Leukocyte-endothelial Cell Adhesion Research Articles

Inhibiting leukocyte-endothelial cell interactions by Chinese medicine Tongxinluo capsule alleviates no-reflow after arterial recanalization in ischemic stroke.

Despite successful vascular recanalization in stroke, one-fourth of patients have an unfavorable outcome due to no-reflow. The pathogenesis of no-reflow is fully unclear, and therapeutic strategies are lacking. Upon traditional Chinese medicine, Tongxinluo capsule (TXL) is a potential therapeutic agent for no-reflow. Thus, this study is aimed to investigate the pathogenesis of no-reflow in stroke, and whether TXL could alleviate no-reflow as well as its potential mechanisms of action. Mice were orally administered with TXL (3.0 g/kg/d) after transient middle cerebral artery occlusion. We examined the following parameters: neurological function, no-reflow, leukocyte-endothelial cell interactions, HE staining, leukocyte subtypes, adhesion molecules, and chemokines. Our results showed stroke caused neurological deficits, neuron death, and no-reflow. Adherent and aggregated leukocytes obstructed microvessels as well as leukocyte infiltration in ischemic brain. Leukocyte subtypes changed after stroke mainly including neutrophils, lymphocytes, regulatory T cells, suppressor T cells, helper T type 1 (Th1) cells, Th2 cells, B cells, macrophages, natural killer cells, and dendritic cells. Stroke resulted in upregulated expression of adhesion molecules (P-selectin, E-selectin, and ICAM-1) and chemokines (CC-chemokine ligand (CCL)-2, CCL-3, CCL-4, CCL-5, and chemokine C-X-C ligand 1 (CXCL-1)). Notably, TXL improved neurological deficits, protected neurons, alleviated no-reflow and leukocyte-endothelial cell interactions, regulated multiple leukocyte subtypes, and inhibited the expression of various inflammatory mediators. Leukocyte-endothelial cell interactions mediated by multiple inflammatory factors are an important cause of no-reflow in stroke. Accordingly, TXL could alleviate no-reflow via suppressing the interactions through modulating various leukocyte subtypes and inhibiting the expression of multiple inflammatory mediators.

Methods for Imaging Inflammation and Transendothelial Migration in Vivo and ex Vivo.

Inflammation is the body's response to injury and harmful stimuli and contributes to a range of infectious and noninfectious diseases. Inflammation occurs through a series of well-defined leukocyte-endothelial cell interactions, including rolling, activation, adhesion, transmigration, and subsequent migration through the extracellular matrix. Being able to visualize the stages of inflammation is important for a better understanding of its role in diseases processes. Detailed in this article are protocols for imaging immune cell infiltration and transendothelial migration in vascular tissue beds, including those in the mouse ear, cremaster muscle, brain, lung, and retina. Also described are protocols for inducing inflammation and quantifying leukocytes with FIJI imaging software. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Induction of croton oil dermatitis Alternate Protocol 1: Induction of croton oil dermatitis using genetically fluorescent mice Basic Protocol 2: Intravital microscopy of the mouse cremaster muscle Support Protocol: Making a silicone stage Basic Protocol 3: Wide-field microscopy of the mouse brain Basic Protocol 4: Imaging the lungs (ex vivo) Alternate Protocol 2: Inflating the lungs without tracheostomy Basic Protocol 5: Inducing, imaging, and quantifying infiltration of leukocytes in mouse retina.

Ginsenoside Rb3 attenuates skin flap ischemia-reperfusion damage by inhibiting STING-IRF3 signaling.

We investigate the protective effect of ginsenoside Rb3 on skin flap microvasculature following ischemia-reperfusion (I/R) injury and its regulatory mechanism. We used a rat model of I/R injury with the right iliolumbar artery and oxidative stress model of human dermal microvascular endothelial cells. The effects of Rb3 on skin flap tissue and endothelial cell survival, STING-IRF3 pathway activation, and endothelial cell adhesion were measured. Following reperfusion, the survival rate of rat perforator flaps in the Rb3-treated group gradually increased with increasing Rb3 concentration. The treatment also reduced the amount of STING protein, phosphorylated IRF3, and P-selectin in skin flap tissue, with this change being most obvious in microvascular endothelial cells. In vitro, activated IRF3 binds to the P-selectin promoter and induces P-selectin expression. Our results suggest that Rb3 plays a role in reducing I/R flap damage through negatively regulating STING-IRF3 activation to limit leukocyte-endothelial cell adhesion.

