences related to the specific environmental cues in each organ that foster tissue-specific macrophage function . Differehi monocytes that originate directly from Ly6c + precursors and non-classical Ly6clow monocytes that derive from Ly6chigh monocytes through the action of the transcription factor Nr4a1 [lo monocytes as “blood macrophages” involved in the maintenance of the integrity of the endothelial barrier [high monocytes contribute to replenishment of tissue macrophages with the rate of replenishment varying among anatomical sites. In the steady state, monocyte-derived tissue macrophages are almost non-existent in the case of brain as microglia are derived from yolk sac precursors. Similar scenarios have been observed in the case of Langerhans cells in the epidermis, alveolar macrophages in the lung and Kupffer cells in the liver. Monocyte-derived resident macrophages are more prevalent in the heart and pancreas (which display a slow replacement rate) and gut and dermis (which display a higher replacement rate) [In addition to tissue-specific environmental cues, tissue-specific characteristics in macrophages can be linked in part to on site differentiation of macrophages from embryonic precursors during development and maintenance of macrophage numbers in tissues through self-renewal , 63 withor Nr4a1 . Further barrier . Ly6Chignt rate) , 63.high monocytes are recruited to tissues under steady state and inflammatory conditions. In the absence of inflammation, Ly6Chigh monocytes sample the tissues and transport material to draining lymph nodes without differentiating into macrophages or dendritic cells [high monocytes differentiate into macrophages and can give rise to tissue macrophages with capacity for self-renewal. This process is highly dependent on the tissue-depletion strategy used and the organ under consideration [Ly6Cic cells . Under ideration .2+ fluxes into and from the extracellular fluid which is required for the maintenance of the levels of calcium in blood [Bone remodelling occurs throughout life and is influenced by physical activity, diet and hormones. Bone generation, promoted by cells of mesenchymal origin termed osteoblasts, is balanced by the action of cells of haematopoietic origin termed osteoclasts that mediate bone reabsorption. Bone formation and degradation need to be balanced as disequilibrium would lead to osteoporosis (loss of bone mass) or osteopetrosis . Further, bone remodelling controls Cain blood . Osteoclin blood . Exchangin blood . For insin blood . As a coin blood .Remodelling deficiencies in the absence of macrophages have been observed in mammary glands, kidney and pancreas, which suggest an important role in morphogenesis; this can be due to the contribution of macrophages to extracellular matrix remodelling and a role in maintenance of the viability and function of stem cells , as obseDefinitive erythropoiesis occurs in the bone marrow; in particular, terminal erythropoiesis takes place in erythroblastic islands where tight clusters can be found between erythroid precursors with a central macrophage . MoleculMicroglia constitute the resident macrophage population in the brain and spinal cord and are yolk sac-derived. In addition to responding to tissue injury, ramified microglia have been shown to contribute to synaptic maturation during brain development . This prAlveoli are lined by the alveolar epithelium which is mainly composed of type I and type II alveolar epithelial cells. Type I epithelial cells cover 90% of the alveoli surface while type II epithelial cells are less abundant and mediate the synthesis, secretion and recycling of lung surfactant , 46. AlvThe involvement of macrophages in metabolic homeostasis was highlighted by major changes in macrophage numbers  and phenotype  in white adipose tissue in obese insulin-resistant animals and humans , 49, 57.hi, VCAMhi iron recycling macrophages in bone marrow and spleen through induction of the transcription factor Spi-c [Resident bone marrow macrophages and red pulp macrophages are essential for the process of erythrophagocytosis to recycle iron. Senescent or damaged erythrocytes are phagocytosed and digested to extract heme . Upon upor Spi-c . Red pulor Spi-c . There aor Spi-c . Other por Spi-c . Splenicor Spi-c .Self-contained inflammation with preservation of organ function depends on multifaceted interactions between macrophages and recruited immune cells, particularly neutrophils and monocytes that ensure timely resolution through changes in the activation state of macrophages (from a M1 to a M2-like phenotype). These constitute a “lipid mediator class switch” which shifts the production of lipid mediators from pro-inflammatory  to pro-resolving lipids , production of proteins such as annexin A1, gaseous mediators (carbon monoxide), chemokine inactivation and release of acetylcholine