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Supplemental Digital Content is available in the text., Background: Cellular diversity of the lung endothelium has not been systematically characterized in humans. We provide a reference atlas of human lung endothelial cells (ECs) to facilitate a better understanding of the phenotypic diversity and composition of cells comprising the lung endothelium. Methods: We reprocessed human control single-cell RNA sequencing (scRNAseq) data from 6 datasets. EC populations were characterized through iterative clustering with subsequent differential expression analysis. Marker genes were validated by fluorescent microscopy and in situ hybridization. scRNAseq of primary lung ECs cultured in vitro was performed. The signaling network between different lung cell types was studied. For cross-species analysis or disease relevance, we applied the same methods to scRNAseq data obtained from mouse lungs or from human lungs with pulmonary hypertension. Results: Six lung scRNAseq datasets were reanalyzed and annotated to identify >15 000 vascular EC cells from 73 individuals. Differential expression analysis of EC revealed signatures corresponding to endothelial lineage, including panendothelial, panvascular, and subpopulation-specific marker gene sets. Beyond the broad cellular categories of lymphatic, capillary, arterial, and venous ECs, we found previously indistinguishable subpopulations; among venous EC, we identified 2 previously indistinguishable populations: pulmonary–venous ECs (COL15A1neg) localized to the lung parenchyma and systemic–venous ECs (COL15A1pos) localized to the airways and the visceral pleura; among capillary ECs, we confirmed their subclassification into recently discovered aerocytes characterized by EDNRB, SOSTDC1, and TBX2 and general capillary EC. We confirmed that all 6 endothelial cell types, including the systemic–venous ECs and aerocytes, are present in mice and identified endothelial marker genes conserved in humans and mice. Ligand-receptor connectome analysis revealed important homeostatic crosstalk of EC with other lung resident cell types. scRNAseq of commercially available primary lung ECs demonstrated a loss of their native lung phenotype in culture. scRNAseq revealed that endothelial diversity is maintained in pulmonary hypertension. Our article is accompanied by an online data mining tool (www.LungEndothelialCellAtlas.com). Conclusions: Our integrated analysis provides a comprehensive and well-crafted reference atlas of ECs in the normal lung and confirms and describes in detail previously unrecognized endothelial populations across a large number of humans and mice.

Objectives Resolution of impaired microvascular flow may lag the normalization of macrocirculatory variables. The significance of microcirculatory dysfunction in critically ill children and neonates is unknown, but microcirculatory variables can be measured using Doppler or videomicroscopy imaging techniques. We outline the current understanding of the role of the microcirculation in critical illness, review methods for its assessment, and perform a systematic review of how it has been monitored in critically ill neonates and children. Design Systematic review (PROSPERO CRD42019117993). Setting Not applicable. Subjects Not applicable. Interventions None. Measurements and results We systematically searched MEDLINE, EMBASE, PubMed, and Web of Science. We included studies of critically ill patients 0 to 18 years old investigating microcirculatory blood flow. Two reviewers analyzed abstracts and articles. Results were qualitatively analyzed due to study heterogeneity. A total of 2,559 abstracts met search criteria, of which 94 underwent full-text review. Of those, 36 met inclusion criteria. Seven studies investigated microcirculatory changes in critically ill children. Twenty studies investigated the microcirculatory changes in neonates with variable diagnoses compared with a diverse set of clinical endpoints. Nine studies assessed the effects of age, sex, and birth weight on microvascular flow in neonates. Across all studies, microcirculatory dysfunction was associated with poor outcomes and may not correlate with observed macrovascular function. Conclusions Assessment of microvascular flow in critically ill children and neonates is possible, although significant challenges remain. In many such patients, microvascular blood flow is disrupted despite medical management targeting normalized macrovascular variables. Future studies are needed to define normal pediatric microvascular flow variables and to assess the impact of patient and treatment factors on its function.