Genetic and small molecule strategies to edit the glycocalyx

The glycocalyx covers all living cells and is a key regulator of cell communication with the extracellular milieu/matrix, other cells, foreign pathogens and the microbiome. A variety of glycoconjugate types decorate mammalian cells including N‐ and O‐linked glycans attached to proteins, glycolipids, and glycosaminoglycans. A complete understanding of the molecular and cellular features controlling living systems is not possible without delineating the structural and functional contributions of the glycocalyx. My laboratory studies this aspect by developing systems based experimental and computational tools to characterize the role of the glycocalyx in normal physiological and selected pathophysiological contexts. For such investigations, we have developed both novel CRISPR‐Cas9 based tools to perturb cell surface glycans and small molecule inhibitors of the same. Additionally, we have developed a Synthetic Glycan toolkit and liquid‐chromatography mass‐spectrometry (LC‐MS) workflow to longitudinally track glycan changes in living cells in a non‐destructive manner. My talk will present the development and application of these technologies in two contexts. First, I will discuss studies of human inflammatory diseases which will present some new molecular players regulating glycosylation and leukocyte‐endothelial cell adhesion mechanics under fluid shear flow. Second, I will present our studies on the role of glycans in regulating SARS‐CoV‐2 viral entry into human cells expressing ACE2. The data demonstrate a critical role for N‐glycans and O‐linked glycans in viral entry and propagation.

Dehydroabietic Acid Attenuates Atherosclerosis in Apolipoprotein E-Deficient Mice and VCAM-1 Expressions in-vitro

Dehydroabietic acid (DHA) is an analog of abietic acid (AA) for cardiovascular disease prevention, known to act pharmacologically against aging, inflammation, bacterial infections and cancer. The current research investigated the molecular mechanisms of DHA on the adhesiveness of endothelial leukocytes and activation of NF-κB in TNF-α treated HAEC and ApoE−/− mice in experimental atherosclerosis. The HAECs were tested for toxicity using MTT assay at various DHA concentrations. The cell-surface expression of CAM against endothelial leukocyte adhesion molecule-1, ICAM -1, or human VCAM-1 was determined by the ELISA test, followed by western blot analysis. Endothelial cell-leukocyte adhesion assay involving U937 cells was carried out followed by the determination of NF-κB p65 expression. ApoE−/− mice were fed with a high cholesterol diet every day followed by oral administration of DHA (10 and 20 mg/kg/day). The DHA (5 and 10 mM) substantially reduced (p < 0.05) human (U937) cell lines binding to TNF-α activated human endothelial aortic cells. The assays involving the 32P-labelled NF-kB as a probe demonstrated that DHA pre-treatment decreased the shifted bands density following the stimulation of TNF-α. Nuclear extracts immune blot assessment and immune fluorescence staining showed a significant decrease (p < 0.05) in the NF-κB p65 concentration in the nuclei with DHA treated human endothelial aortic cells. Together these findings indicate that DHA inhibits nuclear trans localization of TNF-α-induced NF-κB p65, and thus considerably suppresses (p < 0.05) the VCAM-1 expression, which contributes to lower leukocyte adherence suggesting that DHA helps in preventing inflammatory atherosclerosis in vivo.

