Autism spectrum disorders (ASD) are complex neurodevelopmental disorders with unknown heterogeneous aetiologies. Epidemiological studies have found an association between maternal infection and development of ASD in the offspring, and clinical findings reveal a state of immune dysregulation in the pre- and postnatal period of affected subjects. Maternal immune activation (MIA) has been proposed to mediate this association by altering fetal neurodevelopment and leading to autism. Although animal models have supported a causal link between MIA and development of ASD, their validity needs to be explored. Moreover, considering that only a small proportion of affected offspring develop autism, and that MIA has been implicated in related diseases such as schizophrenia, a key unsolved question is how disease specificity and phenotypic outcome are determined. Here, we have integrated preclinical and clinical evidence, including the use of animal models for establishing causality, to explore the role of maternal infections in ASD. A proposed priming/multi-hit model may offer insights into the clinical heterogeneity of ASD, its convergence with related disorders, and therapeutic strategies. Autism spectrum disorders (ASD) are neurodevelopmental disorders characterised by impairments in social interaction and communication along with restricted/repetitive behaviours . DespiteSeveral lines of evidence implicate the prenatal innate and adaptive immune environment in ASD. Meta-analyses and epidemiological studies have reported an association between maternal infection and incidence of ASD in the offspring ,7,8, andHowever, clinical findings only show correlation and cannot establish a causal link between MIA or maternal antibodies and ASD. Supporting this link are animal models of MIA that serve to elucidate the mechanisms by which fetal development can be altered.The PubMed, Embase, and Web of Science databases were searched on 2 May 2020 using the search strategy:  AND immun*) OR (influenza OR flu) OR (polyinosinic:polycytidylic acid OR polyI:C OR poly I:C OR poly(I:C)) OR (lipopolysaccharide OR LPS) OR (interleukin)) AND (autis* OR ASD OR neurodevelopment*). Reference lists of relevant articles were reviewed to locate additional studies that may have been missed during the search. Search results were restricted to English-language studies only.The main paradigms used to model MIA consist of applying live influenza to pregnant rodents or mimicking infection with immunogenic molecules. Influenza models were pioneered by Fatemi et al.  to extenModels based on the bacterial mimic lipopolysaccharide (LPS) or the viral mimic polyinosinic–polycytidylic acid (Poly(I:C)) overcome this limitation and afford precise control of the duration of the immune response. Both induction protocols elicit overlapping findings with maternal influenza injection , suggestAlthough these studies support a causal link between MIA and autism, LPS and poly(I:C) injection differ from a live influenza infection, which is acquired through a respiratory route and induces both innate and adaptive arms of immunity. Moreover, maternal infection might have confounding effects on postpartum behaviour, which can influence offspring phenotype . Litter It is also unclear to what extent these behavioural findings are representative of ASD symptoms. Although ultrasonic vocalisations (USVs) have been used as a measure of communication deficits ,28, theiAnother limitation is that MIA models are restricted by specificity and are unlikely to apply solely to ASD. Most studies have evaluated behaviours that are more relevant to schizophrenia than to autism, such as prepulse inhibition (PPI) and latent inhibition (LI). Behavioural testing is normally conducted during adulthood, which relates more closely to the onset of schizophrenia. Further specificity might be achieved by characterising disease-differentiating features ; such dissociation might be unfeasible since both diseases share overlapping genetic, neuropathological, and symptomatic traits and could lie across a neurodevelopmental spectrum ,30.Future studies could incorporate assays investigating the “theory of mind” deficit in ASD. Mice exhibit greater pain sensitivity after observing cagemates undergo painful stimuli, indicating that some elements of empathy may be testable in mice. Changes in the reward value of social interactions could also be assessed with operant chambers requiring lever presses to access partners. Histological and electrophysiological recordings in the first days of postnatal life are scarce and may inform how neurobiology relates to disease progression. Investigations could also expand to peripheral systems disrupted in ASD patients, such as emerging alterations in gut microbiota ,32.Another promising avenue are primate models, due to their complex social structure and their neuroanatomical homology. Patterson and colleagues found that maternal poly(I:C) injection in rhesus macaques led to abnormal social approach, affiliative vocalisations, and repetitive behaviours, validating the MIA primate model  Table 2Table 2. Epidemiological studies have shown that MIA is a risk factor shared with related disorders including schizophrenia and epilepsy . AlthougTwo hypotheses have been put forward to explain the relationship between MIA and ASD ,35,36:  can bias progression towards a specific disease is compelling, as different immunogens administered at critical developmental windows could lead to pathogenic cascades manifesting as distinct phenotypes.Epidemiological evidence indicates that the nature of the immune challenge is less important than the maternal response, with ASD being associated with both bacterial and viral infections ,8. InterThe relative importance of the maternal response over the pathogen is also highlighted by animal models, which exhibit similar deficits regardless of the immunogen . NeverthThe intensity of the immunogen could also be relevant since different doses may trigger immune responses of varying severity, with disease only occurring when a specific MIA threshold is surpassed. Indeed, rodent models using higher doses of immunogen typically induce more striking behavioural deficits and cytokine alterations in the fetal brain ; Table 3Timing of prenatal exposure is crucial as immune activation at different critical windows could impact disease-specific neurodevelopmental processes. Although several epidemiological studies support an association between prenatal stressors during mid- and late gestational periods and ASD, others have found associations with early gestation or no association ,3,7,8. T26 (The poly(I:C) model is useful to study timing since it induces a controlled immune reaction lasting 24–48 h ,44. Poly26 . By contIt would be informative to interrogate to what extent the neural systems affected at specific stages account for symptom clusters. For example, the hippocampus and corpus callosum develop around E16 in mice. However, although the Black and Tan Brachyury (BTBR) mouse exhibiting agenesis of the corpus callosum is a widely used model of autism , the hipAnother possibility is that MIA only reveals its neuropathological impact in subjects with genetic predisposition. Several cohorts have highlighted that ASD may have a significant genetic component, with heritability estimates ranging from 50% using a Cox model  to 83–97Transcriptomic analyses have found that ASD is correlated with gene expression modules corresponding to immune genes and M2-activated microglia , suggestMultiple multi-trait models have investigated gene-MIA interactions. Ehninger et al. found that MIA resulted in disrupted social behaviour in mice offspring only in the presence of ASD-associated tuberous sclerosis complex 2 (TSC2) mutations . SimilarHowever, these genetic models are confounded by the effects of sex and background strain, which can affect the expression of autism-like phenotypes ,72. AlthTechnologies such as the CRISPR/Cas  system allow targeting multiple genes and could be used to investigate the additive effects of gene variants and their interaction with MIA. However, using transgenic models of common variants of small effect could compromise construct validity. Two categories of genes that could be further examined in combination with MIA are susceptibility factors that do not recapitulate all core symptoms of autism when manipulated alone , methyl CpG binding protein 2 (MECP2)) and immune-related genes associated with ASD (human leukocyte antigen (HLA), phosphatase and tensin homolog (PTEN)).On the other hand, the possibility that MIA may require exposure to an additional environmental insult to trigger full-blown pathology remains underexplored. Animal models combining MIA with postnatal stress have found synergistic effects on PPI deficits and microglia activation, supporting this “two-hit” model . NonetheIn terms of genetic versus environmental contribution, one can propose a framework in which either extreme genetic predisposition or environmental insults can cause ASD, with a proportion of ASD cases resulting from gene-environment interactions in the middle of this spectrum .The hypotheses of disease specificity arising from MIA properties or gene-environment interactions are not mutually exclusive. It is possible to integrate these factors into a single priming/multi-hit model, in which MIA serves as a developmental primer that increases vulnerability towards disease-specific pathways depending on its nature, intensity, and timing. Another layer of phenotypic specificity is provided in the form of genetic, epigenetic, and environmental factors that are required for the progression from vulnerability to full-blown disease . Two metHow does MIA result in autism-like behaviours in the offspring? One hypothesis is that an MIA-induced elevation in proinflammatory cytokines alters fetal neurodevelopment . IL-6 haCytokines could exert their effects at three levels: the maternal circulation, the maternal-fetal interface, and the fetal brain . InjectiA more direct mechanism could involve IL-6 crossing the placenta to enter the fetal circulation. However, the ability of cytokines to cross the placenta is disputed. While some studies have shown that radiolabelled IL-6 crosses the rat placenta and that IL-6 is transported across the human placenta ex vivo , others Elevated cytokine levels are found in the fetal brain , possiblAnother unsolved question is how an acute immune challenge results in permanent behavioural deficits. MIA could lead to chronic neuronal injury underpinning later pathophysiology , or MIA-N-methyl-d-aspartate receptor) NMDAR can influence the behaviour of mice in maternal-to-fetal transfer models [Finally, the evidence that maternal antibodies to membrane proteins such as contactin-associated protein-like 2 (CASPR2) and (r models , suggestThe role of MIA in the development of ASD and related disorders is highlighted by animal models linking prenatal infection and ASD-relevant deficits and clinical studies providing correlative evidence. However, key questions about the validity, mechanism, and the source of disease specificity in this hypothesis remain unsolved. A priming/multi-hit model, in which MIA acts as a primer requiring a substantial genetic contribution and further environmental factors to trigger the clinical manifestation of ASD, could be used in future studies to develop increasingly valid multi-trait animal models. Applying this framework to answer the MIA disease specificity question may offer insight into the clinical heterogeneity of ASD and its convergence with related diseases such as schizophrenia, with the ultimate goal of guiding diagnostic classification and therapeutic strategies in neurodevelopmental psychiatric disorders.


