Despite their humble origins as anuclear fragments of megakaryocytes, platelets have emerged as versatile mediators of thrombosis and immunity. The diverse spectrum of platelet functions are on full display during the host response to severe infection and sepsis, with platelets taking center-stage in the intravascular immune response to blood-borne pathogens. Platelets are endowed with a comprehensive armamentarium of pathogen detection systems that enable them to function as sentinels in the bloodstream for rapid identification of microbial invasion. Through both autonomous anti-microbial effector functions and collaborations with other innate immune cells, platelets orchestrate a complex intravascular immune defense system that protects against bacterial dissemination. As with any powerful immune defense system, dysregulation of platelet-mediated intravascular immunity can lead to profound collateral damage to host cells and tissues, resulting in sepsis-associated organ dysfunction. In this article, the cellular and molecular contributions of platelets to intravascular immune defenses in sepsis will be reviewed, including the roles of platelets in surveillance of the microcirculation and elicitation of protective anti-bacterial responses. Mechanisms of platelet-mediated thromboinflammatory organ dysfunction will be explored, with linkages to clinical biomarkers of platelet homeostasis that aid in the diagnosis and prognostication of human sepsis. Lastly, we discuss novel therapeutic opportunities that take advantage of our evolving understanding of platelets and intravascular immunity in severe infection. Expanding beyond their well-known role in primary hemostasis, much of the recent research in the field of platelet biology has focused on elucidating the contributions of platelets to host defense and immunity. It is now well-established that platelets are integral to the innate immune response to infection and inflammation, both as autonomous effectors as well as collaborative conductors of anti-microbial defenses –6. The oAbnormalities of platelet homeostasis are common during acute infections and sepsis, and clinical monitoring of platelet counts has emerged as one of the most important biomarkers in the management of septic patients . The mosAn acute rise in the circulating platelet count (acute thrombocytosis) is generally interpreted as a manifestation of systemic inflammation, as may be seen in the context of acute infection . HoweverIn addition to being a common finding in sepsis, clinicians have long appreciated that thrombocytopenia represents an ominous sign in the setting of severe infection –20, 22. The strong epidemiological links between abnormalities of platelet homeostasis and outcomes strongly support a role for platelets in the pathogenesis of sepsis and septic shock. The development of thrombocytopenia in sepsis occurs primarily as a result of massive consumption of circulating platelets through interactions with immune cells in the vasculature (described below), with additional contributions from reduced thrombopoiesis, sequestration, thrombotic microangiopathy , direct pathogen-induced thrombocytopenia, immune-mediated thrombocytopenia, drug induced thrombocytopenia, and hemophagocytosis [see recent reviews by  and 28)28)]. AltIntravascular immune sentinels are strategically positioned within the vasculature to rapidly detect and respond to bloodstream invaders . Platelein vitro is significantly higher than that required for activation of neutrophils  has revealed that platelets undergo transient touch-and-go interactions with the vascular endothelium and other intravascular immune cells such as Kupffer cells in the liver . Under hbacteria . It remaThe early phase of sepsis is characterized by a vigorous systemic inflammatory response, during which platelets  are recruited from the circulation and sequestered within highly vascular organs such as the lungs and liver, resulting in consumptive thrombocytopenia , 60–65. in vitro and in vivo via both direct and indirect mechanisms. Interactions between selectins and selectin-ligands are best known for mediating transient low-affinity catch-bonds that support leukocyte rolling on activated endothelium (Mβ2 integrin) can mediate adhesion to platelets via multiple receptors, including GP1bα as well as GPIIbIIIa via a fibrinogen bridge (Lβ2 integrin) and ICAM-2 , the recruitment of platelets and neutrophils within the microcirculation may follow the opposite sequence, that is, initial platelet accumulation upon the endothelium followed by subsequent neutrophil recruitment. This mode of platelet-dependent neutrophil recruitment has been observed in a number of models of inflammatory and thrombotic disease, including cytokine-induce cerebral inflammation , thermalin vivo investigation of neutrophil polarization , and made the striking discovery that platelets dock to the leading-edge of adherent neutrophils in the vasculature, and that this polarized binding was crucial for directional migration (Mβ2 integrin) and CXCL2, generating polarized receptor microdomains that were essential for effective locomotion toward infection and injury. This discovery revealed that adherent neutrophils scan for activated platelets in the vasculature to enable physical interactions that steer neutrophil migration toward appropriate targets.Following adhesion to the endothelium, immune cells migrate through the vasculature guided by a variety of chemotactic cues –86. The igration . Followiin vitro observation for many years  integrin inhibits intravascular platelet locomotion  in response to chemoattractant stimuli. Platelets express a variety of surface receptors for prototypical chemoattractants, as well as the necessary intracellular signal transduction and cytoskeletal machinery required for cell motility. Platelet chemotaxis remained an ny years , until ad sepsis , 88. Dirin vivo host defense remains unknown.Platelets have emerged as versatile effectors of anti-bacterial immunity, contributing both autonomous bacteriocidal/bacteriostatic properties as well as synergistic partnerships with other innate immune cells. In co-culture experiments, platelets can autonomously inhibit the growth of various bacteria –91. The in vivo that arise from synergistic partnerships with other immune cells . NETs are extracellular webs of decondensed chromatin laden with proteolytic enzymes and other anti-bacterial molecules that are expelled from activated neutrophils . NETs arin vivo, it is not sufficient. Using a mouse model of sterile pulmonary inflammation, Rossaint et al. found that a second signal composed of platelet-derived CXCL4/CCL5 heterodimers was essential for NETs release in response to platelet-neutrophil binding (In vitro, β-defensin-1 released from activated human platelets was shown to induce NETs release from neutrophils, but this is yet to be confirmed in vivo (in vivo remains to be elucidated.The molecular mechanisms controlling NETs release in response to platelet-neutrophil interactions are incompletely understood. Blocking the physical engagement of platelets with neutrophils inhibits the release of NETs, suggesting a contact-dependent induction pathway. However, while contact between neutrophils and platelets is essential for intravascular NETs release  binding . In vitr in vivo . A full Finally, although platelet-neutrophil collaboration has been studied more extensively, it should be noted that platelets engage in functional interactions with other innate immune cells in the vasculature during sepsis. Platelet-monocyte complex formation has been observed in the blood of septic patients , 103, anOverall, there is strong evidence that collaboration between platelets, neutrophils, and other antibacterial effector cells within the microvasculature is crucial to protect against blood-borne infections. Unfortunately, as with most powerful immune defense mechanisms, dysregulation of the intravascular immune response during sepsis can result in tremendous collateral damage to host cells and tissue, resulting in organ dysfunction.in vitro and in vivo (The intricate interplay between platelets, neutrophils, and NETs in sepsis represents a classical example of a “double-edged sword”; providing protective host defense while simultaneously causing immune-mediated organ dysfunction . The imp in vivo . In fact in vivo . Further in vivo . Lastly,in vivo has revealed disseminated microvascular coagulation following NETs release, resulting in extensive obstruction of blood flow and ischemic injury to the affected organ (In addition to direct cytotoxicity, a growing body of literature implicates the platelet-neutrophil-NETs axis in sepsis pathogenesis through the induction of intravascular coagulation. Platelet-neutrophil interactions and the subsequent release of NETs coincides with diffuse activation of thrombin and fibrin deposition within the vasculature of multiple organs . Imaginged organ . This wied organ . Conversed organ . There ied organ –119.The pro-coagulant properties of NETs arise from a variety of components that are known to interact with the clotting cascade at multiple levels. For example, histone proteins have been shown to initiate coagulation through the upregulation of tissue factor as well as initiation of the contact-dependent pathway , 82, 120in vivo requires the intact NETs macrostructure. Most notably, experiments using exogenous DNase to dissolve the DNA backbone of NETs have demonstrated marked inhibition of intravascular thrombin activation and fibrin production, despite the fact that individual pro-coagulant NETs components remain in the vasculature (Although a number of components within NETs have the ability to activate coagulation in isolation, emerging evidence suggests that the induction of intravascular coagulation culature , 122. Thin vivo (in vitro (Finally, in addition to stimulating NETs release from neutrophils, platelets also provide synergistic amplification of NET-induced coagulation. While the majority of platelets are sequestered in the microvasculature through interactions with neutrophils, platelets can also bind and aggregate directly on NETs. NETs-mediated platelet aggregation has been demonstrated in a variety of thromboinflammatory disorders, including sepsis, venous thromboembolic disease, and atherosclerotic vascular disease , 125. Duin vivo , and polin vitro . OverallPlatelets are versatile mediators of antimicrobial immunity and host defense within the bloodstream during sepsis . HoweverBM performed the literature review, wrote and edited the manuscript. MD contributed to the literature review, manuscript preparation, and figure development.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The handling Editor declared a shared affiliation, though no other collaboration, with the authors at time of review.