Elucidating mechanisms of genetic cross-disease associations at the PROCR vascular disease locus

Many individual genetic risk loci have been associated with multiple common human diseases. However, the molecular basis of this pleiotropy often remains unclear. We present an integrative approach to reveal the molecular mechanism underlying the PROCR locus, associated with lower coronary artery disease (CAD) risk but higher venous thromboembolism (VTE) risk. We identify PROCR-p.Ser219Gly as the likely causal variant at the locus and protein C as a causal factor. Using genetic analyses, human recall-by-genotype and in vitro experimentation, we demonstrate that PROCR-219Gly increases plasma levels of (activated) protein C through endothelial protein C receptor (EPCR) ectodomain shedding in endothelial cells, attenuating leukocyte–endothelial cell adhesion and vascular inflammation. We also associate PROCR-219Gly with an increased pro-thrombotic state via coagulation factor VII, a ligand of EPCR. Our study, which links PROCR-219Gly to CAD through anti-inflammatory mechanisms and to VTE through pro-thrombotic mechanisms, provides a framework to reveal the mechanisms underlying similar cross-phenotype associations.

Endovascular Biopsy of Vertebrobasilar Aneurysm in Patient With Polyarteritis Nodosa.

Background and Purpose: The management of unruptured intracranial aneurysms remains controversial. The decisions to treat are heavily informed by estimated risk of bleeding. However, these estimates are imprecise, and better methods for stratifying the risk or tailoring treatment strategy are badly needed. Here, we demonstrate an initial proof-of-principle concept for endovascular biopsy to identify the key molecular pathways and gene expression changes associated with aneurysm formation. We couple this technique with single cell RNA sequencing (scRNAseq) to develop a roadmap of the pathogenic changes of a dolichoectatic vertebrobasilar aneurysm in a patient with polyarteritis nodosa.Methods: Endovascular biopsy and fluorescence activated cell sorting was used to isolate the viable endothelial cells (ECs) using the established techniques. A single cell RNA sequencing (scRNAseq) was then performed on 24 aneurysmal ECs and 23 patient-matched non-aneurysmal ECs. An integrated panel of bioinformatic tools was applied to determine the differential gene expression, enriched signaling pathways, and cell subpopulations hypothesized to drive disease pathogenesis.Results: We identify a subset of 7 (29%) aneurysm-specific ECs with a distinct gene expression signature not found in the patient-matched control ECs. A gene set enrichment analysis identified these ECs to have increased the expression of genes regulating the leukocyte-endothelial cell adhesion, major histocompatibility complex (MHC) class I, T cell receptor recycling, tumor necrosis factor alpha (TNFα) response, and interferon gamma signaling. A histopathologic analysis of a different intracranial aneurysm that was later resected yielded a diagnosis of polyarteritis nodosa and positive staining for TNFα.Conclusions: We demonstrate feasibility of applying scRNAseq to the endovascular biopsy samples and identify a subpopulation of ECs associated with cerebral aneurysm in polyarteritis nodosa. Endovascular biopsy may be a safe method for deriving insight into the disease pathogenesis and tailoring the personalized treatment approaches to intracranial aneurysms.

Improved Immunotherapy Efficacy by Vascular Modulation.

Simple SummaryAlthough immunotherapy has given the highest rate of improvement in cancer treatment in recent years, there is an urgent need to further improve its efficacy. Numerous strategies aim to transform non-responsive, immunosuppressive tumours into sensitive, immunopermissive tumours. The modulation of the tumour microenvironment, and especially the tumour vasculature offers opportunities for improved sensitivity to immunotherapy. Modulation of tumour blood vessels can enhance tumour oxygenation and T cell infiltration. Additionally, maturation of tumour blood vessels is thought to be involved in the efficient delivery of therapeutic agents. This review compiles the current strategies of vascular modulation to improve the efficacy of different immunotherapies: PD-1/PD-L1 and CTLA-4 antibodies, CAR T cells and cancer vaccines.Several strategies have been developed to modulate the tumour vasculature for cancer therapy including anti-angiogenesis and vascular normalisation. Vasculature modulation results in changes to the tumour microenvironment including oxygenation and immune cell infiltration, therefore lending itself to combination with cancer therapy. The development of immunotherapies has led to significant improvements in cancer treatment. Particularly promising are immune checkpoint blockade and CAR T cell therapies, which use antibodies against negative regulators of T cell activation and T cells reprogrammed to better target tumour antigens, respectively. However, while immunotherapy is successful in some patients, including those with advanced or metastatic cancers, only a subset of patients respond. Therefore, better predictors of patient response and methods to overcome resistance warrant investigation. Poor, or periphery-limited, T cell infiltration in the tumour is associated with poor responses to immunotherapy. Given that (1) lymphocyte recruitment requires leucocyte–endothelial cell adhesion and (2) the vasculature controls tumour oxygenation and plays a pivotal role in T cell infiltration and activation, vessel targeting strategies including anti-angiogenesis and vascular normalisation in combination with immunotherapy are providing possible new strategies to enhance therapy. Here, we review the progress of vessel modulation in enhancing immunotherapy efficacy.