Approximately 50% of colorectal cancer patients will develop metastases during the course of the disease. Local or locoregional therapies for the treatment of liver metastases are used in the management of oligometastatic colorectal liver disease, especially in nonsurgical candidates. Thermal ablation (TA) is recommended in the treatment of limited liver metastases as free-standing therapy or in combination with surgery as long as all visible disease can be eradicated. Percutaneous TA has been proven as a safe and efficacious therapy offering sustained local tumor control and improved patient survival. Continuous technological advances in diagnostic imaging and guidance tools, the evolution of devices allowing for optimization of ablation parameters, as well as the ability to perform margin assessment have improved the efficacy of ablation. This allows resectable small volume diseases to be cured with percutaneous ablation. The ongoing detailed information and increasing understanding of tumor biology, genetics, and tissue biomarkers that impact oncologic outcomes as well as their implications on the results of ablation have further allowed for treatment customization and improved oncologic outcomes even in those with more aggressive tumor biology. The purpose of this review is to present the most common indications for image-guided percutaneous ablation in colorectal cancer liver metastases, to describe technical considerations, and to discuss relevant peer-reviewed evidence on this topic. The growing role of imaging and image-guidance as well as controversies regarding several devices are addressed. Colorectal cancer (CRC) is the second-most common neoplastic disease in Europe and a leading death cause worldwide, with a new case diagnosed every 3.5 min and a patient dying every 9 min ,2. ApproCurrently, there are several thermal ablation (TA) technologies available, including radiofrequency (RFA), microwave (MWA), laser, or cryoablation (CWA) as well as nonthermal ablation technologies such as irreversible electroporation (IRE), otherwise known as Nanoknife. Thermal TA techniques destroy liver tumors by using different types of thermal energy, by heating , or by freezing (CWA) the target tumor; ablation with clear margins (A0) is a prerequisite for the technique in order to provide local cure and to serve as a surgical alternative. This requires proper lesion and patient selection along with high-standards application of the technique, including real-time imaging confirmation of margins. Several studies support the use of TA in the management of colorectal liver metastatic disease ,10,11,12The purpose of this review is to become familiar with the most common image-guided percutaneous ablation indications for colorectal cancer liver metastatic disease, to learn about different technical considerations, and to review the current evidence. The growing role of imaging and image guidance along with controversies concerning ablation devices are discussed.All heat-based ablation modalities affect cell-death via the common end-points of protein denaturation and coagulative necrosis ,18,19,20Radiofrequency ablation induces thermal damage by utilizing a high-frequency alternating current (375–500 kHz), which causes ionic oscillation, frictional heating, and coagulation necrosis as the end result . TemperaMicrowave ablation works similarly to RFA utilizing an electrical current produced by a 915 MHz or 2.45 GHz generator (at continuous or pulsed delivery modes) in order to cause protein denaturation and coagulative necrosis ,18. A waLaser ablation or laser interstitial thermotherapy (LITT) utilizes a micrometer optical fiber with a bare tip that transmits infrared light (700–1200 nm) for heat production . The fibCryoablation utilizes extreme cold at temperatures of −40 °C in order to cause cellular death. One or more cryoprobes are inserted to the target lesion, aiming to create enough ice to cover the lesion and the necessary margins. Inside the cryoprobe, there is a hollow tube containing a smaller inner and a larger outer tubular part; argon gas travels from the inner to the outer tube at high pressure through a very narrow aperture, where it rapidly expands within the distal tip . Due to IRE is a non-heat-based ablation modality that utilizes electrical energy in terms of high voltage pulses that cause irreversible cellular membrane disruption and cellular death as the end result . AlthougPre-ablation imaging serves for patient staging and eligibility for TA. Most commonly, imaging evaluation of patients with colorectal liver metastatic disease includes a baseline computed tomography exam of the chest, abdomen, and pelvis without and with contrast  ,9. ApartAlmost all diagnostic methods were used either solely or in combination (fusion imaging) for ablation guidance . SolbiatTsitskari et al. reported 96.8%, 68.7%, and 34.3% for 1-, 3-, and 5-year overall survival rates, respectively, for colorectal cancer patients with oligometastatic liver-only disease who underwent percutaneous computed tomography-guided microwave ablation . ImmediaRuers et al. provided level I evidence that, in patients with initially unresectable liver metastatic disease, the addition of local treatment with RFA (with or without resection) to systemic FOLFOX  therapy significantly prolongs survival when compared to those treated with FOLFOX alone ,34. SizeSimilar to the results reported by studies evaluating data from post-surgical resection of CLMs, as far as percutaneous ablation is concerned, the number of metastases  seems to be an independent predictor factor of tumor recurrence; Siperstein et al. evaluated 234 patients reporting a significant overall median survival difference (27 versus 17 months) associated with the presence of fewer or more than three metastases at initial presentation . The sizMolecular and proliferation markers  may contribute to the prediction of outcomes post-ablation, offering a valuable stratification tool for tumor-tailored therapeutic approach . The ideThe safety margin constitutes a significant technical factor governing local tumor control and liver progression-free survival rate; a minimum of 5 mm safety margin is a prerequisite for successful ablation, whilst margins ≥10 mm have been correlated with sustained long-term local tumor progression free survival rates >95%, as reported in the studies by Calandri et al. and Shady et al. ,43,48. SAn assessment of the safety margin is of critical importance; Wang et al. evaluated 73 patients with 94 previously untreated CLM that underwent RFA, concluding that a minimal margin of the ablation zone uniformly larger than 5 mm at 4–8 weeks post-ablation CT is associated with the best local tumor control . Since 2There is currently no clear evidence supporting the superiority of one ablation technique over the other. Microwaves are less affected by the “heat-sink” effect of any kind of impedance-driven performance and achieve temperatures over 100 °C, producing a relatively larger ablation volumes in a shorter period time without the need for grounding pads placement ,55. RadiThroughout the literature, there are limited data supporting IRE application for colorectal liver metastatic disease, focusing mainly on tumors in locations considered high risk for collateral damage if treated by TA or surgery . A recenContraindications to TA techniques for colorectal cancer liver metastatic disease include lesions located at <1 cm distance from the main biliary duct, significant ascites along the applicator path, and untreatable or unmanageable coagulopathy . Common side effects post-ablation of CLMs include pain and post-ablation syndrome . Pain isComplications can be puncture- or ablation-related. Puncture-related complications include intraperitoneal bleeding, pneumothorax, and hemothorax, whilst ablation-related adverse events include bowel perforation; portal vein thrombosis; liver abscess; bile duct injury including, strictures, bilomas, and bile leaks; as well as cholecystitis . BiliaryCombining percutaneous TA with SOC chemotherapy for CLM is supported by the results of the EORTC-CLOCC trial that achieved long-term disease control and significant patient survival benefit (with very low morbidity) that proved the value of ablation as an adjuvant to chemotherapy over chemotherapy alone . The NCCBoth surgery and ablation are recommended and preferred to radiotherapy ,15. ThisThermal ablation is a tumor therapy with local curative intent, offering significant and sustained local tumor control. The addition of thermal ablation to chemotherapy provides survival benefits at lower morbidity, mortality, and cost. The ablation margin is the most important, single technical factor in optimizing local tumor control. Tumor size and number, location, biology, and genetics are additional factors that can affect efficacy and safety. Percutaneous ablation can be compared only to surgical approaches in terms of survival rates offering lower complication rates. Ablation standardization in terms of predictability and reliability with the use of immediate treatment assessments is the quest of our era.


Orbital invasive aspergillosis infection is rare life-threatening infection, most commonly seen in immunocompromised patients and extremely rare in individuals without risk factors. Here we present a rare case of bilateral cellulitis caused by invasive aspergillosis associated with bilateral intraorbital abscesses in a female patient.A 49-year-old woman presented with a 3-month history of painful proptosis and periorbital swelling of bilateral eyes. She was initially diagnosed as bilateral orbital cellulitis complicated with cavernous sinus thrombosis and was treated with antibiotic medication for 1 month, but her symptoms persisted. MRI demonstrated orbital masses behind both globes. The lesion in right orbit was biopsied with a diagnosis of orbital granulomatosis with invasive aspergillosis infection. The patient was healed after receiving antifungal treatment.This is an unusual case about bilateral orbital abscesses with invasive fungal infection. Fungal infection of the orbit should be considered when patient does not respond to combination of anti-inflammatory and antibiotic therapies, even in some cases without any risk factors. Orbital fungal infections are rare and often life-threatening, with mortality ranging from 21 to 80% [To date, there are only a few published reports about individuals having bilateral orbital involvement due to extension of fungal sinusitis , 4. To oA 49-year-old woman presented with a 3-month history of painful proptosis and periorbital swelling of bilateral eyes, accompanying with reduced vision and diplopia. The patient declared no medical history, such as hypertension, diabetes and systemic immunosuppression. She also denied recent trauma. She had been treated with antiviral medication and corticosteroids for 5 days in another hospital as the initial diagnosis was herpes virus infection, but her symptoms deteriorated. The vision at first visit was 20/25 OD and 20/30 OS. The axial and sagittal T1 contrast-enhanced magnetic resonance imaging (MRI) showed bilateral orbital lesions spreading to the cavernous sinus -associated vasculitides . HoweverInvasive fungal infections of the orbit are rare but often fatal, and patients with mucormycosis had a higher mortality than patients with aspergillus . InvasivOrbital imaging of fungal infection commonly demonstrated an ill-defined mass or infiltration of orbit . MRI proThe lack of standard guideline of orbital fungal infection makes it difficult to be handled in clinic, antifungal therapy alone cannot be enough to control some severe cases, so surgery associated with antifungal therapy should be considered in this situation. Prompt treatment is also essential as fungal infection is invasive, which could cause irreversible damage. Management often begins with surgical debridement followed by systemic antifungal drug therapy , 7.In conclusion, fungal infection of the orbit should be considered when patient does not respond well to the combination of anti-inflammatory and antibiotic therapies. Orbital MRI is essential for diagnosis, if the images demonstrate unusual findings, then biopsy is necessary.