Metabolic and genotoxic stresses that arise during tumor progression and anti-cancer treatment, respectively, can impose a selective pressure to promote cancer evolution in the tumor microenvironment. This process ultimately selects for the most “fit” clones, which generally have a cancer stem cell like phenotype with features of drug resistance, epithelial-mesenchymal transition, invasiveness, and high metastatic potential. From a bioenergetics perspective, these cancer stem-like cells (CSCs) exhibit mitochondria-centric energy metabolism and are capable of opportunistically utilizing available nutrients such as fatty acids to generate ATP and other metabolic substances, providing a selective advantage for their survival in an impermissible environment and metabolic context. Thus, diverse therapeutic strategies are needed to efficiently tackle these CSCs and eliminate their advantage. Here, we review the metabolic and bioenergetic characteristics and vulnerabilities specific to CSCs, which can provide an unprecedented opportunity to curb CSC-driven cancer mortality rates. We particularly focus on the potential of a CSC bioenergetics-targeted strategy as a versatile therapeutic component of treatment modalities applicable to most cancer types. A cancer bioenergetics-targeted strategy can expand the inventory of combinatorial regimens in the current anti-cancer armamentarium. Research on cancer cell-specific metabolism has exponentially increased in recent years, with recognition of the discovery of potential therapeutic targets for refractory cancers. This field has illuminated novel aspects of cancer cell biology related to the metabolic reprogramming that occurs during tumorigenesis and metastasis, which has even been referred to as a hallmark of cancer  and highlight the therapeutic opportunities that can arise by identifying metabolic vulnerabilities specific to this deadly subpopulation. In particular, we provide an overview of recent findings implicating mitochondria-centered bioenergetics during cancer malignant evolution in selective tumor environmental context. We then describe the specific metabolic pathways and key molecules enabling metabolic stress adaptation during cancer progression. Finally, we introduce new strategies to pharmacologically intervene with cellular bioenergetics in malignant cancers.Cancer progresses in the defined and adaptive microenvironement. Both the interaction between heterogeneous subpopulations within a given cancer type and that between the cancer cells and surrounding microenvironment influence tumor progression, metastasis, and ultimate drug resistance (Tabassum and Polyak Helicobacter pylori cytotoxin-associated gene A (CagA) increases reactive oxygen species (ROS) production and activates the transcription factor hypoxia inducible factor 1α (HIF-1α), which in turn facilitates the metabolic changes that help cancer cells survive under hypoxia and glucose deprivation   inhibits anabolic processes  according to their specific anabolic and catabolic needs depending on the cell state, i.e., quiescence, pluripotency, and proliferation  itself, which contains genes of the electron transport chain (ETC). Various mtDNA abnormalities and the associated mitochondrial dysfunction have been demonstrated to support cancer progression, contributing to tumorigenesis, metastasis, and treatment resistance   is a transcriptional co-activator that acts as a master regulator of mitochondrial metabolism. In cancer cells, PGC-1α serves as a stress sensor that is activated under nutrient limitation, oxidative stress, and chemotherapy to facilitate mitochondrial biogenesis, OXPHOS, FAO, and ROS detoxification. PGC-1α has been reported to have both oncogenic and tumor-suppressive features: its expression is downregulated in the early stage of carcinogenesis, thereby inducing glycolysis to play an anticancer role, whereas it is upregulated in the late stage of cancer progression, contributing to the metabolic plasticity that facilitates tumor cell growth, metastasis, and drug resistance  derived from FAO supports the transformation of intraductal papillary mucinous neoplasms to pancreatic ductal adenocarcinomas (PDACs). In addition, coordinated triglyceride synthesis and FAO have been described in KRAS-mutant lung cancer  impaired cell viability only in low-grade ccRCC cells  family of nuclear receptors function as transcription factors, and they have recently been shown to play a role in cancer metabolism through FAO. Constitutive activation of PPARα activated FAO, and the subsequent production of ATP and ROS contributed to the pathogenesis of hepatocellular carcinoma (HCC) , generally known as a tumor suppressor, which deacetylates PGC-1α, leading to activation of PPAR signaling to promote FAO, thereby supporting the survival, growth, and malignant phenotype of breast cancer cells  increases mitochondrial mass and FAO in specific cancer cell types. MYC activates mitochondrial biogenesis and fusion by upregulating PGC-1β and phospholipase D family member 6, respectively, to increase both the respiratory and biosynthetic capacity of cancer cells, supporting their rapid proliferation  cation is the most effective method developed to date (Kalyanaraman et al. Targeting mitochondrial biogenesis has demonstrated particular therapeutic efficacy for cancers of malignant phenotypes. For example, inhibition of mitochondrial biogenesis with gamitrinib, a mitochondrial HSP90 inhibitor, effectively eradicated BRAF-mutated melanoma cells, which were resistant to a MAPK inhibitor (Zhang et al. OXPHOS inhibitors are emerging as promising therapeutics of malignant cancers marked by increased mitochondrial respiration Wolf . AntidiaThe therapeutic efficacy of FAO inhibition has been proven in various types of cancer in preclinical studies. Etomoxir, a CPT1 inhibitor, is one of the most well-studied FAO inhibitors. Similar to metformin, etomoxir demonstrated selective efficacy against CSCs and cancers with malignant phenotypes, sensitizing them to conventional treatments (Camarda et al. Various combination strategies are now being attempted to make the best use of drugs targeting cancer bioenergetics. Combination of an OXPHOS/FAO inhibitor with other chemotherapeutic regimens has emerged as a particularly promising strategy. Mitochondrial respiration is a strong candidate to target the vulnerability of drug-resistant cancer cells, and several OXPHOS inhibitors can block their emergence. Various combinations of OXPHOS inhibitors with conventional cancer treatments—both cytotoxic and targeted therapies—proved to have synthetic lethality in pre-clinical models Wolf . For exaThe concurrent inhibition of OXPHOS/FAO with other metabolic pathways is a fascinating approach to completely starve cancer cells to death. Typically, combined targeting of OXPHOS/FAO and glycolysis is an effective strategy. Inhibition of OXPHOS/FAO displayed efficacy against starvation-resistant cancer cells under glucose-deprivation conditions (Isono et al. Detailed studies on the metabolism and bioenergetics of cancer cells are shedding light for the next generation of cancer treatment in the postgenomic era. Although the advent of next-generation sequencing ushered in a huge wave of elucidation of the genome-level anomalies of cancer, resulting in numerous effective drug targets, the great diversity of these genes leaves us with an unsolved problem in this genomic era of effective targeting, especially for CSCs. Thus, the postgenomic era of cancer drug development should be driven by a paradigm shift toward bioenergetics, aiming at the energy metabolism distinctive to cancer cells. This strategy can directly target the fundamental vulnerability of cancer cells, with a focus on drug-resistant cancer cells.Recently, adoption of a systems biology-based approach has effectively facilitated the discovery of feasible strategies to target cancer bioenergetics. For example, a recent study using an integrative analysis of signaling networks and drug functional networks revealed that subtypes of medulloblastoma with a poor prognosis can be targeted by digoxin (Huang et al.


