Deciphering tumour tissue organization by 3D electron microscopy and machine learning.

Despite recent progress in the characterization of tumour components, the tri-dimensional (3D) organization of this pathological tissue and the parameters determining its internal architecture remain elusive. Here, we analysed the spatial organization of patient-derived xenograft tissues generated from hepatoblastoma, the most frequent childhood liver tumour, by serial block-face scanning electron microscopy using an integrated workflow combining 3D imaging, manual and machine learning-based semi-automatic segmentations, mathematics and infographics. By digitally reconstituting an entire hepatoblastoma sample with a blood capillary, a bile canaliculus-like structure, hundreds of tumour cells and their main organelles (e.g. cytoplasm, nucleus, mitochondria), we report unique 3D ultrastructural data about the organization of tumour tissue. We found that the size of hepatoblastoma cells correlates with the size of their nucleus, cytoplasm and mitochondrial mass. We also found anatomical connections between the blood capillary and the planar alignment and size of tumour cells in their 3D milieu. Finally, a set of tumour cells polarized in the direction of a hot spot corresponding to a bile canaliculus-like structure. In conclusion, this pilot study allowed the identification of bioarchitectural parameters that shape the internal and spatial organization of tumours, thus paving the way for future investigations in the emerging onconanotomy field.

High mean arterial pressure target to improve sepsis-associated acute kidney injury in patients with prior hypertension: a feasibility study.

BACKGROUND: The optimal mean arterial pressure (MAP) in cases of septic shock is still a matter of debate in patients with prior hypertension. An MAP between 75 and 85 mmHg can improve glomerular filtration rate (GFR) but its effect on tubular function is unknown. We assessed the effects of high MAP level on glomerular and tubular renal function in two intensive care units of a teaching hospital. Inclusion criteria were patients with a history of chronic hypertension and developing AKI in the first 24 h of septic shock. Data were collected during two 6 h periods of MAP regimen administered consecutively after haemodynamic stabilisation in an order depending on the patient's admission unit: a high-target period (80-85 mmHg) and a low-target period (65-70 mmHg). The primary endpoint was the creatinine clearance (CrCl) calculated from urine and serum samples at the end of each MAP period by the UV/P formula. RESULTS: 26 patients were included. Higher urine output (+0.2 (95%:0, 0.4) mL/kg/h; P = 0.04), urine sodium (+6 (95% CI 0.2, 13) mmol/L; P = 0.04) and lower serum creatinine (- 10 (95% CI - 17, - 3) µmol/L; P = 0.03) were observed during the high-MAP period as compared to the low-MAP period, resulting in a higher CrCl (+25 (95% CI 11, 39) mL/mn; P = 0.002). The urine creatinine, urine-plasma creatinine ratio, urine osmolality, fractional excretion of sodium and urea showed no significant variation. The KDIGO stage at inclusion only interacted with serum creatinine variation and low level of sodium excretion at inclusion did not interact with these results. CONCLUSIONS: In the early stage of sepsis-associated AKI, a high-MAP target in patients with a history of hypertension was associated with a higher CrCl, but did not affect the kidneys' ability to concentrate urine, which may reflect no effect on tubular function.

In vitro comparison of three percutaneous atrial septal defect closure devices for endothelialisation and haemocompatibility.

BACKGROUND: Percutaneous device closure of atrial septal defect (ASD) is the gold-standard treatment, but several delayed complications may occur as a result of incomplete device endothelialisation. AIMS: In this in vitro study, we compared three ASD closure devices [Nit-Occlud® ASD-R (device 1); Hyperion™ ASDO (device 2); and Amplatzer™ Septal Occluder (device 3)] in terms of the endothelialisation process, using human endothelial progenitors cells (EPCs), and haemocompatibility. METHODS: EPCs from umbilical cord blood were extracted, cultured and characterised. Device samples were seeded with 100,000 cells/cm2. EPC adhesion was investigated at 3 and 24hours, and EPC proliferation was monitored, which allowed longitudinal follow-up (days 1-12). Haemocompatibility of device samples was assessed using a complement C3a assay and platelet and coagulation activation. RESULTS: With regard to EPC adhesion and proliferation, no statistically significant differences were found between the three devices. We observed for each device a significant time-dependent EPC proliferation, appearing at day 8 for devices 2 and 3 and day 10 for device 1. No complement or platelet activation occurred within 15minutes of contact with devices. However, there was minimal activation of coagulation for the three devices. CONCLUSIONS: In this in vitro study we showed that, despite the three ASD occluders having different device designs and coatings, adhesion and proliferation of human endothelial cells was similar for all devices. This should be further confirmed by similar studies including shear stress forces and anti-thrombotic treatments.

