Immune dysregulation and autoreactivity correlate with disease severity in SARS-CoV-2-associated multisystem inflammatory syndrome in children.

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.

Single-cell atlas of the human neonatal small intestine affected by necrotizing enterocolitis.

Necrotizing enterocolitis (NEC) is a gastrointestinal complication of premature infants with high rates of morbidity and mortality. A comprehensive view of the cellular changes and aberrant interactions that underlie NEC is lacking. This study aimed at filling in this gap. We combine single-cell RNA sequencing (scRNAseq), T-cell receptor beta (TCRß) analysis, bulk transcriptomics, and imaging to characterize cell identities, interactions, and zonal changes in NEC. We find an abundance of proinflammatory macrophages, fibroblasts, endothelial cells as well as T cells that exhibit increased TCRß clonal expansion. Villus tip epithelial cells are reduced in NEC and the remaining epithelial cells up-regulate proinflammatory genes. We establish a detailed map of aberrant epithelial-mesenchymal-immune interactions that are associated with inflammation in NEC mucosa. Our analyses highlight the cellular dysregulations of NEC-associated intestinal tissue and identify potential targets for biomarker discovery and therapeutics.

An AI-powered patient triage platform for future viral outbreaks using COVID-19 as a disease model.

Over the last century, outbreaks and pandemics have occurred with disturbing regularity, necessitating advance preparation and large-scale, coordinated response. Here, we developed a machine learning predictive model of disease severity and length of hospitalization for COVID-19, which can be utilized as a platform for future unknown viral outbreaks. We combined untargeted metabolomics on plasma data obtained from COVID-19 patients (n = 111) during hospitalization and healthy controls (n = 342), clinical and comorbidity data (n = 508) to build this patient triage platform, which consists of three parts: (i) the clinical decision tree, which amongst other biomarkers showed that patients with increased eosinophils have worse disease prognosis and can serve as a new potential biomarker with high accuracy (AUC = 0.974), (ii) the estimation of patient hospitalization length with ± 5 days error (R2 = 0.9765) and (iii) the prediction of the disease severity and the need of patient transfer to the intensive care unit. We report a significant decrease in serotonin levels in patients who needed positive airway pressure oxygen and/or were intubated. Furthermore, 5-hydroxy tryptophan, allantoin, and glucuronic acid metabolites were increased in COVID-19 patients and collectively they can serve as biomarkers to predict disease progression. The ability to quickly identify which patients will develop life-threatening illness would allow the efficient allocation of medical resources and implementation of the most effective medical interventions. We would advocate that the same approach could be utilized in future viral outbreaks to help hospitals triage patients more effectively and improve patient outcomes while optimizing healthcare resources.

Single-cell longitudinal analysis of SARS-CoV-2 infection in human airway epithelium identifies target cells, alterations in gene expression, and cell state changes.

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.

ArhGEF12 activates Rap1A and not RhoA in human dermal microvascular endothelial cells to reduce tumor necrosis factor-induced leak.

Overwhelming inflammation in the setting of acute critical illness induces capillary leak resulting in hypovolemia, edema, tissue dysoxia, organ failure and even death. The tight junction (TJ)-dependent capillary barrier is regulated by small GTPases, but the specific regulatory molecules most active in this vascular segment under such circumstances are not well described. We set out to identify GTPase regulatory molecules specific to endothelial cells (EC) that form TJs. Transcriptional profiling of confluent monolayers of TJ-forming human dermal microvascular ECs (HDMECs) and adherens junction only forming-human umbilical vein EC (HUVECs) demonstrate ARHGEF12 is basally expressed at higher levels and is only downregulated in HDMECs by junction-disrupting tumor necrosis factor (TNF). HDMECs depleted of ArhGEF12 by siRNA demonstrate a significantly exacerbated TNF-induced decrease in trans-endothelial electrical resistance and disruption of TJ continuous staining. ArhGEF12 is established as a RhoA-GEF in HUVECs and its knock down would be expected to reduce RhoA activity and barrier disruption. Pulldown of active GEFs from HDMECs depleted of ArhGEF12 and treated with TNF show decreased GTP-bound Rap1A after four hours but increased GTP-bound RhoA after 12 h. In cell-free assays, ArhGEF12 immunoprecipitated from HDMECs is able to activate both Rap1A and RhoA, but not act on Rap2A-C, RhoB-C, or even Rap1B which shares 95% sequence identity with Rap1A. We conclude that in TJ-forming HDMECs, ArhGEF12 selectively activates Rap1A to limit capillary barrier disruption in a mechanism independent of cAMP-mediated Epac1 activation.