by the vagus nerve . ResidenThe fate of the recruited monocytes and their contribution to repair depends on the organ and extent of damage. In the heart, recruited monocytes give rise to pro-inflammatory macrophages, while resident macrophages mediate tissue repair. In the spinal cord, both the pro-inflammatory and repair macrophages are monocyte-derived. While the pro-inflammatory macrophages derive from monocytes recruited through the CCR2-CCL2 pathway, repair-promoting macrophages derived from monocytes are recruited through a pathway involving VLA-4, VCAM-1 and CD73 . In otheA key aspect during tissue regeneration and repair is the cross-talk between macrophages and stem cells. For instance, in liver, macrophage depletion has been shown to reduce invasiveness of liver progenitor cells into the parenchyma which is required for differentiation into hepatocytes . In addiScar formation during wound healing can compromise organ function after injury and multiple reports have linked Th2 immunity to the regulation of tissue fibrosis, although the molecular processes of this effect are poorly understood , 74. KniBrown fat, termed brown adipose tissue, generates heat (thermogenesis) and plays an important role in maintaining body temperature. In contrast to white adipocytes, brown adipocytes contain multilocular fat droplets, a large number of mitochondria, and express high levels of UCP1 (uncoupling protein 1). UCP1 is located in the inner membrane of the mitochondria and uncouples electron transport to promote heat instead of ATP in brown adipocytes. In humans, brown adipose tissue was thought to be restricted to neonates but recent data have shown the presence of brown fat in adults , 41, 69.Macrophages are remarkable for their ability to sense their surroundings, produce a wide range of immune mediators alongside growth factors and opsonins, and their endocytic and phagocytic activity. The recognition of macrophages as intrinsic components of organs, adapted to the particular tissue environment and actively involved in homeostatic processes, opens the opportunity for macrophage-centred therapies that can be exploited to steer pathological processes towards outcomes that favour maintenance of organ function. Furthermore, the potential plasticity shown by these cells  makes it


ISD). Dependent on the sensing layer which can be semiconducting polymer, dielectric layer or conducting polymer gate, the PFET sensors can be subdivided into three types. For each type of sensor, we present the molecular structure of sensing polymer, the gas analyte and the sensing performance. Most importantly, we summarize various analyte–polymer interactions, which help to understand the sensing mechanism in the PFET sensors and can provide possible approaches for the sensor fabrication in the future.This review focuses on polymer field-effect transistor (PFET) based gas sensor with polymer as the sensing layer, which interacts with gas analyte and thus induces the change of source-drain current (Δ Organic field-effect transistor (OFET) based gas sensors have attracted great interest due to the high selectivity, repeatable response and low-cost production ,3,4,5,6.μ), threshold voltage (VT), on/off-current (ION/IOFF) of the devices, and the bulk conductivity (σ) of the organic semiconducting film . I. I2/Si sed ROMP, b dielectMetal gate could be replaced with conducting polymer sensing layer, usually in a MOSFET sensor. Much work on conducting polymer gate FETs for vapor sensing has been reported. There are already a few reviews to summarize this topic ,54,55; tThis review summarizes the PFET sensors categorized by sensing layers consisting of semiconducting layer, dielectric layer and conducting gate layer, separately, with the focus on sensing polymers, gas analytes, sensor performance and sensing mechanism. OFETs with polymers as the semiconducting layer have been demonstrated as effective sensors in gas detection. Although discussions on analyte–polymer interaction have been addressed, there is still a lack of systematic rules to predict and instruct the fabrication of OFET sensors. More gas analytes and sensing polymers need to be introduced, which will enrich the sensing database and help to understand the sensing mechanism. OFET sensor array is able to analyze complex analytes and is a promising area in the future. Furthermore, humidity in the air can impair the OFET sensor and this effect should be minimized. Although calibration of sensor measurements by subtracting the humidity effect at various humidity levels is available, improving the stability of sensing polymers is the most efficient approach, and needs much more investigation. Other approaches including mixing sensing polymer semiconductors with dielectrics and modifying the dielectric layers with self-assembled layers should also be developed.