CD154 Induces Interleukin-6 Secretion by Kidney Tubular Epithelial Cells under Hypoxic Conditions: Inhibition by Chloroquine.

Inflammation is a major contributor to tubular epithelium injury in kidney disorders, and the involvement of blood platelets in driving inflammation is increasingly stressed. CD154, the ligand of CD40, is one of the mediators supporting platelet proinflammatory properties. Although hypoxia is an essential constituent of the inflammatory reaction, if and how platelets and CD154 regulate inflammation in hypoxic conditions remain unclear. Here, we studied the control by CD154 of the proinflammatory cytokine interleukin- (IL-) 6 secretion in short-term oxygen (O2) deprivation conditions, using the HK-2 cell line as a kidney tubular epithelial cell (TEC) model. IL-6 secretion was markedly stimulated by CD154 after 1 to 3 hours of hypoxic stress. Both intracellular IL-6 expression and secretion were stimulated by CD154 and associated with a strong upregulation of IL-6 mRNA and increased transcription. Searching for inhibitors of CD154-mediated IL-6 production by HK-2 cells in hypoxic conditions, we observed that chloroquine, a drug that has been repurposed as an anti-inflammatory agent, alleviated this induction. Therefore, CD154 is a potent early stimulus for IL-6 secretion by TECs in O2 deprivation conditions, a mechanism likely to take part in the deleterious inflammatory consequences of platelet activation in kidney tubular injury. The inhibition of CD154-induced IL-6 production by chloroquine suggests the potential usefulness of this drug as a therapeutic adjunct in conditions associated with acute kidney injury.

New factors for protein transport identified by a genome-wide CRISPRi screen in mammalian cells.

Protein and membrane trafficking pathways are critical for cell and tissue homeostasis. Traditional genetic and biochemical approaches have shed light on basic principles underlying these processes. However, the list of factors required for secretory pathway function remains incomplete, and mechanisms involved in their adaptation poorly understood. Here, we present a powerful strategy based on a pooled genome-wide CRISPRi screen that allowed the identification of new factors involved in protein transport. Two newly identified factors, TTC17 and CCDC157, localized along the secretory pathway and were found to interact with resident proteins of ER-Golgi membranes. In addition, we uncovered that upon TTC17 knockdown, the polarized organization of Golgi cisternae was altered, creating glycosylation defects, and that CCDC157 is an important factor for the fusion of transport carriers to Golgi membranes. In conclusion, our work identified and characterized new actors in the mechanisms of protein transport and secretion and opens stimulating perspectives for the use of our platform in physiological and pathological contexts.

Correction to: Blood platelets and sepsis pathophysiology: A new therapeutic prospect in critically ill patients?

Upon publication of the original article [1], it was noticed that the title was incorrect. Instead of 'critical', it should read 'critically', and therefore, the correct title should be.

Unconventional secretion of FABP4 by endosomes and secretory lysosomes.

An appreciation of the functional properties of the cytoplasmic fatty acid binding protein 4 (FABP4) has advanced with the recent demonstration that an extracellular form secreted by adipocytes regulates a wide range of physiological functions. Little, however, is known about the mechanisms that mediate the unconventional secretion of FABP4. Here, we demonstrate that FABP4 secretion is mediated by a membrane-bounded compartment, independent of the conventional endoplasmic reticulum-Golgi secretory pathway. We show that FABP4 secretion is also independent of GRASP proteins, autophagy, and multivesicular bodies but involves enclosure within endosomes and secretory lysosomes. We highlight the physiological significance of this pathway with the demonstration that an increase in plasma levels of FABP4 is inhibited by chloroquine treatment of mice. These findings chart the pathway of FABP4 secretion and provide a potential therapeutic means to control metabolic disorders associated with its dysregulated secretion.

Blood platelets and sepsis pathophysiology: A new therapeutic prospect in critically [corrected] ill patients?

Beyond haemostasis, platelets have emerged as versatile effectors of the immune response. The contribution of platelets in inflammation, tissue integrity and defence against infections has considerably widened the spectrum of their role in health and disease. Here, we propose a narrative review that first describes these new platelet attributes. We then examine their relevance to microcirculatory alterations in multi-organ dysfunction, a major sepsis complication. Rapid progresses that are made on the knowledge of novel platelet functions should improve the understanding of thrombocytopenia, a common condition and a predictor of adverse outcome in sepsis, and may provide potential avenues for management and therapy.