Rationale: BMX (bone marrow kinase on the X chromosome) is highly expressed in the arterial endothelium from the embryonic stage to the adult stage in mice. It is also expressed in microvessels and the lymphatics in response to pathological stimuli. However, its role in endothelial permeability and sepsis remains unknown. Objective: We aimed to delineate the function of BMX in thrombin-mediated endothelial permeability and the vascular leakage that occurs with sepsis in cecal ligation and puncture models. Methods and Results: The cecal ligation and puncture model was applied to WT (wild type) and BMX-KO (BMX global knockout) mice to induce sepsis. Meanwhile, the electric cell-substrate impedance sensing assay was used to detect transendothelial electrical resistance in vitro and, the modified Miles assay was used to evaluate vascular leakage in vivo. We showed that BMX loss caused lung injury and inflammation in early cecal ligation and puncture–induced sepsis. Disruption of BMX increased thrombin-mediated permeability in mice and cultured endothelial cells by 2- to 3-fold. The expression of BMX in macrophages, neutrophils, platelets, and lung epithelial cells was undetectable compared with that in endothelial cells, indicating that endothelium dysfunction, rather than leukocyte and platelet dysfunction, was involved in vascular permeability and sepsis. Mechanistically, biochemical and cellular analyses demonstrated that BMX specifically repressed thrombin-PAR1 (protease-activated receptor-1) signaling in endothelial cells by directly phosphorylating PAR1 and promoting its internalization and deactivation. Importantly, pretreatment with the selective PAR1 antagonist SCH79797 rescued BMX loss-mediated endothelial permeability and pulmonary leakage in early cecal ligation and puncture–induced sepsis. Conclusions: Acting as a negative regulator of PAR1, BMX promotes PAR1 internalization and signal inactivation through PAR1 phosphorylation. Moreover, BMX-mediated PAR1 internalization attenuates endothelial permeability to protect vascular leakage during early sepsis.

Objective Systemic capillary leak syndrome (SCLS) is a rare disorder that presents with episodes of hypovolemic shock. The extent to which genetic abnormalities contribute to SCLS is unknown. We identified pediatric and adult cohorts with characteristic clinical courses. We sought to describe the clinical characteristics of both cohorts, identify a possible genetic contribution to SCLS, and demonstrate that whole-exome sequencing (WES) may be conducted by critical care providers. Design Prospective observational study of WES of nine adult and eight pediatric SCLS patients and available unaffected first-degree relatives. Setting Tertiary children's hospitals and referral research laboratory. Patients Children and adults with SCLS. Interventions None. Measurements Patients and available first-degree relatives underwent WES. Data were analyzed for rare homozygous, biallelic, de novo, and heterozygous variants with allelic enrichment and metabolic pathway analyses. Main results Children with SCLS presented at a younger age with episodes similar to those experienced by adults. All patients and available relatives underwent satisfactory WES. No overlapping gene variants or metabolic pathways were identified across all SCLS patients. Multiple candidate genes with homozygous or biallelic mutations were identified in individual subjects with SCLS. There was no significant enrichment of genes with rare heterozygous variants. Conclusions The clinical characteristics of children and adults with SCLS are similar. We did not identify a uniform germline exomic genetic etiology for SCLS. WES identified several candidate genes in individual patients for future research. WES is a viable way for critical care providers to investigate the etiology of diseases with presumed genetic contributions.

OBJECTIVE Angiopoietins are postulated diagnostic biomarkers in children and adults with severe sepsis and septic shock. The diagnostic value of angiopoietins in children less than 5 years old has not been established, nor has their effect on permeability in the capillary microvasculature. We aim to determine if levels of angiopoietin-1 or -2 (angpt-1, -2) are diagnostic for severe sepsis/shock in young children and whether they affect the permeability of cultured human dermal microvascular endothelial cells (HDMEC). DESIGN Prospective observational study of children < 5 years old. Patients were classified as non-systemic inflammatory response syndrome (SIRS), SIRS/sepsis and severe sepsis/septic shock. SETTING Tertiary care pediatric hospitals. PATIENTS Critically ill children. INTERVENTIONS None. MEASUREMENTS Plasma angpt-1 and -2 levels were measured with enzyme-linked immunoassays. Expression of angpt-2 in endothelial cells was assessed with quantitative polymerase chain reaction. Permeability changes in cultured HDMECs were assessed with transendothelial electrical resistance measurements. RESULTS Angpt-1 levels were significantly higher in younger children compared with levels found in previous study of older children across disease severity (all P