The bone marrow (BM) is the primary site of postnatal hematopoiesis and hematopoietic stem cell (HSC) maintenance. The BM HSC niche is an essential microenvironment which evolves and responds to the physiological demands of HSCs. It is responsible for orchestrating the fate of HSCs and tightly regulates the processes that occur in the BM, including self-renewal, quiescence, engraftment, and lineage differentiation. However, the BM HSC niche is disturbed following hematological stress such as hematological malignancies, ionizing radiation, and chemotherapy, causing the cellular composition to alter and remodeling to occur. Consequently, hematopoietic recovery has been the focus of many recent studies and elucidating these mechanisms has great biological and clinical relevance, namely to exploit these mechanisms as a therapeutic treatment for hematopoietic malignancies and improve regeneration following BM injury. The sympathetic nervous system innervates the BM niche and regulates the migration of HSCs in and out of the BM under steady state. However, recent studies have investigated how sympathetic innervation and signaling are dysregulated under stress and the subsequent effect they have on hematopoiesis. Here, we provide an overview of distinct BM niches and how they contribute to HSC regulatory processes with a particular focus on neuronal regulation of HSCs under steady state and stress hematopoiesis. BM sinusoids form a complex network and are found in the central marrow, operating as the site where hematopoietic cells migrate in and out of the BM4, although a recent study suggested that transcortical vessels in bone represent an additional important migration route5.The bone marrow (BM) is highly vascularized to provide nutrients and oxygen. The nutrient artery and vein infiltrate the compact bone and subsequently branch to form small arterioles. These arterioles connect via transition zone vessels  to the venous sinusoids near the endosteum, which is the interface between the bone surface and the BMNestin-Gfp transgenic mice into two subsets according to their GFP expression: Nes-GFPbright and Nes-GFPdim. Nes-GFPdim cells are located around the sinusoids, and Nes-GFPbright cells are located around the arterioles2 and the transition zone vessels6. Stromal cells can be further divided to neuron-glial antigen (NG2)-expressing cells2, Cxcl12-abundant reticular (CAR) cells7, and cells expressing leptin receptor (LepR)1, all of which overlap with Nes-GFP+ cells to varying degrees9.Each niche likely operates different functions and exhibits its own cellular composition. Key cellular niche components are the stromal cells necessary for producing niche factors that directly act on hematopoietic stem cells (HSCs). Perivascular cells identified by the expression of the intermediate filament protein NESTIN contain BM mesenchymal stem cells (MSCs) and can be divided in+ cells ensheath the arterioles which have been proposed as an important niche for regulating the quiescence of HSCs via the secretion of Cxcl12, whereas others have attributed the same function to LEPR+ cells in the sinusoids12. Most likely, the discrepancies are due to different interpretations of the specificity and recombination efficiency of the Cre lines used, given the large overlap among these cell populations9. On the other hand, CAR cells are defined by Cxcl12 expression, essentially coincide with LEPR+ cells and Nes-GFPdim cells, and are located throughout the BM7.NG213. Sympathetic nerve fibers enter the BM through the nutrient foramen and are closely associated with the blood vessels, before sprouting and innervating different BM regions14, although some nerves may reach the BM associated with transcortical vessels in bone. The sympathetic nervous system (SNS) has been demonstrated to regulate various hematopoietic cell functions directly or indirectly mainly via the stromal cells, mediated by neurotransmitters binding to beta adrenergic receptors (β-ADRs)13. β-ADRs are coupled with Gs trimeric proteins and activate adenylate cyclase, catalyzing the formation of cyclic adenosine monophosphate, which subsequently activates protein kinase A phosphorylation of the receptor15. Contrastingly, the presence of the parasympathetic nervous system (PNS), another branch of the SNS, within the BM is vastly unexplored. The PNS uses acetylcholine (ACh) as the main neurotransmitter, which binds to muscarinic or nicotinic receptors. One study suggested that the PNS may innervate the distal femoral metaphysis16 and another similarly supported cholinergic innervation within the BM of rats17. However, additional neuroanatomical evidence of parasympathetic BM innervation is essentially lacking18. Moreover, the bone anabolic effect of the PNS16 was suggested by another group to be indirectly mediated through the inhibition of central sympathetic tone19. Therefore, clarification on whether the PNS innervates the BM is required. Overall, little is known about how parasympathetic or, more broadly, cholinergic signaling might influence either HSCs or their BM niches.The BM is highly innervated by various types of nerves, of which the autonomic branch is predominant22. In addition, HSCs themselves are functionally and molecularly heterogeneous25, raising the possibility that distinct subpopulations of HSCs are regulated by specialized niches.The dissection of BM niches is still a developing area because of the dynamic features of the niches to meet the physiological demands and their alterations in different scenarios such as aging, malignancies, or response to stress. Single-cell studies have provided insights into the heterogeneity of the stromal cells, forming an increasingly complex picturein vivo homing and reconstitution potential than HSCs located in the central marrow28.It is possible that distinct vascular niches can orchestrate the balance between quiescence and proliferation of HSCs, which is necessary for homeostasis but also regeneration of the BM following injury. Consequently, studies have investigated how the regulation of HSCs differs depending on whether they are located within the endosteal region or the central marrow. In particular, these differences become more apparent under stress conditions. Following irradiation, HSCs tend to home to the endosteal region and HSCs isolated from this region exhibit greater31. The stromal cell populations within the endosteal region better resist myeloablation, and N-cadherin+ MSCs31 and CD73+ MSCs32 have been identified as resistant cell populations that contribute to hematopoietic stem and progenitor cell (HSPC) engraftment and subsequent hematopoietic recovery. Reserve HSCs and primed HSCs have been distinguished by their proliferation and sensitivity to chemotherapy (5-fluorouracil). Notably, whereas primed HSCs tend to be located within the central BM niche, reserve HSCs are preferentially maintained in the endosteal region31. Reserve HSCs are able to resist chemotherapy in part due to N-cadherin+ MSCs, which expand and produce cytokines to aid recovery after myeloablation31. Overall, these studies confirm that the endosteal BM region is important for mediating hematopoietic regeneration after stress.Furthermore, it has been demonstrated that the endosteal region is important to preserve HSC quiescence under proliferative stress and to support regeneration of the HSC pool following injury33. More recently, Golanet al. demonstrated in mice that a morning peak of norepinephrine and TNF induces vascular permeability, temporarily increases reactive oxygen species (ROS) levels and facilitates HSPC proliferation, differentiation and migration. Whereas, at night, a second TNF peak increases melatonin secretion and reduces vascular permeability and HSPC ROS levels, facilitating HSPC maintenance34.Cumulative evidence indicates that the SNS regulates the proliferation and differentiation of HSPCs, and the migration of HSPCs and leukocytes between the BM and extramedullary sites. This was initially suggested because catecholamine levels in the blood and the BM adhered to circadian rhythms that also affected the proliferation of BM cells that expressed catecholamine receptors35. Leukocytes are also recruited to many vital organs, including skeletal muscle, following circadian oscillations of neural activity36. Noradrenaline binding to β3-ADR on stromal cells causes a decrease in the nuclear content of Sp1 transcription factor and finally downregulation of Cxcl1235. The interaction of Cxcl12 expressed by stromal cells with its receptor Cxcr4, located on HSCs and leukocytes, is pivotal for HSC/leukocyte retention in the BM37. It has also been demonstrated that the bone itself is an important transducer of signals emanating from the nervous system leading to HSC mobilization39.A neurally-driven circadian release of HSCs and leukocytes into circulation occurs during the resting period, following photic cues40. At night, the PNS acts centrally to dampen the noradrenergic sympathetic branch and decrease BM egress of HSPCs and leukocytes mediated through β3-ADR40. In contrast, epinephrine released at night in circulation can stimulate β2-ADR and increase vascular adhesion and subsequent BM homing at night40. In the morning, a novel cholinergic sympathetic branch regulates vascular adhesion and β3-ADR expression40. These results illustrate how a master rheostat SNS regulates the daily migration of HSCs and leukocytes.We recently demonstrated how parasympathetic cholinergic signals coordinate with sympathetic signals to regulate the egress and homing of HSPCs and leukocytes in mice41. Non-myelinating Schwann cells wrap around the sympathetic nerves travelling along the vasculature within the BM. Non-myelination Schwann cells have been reported to maintain HSC quiescence via secretion of tumor growth factor beta (TGF-β) activator molecules and induction of TGF-β/SMAD signaling in HSCs41. This signaling contributes to HSC quiescence through increased phosphorylation of Smad2 and Smad341, hence supporting the maintenance and self-renewal of HSCs42.Glial cells supporting BM nerve fibers have also been suggested to regulate HSC proliferation43. Pierceet al. uncovered another pathway connecting the brain with the BM to regulate mobilization of HSCs enforced by granulocyte colony-stimulating factor (G-CSF)44. The authors demonstrated how the muscarinic receptor type 1 (Chrm1) signaling in the hypothalamus promoted G-CSF-induced HSC mobilization via the hypothalamic-pituitary-adrenal (HPA) axis44. Thus, priming HSC migration through glucocorticoid (GC) hormone levels, which exhibit circadian oscillations, and binding to the receptor Nr3c1 regulates G-CSF-induced HSC mobilization44. Whereas these studies have