Consequently, the complexity of reported macrophage phenotypes has implications for our understanding of the metabolic pathways, processes and shifts underpinning their activation and inflammatory status. Using oxidized low density lipoproteins and its oxidized cholesteryl esters and phospholipid constituents to stimulate macrophage has been hugely valuable, however there is now an argument that only working with low complexity lipid species can deliver the most useful information to guide therapies aimed at controlling atherosclerosis and cardiovascular complications.Atherosclerosis is a chronic inflammatory disease and a leading cause of human mortality. The lesional microenvironment contains a complex accumulation of variably oxidized lipids and cytokines. Infiltrating monocytes become polarized in response to these stimuli, resulting in a broad spectrum of macrophage phenotypes. The extent of lipid loading in macrophages influences their phenotype and consequently their inflammatory status. In response to excess atherogenic ligands, many normal cell processes become aberrant following a loss of homeostasis. This can have a direct impact upon the inflammatory response, and conversely inflammation can lead to cell dysfunction. Clear evidence for this exists in the lysosomes, endoplasmic reticulum and mitochondria of atherosclerotic macrophages, the principal lesional cell type. Furthermore, several intrinsic cell processes become dysregulated under lipidotic conditions. Therapeutic strategies aimed at restoring cell function under disease conditions are an ongoing coveted aim. Macrophages play a central role in promoting lesional inflammation, with plaque progression and stability being directly proportional to macrophage abundance. Understanding how mixtures or individual lipid species regulate macrophage biology is therefore a major area of atherosclerosis research. In this review, we will discuss how the myriad of lipid and lipoprotein classes and products used to model atherogenic, proinflammatory immune responses has facilitated a greater understanding of some of the intricacies of chronic inflammation and cell function. Despite this, lipid oxidation produces a complex mixture of products and with no single or standard method of derivatization, there exists some variation in the reported effects of certain oxidized lipids. Likewise, differences in the methods used to generate macrophages  In pigeons, cholesterol trapped in the lysosomes of lesional foam cells remained trapped, even after intentionally reducing plasma cholesterol returned this parameter to normal levels and cytoplasmic CE droplets had been cleared  are recruited to the intimal layer of the medium and large size arteries. Here, they differentiate into macrophages (MO) and encounter a plethora of modified lipid species such as oxidized low-density lipoprotein (oxLDL). MOs employ mainly scavenger receptors to facilitate the uptake of these modified lipoproteins, leading to the formation of foam cells  secretory pathway or by non-conventional secretion. The majority of cytokines expressed in MOs are processed and transported through the constitutive pathway; however, some require non-conventional secretion for cellular release. Secretory lysosomes facilitate this secretion and are also able to degrade inflammatory cytokines to regulate the immune response to an external stimulus, such as lipopolysaccharide (LPS) and adenosine triphosphate (ATP)  and smooth muscle cells (SMC) to secrete proinflammatory cytokines and chemokines Libby, . These c+ efflux from the cell  is mobilized to mitigate ER stress signaling to restore homeostasis. Under pathologically chronic ER stress, cell dysfunction and disease manifest, potentially leading to cell death. Three upstream ER stress transmembrane sensor proteins respond to the presence of misfolded and unfolded proteins by triggering the UPR. They are activating transcription factor 6 (ATF6), inositol-requiring enzyme 1α (IRE1α), and protein kinase R-like endoplasmic reticulum kinase (PERK). The latter augments the translation of ATF4, inducing the production of C/EBP-homologous protein (CHOP), which instigates a number of restorative functions to temper transient ER stress Tabas, . One of Mitochondrial dysfunction is also prominent during atheroprogression. Amongst the multitude of processes that occur within mitochondria, oxidative phosphorylation, through the production of ATP, liberates moderate  levels of superoxide, the bulk of which are converted to hydrogen peroxide by superoxide dismutase. This process remains homeostatic under normal conditions, however, under pathophysiological conditions; chronic overproduction of ROS arises from excessive mitochondrial oxidative stress. The link between mitochondrial oxidative stress (MitoOS)-induced pathology and atherosclerosis was made in an elegant study using transgenic mice in which MitoOS had been suppressed in lesional MOs. These mice exhibited a reduction in lesional area of the aortic root, a lower abundance of Mos in these lesions and a concomitant reduction in Ccl2 and RelA phosphorylation, indicating the NF-κB signaling pathway responsible for proinflammatory cytokine production was also downregulated. Mitochondrial damage or dysfunction delivers, through the production of ROS, another source of “signal 2” to initiate inflammasome assembly  , revealing hitherto unknown levels of diversity amongst these cell types and their distinct subsets in vivo  and human Mo-derived MOs. In both cell types, cholesterol loading followed by Tlr4 stimulation  led to a significant inhibition of Cxcl9 and Cxcl10 expression, compared with KLA-treated cells  of LPS-induced cytokine expression were evidently under circadian control , and are therefore recognized by host pattern recognition receptors (PRRs), similar to how bacterial pattern-associated molecular patterns (PAMPs) are recognized by TLRs  , but not in their stem and progenitor cell populations. In LDLR−/− mice transplanted with ABCDKO bone marrow (BM), both inflammation and atherosclerosis are increased  and chemokine  expression is significantly elevated , to downregulate pro-inflammatory cytokine production  is commonly perceived as being anti-inflammatory, with a wealth of research supporting this view  and a low dose of LPS synergistically increase chemokine release in MO cell lines , formed following polyunsaturated fatty acid oxidation, are another major class of lipids that have been extensively studied in the context of inflammation and atherosclerosis. Their biology is also complicated and they initiate pro-inflammatory responses in a context-dependent manner. The transcriptome and functional phenotype of BMDMs exposed to the oxidation products of 1-palmitoyl-2-arachidonoyl-sn-3-glycero-phosphorylcholine (oxPAPC) is markedly different to that generated by LPS + IFNγ (M1) and IL-4 (M2) polarization stimuli  molecules, which have only come under greater scrutiny over the past decade. They are abundantly present in oxLDL and are bioactive, inflammatory components of mmLDL, able to induce Cxcl2 release in mouse MOs  or L-Cell Conditioned Media  or Granulocyte-MO Colony-Stimulating Factor (GM-CSF) added to human Mo or mouse bone marrow cultures. This is important because all three of these cytokine options produce MOs with markedly different transcriptomes, including differentially expressed inflammatory and lipid handling genes. Studies carried out a decade ago or earlier showed that human Mos differentiated with GM-CSF generate pro-inflammatory (M1-like) MOs, whilst those exposed to M-CSF develop an anti-inflammatory (M2-like) phenotype  or oxLDL-transformed foam cells, substantial differences in cell surface marker, and inflammatory cytokine gene expression levels were evident  or M-CSF or GM-CSF. Each of these types possess a distinct transcriptional phenotype that has some overlap with the other two CSF-derived types , which is found abundantly in oxLDL. Feeding the U937 Mo line either with 7KC or oxLDL induces a loss of cell viability. Both also cause glutathione loss and lead to increased 7KC content in these cells. By contrast, when acLDL is used to deliver this sterol to U937s, its uptake is high yet the toxicity to U937 cells is very low , copper, or osmium tetraoxide amongst others and have been reviewed extensively elsewhere (Levitan et al., in vitro experiments are carried out, by moving away from using complex mixtures and instead switching to individual lipid species or low complexity mixtures enriched with a single lipid component. In doing so, it is going to become easier to associate specific effects with specific lipid structures, which will improve the development of both generic and personalized therapies aimed at reducing or controlling atherosclerosis and other inflammatory diseases.Several of the studies reviewed in this article have highlighted the benefit of assessing the function of individual lipid components. The most striking of these perhaps being where oxPAPC and its minor fractions hyperactivate DCs to release IL-1β, however, only the minor fractions activate MOs (Zanoni et al., in vitro and in vivo (Emanuel et al., By restoring or augmenting cellular function, or by inhibiting the factors that disturb it, numerous therapeutic interventions have been developed to treat atherosclerosis. Anti-inflammatory therapies are particularly ubiquitous and have a range of targets, with variable levels of efficacy. These include nutraceuticals, which are ingested as dietary supplements and have shown beneficial reductions in ROS activity, endothelial exocytosis, MO migration, and promote reverse cholesterol transport (Moss and Ramji, The purpose of this review was to offer a perspective on how cell dysfunction and inflammation contribute to atherosclerosis. We described how critical changes in organelle function, particularly those affecting lysosomes, can transform the inflammatory status of macrophages, the most prominent cell type in atherogenesis and which has a major influence on lesional inflammation, plaque progression and stability. By discussing lipid and non-lipid factors that can regulate inflammation, including the perhaps under appreciated role of circadian rhythms, we outlined some probable targets for more precise therapeutic interventions. Of course a major area of modern biomedical research is understanding the biology of lipid and lipoprotein factors that contribute to atherosclerosis and other chronic diseases, and some of the more prominent compounds studied were reviewed. In carrying out such studies, there are evident discrepancies in the literature, that in many cases are caused by alternative approaches to asking the same question. We made a point of highlighting where we believe these problems exist and hope that some form of standardization or guidelines could be introduced into the field to address them. Finally, we discuss recent advances in generic treatment options for atherosclerosis, which ideally could form one aspect of more personalized future novel therapies.All of the authors listed were involved in the preparation of the manuscript. ND carried out some of the experimental work referred to in the passages describing data produced in our group.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.