Capillary endothelial cells (ECs) maintain a semi-permeable barrier between the blood and tissue by forming inter-EC tight junctions (TJs), regulating selective transport of fluid and solutes. Overwhelming inflammation, as occurs in sepsis, disrupts these TJs, leading to leakage of fluid, proteins, and small molecules into the tissues. Mechanistically, disruption of capillary barrier function is mediated by small Rho-GTPases, such as RhoA, -B, and -C, which are activated by guanine nucleotide exchange factors (GEFs) and disrupted by GTPase-activating factors (GAPs). We previously reported that a mutation in a specific RhoB GAP (p190BRhoGAP) underlays a hereditary capillary leak syndrome. Tumor necrosis factor (TNF) treatment disrupts TJs in cultured human microvascular ECs, a model of capillary leak. This response requires new gene transcription and involves increased RhoB activation. However, the specific GEF that activates RhoB in capillary ECs remains unknown. Transcriptional profiling of cultured tight junction-forming human dermal microvascular endothelial cells (HDMECs) revealed that 17 GEFs were significantly induced by TNF. The function of each candidate GEF was assessed by short interfering RNA depletion and trans-endothelial electrical resistance screening. Knockown of ArhGEF10 reduced the TNF-induced loss of barrier which was phenocopied by RhoB or dual ArhGEF10/RhoB knockdown. ArhGEF10 knockdown also reduced the extent of TNF-induced RhoB activation and disruption at tight junctions. In a cell-free assay, immunoisolated ArhGEF10 selectively catalyzed nucleotide exchange to activate RhoB, but not RhoA or RhoC. We conclude ArhGEF10 is a TNF-induced RhoB-selective GEF that mediates TJ disruption and barrier loss in human capillary endothelial cells.

Multisystem inflammatory syndrome in children (MIS-C) is a life-threatening post-infectious complication occurring unpredictably weeks after mild or asymptomatic SARS-CoV-2 infection. We profiled MIS-C, adult COVID-19, and healthy pediatric and adult individuals using single-cell RNA sequencing, flow cytometry, antigen receptor repertoire analysis, and unbiased serum proteomics, which collectively identified a signature in MIS-C patients that correlated with disease severity. Despite having no evidence of active infection, MIS-C patients had elevated S100A-family alarmins and decreased antigen presentation signatures, indicative of myeloid dysfunction. MIS-C patients showed elevated expression of cytotoxicity genes in NK and CD8+ T cells and expansion of specific IgG-expressing plasmablasts. Clinically severe MIS-C patients displayed skewed memory T cell TCR repertoires and autoimmunity characterized by endothelium-reactive IgG. The alarmin, cytotoxicity, TCR repertoire, and plasmablast signatures we defined have potential for application in the clinic to better diagnose and potentially predict disease severity early in the course of MIS-C., Graphical abstract, Multisystem inflammatory syndrome in children (MIS-C) is a life-threatening and unpredictable condition of unknown etiology. Ramaswamy et al. use peripheral blood single-cell transcriptomic profiling along with other techniques to define key innate and adaptive signatures that characterize MIS-C.

There are currently limited Food and Drug Administration (FDA)-approved drugs and vaccines for the treatment or prevention of Coronavirus Disease 2019 (COVID-19). Enhanced understanding of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infection and pathogenesis is critical for the development of therapeutics. To provide insight into viral replication, cell tropism, and host–viral interactions of SARS-CoV-2, we performed single-cell (sc) RNA sequencing (RNA-seq) of experimentally infected human bronchial epithelial cells (HBECs) in air–liquid interface (ALI) cultures over a time course. This revealed novel polyadenylated viral transcripts and highlighted ciliated cells as a major target at the onset of infection, which we confirmed by electron and immunofluorescence microscopy. Over the course of infection, the cell tropism of SARS-CoV-2 expands to other epithelial cell types including basal and club cells. Infection induces cell-intrinsic expression of type I and type III interferons (IFNs) and interleukin (IL)-6 but not IL-1. This results in expression of interferon-stimulated genes (ISGs) in both infected and bystander cells. This provides a detailed characterization of genes, cell types, and cell state changes associated with SARS-CoV-2 infection in the human airway., Single-cell analysis of human airway epithelial cells infected with SARS-CoV-2 provides novel insights into viral replication, cell tropism, and host-viral interactions. This study reveals novel polyadenylated viral transcripts, preferential tropism for ciliated cells that later extends to other epithelial cell types, and cell-intrinsic expression of type I and type III IFNs and IL6 induced by infection, resulting in expression of interferon-stimulated genes in both infected and bystander cells.