uncovered the influence of muscarinic signaling on HSCs, further investigation of other cholinergic signaling pathways (particularly involving nicotinic receptors) possibly influencing HSCs is warranted.So far, the studies addressing cholinergic regulation of hematopoiesis have focused mostly on muscarinic receptor signaling. One study demonstrated that cholinergic receptor muscarinic 4 (CHRM4) regulated self-renewal of early erythroid progenitors, and muscarinic receptor antagonists have been proposed as a therapy for treating anemiaHematological stress can be caused as a result of a diverse range of factors from psychological stress to hematological malignancies. However, one common consequence is the dysregulation of the SNS, predominantly affecting myelopoiesis. The following sections discuss current studies of neuronal regulation of HSCs under various stress conditions.45. Under stress conditions (e.g. jetlag or transplantation), alterations in these rhythms can have physiological consequences by disrupting hematopoietic cell recruitment and recovery. Therefore, time-based therapeutics for transplantations and inflammatory diseases may prove beneficial36. In addition, leukocyte adhesion to arteries and veins is disproportionately disrupted following an inflammatory response, with arteries driving rhythmic inflammatory responses within the vasculature46. Altogether, these studies suggest an important influence of circadian rhythms in immune response.Leukocytes exit the blood following circadian rhythms and undergo extensive interactions with endothelial cells as they migrate between the BM and extramedullary sites. The expression of adhesion molecules, chemokines and their receptors follow daily rhythms that regulate the migration of leukocyte subsets within distinct vascular beds. Ablation of the transcription factor BMAL1, which is an essential for clock gene, demonstrated that rhythmic leukocyte recruitment is dependent on both cell-autonomous and microenvironmental oscillations50. Recently, they have demonstrated that ChAT is induced in both CD4+ and CD8+ T cells during infection in an interleukin-21 (IL-21)-dependent manner and is key for overcoming infections47. Moreover, they have validated that ChAT is expressed and ACh is produced by B cells following stimulation with sulphated cholecystokinin, resulting in controlled recruitment of neutrophils48. In the proposed circuit, the vagus nerve acts via the splenic nerve, which releases ACh from T cells. ACh binds to the nicotinic ACh receptor α7 subunit on macrophages, causing the inhibition of tumor necrosis factor release, thus regulating inflammation49. The vagus nerve-to-spleen circuit can also be controlled at a central level and can be exploited to suppress pro-inflammatory cytokine release52. Overall, these studies suggest the importance of the cholinergic vagus nerve-to-spleen anti-inflammatory pathways. However, it is important to mention that other groups have suggested alternative ways to explain the cholinergic anti-inflammatory reflex. Particularly, the efferent arm of the inflammatory reflex seems to involve a different splanchnic anti-inflammatory pathway53.Mak and Tracey’s laboratories have pioneered research into how neural signals regulate immunity by showing that norepinephrine-induced T cell-derived ACh regulates immune response2-ADR on osteoblasts regulate their proliferation and control bone formation downstream of leptin54. The SNS favors bone resorption by increasing the expression of Rankl on osteoblast progenitor cells, which regulates osteoclast differentiation. Moreover, leptin regulates the expression of the neuropeptide cocaine amphetamine regulated transcript (CART), which inhibits bone resorption by controlling Rankl expression55.Apart from the direct regulation of hematopoietic cells and their niches, it is likely that circadian oscillation of neural activity indirectly regulates hematopoiesis and immunity through their effects on bone remodeling. Skeletal remodeling comprises two phases: resorption by osteoclasts and formation by osteoblasts, allowing vertebrates to regulate bone mass daily. Osteoblasts are multifunctional cells able to control osteoclast differentiation. Notably, the energy expenditure hormone leptin inhibits bone formation through a neuronal relay. Sympathetic signaling via β19. The skeleton can in turn modulate neural activity through secretion of the hormone osteocalcin, which regulates parasympathetic tone56. Therefore, it is likely there is an interplay between sympathetic and parasympathetic nervous systems to regulate bone remodeling and stress responses. These pathways, which could have potential therapeutic implications for several complex disorders including osteoporosis, chronic fatigue and fracture repair57, may also profoundly impact hematopoiesis.In addition, osteoclast function is inhibited by cholinergic parasympathetic signals that inhibit sympathetic tone centrally61. Therefore, the SNS regulates inflammation in cardiovascular disease by controlling HSPC proliferation and differentiation in response to stress. In addition, it was recently demonstrated that patients who exhibit recurrent myocardial infarction have a dampened emergency hematopoiesis response, due to long-term reprogramming of myeloid progenitors from the first myocardial infarction, resulting in fewer leukocytes being recruited to the site of injury62. Consequently, this is a potentially important factor to consider when selecting therapies for recurrent myocardial infarction62.Ischemic myocardium causes the heart to initiate the influx of circulating myeloid cells to the site of damage. In turn, this results in the SNS signaling to the BM to increase the production of leukocytes to meet the demand, aided by circulating mediators such as granulocyte-macrophage colony-stimulating factor (GM-CSF) and IL-1β produced by the heart64. These effects were reversed with the treatment of propranolol, which is a non-selective beta blocker64. Following on from those studies and previous studies on adrenergic HSC regulation35, chronic psychosocial stress was demonstrated to act on the most primitive progenitors, causing an increase in the proliferation of HSPCs in the BM of mice65. This finding translated to the human setting, where it was observed that chronic stress induced monocytosis and neutrophilia in humans65. This was due to the activation of the SNS, causing an increase in catecholamine levels, which activate the β3-ADR on BM niche cells, resulting in a decrease in CXCL12 levels35. The HSC mobilization to peripheral circulation and the spleen, and the subsequent myeloid expansion in the spleen can aggravate chronic inflammatory diseases such as atherosclerosis66.Both social and chronic psychological stress have been demonstrated to lead to SNS-induced upregulation of myelopoiesis in mice and subsequently to increases in the production of pro-inflammatory cytokines67. Chronic propranolol treatment restored the expansion of these cells but also influenced the myelo-erythroid bifurcation by reducing the granulocyte-monocyte progenitors and increasing megakaryocyte-erythroid progenitor cells in the BM of burn-stressed mice67. These observations translated to the human setting, whereex vivo culture of burn patient peripheral blood mononuclear cells also demonstrated that their commitment stage of erythropoiesis was impaired and could be restored with propranolol67. Consequently, beta-adrenergic blockers exhibit therapeutic value for burn patients by redirecting the hematopoietic commitment toward erythroid lineage via decreased MafB expression in multipotent progenitors, leading to increased erythropoietin responsiveness67. How these forms of stress affect hematopoiesis through the nervous system is summarized inIn the context of burn patients, who receive multiple blood transfusions for the treatment of anemia, increased catecholamine levels induced expansion of HSPCs and increased their myeloid regulatory transcription factor (MafB) expression, causing a myeloid shift at the expense of megakaryocyte-erythrocyte progenitors69. These changes are caused by both cell-intrinsic dysregulation and remodeling of the BM microenvironment71. One of the hallmarks of hematopoietic aging is that the myeloid output of HSCs increases at the expense of lymphopoiesis69. One study carried out transplantations of old HSCs into young recipients and vice versa. The young microenvironment was able to reduce myelopoiesis, confirming the contribution of the microenvironment72. We recently noted that the SNS is actively involved in aging of the hematopoietic system. Our recent study demonstrated that sympathetic noradrenergic fibers marked by tyrosine hydroxylase (Th) increased in the mouse BM with aging6. Increased β2-adrenergic signaling in expanded central BM niches promoted myeloid cell expansion6. A functional switch of neurotransmission, favoring β2-ADR over β3-ADR signaling during aging, appears to favor myeloid cell expansion through the regulation of the BM microenvironment6. An active role for the nervous system in aging is supported by the increased basal sympathetic tone during human aging75 and by a recent study indicating that increased excitatory neurotransmission reduces the life span76. In contrast, another study suggested that BM Th+ fibers were reduced (not increased) during aging and that surgical denervation of young BM increased myelopoiesis77. However, BM noradrenergic nerve fibers appear to decrease from youth to adulthood  but these fibers appear increased (not decreased) in old (20-month-old) mice6. Moreover, the possible contribution of the inflammation caused by experimental surgical denervation to the hematopoietic aging phenotypes77 should be considered.The hematopoietic system is disrupted upon aging, resulting in the increase of HSCs which are functionally impaired79. In humans, cardiovascular diabetic autonomic neuropathy correlates with decreased circulating HSPCs with increased 66-kDa protein from the src homology and collagen homology domain (p66Shc) and reduced expression of sirtuin 1 (Sirt1)80.The disautonomia associated with diabetes has been shown to affect the BM, disrupt the peripheral clock, and compromise G-CSF-induced HSC mobilization in experimental models because of the HSC niche deregulation81 and catecholamines, namely norepinephrine can improve hematopoietic reconstitution following chemotherapy in mice82. Furthermore, G-CSF and GM-CSF are commonly used to accelerate myelopoiesis and minimize the burden of chemotherapy. These cytokines upregulate the expression of neuronal receptors on HSPCs, allowing for them to form a greater response to neurotransmitters, leading to enhanced