Motor capability recovery after ischemic stroke involves dynamic remodeling processes of neural connectomes in the nervous system. Various neuromodulatory strategies combining direct stimulating interventions with behavioral trainings for motor recovery after ischemic stroke have been developed. However, the effectiveness of these interventions varies widely due to unspecific activation or inhibition of undefined neuronal subtypes. Optogenetics is a functional and structural connection-based approach that can selectively activate or inhibit specific subtype neurons with a higher precision, and it has been widely applied to build up neuronal plasticities of the nervous system, which shows a great potential in restoring motor functions in stroke animal models. Here, we reviewed neurobiological mechanisms of enhanced brain plasticities underlying motor recovery through the optogenetic stimulation after ischemic stroke. Several brain sites and neural circuits that have been previously proven effective for motor function rehabilitation were identified, which would be helpful for a more schematic understanding of effective neuronal connectomes in the motor function recovery after ischemic stroke. Ischemic stroke, a leading cause of severe disabilities in the adult population, has an annual economic cost of approximately $34 billion all over the world ; it alsoIn normal conditions, each cortical hemisphere inhibits the other through transcallosal fibers . Stroke As tonic neuronal inhibition increased in the peri-infarct zone after stroke, to help regain the excitatory-inhibitory balance of the ischemic region within the infarct hemisphere, perhaps direct manufacturing of GABAergic neurons in the peri-infarct zone can become a candidate target for optogenetic stimulation in the future , 36.Although iM1 stimulation has demonstrated significant effects in promoting motor function recovery, the overall motor performance of the mice only recovered to about 50% of the prestroke baseline . To opti−2 that reliably activates cortical neurons. As expected, optogenetic stimulation targeting this circuit resulted in a persistent improvement in sensory cortex and paw function. Furthermore, chronic optogenetic stimulation also promotes rewiring of thalamocortical boutons as well as growth and retraction of thalamocortical axon branches [Apart from afferent cLCN, sensory-evoked cortical activity also depends on the integrity of afferent inputs from the thalamus. It has been shown that focal ischemia leads to a loss of thalamic axonal synaptic contacts with the peri-infarct cortex, resulting in a reduction in the excitability of surviving thalamocortical circuits –44, whicbranches . It is w2, which specifically activated corticospinal projecting neurons. The encouraging effects of this stimulation paradigm were comparable to previously established anti-Nogo-A immunotherapy [Ischemic stroke disables not only afferent circuits, but also efferent connections. Experimental studies have shown that a stroke denervated corticospinal neurons in the contralateral cortex. Furthermore, after various therapeutic and rehabilitative interventions, the corticospinal tract (CST) on the contralesional side can sprout and terminate in the denervated hemispinal cord –49. Thusotherapy , verifyiotherapy . Indeed,otherapy . HoweverBrain stimulation strategies based on activity-dependent neuronal plasticity have made great advancements in motor recovery after stroke. Nevertheless, effective treatment of ischemic stroke remains a major challenge mainly because the narrow treatment window of currently available treatment approaches is limited to the acute phase . Recentl2 and a 473 nm-pulse blue laser with 0.5 mW power to specifically inhibit or activate GABAergic neurons in Gad2-Arch-GFP transgenic mice, respectively. It was demonstrated that optogenetic inhibition rather than activation of striatal GABAergic neurons promoted neurogenesis after cerebral ischemia by projecting dendrites and axons to the SVZ [Aside from the stimulation of glutamatergic neurons, GABAergic neurons are known to constitute the majority of striatal neurons . Another the SVZ , leading the SVZ .Altogether, these studies partially illuminate the underlying mechanisms of how altering neuronal activity regulates neurogenesis in a semiphasic synaptic way. This photoactivation of neural regeneration may provide a promising direction in extending the treatment window for stroke.Motor function recovery after stroke depends on the reorganization of neuronal connections throughout the central nervous system (CNS). The contents above have elucidated several potential brain circuits or sites where new connections can grow after optogenetic stimulation. However, the mechanisms of how new functional circuits are formed remain poorly known. We examined three neurobiological mechanisms underlying optogenetic-stimulation-promoted motor function recovery on a synaptic level, including synaptic plasticity, axonal sprouting, and dendritic morphology changes.Among them, synaptic plasticity refers mainly to the function of synaptic circuits or changes in inhibitory control of circuits. While axonal sprouting and dendritic morphology changes are the structural basis of new connection formations, the axonal sprouting in peri-infarct and connected cortical areas is causally associated with motor recovery and can be triggered by synchronized low frequency neuronal activity . AlthougAs with activity-dependent synaptic plasticity, both long-term potentiation (LTP) and long-term depression (LTD) have been proposed to be involved in cortical plasticity , 63. It As to axonal sprouting, several sprouting transcriptomes and molecules like EphA4, ATRX, Lingo1, and IGF have been evaluated to be responsible for axonal sprouting after stroke , 34, 67.The dendritic morphology of a neuron determines where and how the new connections of that neuron will be made . AlthougBased on evidence from the microscale synaptic level to the mesoscale circuit level, optogenetics can be utilized as a useful tool to sculpt brain connectivity after stroke. However, since the establishment of connections requires guiding axons to correct target areas and forming synapses at particular regions on postsynaptic neurons, there is still no clear mechanistic rationale on molecular details of the process. Consequently, it calls for more mechanistic studies of neuronal plasticities to advance in developing effective treatment strategies.Overall, stroke can be viewed as a disruption of old brain connectomes as well as an origin of new neuronal connection reorganization throughout the central nervous system. Optogenetics is a useful and neuron-specific tool in promoting neural plasticity to benefit motor function rehabilitation after ischemic stroke. Several proved-to-be effective neural circuits have been identified by optogenetics. Among these stimulation targets, recovery through the stimulation of the primary site of ischemia iM1 is mainly involved in potentiating synaptic plasticity within the core of the infarcted area and promoting new connections within peri-infarct areas by enhancing axonal sprouting. Stimulation of afferent and efferent circuits are mainly involved in axonal sprouting within the ipsilesional somatosensory cortex, the thalamocortical circuit, and CST fibers in the denervated cervical hemicord. Optogenetic stimulation of SVZ, which is a niche for the genesis of neuroblasts, involves promoting neuroblasts to migrate to the boundaries of the infarct zone, to differentiate into mature neurons, and to integrate into neuronal networks .Optogenetics-enhanced neural plasticities include both long-term potentiation (LTP) and long-term depression (LTD) . At the Apart from these brain targets, it is worth noting that the contralateral hemisphere has always been of great interest as a target of neuromodulatory interventions . In animAlthough optogenetics itself could not be applied directly to clinical patients, these studies can be instructive in identifying specific excitatory or inhibitory circuitry that may work in human patients, thus allowing for more precise and effective outcomes for noninvasive and mature stimulation toolboxes like transcranial magnetic stimulation (TMS) or tDCS.In conclusion, optogenetics is a useful tool in extending the boundaries of poststroke rehabilitation of motor function by providing a conceptual framework to improve established clinical stimulation techniques . The dev


SPAST mutations, the most common cause of HSP.Hereditary spastic paraplegia is an inherited, progressive paralysis of the lower limbs first described by Adolph Strümpell in 1883 with a further detailed description of the disease by Maurice Lorrain in 1888. Today, more than 100 years after the first case of HSP was described, we still do not know how mutations in HSP genes lead to degeneration of the corticospinal motor neurons. This review describes how patient-derived stem cells contribute to understanding the disease mechanism at the cellular level and use this for discovery of potential new therapeutics, focusing on  SPAST gene mutation accounts for up to 40% of autosomal-dominant mutations, making it the most commonly mutated gene in this family of disease [Hereditary spastic paraplegia (HSP) is a neurological disorder in which the corticospinal motor neurons degenerate ,2. It is disease ,7.SPAST, is a protein of the AAA (ATPase associated with diverse cellular activities) protein family [Spastin, encoded by n family ,9. Spastn family , shapingn family , and regn family . Our revSPAST studies) and corpus callosum  [SPAST HSP patients for evidence of non-motor manifestations that are seen in patients with other HSP mutations [In recent studies, cerebral white matter alterations in HSP patients were quantified using diffusion tensor imaging (DTI), a widely used magnetic resonance imaging technique. DTI measures the anisotropic flow of water in white matter tracts . White mstudies) ,23,24,25studies) . These cstudies) ,17,19,24utations . The conPost-mortem studies of six HSP patients showed the loss of small and large fibres of the corticospinal tract leading to significant reductions in axon numbers and tract volumes throughout the spinal cord and into the medulla . These cSPAST HSP patients carrying