SARS-CoV-2, the causative agent of COVID-19, has tragically burdened individuals and institutions around the world. There are currently no approved drugs or vaccines for the treatment or prevention of COVID-19. Enhanced understanding of SARS-CoV-2 infection and pathogenesis is critical for the development of therapeutics. To reveal insight into viral replication, cell tropism, and host-viral interactions of SARS-CoV-2 we performed single-cell RNA sequencing of experimentally infected human bronchial epithelial cells (HBECs) in air-liquid interface cultures over a time-course. This revealed novel polyadenylated viral transcripts and highlighted ciliated cells as a major target of infection, which we confirmed by electron microscopy. Over the course of infection, cell tropism of SARS-CoV-2 expands to other epithelial cell types including basal and club cells. Infection induces cell-intrinsic expression of type I and type III IFNs and IL6 but not IL1. This results in expression of interferon-stimulated genes in both infected and bystander cells. We observe similar gene expression changes from a COVID-19 patient ex vivo. In addition, we developed a new computational method termed CONditional DENSity Embedding (CONDENSE) to characterize and compare temporal gene dynamics in response to infection, which revealed genes relating to endothelin, angiogenesis, interferon, and inflammation-causing signaling pathways. In this study, we conducted an in-depth analysis of SARS-CoV-2 infection in HBECs and a COVID-19 patient and revealed genes, cell types, and cell state changes associated with infection.

Acute lung injury (ALI) causes high mortality and lacks any pharmacological intervention. Here, we found that pazopanib ameliorated ALI manifestations and reduced mortality in mouse ALI models and reduced edema in human lung transplantation recipients. Pazopanib inhibits mitogen-activated protein kinase kinase kinase 2 (MAP3K2)–and MAP3K3-mediated phosphorylation of NADPH oxidase 2 subunit p47(phox) at Ser(208) to increase reactive oxygen species (ROS) formation in myeloid cells. Genetic inactivation of MAP3K2 and MAP3K3 in myeloid cells or hematopoietic mutation of p47(phox) Ser(208) to alanine attenuated ALI manifestations and abrogates anti-ALI effects of pazopanib. This myeloid MAP3K2/MAP3K3-p47(phox) pathway acted via paracrine H(2)O(2) to enhance pulmonary vasculature integrity and promote lung epithelial cell survival and proliferation, leading to increased pulmonary barrier function and resistance to ALI. Thus, pazopanib has the potential to be effective for treating ALI.

Objectives Children undergoing cardiopulmonary bypass develop clinically impactful capillary leak of unclear etiology. A widely held hypothesis that exposure of circulating cells to the cardiopulmonary bypass circuit induces the release of inflammatory mediators that act to disrupt intercellular junctions of capillary endothelial cells inducing paracellular capillary leak either directly or through new gene expression. Design Cohort study. Setting Tertiary pediatric hospital. Patients Twenty children undergoing surgery with cardiopulmonary bypass for congenital heart disease. Serum was collected before cardiopulmonary bypass, 2 hours after cardiopulmonary bypass, and 18 hours after cardiopulmonary bypass. Interventions None. Measurements and main results We analyzed the effects of 10% patient sera on the "function, structure, and gene expression" of cultured human dermal and pulmonary microvascular endothelial cells. Changes in barrier "function" were measured using transendothelial electrical resistance. Associations between changes in transendothelial electrical resistance and subject characteristics were analyzed using linear mixed effects model with area under the resistance curve as outcome. Changes in junctional "structure" were assessed by analyzing the organization of the endothelial cell junctional proteins claudin-5 and VE-cadherin using immunofluorescence microscopy. Changes in inflammatory "gene expression" were measured using real-time quantitative reverse transcription-polymerase chain reaction. All serum samples induced a transient, 120-minute increase in transendothelial electrical resistance followed by persistent loss of barrier function. Unexpectedly, sera collected postcardiopulmonary bypass-induced significantly less loss of barrier function in both dermal and pulmonary capillary endothelial cell compared with precardiopulmonary bypass sera. Consistent with the transendothelial electrical resistance results, claudin-5 and vascular endothelial-cadherin junctional staining showed less disruption in cultures treated with postcardiopulmonary bypass sera. Expression of genes commonly associated with inflammation was largely unaffected by patient sera. Conclusions Contrary to the hypothesis, sera taken from children after cardiopulmonary bypass induces less capillary barrier disruption relative to sera taken from children before cardiopulmonary bypass, and none of the sera induced significant changes in expression of inflammatory genes.