Abstract 102: Apolipoprotein E Receptor 2 In Endothelium Affords Protection From Both Atherosclerosis And Insulin Resistance

Introduction: Apolipoprotein E Receptor 2 (ApoER2) is an LDL receptor family member that mediates the actions of apolipoprotein E (apoE) and other ligands. The LRP8 gene that encodes ApoER2 is a major gene locus for premature atherosclerosis and myocardial infarction. Studies were designed to determine how ApoER2 in endothelial cells impacts cardiovascular and metabolic health. Hypothesis: Based on prior cell culture studies, the hypothesis was that endothelial ApoER2 is atheroprotective. How endothelial ApoER2 impacts adiposity or glucose homeostasis is more difficult to predict. Methods: In vivo studies were done in control ApoER2 floxed mice (ApoER2 fl/fl ) and mice deficient in ApoER2 in endothelial cells (ApoER2 ΔEC ). Studies of endothelial cell insulin uptake and transcytosis were performed in human aortic and skeletal muscle endothelial cells (HAEC and HSMEC). Results: Atherosclerotic lesion severity on LDLR -/- background was increased in ApoER2 ΔEC mice compared to ApoER2 fl/fl , and this was not related to differences in circulating lipids. Intravital microscopy revealed that leukocyte-endothelial cell adhesion is increased in ApoER2 ΔEC , and vascular TNFα and IL-1β expression was exaggerated. There was no impact of endothelial ApoER2 silencing on adiposity. However, standard chow-fed ApoER2 ΔEC mice displayed marked glucose intolerance and insulin resistance. Both pancreatic insulin secretion and hepatic insulin sensitivity were normal. Alternatively, in ApoER2 ΔEC mice skeletal muscle glucose disposal was decreased by 41%, and this was due to a 55% fall in insulin delivery to muscle. Using human apoE3 ex vivo, apoE stimulated a 279% increase in insulin uptake in HAEC, and the finding was confirmed in HSMEC. Insulin transcytosis rose 209% with apoE, and apoE enhancement of insulin transport was fully ApoER2-dependent. Conclusions: Endothelial ApoER2 affords atheroprotection by decreasing leukocyte-endothelial cell adhesion and inflammatory gene expression in the vessel wall. Endothelial ApoER2 also has antidiabetic action, and this is due to the promotion of endothelial insulin transport to skeletal muscle. Targeting of the related mechanisms may provide protection from both cardiovascular and metabolic disease.

Abstract MP40: Breakpoint Cluster Region Protein Function As A Kinase Mediates The Atheroprotective Actions Of Hdl In Vascular Endothelium