Pneumocystis pneumonia in HIV-positive patients. All communities should have access to nonculture fungal diagnostics, which can substantially benefit clinical outcome, antimicrobial stewardship, and control of antimicrobial resistance.Antimicrobial resistance, a major public health concern, largely arises from excess use of antibiotic and antifungal drugs. Lack of routine diagnostic testing for fungal diseases exacerbates the problem of antimicrobial drug empiricism, both antibiotic and antifungal. In support of this contention, we cite 4 common clinical situations that illustrate this problem: 1) inaccurate diagnosis of fungal sepsis in hospitals and intensive care units, resulting in inappropriate use of broad-spectrum antibacterial drugs in patients with invasive candidiasis; 2) failure to diagnose chronic pulmonary aspergillosis in patients with smear-negative pulmonary tuberculosis; 3) misdiagnosis of fungal asthma, resulting in unnecessary treatment with antibacterial drugs instead of antifungal drugs and missed diagnoses of life-threatening invasive aspergillosis in patients with chronic obstructive pulmonary disease; and 4) overtreatment and undertreatment of  Antimicrobial resistance (AMR) is a major public health concern and a major threat to modern medicine  outside ICUs in India are at least twice as common as in ICUs, then >600,000 persons each year in India are estimated to have invasive candidiasis. Assuming these patients are treated, ≈300,000 die each year, but many more die if patients are not treated.Hospitalized patients, especially those in intensive care units (ICUs), are often inappropriately placed on broad-spectrum antibiotic drugs because fungal diseases involving In addition, a high prevalence of candidemia has been reported in children, including neonates, in India and Latin America. Central nervous system involvement is common in premature infants, leading to a high rate of neurologic sequelae   of the 134 patients with precipitating antibody to A. fumigatus (Smear-negative pulmonary tuberculosis (TB) is a problematic area for clinicians and policymakers. Post-TB sequelae are common, are poorly studied, and may be mistaken for active, recurrent TB  directly on respiratory specimens has transformed the rapidity of detecting positive samples, but there remain millions of unwell, smear-negative, PCR-negative patients. Some of these patients have relapsed after anti-TB therapy, and CPA has developed subsequent to cured TB. Among HIV-positive persons, those with smear-negative TB test results have a higher death rate than those with smear-positive results  were hospitalized in 2012 (It is common practice to treat asthma and exacerbations of chronic obstructive pulmonary disease (COPD) with antibiotic drugs and corticosteroids, although guidelines caution against their unnecessary use, especially in patients with COPD  for COPD and asthma exacerbations be evaluated and implemented and that fungal asthma be properly diagnosed and treated.Failure to properly diagnose and treat patients with asthma and COPD who are colonized with Pneumocystis pneumonia (PCP) in AIDS is often diagnosed empirically based on a subacute onset of cough; breathlessness out of proportion to abnormalities seen on chest radiographs; and subtle, bilateral changes seen on chest radiographs, in the context of a low CD4 cell count  or the use of diagnostics to prevent superinfection with fungi that are commonly resistant to antifungal drugs, such as C. glabrata or Rhizopus oryzae. Antifungal drug resistance is problematic in some settings and demands informed prescribing, therapeutic drug monitoring, and development of new antifungal agents . Among those situations are cases of cryptococcal meningitis ; CandidaThe lack of availability and underuse of nonculture fungal diagnostics results in overprescribing, prescription of unduly long courses of antibacterial agents, and excess empirical use of antifungal agents and leaves many millions of patients with undiagnosed fungal infections. This lack and underuse of proper diagnostics squanders resources. The large scale of the problem, even in many of the world’s most advanced medical centers, compromises AMR control. In many countries, the government and private healthcare providers should be actively promoting diagnosis of fungal infections to minimize deaths and illness from fungal disease; such efforts will probably also have a positive benefit on inappropriate antibacterial drug usage and support stewardship programs. Public health authorities must embrace acute and chronic fungal disease as areas of considerable need and seize the opportunity to improve health and preserve what remains of the antimicrobial drug toolbox.