A Role for CD154, the CD40 Ligand, in Granulomatous Inflammation.

Granulomatous inflammation is a distinctive form of chronic inflammation in which predominant cells include macrophages, epithelioid cells, and multinucleated giant cells. Mechanisms regulating granulomatous inflammation remain ill-understood. CD154, the ligand of CD40, is a key mediator of inflammation. CD154 confers a proinflammatory phenotype to macrophages and controls several macrophagic functions. Here, we studied the contribution of CD154 in a mouse model of toxic liver injury with carbon tetrachloride and a model of absorbable suture graft. In both models, granulomas are triggered in response to endogenous persistent liver calcified necrotic lesions or by grafted sutures. CD154-deficient mice showed delayed clearance of carbon tetrachloride-induced liver calcified necrotic lesions and impaired progression of suture-induced granuloma. In vitro, CD154 stimulated phagocytosis of opsonized erythrocytes by macrophages, suggesting a potential mechanism for the altered granulomatous inflammation in CD154KO mice. These results suggest that CD154 may contribute to the natural history of granulomatous inflammation.

CD40 signaling and hepatic steatosis: Unanticipated links.

Obesity predisposes to an increased risk of nonalcoholic fatty liver disease (NAFLD). Hepatic steatosis is the key pathological feature of NAFLD and has emerged as a metabolic disorder in which innate and adaptive arms of the immune response play a central role in disease pathogenesis. Recent studies have revealed unexpected relationships between CD40 signaling and hepatic steatosis in high fat diet rodent models. CD154, the ligand of CD40, is a mediator of inflammation and controls several critical events of innate and adaptive immune responses. In the light of these reports, we discuss potential links between CD40 signaling and hepatic steatosis in NAFLD.

CD154 Induces Matrix Metalloproteinase-9 Secretion in Human Podocytes.

Matrix remodeling is a key feature of glomerulosclerosis secondary to diabetes or hypertension. Podocytes contribute to glomerular basement membrane (GBM) turnover by producing matrix components and matrix remodelling enzymes, including matrix metalloproteinases (MMPs). The CD40/CD154 signaling pathway modulates matrix remodeling through the synthesis of MMPs and tissue inhibitors of MMPs. Platelets are a primary blood reservoir of CD154. Here we studied, the impact of the CD154/CD40 pathway on MMP-9 expression by cultured human podocytes. The role of CD40/CD154 was evaluated upon exposure of podocytes to recombinant human CD154 (rhCD154) or activated platelet supernatants from healthy human subjects. We first showed by protein and mRNA expression that CD40 was synthesized by podocytes and detectable on kidney tissue sections. CD40 expression was acquired during podocyte differentiation and enhanced upon exposure to rhCD154. In podocytes, rhCD154 induced an increase of MMP-9 production as shown by RT-PCR, Western blot and and gelatin zymography. Activated platelet supernatants induced MMP-9 mRNA synthesis in podocytes, an effect reduced by anti-CD40 antibody. Our results underscore a potential role for platelets through the CD40/CD154 signaling pathway in the control of GBM synthesis and degradation, via its regulatory role on MMP-9 production. CD154 secretion by activated platelets may contribute to GBM alterations in proteinuric nephropathies. J. Cell. Biochem. 117: 2737-2747, 2016. © 2016 Wiley Periodicals, Inc.

Beneficial Effect of Covalently Grafted α-MSH on Endothelial Release of Inflammatory Mediators for Applications in Implantable Devices.

Intravascular devices for continuous glucose monitoring are promising tools for the follow up and treatment of diabetic patients. Limiting the inflammatory response to the implanted devices in order to achieve better biocompatibility is a critical challenge. Herein we report on the production and the characterization of gold surfaces covalently derivatized with the peptide α-alpha-melanocyte stimulating hormone (α-MSH), with a quantifiable surface density. In vitro study demonstrated that the tethered α-MSH is able to decrease the expression of an inflammatory cytokine produced by endothelial cells.

Kinetic eGFR and Novel AKI Biomarkers to Predict Renal Recovery.