proliferation and motility of human CD34+ progenitor cells and subsequent repopulation of mouse BM83. Additionally, mitotically active Nestin-GFP+ perisinusoidal niche cells are greatly diminished whereas the Nestin-GFP+ peri-arteriolar niche cells exhibit greater chemoresistance because of their higher quiescence2.Ionizing radiation and chemotherapy used to treat cancer cause BM injury and alter the BM cellular composition. Following chemotherapy, there is an increase in apoptosis of mature cells along with progenitor cells that are cycling. Chemotherapy can damage BM innervation84.Consequently, the use of adrenergic agents as a therapeutic approach should be investigated further, adding to the available evidence on α1-ADR agonists or β-ADR agonists86, but the consequences appear to be different. In MPN, IL-1β produced by the mutant hematopoietic cells damages sensitive HSC niche components, such as neural terminals, Schwann cells, and Nestin+ MSCs86. In contrast, experimental AML causes the reduction in arteriole-associated NG2+ cell numbers and correlates with the expansion of Nestin-GFP+ stromal cells85. In MPN, chronic administration of β3-adrenergic agonists to compensate for the defective innervation can rescue Nestin+ niche cells and improve myelofibrosis  in both mice86 and humans87. Different effects of β3-adrenergic agonists in mouse models and human MPN might be explained by the different drugs or dosing used. In AML, the relevance of sympathetic neuropathy85 remains to be demonstrated in humans. The changes in sympathetic regulation of HSC niches during aging and age-related myeloid malignancies are briefly summarized inThe SNS has also been implicated in the development of hematological malignancies, predominantly the progression of myeloid malignancies. Sympathetic neuropathy occurs in the development of both acute myelogenous leukemia (AML) and myeloproliferative neoplasms (MPNs)40. However, the roles of this cholinergic branch (sympathetic or parasympathetic) in the regulation of hematopoiesis remain largely unexplored.The BM is regulated by neural signals principally emerging from the autonomic nervous system. The sympathetic noradrenergic branch has been much more explored than the parasympathetic (cholinergic) branch, both under steady state and during stress hematopoiesis. The data available suggest that sympathetic innervation regulates BM homeostasis but is especially important to respond to stress scenarios. Recent evidence suggests that the cholinergic branch of the autonomic nervous system contributes to this regulationDuring chronic inflammation, cardiovascular disease, and short-term social and psychological stress, beta blockers have been demonstrated to revert excessive myelopoiesis. Whether a similar strategy could be proposed to prevent excessive myeloid cell production during aging or age-related myeloid malignancies requires further investigation. The possible contribution of other adrenergic or cholinergic signaling pathways to the progression of hematological disorders is an exciting area for future investigation.ACh, acetylcholine; ADR, adrenergic receptor; AML, acute myelogenous leukemia; BM, bone marrow; CAR, Cxcl12-abundant reticular; ChAT, choline acetyltransferase; G-CSF, granulocyte colony-stimulating factor; HSC, hematopoietic stem cell; HSPC, hematopoietic stem and progenitor cell; IL, interleukin; LepR, leptin receptor; MafB, myeloid regulatory transcription factor; MPN, myeloproliferative neoplasm; MSC, mesenchymal stem cell; NG2, neuron-glial antigen; PNS, parasympathetic nervous system; SNS, sympathetic nervous system; Th, tyrosine hydroxylase


We included 81 healthy controls, 47 women with PE, 65 with SGA/FGR and 40 with PE + SGA/FGR. Maternal CO was inversely related to fetal UA PI and positively related to MCA PI; the opposite was observed for PVR, which was also positively associated with increased uterine artery impedance. CO was lower  and PVR higher  with UA ARED flow. We report that maternal CV dysfunction is associated with fetal vascular changes, namely raised impedance in the fetal-placental circulation and low impedance in the fetal cerebral vessels. These findings are most evident with critical UA Doppler changes and represent a potential mechanism for therapeutic intervention.We investigate the relationship between maternal cardiovascular (CV) function and fetal Doppler changes in healthy pregnancies and those with pre-eclampsia (PE), small for gestational age (SGA) or fetal growth restriction (FGR). This was a three-centre prospective study, where CV assessment was performed using inert gas rebreathing, continuous Doppler or impedance cardiography. Maternal cardiac output (CO) and peripheral vascular resistance (PVR) were analysed in relation to the uterine artery, umbilical artery (UA) and middle cerebral artery (MCA) pulsatility indices (PI, expressed as  Maternal cardiovascular function changes profoundly throughout pregnancy . CardiacSeveral non-invasive techniques are available for the measurement of cardiovascular parameters in pregnant women. There currently is no gold standard: the choice depends on factors such as the availability of techniques, training of the operators and costs. Some techniques can be used only in the maternal lying position , others In this study, we analysed cardiac output and peripheral vascular resistance in a cohort of pregnant women with or without pre-eclampsia or FGR. We used three different techniques for cardiovascular assessment in pregnancy, based on inert gas rebreathing ,11, contThis is a prospective cohort observational study performed in three European University Hospitals, located in London, UK (Centre A), Hasselt, Belgium (Centre B) and Rome, Italy (Centre C), and included women with singleton pregnancies at or above 24 weeks’ gestation, who were healthy or affected by one of the following conditions: pre-eclampsia alone; small for gestational age (SGA) fetuses/FGR alone; or the combination of both pre-eclampsia and SGA/FGR.Before the combined analysis of the datasets, the study analysis plan was to examine maternal cardiac output and peripheral vascular resistance in relation to maternal (uterine) and fetal  Doppler impedance indices.The data for the women in Centre A have been recently reported , though Women were recruited from January 2013 and the studies were individually approved by each local Research Ethics Committee . Exclusion criteria were the presence of multiple pregnancy, fetal malformations and maternal cardiovascular comorbidities, such as smoking or chronic hypertension.Pre-eclampsia was defined as a maternal blood pressure at diagnosis of >140/90 mm Hg and a urine protein creatinine ratio of >30; SGA was defined as abdominal circumference or estimated fetal weight <10th centile with normal umbilical artery Doppler. The presence of abnormal umbilical artery Doppler (above the 95th percentile) in an SGA fetus denoted the presence of FGR ,16. Pre-All women underwent a fetal ultrasound scan and cardiovascular assessment within 72 h of the scan, as detailed below.Fetal ultrasound scans were performed using Samsung WS80  in Centre A, General Electric Voluson E8 Expert  in Centre B and General Electric Voluson E6  in Centre C. Measurements of fetal biparietal diameter, head circumference, abdominal circumference and femur length were obtained, and fetal growth was assessed using local growth charts.z-score by gestational week as previously described [With regards to Doppler assessment, uterine artery mean pulsatility index (PI), umbilical artery PI and middle cerebral artery (MCA) PI were collected. Doppler parameters were analysed in all groups ; umbilicescribed . UterineCardiac output and peripheral vascular resistance were measured with different devices according to the technique available at each centre after 5 to 10 min of rest, with the participant in the left lateral lying position to avoid aortocaval compression. Standing measurements were made where this was feasible, depending on the device used, again after a period of rest for stabilization of cardiovascular function.Centre A: an operator-independent device based on inert gas rebreathing  was used for the measurement of cardiac output as previously described ,18. ThisCentre B: an automated and operator-independent device measured both blood pressure through oscillometry and cardiac output through impedance cardiography . Inter- and intra-observer variation as well as normal reference values have been reported elsewhere ,20,21. BPeripheral vascular resistance was calculated from mean arterial pressure and cardiac output as per the formula above. Both lying and standing measurements were obtained with this device.Centre C: cardiac output was measured with a continuous Doppler-based device , which is non-invasive, though not operator-independent. A non-imaging continuous-wave Doppler transducer was placed at the suprasternal notch to measure transaortic blood flow. After manual entry of the patient’s weight and height, the device computed cardiac output from the velocity time integral of the blood flow through the aortic valve . Blood pz-score for gestational week. For each week, the mean and standard deviation used as references for z-scoring were calculated from those obtained in a group of women with healthy pregnancies assessed in the same centre and using the same methodology [In order to compare the measurements of cardiac output and peripheral vascular resistance obtained with the three different techniques, for each subject the absolute values of these parameters were transformed into the corresponding hodology ,4.t-test was used to compare means.Statistical analysis was performed with SPSS statistical software . Continuous variables were described as mean (standard deviation) or median (range) where appropriate. Demographic characteristics and maternal cardiovascular indices between controls and pathology groups were compared with the Kruskal–Wallis test. A further analysis was performed for comparison of cardiovascular measurements between those cases with positive umbilical artery end diastolic flow (EDF) and those with ARED; p-value of less than 0.05 was considered statistically significant; in order to control the Type I error, we used the Bonferroni correction in the case of multiple comparisons.Associations between maternal cardiovascular parameters and Doppler indices were assessed using polynomial regression analyses. By using fitted curves, we chose quadratic models to describe these relationships. A z-score of 10.6, and two cases with peripheral vascular resistance z-scores of 10.99 and 34.77.For all analyses, we removed 3 cases with implausible outlying maternal cardiovascular values: one