missense and frameshift mutations had axonal swellings in the descending axons of the corticospinal tract and dorsal column of the spinal cord [SPAST HSP cases showed altered mitochondrial distribution in the cell body of spinal cord lower and upper motor neurons, with mitochondria being restricted to the periphery of the neuronal soma, in contrast to uniformly distributed mitochondria in control cells [Post-mortem analysis of spinal cord sections at the cervical and lumbar level of two nal cord . The axool cells .SPAST mouse models do not exhibit the characteristic corticospinal degeneration, but retain the neuronal defects of axonal swellings and altered organelle distribution. In a mouse model with exon 5–7 deletion in SPAST (leading to the lack of the AAA domain in spastin), spinal cord sections of 12 and 24 months homozygous mouse at the cervical and lumbar level did not show any obvious degeneration of the corticospinal or dorsal columns [SPAST mutations [ columns . Spinal utations .SPAST, spinal cord sections of three month old mice with heterozygous and homozygous mutations mouse there were axonal swellings in the white matter [SPAST mutant mouse or was present, regardless. Although the two mouse studies agree on the axonal defect of axonal swellings in HSP, there were inconsistencies. In the former model, the swellings were specific to distal regions of the axons [Homozygous mouse spinal cord sections also showed abnormal accumulation of neurofilaments and cellular organelles in the axonal swellings, including mitochondria and peroxisomes , as obsee matter . The stuhe axons , whereashe axons .SPAST HSP patients [In summary, although the mouse models show swelling defects in corticospinal axons that are similar to corticospinal tract axons in patients , there ipatients . This supatients  without Functional studies in Drosophila and zebrafish have shown that spastin function is essential for motor neurons ,34,35,36Genetic manipulation in animal models can help in our understanding of the molecular consequences of spastin mutations, but it is still a challenge to understand the disease mechanism in humans. Perhaps this is because animal models, unlike human patients, are genetically homogeneous and there are major species-specific differences at all levels . Genetically modified animal models invariably fail to fully replicate human disease deficits and when used for drug development produce, therapeutics that work well in these animal models, work poorly in humans, which is a common problem in neuroscience therapeutic development ,38,39. AInduced pluripotent stem (iPS) cells  are geneSPAST HSP patient-derived iPS cells have reported axonal swellings and axonal transport deficits. Glutamatergic neurons from one iPS cell line carrying the heterozygous SPAST c.683-iG > T mutation showed increased axonal swellings and altered mitochondria transport with reductions in the percentage of microtubule-dependent mitochondria, reduced retrograde, and unchanged anterograde transport [SPAST mutation c.1684C > T showed alterations in neuron morphology with a reduced number and length of neurites; increased axonal swellings and altered mitochondria transport with reduced retrograde and increased anterograde transport [SPAST iPS-derived neurons can identify HSP disease-associated neuronal deficits, they do not suggest the mechanism of how the observed axonal defects could lead to axonal degeneration in HSP patients. Two studies of neurons generated from ransport . Anotherransport ,30. AlthiPS cells hold great potential use in disease modelling and drug screening of human diseases because they provide an unlimited source of tissue-specific cells, but there are still technical challenges for their widespread use as disease models . DevelopPatient-derived olfactory cells and tissues are increasingly used for investigating neurodegenerative and neuropsychiatric diseases for which they have certain advantages over iPS cell models . The olfSPAST mutations and ten healthy controls. By comparing the differences between patient- and control-derived cells, we identified altered cell functions in patient cells and identified drugs that restored their function, thus making them potential new therapeutics for HSP. In discovery phase, an initial unbiased transcriptomics analysis showed that 57% of all mRNA transcripts were affected in patient cells suggesting that SPAST mutations cause major changes in cellular homeostasis [SPAST patient cells. Further analysis of microtubule-associated proteins showed a 50% reduction in acetylated α-tubulin, a marker of stable microtubules and substrate of spastin, and a 150% increase in stathmin, a microtubule destabilising enzyme. This reduction in stabilised microtubules when spastin is reduced may be explained by increased expression of stathmin, as a consequence of homeostatic feedback regulation of microtubule dynamics.As cell models of HSP, we generated ONS cell lines from nine patients with heterozygous eostasis . ConsistQuantitative automated microscopic analysis confirmed the loss of stable microtubules throughout the patient cells and altered the distributions of peroxisomes (more numerous at the periphery of patient cells compared to control cells) and mitochondria (less numerous at the periphery), suggesting that organelle transport was impaired in patient cells . DisplacIntracellular microtubule-dependent organelle transport is essential in neuronal axons that depend solely on microtubules for intracellular transport to and from their distally located synapses  . We testSPAST patient ONS cells that are under oxidative stress when compared to control cells, and more sensitive to hydrogen peroxide [SPAST HSP patient-derived cells and it links SPAST mutations with the induction of cell death via a reduction in stabilised microtubules. This mechanism was confirmed in patient cells when acetylated α-tubulin levels were restored pharmacologically to control levels and their oxidative response to hydrogen peroxide was also restored [Peroxisomes are important regulators of oxidative state in cells, especially the metabolism of hydrogen peroxide . So, defperoxide . In patirestored .SPAST HSP, dysfunctions in microtubule-dependent transport of peroxisomes and mitochondria could have profound consequences for many cell functions, requiring dynamic homeostatic feedback. This may help to explain the need for the genome-scale changes that were observed in the transcriptome of SPAST patient cells [SPAST mutations would affect organelle transport in all cells of the body, but with more profound effects in long axons that depend on microtubules alone for organelle transport. In agreement with this, is evidence that SPAST patient muscle is morphologically normal, despite similarly large magnitude changes in the transcriptome and disruption of microtubule pathways [Peroxisomes and mitochondria interact to regulate responses to oxidative stress and altered biogenesis, dynamics, or metabolism in one organelle can affect the other . Mitochont cells . Presumapathways .SPG7 HSP patient-derived fibroblasts are more sensitive to hydrogen-peroxide with lower ATP production and viability when compared to control fibroblasts [Peroxisome and mitochondria gene mutations are associated with numerous developmental and other deficits in several neurological diseases ,71,72,73roblasts .SPAST HSP patients whose initially moderate symptoms increase in severity over time as initial axon impairment leads increasingly to axon death and die-back.Neurons are particularly vulnerable to oxidative stress, related to the unique functional and structural organisation of neurons. Neurons have a very high requirement for oxygen for intracellular ion homoeostasis, which is essential for propagation of action potentials ,78. NeurSPAST patients and three healthy controls and differentiated them into cortical neurons (unpublished data) [SPAST patient-derived neurons had reduced spastin, reduced acetylated α-tubulin, reduced transport of peroxisomes, and were more vulnerable to hydrogen peroxide-induced oxidative stress demonstrated by increased axonal degeneration (unpublished data) [Patient and control-derived ONS cells are relatively easy to generate and freeze/thaw in vitro, making them suitable for multiple high-through put assays for hypothesis generation and the discovery of disease-associated cell dysfunctions. ONS cells are neural progenitors but they are not neurons and so may not reveal neuron-specific dysfunctions resulting from disease-associated mutations. Our disease-modelling approach is to use patient- and control-derived ONS cells as discovery tools to generate hypothesis and to understand the domain of deficits that are associated with HSP mutations and to use this understanding to screen for possible therapeutic drug candidates. Specific hypotheses are then validated in neurons differentiated from patient-derived iPS cells, keeping those experiments to a minimum because they are more time-consuming to generate and expensive to maintain than ONS cells. We see this two-stage approach as the fastest and most inexpensive way to use patient-derived stem cells for drug discovery and clinical outcomes. We generated 11 iPS cell lines from three ed data) . Like ONed data) .SPAST patient-derived ONS cells and patient-derived neurons demonstrate reduced levels of stabilised microtubules, impaired peroxisome transport, increased oxidative stress, and sensitivity to hydrogen peroxide, leading to reduced cell viability and increased axon degeneration  that increased the levels of acetylated α-tubulin in patient ONS cells to levels in control ONS cells . We then for HSP . EpothilSPAST mutations. This mechanism may also apply to mitochondria whose transport is impaired in other SPAST patient-derived cells [Deficits in microtubule-associated proteins and impaired organelle transport are emerging as common mechanisms in several motor neuron diseases and neurodegenerative disorders ,90,91, wed cells ,30.The tubulin-binding drugs that we used in our studies have been “re-purposed” from cancer therapies and large doses have passed phase I clinical trials, demonstrating their safety. The drug doses in this study are much lower than used clinically  and did SPAST HSP. As discussed, it is expensive in time and money to generate iPS cells and this may be limiting for research on rare diseases like HSP. The strategy illustrated here, using two patient-derived stem cell models, olfactory neural stem cells and induced pluripotent stem cells, provides an efficient system to understand the disease mechanism at a cellular level and to develop therapeutics with the cheaper and easier ONS cells that are used for hypothesis generation and testing and iPS cell-derived neurons used for validation. With SPAST HSP, the most common form, as a starting point; the way is now open to emulate this research strategy for other forms. As knowledge builds for the effects of different mutations on cell phenotype, common and discrete disease mechanisms will be revealed and appropriate therapeutics can be devised.With the many genes that can cause HSP, there is a real challenge in understanding the genetic and cellular mechanisms of disease and in devising individual therapies. It is not obvious from the causative mutated genes if there are any common cellular mechanisms that can be targeted for therapy. These might be revealed by the use of patient-derived stem cells, as outlined here for