We describe a case of succinyl-CoA:3-oxoacid CoA transferase (SCOT) deficiency in an otherwise healthy 14 month-old female. She presented with lethargy, tachypnea, and hyperpnea with hypoglycemia and a severe anion gap metabolic acidosis. Early management included correction of the acidosis and metabolic support with dextrose and insulin. Inborn errors of metabolism are rare outside the neonatal period. However, SCOT deficiency may present at older ages. Maintaining a high index of suspicion, immediate transfer to a pediatric intensive care unit, and prompt metabolic support are key to achieving a favorable outcome.

Pierce et al. describe a pediatric patient with a fatal systemic capillary leak syndrome (Clarkson’s disease). They identify a point mutation in p190BRhoGAP and show that patient-derived microvascular endothelial cells show prolonged activation RhoB that correlates with impaired barrier recovery after treatment with TNF compared with control cultures., We describe a fatal case of pediatric systemic capillary leak (Clarkson’s disease) associated with a point mutation in p190BRhoGAP. Dermal microvascular endothelial cells (ECs) isolated from this patient form monolayers with similar levels and distribution of junctional proteins and transendothelial electrical resistance compared with normal human dermal microvascular ECs. However, patient-derived ECs demonstrate a greater increase in permeability and impaired recovery of barrier function in response to tumor necrosis factor (TNF) compared with normal donor EC cultures. TNF transiently activates RhoB in ECs coincident with developing leak, and inactivation of RhoB correlates with barrier recovery. The mutation in p190BRhoGAP impairs RhoB inactivation, and the mutant phenotype of patient-derived ECs is replicated by siRNA knockdown of p190BRhoGAP in normal ECs. These data suggest a previously unknown function for p190BRhoGAP in control of capillary EC barrier function that may also be important in acquired systemic capillary leak associated with critical illness in humans.

The purpose of this review is to highlight the significant advances in the testing, interpretation, and diagnosis of genetic abnormalities in critically ill children and to emphasize that pediatric intensivists are uniquely positioned to search for genetic diagnoses in these patients.Ten years following the first clinical diagnosis made through whole exome sequencing, we remain in the dark about the function of roughly 75% of our genes. However, steady advancements in molecular techniques, particularly next-generation sequencing, have spurred a rapid expansion of our understanding of the genetic underpinnings of severe congenital diseases. This has resulted in not only improved clinical diagnostics but also a greater availability of research programs actively investigating rare, undiagnosed diseases. In this background, the scarcity of clinical geneticists compels nongeneticists to familiarize themselves with the types of patients that could benefit from genetic testing, interpretations of test results as well as the available resources for these patients.When caring for seriously ill children, critical care pediatricians should actively seek the possibility of an underlying genetic cause for their patients' conditions. This is true even in instances when a child has a descriptive diagnosis without a clear underlying molecular genetic mechanism. By promoting such diagnostics, in both clinical and research settings, pediatric intensivists can advance the care of their patients, improve the quality of information provided to families, and contribute to the knowledge of broad fields in medicine.

Tissue engineering may address organ shortages currently limiting clinical transplantation. Off-the-shelf engineered vascularized organs will likely use allogeneic endothelial cells (ECs) to construct microvessels required for graft perfusion. Vasculogenic ECs can be differentiated from committed progenitors (human endothelial colony-forming cells or HECFCs) without risk of mutation or teratoma formation associated with reprogrammed stem cells. Like other ECs, these cells can express both class I and class II major histocompatibility complex (MHC) molecules, bind donor-specific antibody (DSA), activate alloreactive T effector memory cells, and initiate rejection in the absence of donor leukocytes. CRISPR/Cas9-mediated dual ablation of β(2)-microglobulin and class II transactivator (CIITA) in HECFC-derived ECs eliminates both class I and II MHC expression while retaining EC functions and vasculogenic potential. Importantly, dually ablated ECs no longer bind human DSA or activate allogeneic CD4(+) effector memory T cells and are resistant to killing by CD8(+) alloreactive cytotoxic T lymphocytes in vitro and in vivo. Despite absent class I MHC molecules, these ECs do not activate or elicit cytotoxic activity from allogeneic natural killer cells. These data suggest that HECFC-derived ECs lacking MHC molecule expression can be utilized for engineering vascularized grafts that evade allorejection.