Despite substantial research on school-based obesity prevention programs, it is unclear how widely they are disseminated. It is also unknown whether schools use obesity programs that inadvertently promote weight stigma or disordered weight-control behaviors.In spring 2016, we distributed an online survey about school wellness programming to a simple random sample of US public school administrators . We analyzed survey responses and conducted immersion/crystallization analysis of written open-ended responses.Slightly less than half  of schools offered any obesity prevention program. Only 17 (6.9%) reported using a predeveloped program, and 7 (2.8%) reported using a program with evidence for effectiveness. Thirty-seven schools (15.0%) reported developing intervention programs that focused primarily on individual students’ or staff members’ weight rather than nutrition or physical activity; 28 schools  used staff weight-loss competitions. School administrators who reported implementing a program were more likely to describe having a program champion and adequate buy-in from staff, families, and students. Lack of funding, training, and time were widely reported as barriers to implementation. Few administrators used educational  or scientific  literature for wellness program decision making.Evidence-based obesity prevention programs appear to be rarely implemented in US schools. Schools may be implementing programs lacking evidence and programs that may unintentionally exacerbate student weight stigma by focusing on student weight rather than healthy habits. Public health practitioners and researchers should focus on improving support for schools to implement evidence-based programs. Implementing school-based obesity prevention programs is a key public health strategy for countering the childhood obesity epidemic ,2. In adAs the American Academy of Pediatrics recently highlighted, a concern with obesity prevention programs is the potential for triggering unhealthy weight-control behaviors or exacerbating weight stigma . AdolescIt is unclear how broadly evidence-based programs have been disseminated and whether in the absence of effective dissemination schools instead use ineffective or even harmful programs. The Diffusion of Innovations Model , which sThis cross-sectional pilot study was conducted from February to June 2016. To reach the target population of US public school principals, we downloaded a list of all public kindergarten through twelfth-grade schools in the United States from the National Center for Education Statistics  and randWe developed a brief survey  to gatheBrain BreaksbSAFE-bFITaCoordinated Approach to Children’s Health (CATCH)COPEFOOD PLAYHealth Ahead/Heart SmartJust for Kids!KaBoomKID-FITaNew MovesaPlanet HealthPresident’s Challenge Physical Activity and FitnessProject Fit AmericaaSPARKStart For LifeaTAKE10Other (write in name)Alliance for a Healthier GenerationEat Smart/Move MoreBuild Our Kids Success (BOKS)Counselor-sponsored small groupsFitnessGramGo NoodleNutrition NetworkaSafe Routes to Schoola Programs categorized as having peer-reviewed evidence in support of the program’s efficacy.We estimated the frequency of responses to each survey item for the entire sample. To explore potential disparities in access to wellness programs, we constructed 2 multivariate logistic regression models that tested whether an association existed between schools’ percentage of students receiving free or reduced-price lunch, percentage of white students, grade level, or Census region  and the the likelihood of 1) having any wellness programs, and 2) having an evidence-based intervention program. Analyses were conducted using Stata, Release 13 (StataCorp LP).To analyze themes in the text written by study participants for the open-ended survey questions about facilitators or barriers for program adoption, the researchers used an immersion/crystallization approach . This apThe 247 survey respondents represented public school administrators from 48 states; approximately one-sixth of respondents were from the Northeast census region , a similar proportion were from the West , one-third were from the South , and more than one-third were from the Midwest  . Most reA minority of survey respondents ranked obesity, nutrition, or physical activity as among their top 3 health concerns for their school populations . Eighty-Fewer than half of respondents  indicated that their schools offered a school wellness or obesity prevention activity of any kind (including broad policies such as competitive foods standards and more structured programs) . AlthougAmong the 117 schools that reported implementing any type of wellness and obesity prevention activity, survey respondents, using a checklist of potential partners in decision making, ranked physical education teachers  most frequently as people involved in the program development or selection process. Participants whose schools offered obesity prevention activities indicated that recommendations from a district administrator , the fact that other schools in the district were using the same program , and recommendation from a teacher at their own school  were common factors in programming decisions. A handful of participants at schools with obesity prevention activities reported that their school programming decisions were influenced by reviews of educational  or scientific  literature. More than half of administrators at schools with wellness activities  perceived a positive impact of their activities on overall student health, and nearly two-thirds  perceived a positive