Gestational age-dependent immune intolerance at the maternal-fetal interface might be a contributing factor to placental pathology and adverse pregnancy outcomes. Although the intrauterine setting is highly choreographed and considered to be a protective environment for the fetus, unscheduled inflammation might overwhelm the intrauterine milieu to cause a cascade of events leading to adverse pregnancy outcomes. The old paradigm of a sterile intrauterine microenvironment has been challenged, and altered microflora has been detected in gestational tissues and amniotic fluid in the absence of induction of significant inflammation. Is there a role for endotoxin tolerance at the maternal-fetal interface? Endotoxin tolerance is a phenomenon in which tissues or cells exposed to the bacterial product, particularly lipopolysaccharide, become less responsive to subsequent exposures accompanied by decreased expression of pro-inflammatory mediators. This could also be related to trained or experienced immunity that leads to the successful outcome of subsequent pregnancies. Adaptation to endotoxin tolerance or trained immunity might be critical in preventing rejection of the fetus by the maternal immune system and protecting the fetus from excessive maternal inflammatory responses to infectious agents; however, to date, the exact mechanisms contributing to the establishment and maintenance of tolerance at the maternal-fetal interface remain incompletely understood. There is now extensive evidence suggesting that microRNAs (miRNAs) play important roles in the maintenance of a healthy pregnancy. miRNAs not only circulate freely in extracellular fluids but are also packaged within extracellular vesicles (EVs) produced by various cells and tissues. The placenta is a known, abundant, and transient source of EVs; therefore, our proposed model suggests that repeated exposure to infectious agents induces a tolerant phenotype at the maternal-fetal interface mediated by specific miRNAs mostly contained within placental EVs. We hypothesize that impaired endotoxin tolerance or failed trained immunity at the maternal-fetal interface will result in a pathological inflammatory response contributing to early or late pregnancy maladies. Our current understanding of immunity at the maternal-fetal interface has been shaped by acceptance of periodic concepts and approaches formed based on tissue-allograft studies in non-pregnant individuals, male or female, and by the paradigm suggesting the uterus as a sterile organ , 2. HoweDespite constant exposure to hygienic challenges and infection during pregnancy, most of the 4 million annual deliveries in the United States have a successful outcome without presenting any clinical evidence of intrauterine infection . In thisAmong various models of immune tolerance, microRNAs (miRNAs) are implicated as mediators, as well as markers of immune tolerance in various tissues; however, the mechanisms associated with miRNA-mediated immune tolerance in gestational tissues are not well-understood. Because miRNAs are also included as cargo in EVs, do placental EVs and their embedded miRNA cargo play a role in immune tolerance at the maternal-fetal interface? This review focuses on immune tolerance at the maternal-fetal interface and reviews the role of miRNA in mediating immune tolerance via EVs in gestational tissues.+ T cells into Tregs, which are then recruited to the endometrium. Tregs and decidual natural killer (dNK) cells increase during early pregnancy and play a vital role in implantation and maintenance of pregnancy (in vitro fertilization (IVF) pregnancies involving fertilized eggs with washed semen do occur, Treg recruitment to the implantation site appears to be a physiologically helpful and critical event for implantation and pregnancy maintenance , active gestation (anti-inflammatory), and parturition (inflammatory) , 23. At per se  and alien (non-self) sources, also described as “danger” and “stranger” stimuli , that inDAMPs are a group of endogenous intracellular molecules released in the early stages of unplanned cell death to signal cell injury . The mosPRRs include Toll-like receptors (TLRs) 1 through TLR13, C-type lectins, scavenger receptors, and nucleotide-binding oligomerization-domain-like receptors, all of which operate transduction pathways resulting in cytokine-mediated inflammatory responses. PRRs are expressed in multiple human cells, including decidua, placenta, membranes, and myometrium, throughout pregnancy and in immune and non-immune cells , 40–42. The systemic immune system is regulated and dominated by T lymphocytes and adaptive immunity. On the other hand, the decidual leukocyte population in the pregnant uterus is replete with NK cells 65–70%) and antigen presenting cells (macrophages and dendritic cells 10–20%), both contributors to innate immunity , 5. Othe–70% and The molecular mechanisms associated with endotoxin tolerance are believed to be multifactorial, with multiple levels of negative feedback to blunt the inflammatory response. Mice deficient in IL-10 are more susceptible to endotoxic shock following repeated exposure to LPS, suggesting a role for IL-10 in endotoxin tolerance . In gestmiRNAs are small (18–22 nucleotides), non-coding RNA sequences that play an important role in regulating multiple cellular processes critical for development, differentiation, and organ function and are associated with marked biological consequences in health and disease . miRNAs Multiple organs harbor specific miRNAs that play vital roles. miR-122 is associated with cholesterol and lipid metabolism in the liver , whereasIn addition to regulating multiple cellular processes, various miRNAs are also implicated in regulating the immune system, including the differentiation and function of innate immune cells , 64. miRMultiple miRNAs are up-regulated via TLR-ligand stimulation in monocytes  and suggIncreases in miR-146a levels in response to LPS stimulation was observed in THP1 cells , 71, 72.miRNAs are also being studied as diagnostic and therapeutic tools . A recenThe human placenta produces numerous miRNAs. miRNAs play roles in various key steps in pregnancy, including implantation, maintenance, and labor –84. miRNinsulin-like growth factor receptor-1, whereas miR-675 deletion is associated with placental overgrowth  without consequence . Additioergrowth . Moreoveergrowth , 89, andergrowth . Furtherergrowth . AlthougThe majority of miRNAs function in the cell of origin by regulating mRNA levels and translation; however, some miRNAs are selectively secreted by cells into the extracellular space (mainly packaged within EVs) to regulate intercellular signaling of distant “target” cells. Analyses of miRNA from EVs show that EVs' miRNAs content is distinct from that found in the cytoplasm of donor cells from which they were derived. This suggests active miRNA sorting into these vesicles –95, althEVs are membrane vesicles of various sizes that are secreted by almost every cell type and multiple organisms ranging from bacteria to humans. EVs are broadly classified according to size and origin into two different categories: exosomes and microvesicles  , 96. MVs+ exosomes have only been described in the peripheral circulation of pregnant women (+) and placental exosomes (PLAP+) present in maternal plasma increases exponentially in the second and third trimesters  to unique RNA substrates, and exosome cofactors, such as the TRAMP-like protein complex, localize to the cytoplasm and recruit exosomes to specific viral RNA for degradation . Other eOur lab demonstrated that endotoxin tolerance exists in placental tissues  and we ptolerized cells). However, in the absence of these specific miRNAs, subsequent exposure to LPS would result in failure of immune tolerance to repeated infection resulting in exaggerated inflammation at the maternal-fetal interface . nterface . We specIn summary, we propose that miRNAs play a vital role in mediating immune tolerance at the maternal-fetal interface by attenuating immune responses following repeated exposure to inflammatory insult. Packaging and transport of miRNAs by EVs is suggested as a “smart” process specifically intended to address the potential requirements of the target cell/tissue. The success of this activity is dependent upon the homeostasis of the origin cell . Therefore, normal or pathologic conditions in the placenta might affect EVs composition and number, resulting in an altered response to endotoxins or other inflammatory stimuli. Additionally, we suggest that the target cells are not randomly selected and are, in fact, pre-identified according to specific markers presented on the EV surface to allow specific identification of target cell/tissue. Perturbations in this process can result in the failure of miRNA-mediated endotoxin tolerance and an imbalanced proinflammatory state leading to adverse pregnancy outcomes, including preterm labor or preeclampsia. The identification of placenta-specific miRNAs and EV markers will promote identification of novel molecules as potential biomarkers for further study of endotoxin tolerance, as well as possible molecular