A classical hallmark of acute inflammation is neutrophil infiltration of tissues, a multistep process that involves sequential cell–cell interactions of circulating leukocytes with IL-1– or TNF-activated microvascular endothelial cells (ECs) and pericytes (PCs) that form the wall of the postcapillary venules. The initial infiltrating cells accumulate perivascularly in close proximity to PCs. IL-17, a proinflammatory cytokine that acts on target cells via a heterodimeric receptor formed by IL-17RA and IL-17RC subunits, also promotes neutrophilic inflammation but its effects on vascular cells are less clear. We report that both cultured human ECs and PCs strongly express IL-17RC and, although neither cell type expresses much IL-17RA, PCs express significantly more than ECs. IL-17, alone or synergistically with TNF, significantly alters inflammatory gene expression in cultured human PCs but not ECs. RNA sequencing analysis identifies many IL-17–induced transcripts in PCs encoding proteins known to stimulate neutrophil-mediated immunity. Conditioned media from IL-17–activated PCs, but not ECs, induce pertussis toxin–sensitive neutrophil polarization, likely mediated by PC-secreted chemokines, and they also stimulate neutrophil production of proinflammatory molecules, including TNF, IL-1α, IL-1β, and IL-8. Furthermore, IL-17–activated PCs, but not ECs, can prolong neutrophil survival by producing G-CSF and GM-CSF, delaying the mitochondrial outer membrane permeabilization and caspase-9 activation. Importantly, neutrophils exhibit enhanced phagocytic capacity after activation by conditioned media from IL-17–treated PCs. We conclude that PCs, not ECs, are the major target of IL-17 within the microvessel wall and that IL-17–activated PCs can modulate neutrophil functions within the perivascular tissue space.

Background Epithelial and endothelial barrier integrity, essential for homeostasis, is maintained by cellular boarder structures known as tight junctions (TJs). In critical illness, TJs may become disrupted, resulting in barrier dysfunction manifesting as capillary leak, pulmonary edema, gut bacterial translocation, and multiple organ failure. We aim to provide a clinically focused overview of TJ structure and function and systematically review and analyze all studies assessing markers of endothelial and epithelial TJ breakdown correlated with clinical outcomes in critically ill humans. Methods We systematically searched MEDLINE, EMBASE, and PubMed. Additional articles were identified by targeted searches. We included studies that looked at the relationship between biomarkers of endothelial or epithelial TJ structure or function and critical illness. Results were qualitatively analyzed due to sample size and heterogeneity. Results A total of 5297 abstracts met search criteria, of which 150 articles met requirements for full text review. Of these, 30 studies met inclusion criteria. Fifteen of the 30 reports investigated proteins of endothelial tight junctions and 15 investigated epithelial TJ markers, exclusively in the gastrointestinal epithelium. No studies investigated TJ-derived proteins in primary cardiac or pulmonary pathology. Conclusions TJ integrity is essential for homeostasis. We identified multiple studies that indicate TJs are disrupted by critical illness. These studies highlight the significance of barrier disruption across many critical disease states and correlate TJ-associated markers to clinically relevant outcomes. Further study on the role of multiple tissue-specific claudins, particularly in the setting of respiratory or cardiac failure, may lead to diagnostic and therapeutic advances. Systematic review registration This systematic review is registered in the PROSPERO database: CRD42017074546. Electronic supplementary material The online version of this article (10.1186/s40635-018-0203-4) contains supplementary material, which is available to authorized users.

Endothelial cells (ECs) line the lumen of the entire vascular system and actively regulate blood flow; maintain blood fluidity; control water, solute, and macromolecular transfer between blood and tissue; and modulate circulating immune cell recruitment and activation. These vital functions, combined with the broad anatomic distribution of ECs, implicate them in all forms of critical illness. The present article discusses how ECs adapt and break down during the course of critical illness. We first review the biology of ECs, highlighting the vascular segmental differences and their specific roles in the maintenance of homeostasis. We then discuss how ECs acquire new functions to restore local and systemic homeostasis (activation) as well as how breakdowns in EC functions (dysfunction) contribute to local and systemic pathologic responses, with clinical correlations. Lastly, how these processes have been studied in critically ill children is discussed.