case with a cardiac output A total of 233 participants were included, 107 in Centre A, 98 in Centre B and 28 in Centre C. Of those, 81 were women with control  pregnancies and 152 had pathological outcomes . The trends with gestational age for CO and PVR are shown in No differences were found between the pathological pregnancy outcome groups with regards to maternal age, body mass index and gestational age at cardiovascular and scan investigations .p < 0.001 for all groups vs. controls). Although not significant, median gestational age at cardiovascular and scan assessment, gestational age at delivery and birthweight were lower in the pre-eclampsia with SGA/FGR group.Gestational age at delivery and birthweight in women with pathological pregnancy outcomes were significantly lower than for the controls .A total of 231 lying and 205 standing cardiac output z-score values were analysed. Women with pathological outcomes showed higher lying peripheral vascular resistance than controls. Standing vascular resistance z-scores were also significantly higher in pre-eclampsia + SGA/FGR compared to controls and SGA/FGR .For peripheral vascular resistance, 229 lying and 205 standing z-scores were compared in the whole study population, divided into two subgroups characterized by a different EDF pattern in the umbilical artery. Cardiac output was lower and vascular resistance higher in the ARED group, both in lying and standing positions . Middle cerebral artery PI z-score was positively associated with lying cardiac output z-score . No associations were detected between uterine artery mean PI and cardiac output.Umbilical artery PI The relationship between umbilical, middle cerebral and uterine artery Doppler PI and peripheral vascular resistance are shown in z-scores . The same association was noted for umbilical artery PI z-score . Middle cerebral artery PI z-score was inversely associated with changes in lying peripheral vascular resistance z-score .Uterine artery PI showed a significant and non-linear association with both lying and standing peripheral vascular resistance In this exploratory study, fetal-placental Doppler indices are associated with maternal cardiac output and vascular resistance both in healthy pregnancies and those with pathological outcomes. The importance of this study is that it utilizes data from three distinct populations and three different techniques for generalizability. Previously unreported is that an abnormal maternal cardiovascular function is associated with reduced impedance in fetal cerebral vessels. Cerebral vasodilatation is the fetal compensatory response to hypoxia detected by direct oxygen sensing of the cerebral vasculature, not mediated through the autonomic system. It is therefore plausible that cerebral redistribution is associated with restricted uteroplacental perfusion secondary to impaired maternal cardiovascular function, hence manifesting as fetal hypoxia. Though the absolute magnitude of the associations seen is small, in the context of fetal growth restriction and pre-eclampsia, marginal changes in the utero-placental circulation may lead to major perturbations in the fetal circulation.The most severe changes in maternal cardiovascular parameters are observed when ARED flow is seen in the fetal umbilical circulation. This is commonly seen in women with the dual pathology of both pre-eclampsia and SGA/FGR, where maternal cardiac output is low and vascular resistance is high. Analysis of cardiovascular parameters in women with or without positive EDF in the umbilical artery revealed that maternal cardiac output was significantly lower where the fetal umbilical artery blood flow was severely compromised showing ARED flow, and these women also had the highest vascular resistance. These Doppler changes in pre-eclampsia, particularly with FGR, have hitherto been ascribed to placental damage and abnormal development . VillousWe assessed cardiac output and peripheral vascular resistance in three centres with different methodologies. It is likely that the magnitude of the findings that we report is attenuated as the absolute CO and PVR values were different in the three centres. However, the trends with gestational age were similar , thus arOur findings provide an explanation for previously reported observations. Beta blockers, which have negative chronotropic effects on maternal cardiac output, are associated with FGR  and stilLower maternal cardiac output and higher peripheral vascular resistance are associated with the fetal circulatory changes that occur in response to hypoxia, namely an increased impedance in the umbilical vessels and reduced impedance in the cerebral arteries. This becomes particularly obvious when umbilical Doppler findings are critically abnormal. This relationship raises the potential for therapeutic manipulation of maternal cardiovascular function in order to improve pathologically abnormal uteroplacental function, and hence fetal condition.


His-bundle pacing (HBP) has emerged as an alternative to conventional ventricular pacing because of its ability to deliver physiological ventricular activation. Pacing at the His bundle produces different electrocardiographic (ECG) responses: selective His-bundle pacing (S-HBP), non-selective His bundle pacing (NS-HBP), and myocardium-only capture (MOC). These 3 capture types must be distinguished from each other, which can be challenging and time-consuming even for experts.The purpose of this study was to use artificial intelligence (AI) in the form of supervised machine learning using a convolutional neural network (CNN) to automate HBP ECG interpretation.We identified patients who had undergone HBP and extracted raw 12-lead ECG data during S-HBP, NS-HBP, and MOC. A CNN was trained, using 3-fold cross-validation, on 75% of the segmented QRS complexes labeled with their capture type. The remaining 25% was kept aside as a testing dataset.P <.0001), with an overall accuracy of 75%. The CNN’s accuracy in the 17-patient testing set was 67% for S-HBP, 71% for NS-HBP, and 84% for MOC.The CNN was trained with 1297 QRS complexes from 59 patients. Cohen kappa for the neural network’s performance on the 17-patient testing set was 0.59 (95% confidence interval 0.30 to 0.88; We demonstrated proof of concept that a neural network can be trained to automate discrimination between HBP ECG responses. When a larger dataset is trained to higher accuracy, automated AI ECG analysis could facilitate HBP implantation and follow-up and prevent complications resulting from incorrect HBP ECG analysis.  These responses must be distinguished from each other when performing His-bundle lead implantation and follow-up.•His-bundle pacing electrocardiogram (ECG) response discrimination can be automated by training a neural network on labeled cases, a form of supervised machine learning.•The neural network was best at differentiating between selective His-bundle pacing and myocardium-only capture and worst at differentiating between non-selective His-bundle pacing and myocardium-only capture, which is similar to what is expected from their distinguishing ECG features.•Saliency mapping can reveal insights into how the neural network performs classification.His-bundle pacing (HBP), in which the His-Purkinje cardiac conduction system is directly stimulated by a permanent pacing lead,6The electrocardiogram (ECG) is a key tool for identifying successful pacing. Conventional ventricular pacing produces characteristic, easily discernible changes in the 12-lead ECG. RV apical pacing results in broad QRS complexes with left bundle branch block (LBBB) morphology.•Selective HBP (S-HBP) occurs when the His bundle is captured alone without any local myocardial capture.•Non-selective HBP (NS-HBP) occurs when both the His bundle and local myocardium adjacent to the lead tip are captured.•Myocardium-only capture (MOC) occurs when the His bundle is not captured and only the local myocardium is captured; this is also termed myocardial capture or septal capture.The emergence of HBP, however, has been accompanied by considerable challenges in ECG interpretation. Several ECG responses are potentially observed with HBP, and their differences can be subtle to the untrained eye .•SelectiDiscriminating among these 3 ECG responses to HBP is more complex than ECG interpretation of conventional pacing, in which only the presence of ventricular capture needs to be identified. This complexity is compounded by a lack of expertise. Although its global uptake has been rapid, as a relatively recent development HBP implantation and follow-up are performed at only a small number of centers. Discriminating among the 3 responses has crucial clinical importance, as misdiagnosis can have adverse consequences. During HBP lead implantation, operators must rapidly determine whether the achieved lead position is satisfactory (capturing at a reasonable threshold) or the lead must be repositioned. At follow-up, appropriate pacing output and configuration must be selected to achieve the desired capture type.Machine learning is a form of artificial intelligence (AI) that allows automation of tasks that otherwise would require human expertise, including ECG classification.The database of electrophysiological (EP) procedures performed at Hammersmith Hospital, London, United Kingdom, was searched for HBP procedures. The research reported in this study adhered to the Helsinki Declaration as revised in 2013. Cases in which an EP system was used  during His-bundle lead implantation were identified. Twelve-lead surface ECG recordings from the cases were inspected for paced QRS complexes, which were reviewed and classified into S-HBP, NS-HBP, or MOC. Cases were randomly assigned in a 3:1 ratio to the training or testing datasets . This alAll types of intrinsic QRS morphology were included, including narrow QRS, LBBB, and right bundle branch block (RBBB). In cases of BBB, paced QRS complexes could display any degree of resynchronization of BBB, including failed correction. All indications for HBP were included. Rare or intermediate forms of capture  and left bundle branch pacing QRS complexes were excluded. Patients younger than 18 years also were excluded.,S-HBP, NS-HBP, and MOC were diagnosed by visual inspection and electronic caliper measurements of a combination of digital 12-lead surface ECGs and His-bundle lead electrograms (EGMs) during intrinsic rhythm and His-bundle lead threshold check, which included programmed stimulation when available. This was performed by a single expert in HBP ECG analysis. Criteria for diagnosing ECG responses were derived from published standardized definitionsRaw, digital 12-lead surface ECG data at 1000-Hz sampling frequency were extracted