targets for controlling injury from failed immune tolerance.All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The brainstem conveys sensory and motor inputs between the spinal cord and the brain, and contains nuclei of the cranial nerves. It controls the sleep-wake cycle and vital functions via the ascending reticular activating system and the autonomic nuclei, respectively. Brainstem dysfunction may lead to sensory and motor deficits, cranial nerve palsies, impairment of consciousness, dysautonomia, and respiratory failure. The brainstem is prone to various primary and secondary insults, resulting in acute or chronic dysfunction. Of particular importance for characterizing brainstem dysfunction and identifying the underlying etiology are a detailed clinical examination, MRI, neurophysiologic tests such as brainstem auditory evoked potentials, and an analysis of the cerebrospinal fluid. Detection of brainstem dysfunction is challenging but of utmost importance in comatose and deeply sedated patients both to guide therapy and to support outcome prediction. In the present review, we summarize the neuroanatomy, clinical syndromes, and diagnostic techniques of critical illness-associated brainstem dysfunction for the critical care setting. The brainstem is the caudal portion of the brain that connects the diencephalon to the spinal cord and the cerebellum . The braIn the present review, we describe brainstem functional neuroanatomy, clinical syndromes, and assessment methods before addressing the concept of critical illness-associated brainstem dysfunction.The brainstem can be categorized into three major parts: midbrain, pons, and medulla oblongata Figs.  and 2. TBrainstem pathology should be considered in cases of (a) sensory or motor deficits combined with cranial nerve palsy, (b) impairment of consciousness, (c) dysautonomia, or (d) neurological respiratory failure.The pyramidal and extrapyramidal tracts connect the upper motor neurons and the extrapyramidal nuclei with the lower motor neurons located in either the brainstem or the spinal cord . While tThe testing of the cranial nerves and brainstem reflexes is described in Table When suspecting brainstem lesions, MRI will have the highest yield to further localize and characterize brainstem lesions  Table 44. EvokedThe ARAS controls the sleep-wake cycle and includes several nuclei mainly located in the pontine and midbrain tegmentum  Table 2 and 2: tDisorders of consciousness can be organized between acute and subacute or chronic . Acute iSubacute or chronic disorders of consciousness include the vegetative state  defined as state of unresponsiveness in which the patient shows spontaneous eye opening without any behavioral evidence of self or environmental awareness . The minN-methyl-d-aspartate (NMDA) receptors [In addition to these classical syndromes, other consciousness impairments have been described. Peduncular lesions can cause hallucinations  which maeceptors .Assessments of consciousness are based on neurological examination to confirm the diagnosis, determine the underlying cause, and evaluate the prognosis. In clinical practice, this assessment most commonly relies on the Glasgow Coma Scale (GCS) . FocusinPatients with severe critical illness may be comatose due to sedation, which in clinical practice can be assessed using the RASS  . In deepComa due to structural brainstem lesions is predominantly related to pedunculo-pontine tegmental lesions, usually detected on MRI  Table 44. NeuropThe intracranial conduction time and intrapontine conduction time are assessed by measures of the P14–N20 inter-peak latency on SSEP and the III–V inter-peak latency on BAEP . The proReactivity on EEG to auditory, visual, or nociceptive stimuli is important to assess after cardiac arrest because its absence is associated with poor outcome , 39. AbsThe ANS plays a key role in homeostasis and allostasis by controlling vital functions and the immune system  and is cBrainstem injury may cause dysautonomic symptoms, which can be life-threatening  Table 22. CardiaThere are two types of muscles that play a major role in the respiratory system, dilatator muscles of the superior airway that are innervated by the brainstem via cranial nerves  and contractor/pump muscles  that are innervated by spinal motor neurons. They are controlled by bulbospinal (automatic command) and corticospinal (voluntary command) pathways. The respiratory drive originates from neurons of the latero-rostro-ventral medulla oblongata, which includes the pre-Botzinger complex and the parafacial respiratory group that control inspiration and expiration, respectively  Table 22. This cAutomatic and voluntary control of respiratory motor neurons can be injured together or separately. For instance, automatic control is impaired in central congenital and acquired hypoventilation syndrome , while voluntary control is preserved . AcquireVentilator management may be significantly affected by brainstem lesions, and importantly, clinical features of neurological respiratory dysfunction are related to the localization of brainstem injury. The more caudal the lesion is, the more it is associated with an impairment of the respiratory drive. Midbrain injuries do not usually affect the respiratory rate (RR). Injuries to the upper pons increase the tidal volume and decrease the RR, while injuries of the lower pons are associated with respiratory asynchrony . Ataxic breathing (irregular pauses and apnea periods) and central apnea are observed in rostro-ventral medulla oblongata injuries and associated with poor outcomes. Central neurogenic hyperventilation results from activation of the medullary respiratory center. Finally, yawning or refractory hiccups may be seen with lesions of the posterolateral medulla oblongata . SwallowThere are various structural and non-structural causes of neurological respiratory dysfunction, including infratentorial lesions, drug toxicity, heart failure, and sepsis –61. DiagThe leading causes of primary brainstem dysfunction are summarized in Table The “brainstem dysfunction” hypothesis originates from our study on usefulness of neurological examination in non-brain-injured critically ill patients who required deep sedation. These patients have usually a severe critical illness and therefore a higher risk to develop severe secondary brain insult , 29. FurIn deeply sedated non-brain-injured critically ill patients, the cessation of brainstem responses follows two distinct patterns. The first is characterized by a depression of whole brainstem responses (similar to Guedel’s description), and the second is characterized by a preferential impairment of the corneal reflex, the pupillary light reflex, and to a lesser extent the cough reflex, with paradoxical preservation of the oculocephalic response. The latter profile is associated with the severity of critical illness and the depth of sedation. Interestingly, this pattern cannot be ascribed to a unique focal brainstem lesion which most likely relies on a functional rather than a structural origin. This suggests that some neuroanatomical centers are more sensitive to deep sedation, critical illness, or both. Opioids might also contribute to brainstem dysfunction, as they depress the ARAS, respiratory centers, and brainstem reflexes (notably pupillary light and cough reflexes). However, morphine infusion rates did not differ in our study between the two cessation patterns of brainstem reflexes .To assess brainstem reactivity in deeply sedated critically ill patients, we developed the BRASS  Table 55. The prNeurophysiological tests provide further arguments for brainstem dysfunction in critically ill patients without primary brainstem injury. For instance, EEG is not reactive in 25% of patients with sepsis , 43, knoCritical illness is also associated with decreased variability in HR and BP, with an impaired sympathetic tone and baroreflex , 50 and ClinicalTrials.gov: NCT02395861) aims to evaluate a multimodal approach based on neurological examination and neurophysiological tests.Thus, a multimodal assessment of brainstem dysfunction in critical illness is warranted. The undergoing multicenter PRORETRO study (Neuroimaging and neuropathological studies show that the brainstem is prone to vascular, inflammatory, and excitotoxic insults . For insarea postrema (Fig. Both humoral and neural pathways can induce a neuro-inflammatory process. The former involves the ema Fig. , which aFinally, metabolic processes can be involved. It is well known that electrolyte disturbances but also renal and liver failure impair brainstem responses, as illustrated by centro-pontine myelinolysis or by usefulness of FOUR score in hepatic encephalopathy .The predictive value of the neurological examination findings and neurophysiological responses has been assessed in critically ill patients. There is a proportional relationship between the BRASS value and mortality. Interestingly, absence of a grimacing response associated with preserved oculocephalic responses is the most predictive of mortality , suggestThere are arguments for a relationship between delirium and brainstem dysfunction. The drugs currently used for treating delirium are involving brainstem receptors. Thus, neuroleptics are antagonists of the dopamine D2 and serotoninergic 5HT2A receptors that are prevalent in the brainstem . DexmedeAnother contributing factor of the brainstem dysfunction in critical illness course might be the impaired sympatho-vagal control of the inflammatory response. The vagus nerve first senses and modulates peripheral inflammation, constituting the so-called cholinergic reflex ; second,Brainstem dysfunction can present with central sensory and motor deficits, cranial nerve palsies and abnormal brainstem reflexes, disorders of consciousness, respiratory failure, and dysautonomia. Clinical examination is essential for detecting a brainstem dysfunction that may be supported by neuroimaging, electrophysiological, autonomic, and respiratory assessments. Brainstem dysfunction mainly results from secondary insult and might contribute to critical illness-related mortality, organ dysfunction, immune dysregulation, delayed awakening, and delirium. The assessment of the brainstem should then be included in the routine neuromonitoring of critically ill patients.