BACKGROUND AND OBJECTIVES: Prompt recognition of severe renal impairment could improve the early management of critically ill patients. We compared the value of kinetic eGFR, plasma neutrophil gelatinase-associated lipocalin (NGAL), and urine tissue inhibitor of metalloproteinase-2 and urine insulin-like growth factor-binding protein 7 ([TIMP-2]*[IGFBP7]) in predicting short-term recovery from AKI and major adverse kidney events. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS: During the 6-month study period, 245 patients were admitted to our intensive care unit. This study included 57 consecutive patients presenting with AKI within the first 24 hours after admission. AKI markers were evaluated at inclusion (day 0) and 24 hours later (day 1). Kinetic eGFR was calculated on day 1 according to serum creatinine evolution. Renal recovery was defined as normalization of serum creatinine with reversal of oliguria within 48 hours. Major adverse kidney events included death, need for RRT, or persistence of renal dysfunction at hospital discharge. RESULTS: Plasma NGAL and [TIMP-2]*[IGFBP7] predicted renal recovery, with area under the receiver-operating characteristic curve (AUC-ROC) values between 0.70 and 0.79 at inclusion. Although plasma NGAL values frequently reached the maximal measurement range, their decrease on day 1 predicted recovery. The kinetic eGFR calculation after initial resuscitation provided the best AUC-ROC value for renal recovery, at 0.87. The best predictions for major adverse kidney events were provided by [TIMP-2]*[IGFBP7] and kinetic eGFR (equal AUC-ROCs of 0.81). Combining AKI markers in addition to clinical prediction models improved the discrimination and reclassification of patients who will recover from AKI or suffer from major adverse kidney events. CONCLUSIONS: Biomarkers of kidney damage predicted short-term renal recovery and major adverse kidney events for an unselected cohort of critically ill patients. Calculating the kinetic eGFR imposed a delay after initial resuscitation but provided a good diagnostic and prognostic approach. The utility of functional and damage AKI marker combinations in addition to clinical information requires validation in larger prospective studies.

New frontiers for platelet CD154.

The role of platelets extends beyond hemostasis. The pivotal role of platelets in inflammation has shed new light on the natural history of conditions associated with acute or chronic inflammation. Beyond the preservation of vascular integrity, platelets are essential to tissue homeostasis and platelet-derived products are already used in the clinics. Unanticipated was the role of platelets in the adaptative immune response, allowing a renewed conceptual approach of auto-immune diseases. Platelets are also important players in cancer growth and dissemination. Platelets fulfill most of their functions through the expression of still incompletely characterized membrane-bound or soluble mediators. Among them, CD154 holds a peculiar position, as platelets represent a major source of CD154 and as CD154 contributes to most of these new platelet attributes. Here, we provide an overview of some of the new frontiers that the study of platelet CD154 is opening, in inflammation, tissue homeostasis, immune response, hematopoiesis and cancer.

IQ domain GTPase-activating protein 1 is involved in shear stress-induced progenitor-derived endothelial cell alignment.

Shear stress is one of mechanical constraints which are exerted by blood flow on endothelial cells (ECs). To adapt to shear stress, ECs align in the direction of flow through adherens junction (AJ) remodeling. However, mechanisms regulating ECs alignment under shear stress are poorly understood. The scaffold protein IQ domain GTPase activating protein 1 (IQGAP1) is a scaffold protein which couples cell signaling to the actin and microtubule cytoskeletons and is involved in cell migration and adhesion. IQGAP1 also plays a role in AJ organization in epithelial cells. In this study, we investigated the potential IQGAP1 involvement in the endothelial cells alignment under shear stress. Progenitor-derived endothelial cells (PDECs), transfected (or not) with IQGAP1 small interfering RNA, were exposed to a laminar shear stress (1.2 N/m(2)) and AJ proteins (VE-cadherin and ß-catenin) and IQGAP1 were labeled by immunofluorescence. We show that IQGAP1 is essential for ECs alignment under shear stress. We studied the role of IQGAP1 in AJs remodeling of PDECs exposed to shear stress by studying cell localization and IQGAP1 interactions with VE-cadherin and ß-catenin by immunofluorescence and Proximity Ligation Assays. In static conditions, IQGAP1 interacts with VE-cadherin but not with ß-catenin at the cell membrane. Under shear stress, IQGAP1 lost its interaction from VE-cadherin to ß-catenin. This "switch" was concomitant with the loss of ß-catenin/VE-cadherin interaction at the cell membrane. This work shows that IQGAP1 is essential to ECs alignment under shear stress and that AJ remodeling represents one of the mechanisms involved. These results provide a new approach to understand ECs alignment under to shear stress.

Doppler resistive index to reflect regulation of renal vascular tone during sepsis and acute kidney injury.