RRM2B encodes the crucial p53-inducible ribonucleotide reductase small subunit 2 homolog (p53R2), which is required for DNA synthesis throughout the cell cycle. Mutations in this gene have been associated with a lethal mitochondrial depletion syndrome. Here we present the case of an infant with a novel homozygous p.Asn221Ser mutation in RRM2B who developed hypotonia, failure to thrive, sensorineural hearing loss, and severe metabolic lactic acidosis, ultimately progressing to death at 3 months of age. Through molecular modeling using the X-ray crystal structure of p53R2, we demonstrate that this mutation likely causes disruption of a highly conserved helix region of the protein by altering intramolecular interactions. This report expands our knowledge of potential pathogenic RRM2B mutations as well as our understanding of the molecular function of p53R2 and its role in the pathogenesis of mitochondrial DNA depletion.

Zonisamide is a sulfonamide drug used primarily for the treatment of partial seizures in adults. We describe the case of a 15-year-old woman with a mood disorder who survived without complications after ingestion of an estimated 7.5 g of zonisamide. To the best of our knowledge, there are 4 case reports of individuals with intentional ingestion of more than 4 g of zonisamide as a single agent. Our patient developed coma and hypotension 4 hours after ingestion and was treated with a catecholamine infusion, endotracheal intubation, and mechanical ventilation. She had mild electrocardiographic abnormalities and fully recovered after 4 days. This report contributes to the understanding of acute zonisamide poisoning.

Essentials It is unclear if thrombophilia increases the risk of catheter-associated thrombosis in children. We conducted a meta-analysis on thrombophilia and pediatric catheter-associated thrombosis. Presence of ≥1 trait confers additional risk of venous thrombosis in children with catheters. Limitations of included studies preclude us from recommending routine thrombophilia testing. SummaryBackground The association between thrombophilia and deep vein thrombosis (DVT) associated with central venous catheter (CVC) use, the most important pediatric risk factor for thrombosis, is unclear in children. Pediatric studies with small sample sizes have reported conflicting results. We sought to evaluate whether, among children with CVCs, thrombophilia increases the risk of CVC-associated DVT (CADVT). Materials and methods We systematically searched MEDLINE, EMBASE, the Web of Science, the Cochrane Central Register for Controlled Trials, PubMed and reference lists for controlled studies published from the inception of the database until September 2015. Included were studies of children aged

Purpose To determine provider opinions on factors most commonly used to assess the presence and severity of pathologic capillary leak in critically ill children. Methods We conducted an electronic survey of pediatric critical care providers. Patient scenarios were presented to assess opinions on the risk, presence, and clinical significance of capillary leak. Responses were obtained using Likert scales and multiple-choice questions. Results A total of 160 responses were analyzed. Respondents agreed that capillary leak is present in the scenario with septic shock while respondents somewhat agreed that it is also present with poly-trauma, cardiac arrest, or cardiopulmonary bypass. They agreed that physical exam, but neither agreed nor disagreed that laboratory tests, can be used to assess and follow the severity of capillary leak in these children. Generalized edema, increase in weight, and pulmonary crackles were commonly identified parameters for assessing capillary leak. The patient factor most commonly identified with capillary leak was presence of infection, while treatment factors most commonly identified were cardiopulmonary bypass and general anesthesia. Conclusion There is agreement that capillary leak is common in critically ill children and exacerbates disease. The parameters identified in this study may facilitate a more standardized clinical evaluation of pathologic capillary leak for future studies.

Tintinnid species are traditionally distinguished via lorica features. Recently, sequencing has revealed polymorphism, i.e., genetically identical individuals with distinct lorica morphologies. One such polymorphic species is Cyttarocylis ampulla; individuals can display lorica morphologies of formally different species of Cyttarocylis and Petalotricha, well-represented in the literature. We compiled and analysed a global database of species records to determine if there is a main form and if different morphotypes have distinct temporal or spatial distributions. The two genera show very similar widespread distributions but with some statistical evidence of spatial segregation. Examining co-occurrence among the common `species' we found most were rarely found alone, only 6-14% of the records for all species except for 2 forms: C. eucecryphalus and P. ampulla reported alone in 34% and 43%, respectively, of their records. We identify them as the main forms and analysed data of global distributions, spatial distribution across the Mediterranean in summer and winter and temporal distributions from a site in the Adriatic. The two main forms show frequent co-occurrence, similar lack of strong seasonality and widespread geographic distributions. We tentatively conclude that the different lorica morphologies may only reflect conditions of high temporally variability such as quantities and composition of prey. Directions for further research are suggested. (C) 2013 Elsevier GmbH. All rights reserved.