Introduction: HDL has direct atheroprotective actions on endothelial cells (EC) that include the attenuation of vascular inflammation and promotion of endothelial monolayer integrity. These processes are mediated by scavenger receptor class B, type 1 (SR-BI) and its adaptor protein PDZK1. We have identified the breakpoint cluster region protein (BCR) as a PDZK1 interacting protein in EC. Best known as a gene in the BCR-ABL complex associated with the Philadelphia chromosome, BCR structure predicts both adaptor protein and kinase functions. Hypothesis: Via interaction with PDZK1 and kinase activity, BCR mediates the atheroprotective actions of HDL on EC. Methods: Newly-developed kinase assays that employ time-resolved Forster resonance energy transfer were used to detect kinase activity and identify kinase substrates. In vivo studies were done in ApoAI -/- versus ApoAI -/- ;BCR -/- mice administered control AAV or AAV-ApoAI. Leukocyte-EC interaction and carotid artery reendothelialization were evaluated by intravital microscopy and thermal injury, respectively. Atherosclerosis was invoked using AAV8-PCSK9. BCR expression was compared in human atherosclerotic and normal arteries in two independent publicly available patient cohorts. Results: In cultured EC, HDL activation of eNOS, blunting of EC-monocyte adhesion, and promotion of EC migration all required BCR, and reconstitution studies with BCR mutants revealed requirements for both BCR-PDZK1 interaction and kinase activity. In response to HDL, BCR directly phosphorylated Akt-Ser473 and it provoked Akt-Thr308 phosphorylation by PDK1. ApoAI restoration in vivo in ApoAI -/- mice prompted SR-BI-PDZK1-BCR protein complex formation and BCR phosphorylation of Akt-Ser473 in mouse aorta. It also blunted leukocyte-endothelial cell adhesion and enhanced carotid artery reendothelialization in BCR +/+ , but not in BCR -/- mice. Furthermore, the atheroprotective effect of ApoAI restoration was demonstrable in BCR +/+ , but not in BCR -/- mice. In both human cohorts BCR expression was decreased in atherosclerotic versus normal arteries. Conclusion: BCR function as a novel direct and indirect kinase of Akt mediates the atheroprotective actions of HDL in vascular endothelium.

ZNF667 attenuates leukocyte-endothelial adhesion via downregulation of P-selectin in skin flap following remote limb ischemic preconditioning.

We assessed the effects and potential mechanism of romote ischemic preconditioning (RIPC) on leukocytes-endothelium cell adhesion in the flap microvessel after ischemia-reperfusion (I/R) injury. Eight hours after reperfusion, edema and intravascular leukocyte aggregation were reduced and microvessels were more obvious in the group with superficial inferior epigastric artery (SIEA) perforator flap (SIEA-flap) subjected to RIPC than in the I/R group. Zinc finger protein 667 (ZNF667) was significantly increased but P-selectin was decreased in the flaps subjected to RIPC, compared to those in the I/R group. The low expression of P-selectin was associated with ZNF667 expression and activation in human dermal microvascular endothelial cells in response to hypoxic preconditioning. ZNF667 bound to the P-selectin promoter region, suppressing its transcription through a special core sequence. The ablation of P-selectin by small interfering RNA effectively prevented the leukocytes-endothelium cell adhesion effect of ZNF667-knockdown. ZNF667 upregulation attenuates leukocyte-endothelial cell adhesion by negatively regulating the expression of P-selectin in SIEA-flap subjected to RIPC.

Abstract P778: Hypertension and Vascular Cell Adhesion Molecule 1 Relate To % Change in National Institutes of Health Stroke Scale Score in Ischemic Stroke After Mechanical Thrombectomy

Introduction: The University of Kentucky Blood and Clot Thrombectomy Registry and Collaboration (BACTRAC) protocol utilizes thrombectomy to isolate intracranial (i.e. distal to thrombus) arterial blood and systemic (i.e. carotid) arterial blood from thrombectomy procedures to study stroke. Here, we investigate the relationship among Vascular Cell Adhesion Molecule 1 (VCAM1), hypertension (HTN), and stroke recovery in patients undergoing mechanical thrombectomy for emergent large vessel occlusion (ELVO) stroke. Methods: Intracranial and systemic plasma samples from 25 subjects underwent cardiometabolic proteomic analysis at Olink Proteomics. Demographic data including HTN status and both admission NIHSS and discharge NIHSS were included. Linear regression analysis was run on both intracranial and systemic VCAM1 expression levels against % change in NIHSS ((Admittance NIHSS - Discharge NIHSS)/Admittance NIHSS)) and two-tailed t-tests were run assessing VCAM1 expression with HTN vs. no HTN. Results: Increased expression of intracranial VCAM1 significantly correlated with a smaller % change in NIHSS (p=0.001). Similarly, increased systemic VCAM1 expression was also found to have a significant relationship with smaller % change in NIHSS (p=0.005). Subjects with hypertension had significantly higher intracranial (p=0.03) and systemic (p=0.001) VCAM1 levels compared to those without HTN. Discussion: VCAM1 mediates leukocyte-endothelial cell adhesion and has been shown to play a role in stroke. This study takes a novel approach of sampling both intracranial and systemic arterial blood during an ELVO stroke. We found increased intracranial and systemic VCAM1 independently correlate with a smaller % change in NIHSS, an indicator of poorer initial recovery. Although preliminary, these results suggest an informative role of VCAM1 levels at the time of infarct. Additionally, those with HTN had higher levels of intracranial and systemic VCAM1. These data are in line with previous studies suggesting VCAM1 is a marker of endothelial damage due to HTN leading to negative clinical outcomes. To better understand our findings, we plan to perform subset analyses investigating VCAM1 levels in relation to dyslipidemia, infarct time and infarct volume.