INTRODUCTION: Renal resistive index (RI), determined by Doppler ultrasonography, directly reveals and quantifies modifications in renal vascular resistance. The aim of this study was to evaluate if mean arterial pressure (MAP) is determinant of renal RI in septic, critically ill patients suffering or not from acute kidney injury (AKI). METHODS: This prospective observational study included 96 patients. AKI was defined according to RIFLE criteria and transient or persistent AKI according to renal recovery within 3 days. RESULTS: Median renal RIs were 0.72 (0.68-0.75) in patients without AKI and 0.76 (0.72-0.80) in patients with AKI (P = 0.001). RIs were 0.75 (0.72-0.79) in transient AKI and 0.77 (0.70-0.80) in persistent AKI (P = 0.84). RI did not differ in patients given norepinephrine infusion and was not correlated with norepinephrine dose. RI was correlated with MAP (ρ = -0.47; P = 0.002), PaO2/FiO2 ratio (ρ = -0.33; P = 0.04) and age (ρ = 0.35; P = 0.015) only in patients without AKI. CONCLUSIONS: A poor correlation between renal RI and MAP, age, or PaO2/FiO2 ratio was found in septic and critically ill patients without AKI compared to patients with AKI. These findings suggest that determinants of RI are multiple. Renal circulatory response to sepsis estimated by Doppler ultrasonography cannot reliably be predicted simply from changes in systemic hemodynamics. As many factors influence its value, the interest in a single RI measurement at ICU admission to determine optimal MAP remains uncertain.

IQGAP1 interacts with components of the slit diaphragm complex in podocytes and is involved in podocyte migration and permeability in vitro.

IQGAP1 is a scaffold protein that interacts with proteins of the cytoskeleton and the intercellular adhesion complex. In podocytes, IQGAP1 is associated with nephrin in the glomerular slit diaphragm (SD) complex, but its role remains ill-defined. In this work, we investigated the interaction of IQGAP1 with the cytoskeleton and SD proteins in podocytes in culture, and its role in podocyte migration and permeability. Expression, localization, and interactions between IQGAP1 and SD or cytoskeletal proteins were determined in cultured human podocytes by Western blot (WB), immunocytolocalization (IC), immunoprecipitation (IP), and In situ Proximity Ligation assay (IsPL). Involvement of IQGAP1 in migration and permeability was also assessed. IQGAP1 expression in normal kidney biopsies was studied by immunohistochemistry. IQGAP1 expression by podocytes increased during their in vitro differentiation. IC, IP, and IsPL experiments showed colocalizations and/or interactions between IQGAP1 and SD proteins (nephrin, MAGI-1, CD2AP, NCK 1/2, podocin), podocalyxin, and cytoskeletal proteins (α-actinin-4). IQGAP1 silencing decreased podocyte migration and increased the permeability of a podocyte layer. Immunohistochemistry on normal human kidney confirmed IQGAP1 expression in podocytes and distal tubular epithelial cells and also showed an expression in glomerular parietal epithelial cells. In summary, our results suggest that IQGAP1, through its interaction with components of SD and cytoskeletal proteins, is involved in podocyte barrier properties.

A protective role for CD154 in hepatic steatosis in mice.

UNLABELLED: Inflammation and lipid metabolism pathways are linked, and deregulation of this interface may be critical in hepatic steatosis. The importance of the dialog between inflammatory signaling pathways and the unfolded protein response (UPR) in metabolism has been underlined. Herein, we studied the role of CD154, a key mediator of inflammation, in hepatic steatosis. To this end, Balb/c mice, wild-type or deficient in CD154 (CD154KO), were fed a diet rich in olive oil. In vitro, the effect of CD154 was studied on primary hepatocyte cultures and hepatocyte-derived cell lines. Results showed that CD154KO mice fed a diet rich in olive oil developed hepatic steatosis associated with reduced apolipoprotein B100 (apoB100) expression and decreased secretion of very low-density lipoproteins. This phenotype correlated with an altered UPR as assessed by reduced X-Box binding protein-1 (XBP1) messenger RNA (mRNA) splicing and reduced phosphorylation of eukaryotic initiation factor 2α. Altered UPR signaling in livers of CD154KO mice was confirmed in tunicamycin (TM) challenge experiments. Treatment of primary hepatocyte cultures and hepatocyte-derived cell lines with soluble CD154 increased XBP1 mRNA splicing in cells subjected to either oleic acid (OA) or TM treatment. Moreover, CD154 reduced the inhibition of apoB100 secretion by HepG2 cells grown in the presence of high concentrations of OA, an effect suppressed by XBP1 mRNA silencing and in HepG2 cells expressing a dominant negative form of inositol requiring ER-to-nucleus signaling protein-1. The control of the UPR by CD154 may represent one of the mechanisms involved in the pathophysiology of hepatic steatosis. CONCLUSION: Our study identifies CD154 as a new mediator of hepatic steatosis.