impact on student physical health. School administrators generally perceived that their wellness programs did not increase eating disorders . Thirty-eight (32.5%) participants thought that their programming led to a decrease in unhealthy weight management by students.Results from the logistic regression models testing whether the school’s region or sociodemographic makeup predicted likelihood of having 1) any wellness program or 2) an evidence-based intervention suggested that none of these variables  were significantly associated with either outcome.Most survey respondents  offered commentary in response to open-ended questions about barriers and supports to implementing obesity prevention or nutrition and physical activity programs. Across schools, respondents frequently mentioned financial resources, time and curricular priorities, and staffing and training as key factors in determining uptake of a wellness program; some noted that financial resources and training opportunities were instrumental in facilitating the program, and others reported that a lack of these resources prohibited program adoption. One administrator noted, “Being grant-funded [by the state], our available resources are extremely limited. Once classrooms are staffed and materials for teaching purchased for core subject areas, very little is left,” echoing the comments of many administrators whose schools were lacking funding, staffing, and time to focus on wellness and obesity prevention. Others described district-level coordination and collaboration, and state and federal standards and requirements, as key structural facilitators and barriers to the success of their wellness programmingStratifying the qualitative data by whether the school used any kind of predeveloped program, we found that respondents from schools with such programs were more likely to raise the themes of having an important program champion, substantial buy-in for the program within the school community, or both.In this study of a national random sample of US public primary and secondary schools, we found that, despite the substantial amount of scientific research devoted to developing and testing school-based obesity prevention and nutrition or physical activity promotion programs , very feIn our sample of US kindergarten through twelfth-grade public schools, programs emphasizing weight loss were more commonly used than evidence-based, effective programs for promoting healthy eating and physical activity. Although programs that focus on improved nutrition, physical activity, and screen time behaviors generally prevent obesity and improve health, programs that focus instead on weight status and weight loss can exacerbate both weight stigma and unhealthy weight-control behaviors . ParticuThe themes that emerged from survey respondents’ comments about school wellness programs also suggest that schools need more support to adopt effective programs. Schools that had adopted any program, whether evidence-based or not, were more likely to describe as key facilitators having a program champion and adequate buy-in from stakeholders; a prior study of the postresearch sustainability of one evidence-based program, “New Moves,” also found buy-in, funding, and time to be critical implementation factors . These fStrengths of this study are its random sampling approach, which reduces sampling bias, and its use of mixed methods to gather a nuanced picture of school-based wellness programming. The study also has several limitations. Although a random sample allowed for a broad cross-section of US schools, we found that participating schools had slightly higher percentages of white students than did nonparticipating schools, suggesting that the results may not be generalizable to all US public schools. Schools that responded to our survey may have also differed from nonresponding schools in other ways (such as having more resources), raising the risk of response bias. Given study resource constraints, our sample was also small, raising the risk of error in our estimates. However, the margin of error for our findings on the percentage of schools using evidence-based programs was 3.2%, indicating that the study’s overall conclusions about low levels of dissemination of these programs are likely generalizable. Our small sample size may have precluded us from detecting regional or sociodemographic differences; future research, using larger samples, should further evaluate possible predictors of dissemination. The sample also did not include private or parochial schools. Additionally, the study relied on school administrators’ reports of school wellness programming, the validity of which are unknown; it is possible that respondents may have either overestimated the efficacy of wellness programs at their schools or overlooked them entirely or that school principals may have been unaware of some programs implemented in their school. Future research could compare school policies and curricular materials with school administrator reports to ascertain their accuracy.Few schools in this study implemented nutrition, physical activity, or obesity prevention programs that known to be effective. In the absence of evidence, many schools appear to be trying to address obesity prevention and wellness on their own, unintentionally implementing potentially ineffective or harmful programs. Public health agencies and funding bodies should focus on supporting schools in the dissemination and adoption of safe and effective programs for promoting healthy eating and physical activity and preventing obesity.