Reelin Depletion Protects Against Atherosclerosis by Decreasing Vascular Adhesion of Leukocytes.

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P-selectin glycoprotein ligand 1 deficiency prevents development of acute pancreatitis by attenuating leukocyte infiltration.

BACKGROUNDAcute pancreatitis (AP) is rapid-onset pancreatic inflammation that causes local and systemic inflammatory response syndrome (SIRS) with high morbidity and mortality, but no approved therapies are currently available. P-selectin glycoprotein ligand 1 (PSGL-1) is a transmembrane glycoprotein to initiate inflammatory responses. We hypothesized that PSGL-1 may be involved in the development of AP and would be a new target for the treatment of AP.AIMTo investigate the role and mechanism of PSGL-1 in the development of AP.METHODSThe PSGL-1 expression on leukocytes was detected in peripheral blood of AP patients and volunteers. Pancreatic injury, inflammatory cytokines expression, and inflammatory cell infiltration was measured in AP mouse models induced with PSGL-1 knockout (PSGL-1-/-) and wild-type (PSGL-1+/+) mice. Leukocyte-endothelial cell adhesion was measured in a peripheral blood mononuclear cell (PBMC)-endothelial cell coculture system.RESULTSThe expression of PSGL-1 on monocytes and neutrophils was significantly increased in AP patients. Compared with PSGL-1+/+ mice, PSGL-1-/- AP mice induced by caerulein exhibited lower serum amylase, less Interleukin-1beta (IL-1beta) and Interleukin-6 (IL-6) expression, less neutrophil and macrophage infiltration, and reduced peripheral neutrophil and monocyte accounts. PSGL-1 deficiency alleviated leukocyte-endothelial cell adhesion via IL-6 but not IL-1beta.CONCLUSIONPSGL-1 deficiency effectively inhibits the development of AP by preventing leukocyte-endothelial cell adhesion via IL-6 stimulation and may become a potential therapeutic target for treating AP.

The endocannabinoid 2-arachidonoylglycerol inhibits endothelial function and repair

BackgroundEndothelial dysfunction promotes atherogenesis, vascular inflammation, and thrombus formation. Reendothelialization after angioplasty is required in order to prevent stent failure. Previous studies have highlighted the role of 2-arachidonoylglycerol (2-AG) in murine experimental atherogenesis and in human coronary artery disease. However, the impact of 2-AG on endothelial repair and leukocyte–endothelial cell adhesion is still unknown. MethodsEndothelial repair was studied in two treatment groups of wildtype mice following electrical injury of the common carotid artery. One group received the monoacylglycerol lipase (MAGL)-inhibitor JZL184, which impairs 2-AG degradation and thus causes elevated 2-AG levels, the other group received DMSO (vehicle). The effect of 2-AG on human coronary artery endothelial cell (HCAEC) viability, leukocyte–endothelial cell adhesion, surface expression of adhesion molecules, and expression of endothelial NO synthase (NOS3) was studied in vitro. ResultsElevated 2-AG levels significantly impaired reendothelialization in wildtype mice following electrical injury of the common carotid artery. In vitro, 2-AG significantly reduced viability of HCAEC. Additionally, 2-AG promoted adhesion of THP-1 monocytes to HCAEC following pre-treatment of the HCAEC with 2-AG. Adhesion molecules (E-selectin, ICAM-1 and VCAM-1) remained unchanged in arterial endothelial cells, whereas 2-AG suppressed the expression of NOS3 in HCAEC. Conclusion and translational aspectElevated 2-AG levels hamper endothelial repair and HCAEC proliferation, while simultaneously facilitating leukocyte–endothelial cell adhesion. Given that 2-AG is elevated in patients with coronary artery disease and non-ST-segment elevation myocardial infarction, 2-AG might decrease reendothelialization after angioplasty and thus impact the clinical outcomes.