Outcomes of light and midweight synthetic mesh use in clean-contaminated and contaminated ventral incisional hernia repair: an ACHQC comparative analysis.

BACKGROUND: Use of macroporous synthetic mesh in contaminated ventral hernia repair has become more frequent. The objective of this study is to compare the outcomes of ventral incisional hernia repair with permanent synthetic mesh in contaminated fields to those in a clean field. METHODS: The Abdominal Core Health Quality Collaborative registry, a prospectively updated longitudinal hernia-specific national database, was retrospectively queried for adults who underwent open ventral incisional hernia repair using light or medium-weight synthetic mesh and classified as clean (CDC Class I) or contaminated (CDC Class II/III). Univariate analysis was used to compare demographic information, hernia characteristics, and operative details. Odds ratios (OR) were calculated using multivariable logistic regression for the primary outcome of 30-day surgical site infection (SSI) and secondary outcomes of 30-day surgical site occurrence (SSO), SSO requiring procedural intervention (SSO-PI), and clinical recurrence at one year. RESULTS: 7219 cases met criteria for inclusion; 13.2% of these were contaminated. 83.4% of patients had follow-up data at 30 days and 20.8% at 1 year. The adjusted OR for 30-day SSI in contaminated fields compared to clean was 2.603 (95% CI 1.959-3.459). OR for 30-day SSO was 1.275 (95% CI 1.017-1.600) and 2.355 (95%CI 1.817-3.053) for 30-day SSO-PI. OR for recurrence at one year was 1.489 (95%CI 0.892-2.487). Contaminated cases had higher rates of mesh infection (3.9% vs 0.8%, p < 0.001) and mesh removal (7.3 vs 2.5%, p < 0.001) at 1 year. CONCLUSIONS: After adjusting for baseline differences, patients undergoing ventral incisional hernia repair using light or midweight synthetic mesh in contaminated fields have higher odds of 30-day SSI, SSO, and SSO-PI than those performed in clean wounds. The odds of recurrence did not statistically differ and further studies with long-term outcomes are needed to better evaluate the best treatment options for this patient population.

Integrated Analysis of Tracheobronchial Fluid from Before and After Cardiopulmonary Bypass Reveals Activation of the Integrated Stress Response and Altered Pulmonary Microvascular Permeability.

Objective: We aim to comprehensively describe the transcriptional activity and signaling of pulmonary parenchymal and immune cells before and after cardiopulmonary bypass (CPB) by using a multi-omic approach coupled with functional cellular assays. We hypothesize that key signaling pathways from specific cells within the lung alter pulmonary endothelial cell function resulting in worsening or improving disease. Methods: We collected serial tracheobronchial lavage samples from intubated patients less than 2-years-old undergoing surgery with CPB. Samples were immediately processed for single cell RNA sequencing (10x Genomics). Cell clustering, cell-type annotation, and visualization were performed, and differentially expressed genes (DEG) between serial samples were identified. Metabolomic and proteomic analyses were performed on the supernatant using mass spectrometry and a multiplex assay (SomaScan) respectively. Functional assays were done using electric cell-substrate impedance sensing to measure resistance across human pulmonary microvascular endothelial cells (HPMECs). Results: Analysis of eight patients showed a heterogeneous mixture of pulmonary parenchymal and immune cells. Cell clustering demonstrated time-dependent changes in the transcriptomic signature indicating altered cellular phenotypes after CPB. DEG analysis was represented by genes involved in host defense, innate immunity, and the mitochondrial respiratory transport chain. Ingenuity pathway analysis showed upregulation of the integrated stress response across all cell types after CPB. Metabolomic analysis demonstrated upregulation of ascorbate and aldarate metabolism. Unbiased proteomic analysis revealed upregulation of proteins involved in cytokine and chemokine pathways. Post-CPB patient supernatant improved HMPEC barrier function, suggesting a protective cellular response to CPB. Conclusion: Children who undergo CPB for cardiac surgery have distinct cell populations, transcriptional activity, and metabolism that change over time. The response to ischemia-reperfusion injury in the lower airway of children appears to be protective, with the need to identify potential targets through future investigations.

Integrated Single-Cell Atlas of Endothelial Cells of the Human Lung.