from the EP system recordings for each case. Exactly 5 beats were extracted per beat class for testing set cases to ensure a balanced number of unique ECGs for the final analysis (more cases were extracted for training set cases to a maximum of 10). Each period was manually segmented using custom softwareIn this study, we used a convolutional neural network (CNN).This study used a 1-dimensional CNN, in which the kernels convolve through the data in only 1 dimension, that is, they slide through the ECG over time. It was inspired by the ResNet34 architecture,We used the training data to train 3 neural networks in a process termed 3-fold cross-validation. This involved splitting the training data into 3 smaller datasets before each of the 3 neural networks was trained by using two-thirds to train the network and one-third to validate it. A different validation third was used for each of the 3 networks. The final performance of the system combined the predictions of the 3 networks (ensembling) on the unseen test set . All theExamples from the testing dataset were processed to provide saliency maps.The performance of the neural network was assessed using the Cohen kappa, which is a measure of agreement between the neural network and the true labels that adjusts for imbalance in the size of the classes. Accuracies are reported separately for each class of beats, with confidence intervals (CIs) calculated using the binomial distribution with continuity correction. Statistical analysis was performed using the scikit-learn Python package .Seventy-six unique patients were eligible for analysis. Fifty-nine patients were randomly assigned to the training set, and the remaining 17 patients were assigned to the testing set. Baseline characteristics are given in P <.0001). Accuracies across the 3 classes were 84% (95% CI 70% to 92%) for S-HBP, 71% (95% CI 59% to 80%) for NS-HBP, and 67% (95% CI 39% to 87%) for MOC. Overall accuracy was 75%. Only 1 (2%) of the S-HBP ECGs was incorrectly classified as MOC, and no MOC beats were incorrectly classified as S-HBP. The confusion matrix is shown in The ensemble predictions of the 3 neural networks on the final testing set yielded a Cohen kappa of 0.59 , this reasonably high level of accuracy was achieved in the context of a relatively small dataset of 82 patients with intraclass heterogeneity of paced QRS morphology (varying degrees of left and right bundle recruitment). Human expert diagnosis of HBP ECGs can involve comparing the intrinsic rhythm ECG to the paced ECG, checking for transitions between capture types during threshold checks, performing EP pacing maneuvers, as well as carrying out manual measurements of key ECG intervals.Physiological activation of the ventricles with a normal, narrow QRS in an unpaced intrinsic rhythm occurs due to rapid activation of the ventricles via the insulated His-Purkinje cardiac conduction system fibers. The variation in paced ECG responses seen with HBP are due to HBP’s fundamental property of activating the ventricles in the same way. Altering the energy output through a conventional ventricular pacing lead generally does not alter QRS morphology because only one kind of tissue can be captured: ventricular myocardium. The local myocardium activation propagates to the remainder of the ventricular myocardium nonphysiologically through slow cell-to-cell myocardial conduction. Leads positioned at the His bundle can capture 2 different tissues: conduction system tissue (the His bundle itself) and myocardium local to the lead tip. The difference in capture threshold between these 2 tissues results in the 3 potential capture types: either tissue alone (S-HBP or MOC) or both together (NS-HBP), each with different QRS characteristics . CorrectMOC does not constitute HBP and thus is rarely a desired capture type for a patient with a His-bundle pacemaker. The slow, dyssynchronous activation pattern produced is similar to conventional RV pacing.Distinguishing MOC from NS-HBP usually is the hardest HBP ECG distinction for humans to make based on the paced ECG alone. Without access to threshold checks, programmed stimulation, or the intrinsic ECG, no unique feature easily discriminates the 2 capture types, unlike the isoelectric segment in S-HBP. This may offer an explanation for why NS-HBP/MOC mistakes were the most common error . MOC was also the rarest of the 3 capture types in the training set, potentially making it more difficult for the network to learn its features. This reflects the prevalence of variations in underlying anatomy. A His bundle buried deeply within the septum covered by a thick myocardium layer is a less common anatomic variantMOC and S-HBP usually are the 2 capture types most easily distinguished from each other by human analysis, particularly when the intrinsic QRS is narrow. Due to the absence of myocardial capture, S-HBP produces a characteristic isoelectric segment before QRS onset that is easier to identify than the subtle morphologic changes or cumbersome measurements that distinguish MOC from NS-HBP. In combination with this difference, the QRS of S-HBP is more clearly different from MOC than it is from NS-HBP due to the considerable difference in ventricular activation pattern. Furthermore, due to the nature of myocardial and conduction system thresholds  or, conversely, a definitively flat, physiological isoelectric stimulus-V segment in all leads. However, pre-excitation can be almost imperceptibly subtle, and a wandering baseline can mimic pre-excitation.CNNs have been used successfully in the past to classify biological waveform data such as ECGs,It is intuitive to explain the network’s variable predictive ability as mimicking human performance. First, the neural network’s learning process is thought to be analogous to that of the human brain. Second, humans find certain classifications easier than others due to inherent features and defining criteria of those classes . However, there are other potential reasons for the network’s performance being in line with expected human performance. Class imbalance might hamper the network’s ability to learn less common classes and may bias the network toward predicting more common classes. When humans find some classification tasks difficult and the labels for network training and testing are based on human assessment, labeling errors will occur at a higher rate in the classification tasks that are more difficult for humans . This will worsen network performance for those classifications.,The nature of neural network machine learning has been characterized as a “black box.”end time. However, most HBP implantations are performed in catheterization laboratories that are not dedicated to EP procedures, so the 12-lead ECG is not visualized alongside the EGM. Making measurements in this scenario can be cumbersome. Automated analysis would greatly facilitate intraprocedural diagnosis in such laboratories, potentially shortening procedural time. HBP follow-up also requires accurate diagnosis of ECGs, which currently restricts practice to centers with considerable HBP expertise. Automated diagnosis could help to democratize HBP to be practiced at any center. Finally, even experts occasionally make errors that lead to inappropriate programming, with potentially detrimental clinical consequences. Neural networks are not prone to errors that humans might make due to tiredness, distraction, or lack of concentration, minimizing errors overall.A neural network trained to very high accuracy has several potential applications and benefits. The HBP procedure can be performed in catheterization laboratories with EP systems, where His-bundle lead EGMs are visualized alongside 12-lead ECGs, and electronic caliper measurements can be made across EGMs and ECGs, including the important H-QRSECG diagnosis for label determination was performed by a single expert. Although objective HBP criteria have been set out in detail and are recognized internationally as the gold standard, cases remain for which diagnosis is uncertain even when experts adhere strictly to these criteria. For example, NS-HBP with partial correction of LBBB can be challenging to distinguish from MOC. Both exhibit LBBB-like morphology, and their ECG capture measurement ranges overlap  within the training and testing datasets. Although the Cohen kappa performance metric accounts for such imbalances when reporting results, these disparities nevertheless will compromise the neural network’s ability to learn the rarer classes of data.This iteration of the machine learning algorithm required manual QRS segmentation, but this process can be automated through conventional computational means or machine learning. Raw digital ECG data were the data source in this study, but images of ECGs could be automatically transformed into equivalent signals to be input into the network. A data pipeline for this is under development.This was a single-center retrospective study design with a relatively small dataset, and the cases included were biased toward certain indications due to ongoing research into HBP during the study period. The most common indication for HBP in international multicenter registries is high-degree atrioventricular block, but this was the least common indication in our study. This was mitigated by the balanced proportions of intrinsic RBBB, LBBB, and narrow QRS within and between the training and test sets. This study demonstrated proof of concept justifying dataset expansion. Indeed, at present the threshold of accuracy (or kappa) such a classifier must obtain to be clinically superior to an expert human is unknown, as no data exist demonstrating the performance of humans at this task outside of the research environment. A highly accurate, fully automated, machine learning algorithm trained on large-scale, multiple rater–labeled data will be tested in a prospective, multicenter validation study on consecutive cases and compared to human expert analysis.We demonstrated proof of concept that discrimination between HBP ECG responses can be automated using machine learning. When a larger dataset is used to train the network to higher accuracy, automated AI ECG analysis could facilitate HBP implantation and follow-up and prevent complications from HBP ECG misdiagnosis.Dr Arnold (P74166) acknowledges support from the National Institute of Health Research (NIHR) Imperial Biomedical Research Centre (BRC) and the British Heart Foundation (BHF) Imperial Centre of Research Excellence (RE/18/4/34215). Dr Howard is supported by the Wellcome Trust (212183/Z/18/Z). Prof Francis (FS 04/079) and Dr Whinnett (FS/13/44/30291) have received individual funding from the BHF.The authors have no conflicts of interest to disclose.