Effects of a synbiotic on symptoms, and daily functioning in attention deficit hyperactivity disorder – A double-blind randomized controlled trial

Some prebiotics and probiotics have been proposed to improve psychiatric symptoms in children with autism. However, few studies were placebo-controlled, and there is no study on persons with an attention deficit hyperactivity disorder (ADHD) diagnosis. Our aim was to study effects of Synbiotic 2000 on psychiatric symptoms and functioning in children and adults with ADHD without an autism diagnosis. Children and adults (n = 182) with an ADHD diagnosis completed the nine weeks randomized double-blind parallel placebo-controlled trial examining effects of Synbiotic 2000 on the primary endpoints ADHD symptoms, autism symptoms and daily functioning, and the secondary endpoint emotion regulation, measured using the questionnaires SNAP-IV, ASRS, WFIRS, SCQ, AQ and DERS-16. Levels at baseline of plasma C-reactive protein and soluble vascular cell adhesion molecule-1 (sVCAM-1), central to leukocyte-endothelial cell adhesion facilitating inflammatory responses in tissues, were measured using Meso Scale Discovery. Synbiotic 2000 and placebo improved ADHD symptoms equally well, and neither active treatment nor placebo had any statistically significant effect on functioning or sub-diagnostic autism symptoms. However, Synbiotic 2000, specifically, reduced sub-diagnostic autism symptoms in the domain restricted, repetitive and stereotyped behaviors in children, and improved emotion regulation in the domain of goal-directed behavior in adults. In children with elevated sVCAM-1 levels at baseline as well as in children without ADHD medication, Synbiotic 2000 reduced both the total score of autism symptoms, and the restricted, repetitive and stereotyped behaviors. In adults with elevated sVCAM-1 at baseline, Synbiotic 2000 significantly improved emotion regulation, both the total score and four of the five subdomains. To conclude, while no definite Synbiotic 2000-specific effect was detected, the analysis of those with elevated plasma sVCAM-1 levels proposed a reduction of autism symptoms in children and an improvement of emotion regulation in adults with ADHD.Trial registration number: ISRCTN57795429.

Enhancing Antitumor Efficacy of Heavily Vascularized Tumors by RAMBO Virus through Decreased Tumor Endothelial Cell Activation.

Vascularization is a common pathology for many solid tumors, and therefore anti-angiogenic strategies are being investigated as a therapeutic target for treatment. Numerous studies are also being conducted regarding the effects of oncolytic viruses, including ImlygicTM, an FDA approved oncolytic herpes simplex virus-1 (oHSV) for the treatment of highly vascularized tumors such as Kaposi sarcoma (NCT04065152), and brain tumors. To our knowledge, the effects of combining oncolytic HSV with angiogenesis inhibition on endothelial cell activation has not been previously described. Here, we tested the effects of Rapid Antiangiogenesis Mediated By Oncolytic Virus (RAMBO), an oHSV which expresses a potent anti-angiogenic gene Vasculostatin on endothelial cell activation in heavily vascularized solid tumors. oHSV treatment induces endothelial cell activation, which inhibits virus propagation and oncolysis in adjacent tumor cells in vitro. Consistently, this was also observed in intravital imaging of intracranial tumor-bearing mice in vivo where infected tumor endothelial cells could efficiently clear the virus without cell lysis. Quantitative real-time PCR (Q-PCR), leukocyte adhesion assay, and fluorescent microscopy imaging data, however, revealed that RAMBO virus significantly decreased expression of endothelial cell activation markers and leukocyte adhesion, which in turn increased virus replication and cytotoxicity in endothelial cells. In vivo RAMBO treatment of subcutaneously implanted sarcoma tumors significantly reduced tumor growth in mice bearing sarcoma compared to rHSVQ. In addition, histological analysis of RAMBO-treated tumor tissues revealed large areas of necrosis and a statistically significant reduction in microvessel density (MVD). This study provides strong preclinical evidence of the therapeutic benefit for the use of RAMBO virus as a treatment option for highly vascularized tumors.