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.

Tumor necrosis factor-induced ArhGEF10 selectively activates RhoB contributing to human microvascular endothelial cell tight junction disruption.

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.

A retrospective cohort analysis of the Yale pediatric genomics discovery program.

The Pediatric Genomics Discovery Program (PGDP) at Yale uses next-generation sequencing (NGS) and translational research to evaluate complex patients with a wide range of phenotypes suspected to have rare genetic diseases. We conducted a retrospective cohort analysis of 356 PGDP probands evaluated between June 2015 and July 2020, querying our database for participant demographics, clinical characteristics, NGS results, and diagnostic and research findings. The three most common phenotypes among the entire studied cohort (n = 356) were immune system abnormalities (n = 105, 29%), syndromic or multisystem disease (n = 103, 29%), and cardiovascular system abnormalities (n = 62, 17%). Of 216 patients with final classifications, 77 (36%) received new diagnoses and 139 (64%) were undiagnosed; the remaining 140 patients were still actively being investigated. Monogenetic diagnoses were found in 67 (89%); the largest group had variants in known disease genes but with new contributions such as novel variants (n = 31, 40%) or expanded phenotypes (n = 14, 18%). Finally, five PGDP diagnoses (8%) were suggestive of novel gene-to-phenotype relationships. A broad range of patients can benefit from single subject studies combining NGS and functional molecular analyses. All pediatric providers should consider further genetics evaluations for patients lacking precise molecular diagnoses.

BMX Represses Thrombin-PAR1-Mediated Endothelial Permeability and Vascular Leakage During Early Sepsis.

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.

Characterization of Atmospheric Processes of Brevetoxins in Sea Spray Aerosols from Red Tide Events.

Atmospheric processes can affect the longevity of harmful toxins in sea spray aerosols (SSA). This study characterized the degradation of brevetoxin (BTx) in SSA under different environmental conditions. The samples of seawater collected during a Karenia brevis bloom in Manasota, Florida, were nebulized into a large outdoor photochemical chamber to mimic the atmospheric oxidation of aerosolized toxins and then aged in the presence or absence of sunlight and/or O3. Aerosol samples were collected during the aging process using a Particle-Into-Liquid Sampler. Their BTx concentrations were measured using an enzyme-linked immuno-sorbent assay (ELISA) and high-performance liquid chromatography/tandem mass spectroscopy. The BTx ozonolysis rate constant measured by ELISA was 5.74 ± 0.21 × 103 M-1 s-1. The corresponding lifetime for decay of 87.5% BTx in the presence of 20 ppb of O3 was 7.08 ± 0.26 h, suggesting that aerosolized BTx can still travel long distances at night before SSA deposition. BTx concentrations in SSA decreased more rapidly in the presence of sunlight than in its absence due to oxidation with photochemically produced OH radicals.

Relevance of Microvascular Flow Assessments in Critically Ill Neonates and Children: A Systematic Review.

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.

Methylene Blue for Refractory Shock in Children: A Systematic Review and Survey Practice Analysis.

OBJECTIVES: Shock refractory to fluid and catecholamine therapy has significant morbidity and mortality in children. The use of methylene blue to treat refractory shock in children is not well described. We aim to collect and summarize the literature and define physicians' practice patterns regarding the use of methylene blue to treat shock in children. DESIGN: We conducted a systematic search of MEDLINE, Embase, PubMed, Web of Science, Cochrane for studies involving the use of methylene blue for catecholamine-refractory shock from database inception to 2019. Collected studies were analyzed qualitatively. To describe practice patterns of methylene blue use, we electronically distributed a survey to U.S.-based pediatric critical care physicians. We assessed physician knowledge and experience with methylene blue. Survey responses were quantitatively and qualitatively evaluated. SETTING: Pediatric critical and cardiac care units. PATIENTS OR SUBJECTS: Patients less than or equal to 25 years old with refractory shock treated with methylene blue. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: One-thousand two-hundred ninety-three abstracts met search criteria, 139 articles underwent full-text review, and 24 studies were included. Studies investigated refractory shock induced by a variety of etiologies and found that methylene blue was generally safe and increased mean arterial blood pressure. There is overall lack of studies, low number of study patients, and low quality of studies identified. Our survey had a 22.5% response rate, representing 125 institutions. Similar proportions of physicians reported using (40%) or never even considering (43%) methylene blue for shock. The most common reasons for not using methylene blue were unfamiliarity with this drug, its proper dosing, and lack of evidentiary support. CONCLUSIONS: Methylene blue appears safe and may benefit children with refractory shock. There is a stark divide in familiarity and practice patterns regarding its use among physicians. Studies to formally assess safety and efficacy of methylene blue in treating pediatric shock are warranted.