Little is known about how patients and healthcare providers (HCPs) perceive the impact of asthma-related exacerbations. This study examined the impact of asthma-related exacerbations on patients’ lives from these different perspectives.Web-based surveys were administered to a US sample of adult patients with asthma, and HCPs. Participants reviewed six vignettes describing two hypothetical patients with asthma  experiencing mild, moderate, or severe exacerbations and rated the impact on eight measures: EuroQoL-5 Dimensions , sleep, household costs, and medical costs. The proportions reporting impact for each measure were calculated for each vignette; and patient responses were compared with HCP responses.302 patients with asthma and 300 HCPs completed the survey. As exacerbation severity increased, a higher proportion of patients and HCPs reported impact of exacerbations on patients with asthma. Compared with HCPs, a greater proportion of patients reported problems with pain/discomfort related to mild and moderate exacerbations. Compared with patients, HCPs were more likely to indicate sleep impact, mobility problems, and financial burden across all exacerbation severity levels; self-care problems with moderate and severe exacerbations; and problems with usual activities and anxiety/depression for severe exacerbations.Understanding the distinctions between how patients and HCPs perceive the impact of exacerbations is important for optimizing patient care. HCPs may be less aware of patient’s concerns about exacerbation-related pain/discomfort. Studies are needed to further understand patient-HCP interactions regarding asthma-related exacerbations.The online version of this article (10.1186/s40248-019-0196-1) contains supplementary material, which is available to authorized users. Asthma is a common chronic inflammatory disorder of the airways, affecting over 8% of the population of the United States (US) . The priPoorly controlled asthma is associated with reduced HRQoL and increased healthcare resource utilization and costs compared with well-controlled asthma –9. PreveThis study used a vignette-based research design to examine the perceived impact of asthma-related exacerbations on patients with asthma, from the perspective of both the patients themselves and from HCPs, and the relationship between these perceptions.This non-interventional study utilized internet-based cross-sectional surveys administered to two independent US cohorts: adult patients with asthma (patient cohort) and HCPs who treat patients with asthma (HCP cohort). Participant recruitment and pilot testing were conducted by Research Now , a global research service and online panel provider. The study was conducted using a multi-step approach. The first step was development of the initial vignette-based survey instrument, which was guided by online bulletin board focus groups consisting of 14 patients and 11 HCPs who were members of an opt-in online research panel . The instrument was then pre-tested in a sample of 6 patients and 7 HCPs, followed by pilot testing of the revised instrument in a cohort of 30 patients and 30 HCPs prior to implementation of the full survey.Study participants were enrolled via email invitations that contained a link to cohort-specific internet-based surveys, which were conducted between May 25, 2017 and June 2, 2017. Prior to completing the survey, all participants completed cohort-specific screening questions to confirm eligibility. The invitation email included an Institutional Review Board (IRB)-approved informed consent statement, with participants providing consent by completing the survey. Participants in the patient cohort were required to be ≥18 years of age with a self-reported diagnosis of asthma. Participants in the HCP cohort were required to be HCPs who were currently treating patients with asthma, including primary care practitioners, allergists, pulmonologists, and nurses. An additional cohort of adults moderately to very involved in the informal care of patients diagnosed with asthma (caregiver cohort) was included in the development and administration of the survey (results not shown). To ensure the cohorts were representative of asthma distribution in the US population, enrollment targets included ≥60% female participation in the patient cohort and ≥ 60% primary care or family practice participants in the HCP cohort. After the targeted number of 300 surveys per cohort (patient and HCP) was reached, the survey website was closed. Patients received $5.50 in the form of ‘e-rewards’ and HCPs were paid $32.50 for their participation in the study.Vignettes were developed using guidance from previously-published vignette-based analyses –15 and dThe terms ‘problems’, ‘impact’, and ‘burden’ are all used to describe the various issues that patients with asthma face; however, in this manuscript we have used the term ‘impact’ as a broad term to describe these issues as a whole, except when discussing a specific factor (e.g. economic burden).Patients self-reported their asthma history, comorbid conditions, exacerbation history and current asthma medication use, and completed several patient-reported outcomes (PROs) including the EQ-5D-5 L   plus theHCP demographics and practice characteristics were collected, including gender, specialty, years in practice, degree, practice setting, number of patients with asthma that were treated in the past year, severity of asthma across all patients treated in the past year, and number of patients treated for exacerbations in the prior month, by severity. HCPs were also asked if they agree with several belief statements regarding asthma exacerbations, including: all patients with asthma are at risk of developing an exacerbation; asthma exacerbations should be categorized by their severity ; asthma exacerbations have an impact on their patients’ lives; asthma exacerbations can have a varying impact on their patients’ lives, depending on their severity; and they believe their patients worry about having an asthma exacerbation.t-test, chi-square test) based on the distribution of the measure. A p ≤0.05 was considered significant; p were not adjusted for multiplicity.All variables were analyzed descriptively using SAS statistical software package version 9.4 . For each vignette, the proportions of participants reporting impact for the EQ-5D-5 L domains, sleep, and financial burden measures were calculated and compared between patients and HCPs using appropriate tests  or moderate exacerbations   increased with increasing severity of exacerbations. Significantly more patients than HCPs indicated problems with pain/discomfort for the vignettes of the 25-year-old patient experiencing a mild exacerbation  treatment guidelines to improve symptom control; however, differences in perspectives between patients and HCPs are common and may contribute to poorer outcomes in patients with asthma , 20. In 18 treatmPotential reasons for the differences observed between patient and HCP perceptions may include miscommunication, patients downplaying symptoms, or patients not reporting all exacerbations (particularly mild or moderate) to their HCPs. As such, patients may have a more holistic view of the burden attributed to exacerbations, whereas HCPs are likely only aware of the events for which patients have sought treatment. Both groups agree on the substantial impact of severe exacerbations; however, this study has highlighted that it is important for patients who are experiencing mild, and particularly moderate, exacerbations to see their physicians so that exacerbation episodes can be effectively managed before they become more severe and so the impact of their current exacerbation and risk of future exacerbations are minimized.One factor which may contribute to the gaps in communication between patients and HCPs is the lack of clearly defined levels of severity for asthma-related exacerbations in clinical practice. Exacerbations are currently defined based on healthcare utilization and not patient experience, and the lack of ability to link healthcare utilization to patient experience presents a specific challenge. This study used its own definition for asthma-related exacerbations, operationalizing a picture of what a mild, moderate, or severe exacerbation looked like using healthcare use as the marker of severity. Both patients and HCPs agreed that categorizing exacerbations by severity was helpful as they can have varying impacts  on the patients’ lives according to severity. This highlights a need for a clear definition of asthma-related exacerbations rooted in patient experience in clinical practice and which considers patient-reported experiences such as HRQoL and impact on work.Limitations of this study included those typically associated with survey-based studies. The study relied upon self-reported diagnoses of asthma, which were not confirmed by medical record review. In addition, participants were members of opt-in online panels who participated in survey research, so may not be fully representative of all patients with asthma or all HCPs who treat patients with asthma. The patient sample in this study appears to be more representative of patients with mild asthma and was more closely matched in terms of age to Patient B than Patient A; these factors may have impacted the vignette responses. Similarly, 24% of responding patients reported never having experienced an exacerbation; however, 91% of patients who completed the vignette exercise also indicated that they were familiar with the term and need to categorize exacerbations by severity (97%) and were therefore likely to understand the potential impact of these exacerbations. Furthermore, the population of HCPs who participated were largely family/primary care practitioners and therefore may not be representative of asthma care by respiratory specialists. Finally, this study did not adjust for multiplicity and multiple comparisons could increase the likelihood of statistically significant results; however, as this was a descriptive, hypothesis-generating study, this was not considered necessary for the intended analysis.Asthma-related exacerbations can have a significant impact on patients’ lives; however, the perceived impact of these exacerbations differs between patients and HCPs and this may contribute to suboptimal clinical outcomes. Across all exacerbation severity levels, HCPs perceived a higher degree of impact than patients; however, they may be less aware of their patients’ concerns regarding pain and discomfort when experiencing mild or moderate exacerbations. This study highlights a need for a clear definition for asthma-related exacerbations in clinical practice that considers patient-reported experiences. Further studies are needed to understand differences in patient and HCP perspectives and patient-HCP interactions regarding asthma-related exacerbations, which may be important considerations for optimizing patient care.Additional file 1:Vignette case descriptions. List of case descriptions. (DOCX 31 kb)Additional file 2:Exacerbation definitions. Table of exacerbation definitions. (DOCX 18 kb)


We report a case of an intraabdominal desmoid tumor that occurred at a gastro-pancreatic lesion with spontaneous cystic features, and present the successful laparoscopic resection of the tumor.A 20-mm retroperitoneal cystic mass with a solid component was found adjacent to the stomach and pancreatic body in a 52-year-old woman with no history of familial adenomatous polyposis. Laparoscopic spleen-preserving distal pancreatectomy with wedge resection of the stomach was performed, and complete resection was achieved without intraoperative and postoperative complications. Histopathological examination by immunohistochemistry enabled diagnosis of a desmoid tumor that had originated from the stomach and invaded the pancreatic parenchyma with a spontaneous cystic change. We herein report an extremely rare case of an intraabdominal desmoid tumor with a spontaneous cystic change.Regardless of its rarity, desmoid tumor should be included in the preoperative differential diagnosis of a cystic intraabdominal mass, and laparoscopic function-preserving surgery may be an optimal treatment option. Desmoid tumors  are locally aggressive tumors with no potential for metastasis or dedifferentiation. Radiologically, they are usually non-specific, slow-growing solid masses that are difficult to distinguish from other soft tissue tumors, but they rarely show cystic lesions. Sporadic desmoid tumors can occur at any site in the body, and anatomic locations are classified into 3 regions: trunk/extremity, abdominal wall, and intraabdominal. Complete surgical resection is the only curative option, but intraabdominal desmoid tumors are often unresectable, because they often infiltrate the mesentery. Here, we report a case of an intraabdominal desmoid tumor that occurred at a gastro-pancreatic lesion with cystic features, and presents the successful laparoscopic resection of the tumor.An intraabdominal cystic mass was incidentally detected between the stomach and the pancreas in a 52-year-old woman undergoing postoperative follow-up computed tomography (CT) for chondrosarcoma of a rib. Her family history was unremarkable. Blood counts and serum biochemistry results were within normal limits. An abdominal CT scan revealed a 20 × 18-mm well-defined cystic mass with a solid component  gene [Desmoid tumors are rare, and their estimated incidence in the general population is 2–4 per million per year . Individli) gene .Desmoid tumors potentially show cystic changes with medical treatment or abscess formation , but a sThe mesentery and retroperitoneum are common anatomic locations of intraabdominal desmoid tumors. In the current case, the tumor originated from the stomach, and locally invaded the retroperitoneum and pancreas body, based on the histopathology of the resected specimen. Desmoid tumors can theoretically arise from any part of the body, but those arising from the stomach are extremely rare ; thus, mIntraabdominal desmoid tumors rarely metastasize, but may show local invasion and recurrence . The recIn conclusion, we encountered an extremely rare cystic desmoid tumor at a gastro-pancreatic retroperitoneal site that was difficult to diagnose preoperatively. Laparoscopic SPDP with wedge resection of the stomach was performed safely; such procedures could offer an optimal method to achieve both curability and preservation of function.