Doxycycline-Dependent Self-Inactivation of CRISPR-Cas9 to Temporally Regulate On- and Off-Target Editing.

Exome and deep sequencing of cells treated with a panel of lentiviral guide RNA demonstrate that both on- and off-target editing proceed in a time-dependent manner. Thus, methods to temporally control Cas9 activity would be beneficial. To address this need, we describe a "self-inactivating CRISPR (SiC)" system consisting of a single guide RNA that deactivates the Streptococcus pyogenes Cas9 nuclease in a doxycycline-dependent manner. This enables defined, temporal control of Cas9 activity in any cell type and also invivo. Results show that SiC may enable a reduction in off-target editing, with less effect on on-target editing rates. This tool facilitates diverse applications including (1) the timed regulation of genetic knockouts in hard-to-transfect cells using lentivirus, includinghuman leukocytes for the identification of glycogenes regulating leukocyte-endothelial cell adhesion; (2) genome-wide lentiviral sgRNA (single guide RNA) library applications where Cas9 activity is ablated after allowing pre-determined editing times. Thus, stable knockout cell pools are created for functional screens; and (3) temporal control of Cas9-mediated editing of myeloidand lymphoid cells invivo, both in mouse peripheralblood and bone marrow. Overall, SiC enables temporal control of gene editing and may be applied in diverse application including studies that aim to reduce off-target genome editing.

Update on C1 Esterase Inhibitor in Human Solid Organ Transplantation.

Complement plays important roles in both ischemia-reperfusion injury (IRI) and antibody-mediated rejection (AMR) of solid organ allografts. One approach to possibly improve outcomes after transplantation is the use of C1 inhibitor (C1-INH), which blocks the first step in both the classical and lectin pathways of complement activation and also inhibits the contact, coagulation, and kinin systems. C1-INH can also directly block leukocyte-endothelial cell adhesion. C1-INH contrasts with eculizumab and other distal inhibitors, which do not affect C4b or C3b deposition or noncomplement pathways. Authors of reports on trials in kidney transplant recipients have suggested that C1-INH treatment may reduce IRI and delayed graft function, based on decreased requirements for dialysis in the first month after transplantation. This effect was particularly marked with grafts with Kidney Disease Profile Index ≥ 85. Other clinical studies and models suggest that C1-INH may decrease sensitization and donor-specific antibody production and might improve outcomes in AMR, including in patients who are refractory to other modalities. However, the studies have been small and often only single-center. This article reviews clinical data and ongoing trials with C1-INH in transplant recipients, compares the results with those of other complement inhibitors, and summarizes potentially productive directions for future research.

Nitric oxide pathology and therapeutics in sickle cell disease.

Sickle cell disease is caused by a mutant form of hemoglobin that polymerizes under hypoxic conditions which leads to red blood cell (RBC) distortion, calcium-influx mediated RBC dehydration, increased RBC adhesivity, reduced RBC deformability, increased RBC fragility, and hemolysis. These impairments in RBC structure and function result in multifaceted downstream pathology including inflammation, endothelial cell activation, platelet and leukocyte activation and adhesion, and thrombosis, all of which contribute vascular occlusion and substantial morbidity and mortality. Hemoglobin released upon RBC hemolysis scavenges nitric oxide (NO) and generates reactive oxygen species (ROS) and thereby decreases bioavailability of this important signaling molecule. As the endothelium-derived relaxing factor, NO acts as a vasodilator and also decreases platelet, leukocyte, and endothelial cell activation. Thus, low NO bioavailability contributes to pathology in sickle cell disease and its restoration could serve as an effective treatment. Despite its promise, clinical trials based on restoring NO bioavailability have so far been mainly disappointing. However, particular "NO donating" agents such as nitrite, which unlike some other NO donors can improve sickle RBC properties, may yet prove effective.