These interactions can trigger a wide range of cellular functions, including altering cellular metabolism, regulation of barrier function and the generation of innate immune responses designed to limit infections. The cellular consequences of infection can, however, also exacerbate disease states. The current special issue of the journal covers a number of specific aspects of the interactions of infectious agents with epithelial cells at mucosal surfaces. The papers include insightful review articles as well as a number of original investigations.Mucosal epithelial cells of the respiratory, gastrointestinal, and genital tracts are the initial site of interaction with pathogens. Although initially viewed primarily as functioning as physical barriers to infection, the past several decades have shown a far more complex set of interactions. Pathogens can interact directly with mucosal epithelia, either Ganjian et al. provides an overview of rhinoviruses and how they are recognized by human airway epithelial cells (HAE). This includes recognition of HRV replication intermediates by PRR and the proinflammatory and antiviral responses triggered by such interactions. The review also considers various signaling pathways that regulate epithelial responses to HRV infections and discusses differences in responses between cells from normal subjects and those with lower airway diseases, including potential differences in antiviral responses between patient populations.Six out of the eight manuscripts included in this Research Topic examine the role of airway epithelial cells in the response to inhaled pathogens. Human rhinoviruses (HRV) infections are a major trigger of acute exacerbations of asthma, chronic obstructive pulmonary disease, and cystic fibrosis . The revJamieson et al. focus on the role of epithelial IL-17C, a novel member of the IL-17 family of cytokines, in response to HRV infection. Using highly differentiated HAE they demonstrate that HRV infection triggers production of IL-17C exclusively in the basolateral direction. They further show that basolateral, but not apical, stimulation of HAE with IL-17C led to basolateral release of the neutrophil chemoattractant CXCL1. This suggests that basolateral release of IL-17C during HRV infections can feedback to induce basolateral release of CXCL1 leading to neutrophil recruitment. Since neutrophils correlate with disease severity, IL-17C may be a contributor to increased inflammation and symptoms during infections.Wang et al. showed that infection of differentiated HAE with either of two different strains of HRV increased gene expression of the mucins MUC5AC and MUC5B and caused increased secretion of mucin proteins. They further show that HRV-induced mucin expression appears to be regulated by the transcription factor SAM-pointed domain-containing Ets-like factor (SPDEF) and SPDEF-regulated genes, as well as via purinergic receptor signaling in response to extracellular ATP. Finally, they showed that the muscarinic receptor antagonist, tiotropium bromide, as well as the glucocorticoid, fluticasone propionate, inhibit HRV induced mucin expression without affecting viral loads. These effects may underlie the beneficial effects of these drugs in treating exacerbation of lower airway diseases.An important component of acute exacerbations of lower airway diseases is mucus hypersecretion. Marsh et al. show here that Pellino-1 is expressed in the airway epithelium and plays a role in regulating responses to viral infection. Although knockdown of Pellino-1 in primary bronchial epithelial cells led to reduced production of proinflammatory cytokines in response to the synthetic double-stranded RNA (dsRNA) mimic, Poly I:C, the authors found that Pellino-1 knockout mice actually showed enhanced inflammatory cytokine production upon infection with viruses.It has previously been shown that knockdown of the E3-ubiquitin ligase, Pellino-1, reduces the inflammatory response in sepsis . Marsh eCao et al. focused on the role of a specific ATP binding cassette (ABC) transporter, ABCF1, in epithelial responses to dsRNA mimics. This ABC transporter has been identified as a cytosolic nucleic acid sensor. Here it shown that ABCF1 is expressed in epithelial cells and mediates the production of CXCL10 by dsRNA mimics. Knockdown experiments and gene ontogeny analysis suggest that, while ABCF1 does play a role in regulation of antiviral responses, it is most strongly associated with Toll-like receptor signaling, suggesting a multifactorial role in epithelial innate immunity.Ahn and Prince review the role of IL-22 and IFN-λ in regulating epithelial responses to bacterial infection. These two cytokines both signal through the cognate receptor IL-10RB and have been linked to regulation of epithelial barrier function. IL-22 induces production of antimicrobial peptides and plays a role in increasing junctional proteins. By contrast, IFN-λ has known antiviral and proinflammatory proteins and reduces junctional integrity, which would be expected to facilitate bacterial and immune cell translocation. Because the actions of these cytokines can vary depending on the nature of the pathogen and the specific tissue microenvironment, additional studies are needed to fully understand the role of these cytokines in airway defense and to identify targets that may have therapeutic potential.Airway bacterial infections can also cause substantial morbidity and mortality. Helicobacter pylori (H. pylori) infection in humans and mice . HPV replication y modulates genes involved in stem-cell maintenance in cervical mucosal SCJ, which further leads to HPV-mediated cervical malignancy. Collective evidence highlights SCJ mucosa being a vulnerable site for HPV infection due to its unique anatomical location, altered host-defensin production, lack of immune cells and presence of cancer stem cell niche.Human papillomavirus (HPV) infections are responsible for 99% of cervical cancer . BudhwanRW and DP wrote the editorial together. All authors contributed to the article and approved the submitted version.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.


Excess body weight has been identified as a risk factor for many types of cancers, and for the majority of cancers, it is associated with poor outcomes. In contrast, there are cancers in which obesity is associated with favorable outcomes and this has been termed the “obesity paradox”. In melanoma, the connection between obesity and the increased incidence is not as strong as for other cancer types with some but not all studies showing an association. However, several recent studies have indicated that increased body mass index (BMI) improves survival outcomes in targeted and immune therapy treated melanoma patients. The mechanisms underlying how obesity leads to changes in therapeutic outcomes are not completely understood. This review discusses the current evidence implicating obesity in melanoma progression and patient response to targeted and immunotherapy, and discusses potential mechanisms underpinning these associations. Cutaneous melanoma is a cancer of melanocytes, the pigment producing cells of the skin. The global incidence of cutaneous melanoma is consistently rising, and Australia has one of the highest rates of melanoma incidences worldwide, with an estimated 15,000 new cases in 2019 . Genetic, host and environmental risk factors have been associated with melanoma incidence. The major environmental risk factor for cutaneous melanoma, is ultraviolet (UV) radiation especially with intermittent patterns of sun exposure ,4, and aThe exact molecular mechanisms associated with obesity that lead to cancer development or changes in patient outcomes are not completely understood . ObesityIn melanoma, obesity is associated with worse outcomes after resection  and incrCell intrinsic alterations in metabolism are a hallmark of many cancers, including melanoma. While initial studies focused on changes in aerobic glycolysis first identified by Otto Warburg, a growing body of evidence now supports critical roles for other metabolic pathways, including fatty acid synthesis and oxidation . Fatty aV600E oncogene to accelerate melanoma development in transgenic zebrafish and mouse xenograft studies [V600E oncogene to promote tumor growth in human melanoma cells implanted into mice [V600E mutant-dependent mitogen activated protein kinase kinase  activation [+ metastasis-initiating cells in a CD36-dependent manner, suggesting that metastasis-initiating cells may rely on dietary lipids to promote metastasis. However, this specific association between dietary fat, CD36 and metastasis remains to be tested in melanoma.More recently cell extrinsic factors such as adipocyte-derived lipids have been uncovered as a potential fuel source that can also drive melanoma progression ,49. Advanto mice . Mechanitivation  , have revolutionised melanoma treatment. With the discovery of the prevalence of a specific activating mutation in BRAF (V600)  selectivCurrently, the standard-of-care for BRAF-mutant melanoma is an upfront combination of a BRAF and MEK inhibitor (BRAF/MEKi), of which there are three combinations approved by the Food and Drug Administration (FDA): dabrafenib/trametinib, vemurafenib/cobimetinib and encorafenib/binimetinib. This combinational therapy is highly tolerated and the initial response rates are remarkable with 90% of patients having some degree of response  that is The mutation in BRAF locks the kinase into an active conformation leading to constitutive activation of the MAPK pathway and as consequence increased cell growth, proliferation and survival . ResistaA recent study in metastatic melanoma patients demonstrated that obesity is associated with significantly improved responses to both targeted and immunotherapy in male but not female patients. At the 2-year mark, male obese patients on targeted dabrafenib and trametinib therapy had an overall survival of 64% compared to 35% in normal weight male patients with overweight patients falling in between. Furthermore, progression free survival was also improved, 15.7 months for obese individuals versus 9.6 months for normal . How obeV600E melanoma cells with BRAF and MEK targeted therapies extensively reprograms cell intrinsic metabolism and this influences clinical response. During the initial phase of treatment, a dramatic decrease in glucose utilisation and glycolytic flux is observed [V600E melanomas with both de novo and acquired resistance to MAPK pathway inhibitors [V600E signaling via the FATP/ SLC27A fatty acid transporters would be predicted to abrogate response of obese patients to MAPK pathway targeted therapies based on potentiation of BRAFV600E driven melanoma progression by adipocyte-melanoma cell crosstalk [V600E-expressing melanoma [Treatment of BRAFobserved , and thiobserved . Inductihibitors , and inhhibitors . These dhibitors , and thirosstalk . Similarmelanoma  which woBeyond cell intrinsic metabolism, alterations in systemic metabolism can influence patient responses to targeted therapies. In normal physiological settings, the PI3K pathway functions as a nutrient sensor and is activated by binding of insulin and IGF to cell-surface receptors. Systemic regulation of this pathway is altered in the setting of obesity, frequently manifesting in insulin resistance. This pathway is also frequently activated in many types of cancer, and PI3K pathway inhibitors are under investigation as a cancer treatment. Interestingly, efficacy of PI3K inhibitors appears to be limited due to systemic glucose-insulin feedback caused by targeted inhibition of the PI3K pathway, that is sufficient to reactivate PI3K signalling in tumor cells across multiple tumor models in mice . This inAnother critical aspect of targeted therapy response in melanoma patients is the upregulation of antigen expression and increased infiltration of T cells which is associated with tumor shrinkage ,119. IntThere are currently seven FDA-approved immunotherapy options for melanoma that fall into two classes, oncolytic virus therapy and immune modulators, which includes the immune checkpoint inhibitors that block key immunological pathways that mediate tumor escape. Of these therapies the immune checkpoint inhibitors have shown excellent efficacy in melanoma with overall survival of 52% at 5-years when treated with a combination of ipilimumab, that targets CTLA-4 and nivolumab that targets PD-1 . CTLA-4 The role of obesity in immune checkpoint therapy efficacy is not clear. Anti-PD-1 therapy is more efficacious in melanoma-bearing obese mice compared to their normal-weight counterparts . The impThe more favourable prognosis of female melanoma patients compared to males ,130,131 Excess body fat is a complex metabolic disorder that can potentially impact on melanoma growth and immune surveillance at many levels. Why obese melanoma patients respond better to both targeted- and immune-therapy remains unclear. Many factors increased in obesity would promote melanoma growth and similar to the concept of “oncogenic addiction” this could make them more susceptible to targeted therapy. Similarly, the immune-suppressive microenvironment induced by obesity could confer greater sensitivity to immune checkpoint inhibitors. Many additional complex cellular processes induced in obesity may also impact on melanoma, including gender and the gut microbiome.