Rethinking what constitutes a diagnosis in the genomics era: a critical illness perspective.

PURPOSE OF REVIEW: 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. RECENT FINDINGS: 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. SUMMARY: 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.

IL-17 Promotes Neutrophil-Mediated Immunity by Activating Microvascular Pericytes and Not Endothelium.

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-1ß, 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.

Heller myotomy versus Heller myotomy with Dor fundoplication for achalasia: long-term symptomatic follow-up of a prospective randomized controlled trial.

BACKGROUND: Our prior randomized controlled trial of Heller myotomy alone versus Heller plus Dor fundoplication for achalasia from 2000 to 2004 demonstrated comparable postoperative resolution of dysphagia but less gastroesophageal reflux after Heller plus Dor. Patient-reported outcomes are needed to determine whether the findings are sustained long-term. METHODS: We actively engaged participants from the prior randomized cohort, making up to six contact attempts per person using telephone, mail, and electronic messaging. We collected patient-reported measures of dysphagia and gastroesophageal reflux using the Dysphagia Score and the Gastroesophageal Reflux Disease-Health-Related Quality of Life (GERD-HRQL) instrument. Patient-reported re-interventions for dysphagia were verified by obtaining longitudinal medical records. RESULTS: Among living participants, 27/41 (66%) were contacted and all completed the follow-up study at a mean of 11.8 years postoperatively. Median Dysphagia Scores and GERD-HRQL scores were slightly worse for Heller than Heller plus Dor but were not statistically different (6 vs 3, p = 0.08 for dysphagia, 15 vs 13, p = 0.25 for reflux). Five patients in the Heller group and 6 in Heller plus Dor underwent re-intervention for dysphagia with most occurring more than five years postoperatively. One patient in each group underwent redo Heller myotomy and subsequent esophagectomy. Nearly all patients (96%) would undergo operation again. CONCLUSIONS: Long-term patient-reported outcomes after Heller alone and Heller plus Dor for achalasia are comparable, providing support for either procedure.

Sera From Children After Cardiopulmonary Bypass Reduces Permeability of Capillary Endothelial Cell Barriers.

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.

Complete Recovery After Acute Zonisamide Overdose in an Adolescent Female.

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.

Current trends in the practice of endoscopy among surgeons in the USA.

BACKGROUND: The diagnostic and therapeutic roles for endoscopic intervention are expanding. To continue emphasis on endoscopy in surgical training, The Society of American Gastrointestinal and Endoscopic Surgeons has developed the Fundamentals of Endoscopic Surgery (FES) course to standardize and assess endoscopy training. However, little demographic information exists about the current practice of endoscopy by general surgeons and how to best integrate endoscopic skills into surgical training. METHODS: A survey to collect data regarding the current practice patterns of endoscopy was sent to surgeons with a valid email address in the American Medical Association masterfile. Information regarding the type of training (academic vs. community general surgery residency) and current practice environment (academic medical center vs. community hospital) was collected. The respondents' current practice volume of upper endoscopy and colonoscopy over the prior year was stratified into three groups: rare (<1 per month), moderate (1-10 per month), and frequent (>10 per month). Pearson's Chi-squared test was used to analyze the data. RESULTS: The survey was sent to 9902 general surgeons. There were 767 who provided answers regarding their current practice of endoscopy. Mean time in practice was 18 ± 10 years, 87 % were male, and 83 % practiced in a metropolitan area. Respondents who trained at academic general surgery programs were less likely than those at community programs to frequently perform colonoscopy (17.3 vs. 27.9 %, p < 0.05) and upper endoscopy (11.8 vs. 17.1 %, p < 0.05). Those who currently practice in academic medical centers were also less likely to be frequent performers of colonoscopy (5.6 vs. 24.7 %, p < 0.05) and upper endoscopy (9.8 vs. 14.8 %, p < 0.05) than those who practice at community hospitals. CONCLUSIONS: The type of residency training and current practice setting of general surgeons has a significant influence on the volume of endoscopic procedures performed. This study identifies areas where more emphasis on endoscopic skills training is needed, such as FES.