Shiga toxin down-regulates ERG protein in endothelial cells and impairs angiogenesis.

BACKGROUND: Shiga toxin (Stx) can activate inflammatory signaling, leading to vascular dysfunction and promotion of a pro-thrombotic tissue microenvironment. Stx can trigger the development of the enterohemorrhagic (childhood) hemolytic uremic syndrome (eHUS), a triad of thrombocytopenia, hemolytic anemia, and acute kidney injury, often requiring dialysis. Additional features may include damage to other organs, including the gastrointestinal tract, pancreas, brain and cardiovascular system; death occurs in 2-5 %. eHUS is a thrombotic microangiopathy; thus, endothelial cell (EC) injury and platelet fibrin thrombus formation in glomerular arterioles and in the arterioles of other affected organs are likely. To elucidate mechanisms of this microangiopathy, we examined in human ECs the regulation of the platelet adhesion proteins P-selectin and von Willebrand factor (VWF), along with the downregulation of erythroblast-transformation-specific transcription factor (ERG) a key regulator of angiogenesis and megakaryocyte development. METHODS: VWF, P-selectin, and ERG levels were determined using immunofluorescence and Western blot in human umbilical endothelial cells (HUVECs). HUVECs were treated with tumor necrosis factor-alpha (TNF-α), Stx-1 or both, versus normal controls. Capillary morphogenesis on Matrigel was performed using HUVECs treated, for 22 h with TNF-α, Stx-1, or both, or treated 4 h with Stx-1 alone or in combination with TNF-α for 22 h. RESULTS: Stx-1 significantly reduced ERG and VWF expression on HUVECs, but upregulated P-selectin expression. ERG levels decreased with Stx-1 alone or in combination with TNF-α, in the nuclear, perinuclear and cytoplasmatic regions. Stx-1 reduced capillary morphogenesis, while Stx-1-TNF-α combined treatment reduced capillary morphogenesis still further. CONCLUSIONS: In the presence of Stx-1 or TNF-α or both treatments, ECs were activated, expressing higher levels of P-selectin and lower levels of VWF. Our findings, further, provide evidence that Stx-1 downregulates ERG, repressing angiogenesis in vitro.

Safety, tolerability, and effectiveness of anticoagulation and antiplatelet therapy in hereditary hemorrhagic telangiectasia.

BACKGROUND: Antithrombotic therapy (anticoagulation and antiplatelet therapy) is frequently needed in patients with hereditary hemorrhagic telangiectasia (HHT); however, data describing and guiding its use are very limited. OBJECTIVES: To investigate the safety, tolerability, and effectiveness of antithrombotic therapy in HHT in a cohort large enough to compare agents, evaluate for baseline predictors of premature discontinuation, and evaluate hematologic support requirements and healthcare utilization before and after antithrombitc therapy initiation. METHODS: We performed a multicenter observational cohort study characterizing the outcomes of antithrombic therapy in adults with HHT. RESULTS: A total of 119 patients with HHT with 187 discrete antithrombotic therapy episodes were included. Of these, 59 patients (50%) dose-reduced and/or prematurely discontinued therapy (including 52 patients [44%] who discontinued) due to worsened bleeding complications. Initiation at reduced dose intensity had a similar premature discontinuation rate (49%) as initiation at standard dose intensity (43%). In a multivariable logistic model, a history of gastrointestinal bleeding was associated with 3.25-fold odds of discontinuation (p = .001). Hemoglobin was significantly lower (10.8 g/dL vs 12.2 g/dL, p < .001), and the need for hematologic support (intravenous iron and/or red blood cell transfusion) was significantly higher (29 patients vs 12 patients, p = .004) in the 3 months after antithrombotic therapy initiation vs the 3 months before; emergency department visits and hospital admissions due to bleeding also increased. The rates of dose-reduction and/or premature discontinuation were similar regardless of the anticoagulant class (warfarin, 46%; heparin-based, 48%; direct oral anticoagulants, 44%) or with multiple simultaneous agents (44%) but were slightly lower with single-agent antiplatelet therapy (37%). Thromboembolism despite receiving antithrombotic therapy was common (18 patients, 15%) with varying outcomes. CONCLUSION: Antithrombotic therapy is challenging in HHT, resulting in objectively higher morbidity and health care utilization from worsened bleeding. Discontinuation rates approached 50% regardless of the dose intensity at initiation or type of antithrombotic agent used and were higher in patients with a gastrointestinal bleeding history.

Presentation, Management and Outcomes of Pediatric Pulmonary Embolus: A Retrospective Review.

OBJECTIVES: To review the presentation, management, and outcomes of pediatric pulmonary embolism (PE) patients treated at a single institution over 10 years to determine whether laboratory findings and clinical presentation predict disease severity. METHODS: We performed a retrospective chart review of patients treated for PE in a 14-bed pediatric intensive care unit from January 1, 2008, to December 31, 2018. Associations between clot burden and disease severity, clinical risk factors (body mass index, recent hospitalization, estrogen use), clinical presentation (heart rate, oxygen saturation), and laboratory values (white blood cell count, D-Dimer, troponin, proBNP) were performed using Student t test, χ2 tests, and 1-way analysis of variance. Patients were risk stratified by American Heart Association guidelines. RESULTS: Eighteen (72%) patients (girls) were treated for PE. Common risk factors included recent hospitalization (67%) and oral contraceptives (62%). Risk factors, clinical presentation (including hypoxemia and tachypnea), and laboratory studies did not correlate with disease severity or clot burden. Electrocardiogram and radiographic findings were non-specific. Computer tomography pulmonary angiography (CTPA) was required to diagnose 94%. Sixteen received unfractionated heparin, and 5 required additional intervention. Risk factors, clinical features, and laboratory studies did not predict who required intervention. CONCLUSIONS: Of 18 pediatric patients treated for PE at a single institution over 10 years, vital signs and laboratory data did not predict disease severity or clot burden, and CTPA was required for diagnosis in all but 1. Emergency room providers must have a high index of suspicion for diagnosis and cannot be reassured by normal electrocardiogram or plain film findings. At a time when pediatric providers are under pressure to minimize unnecessary radiation exposure, this lack of correlation of clinical presentation and laboratory findings highlights the importance of considering CTPA when PE is suspected.

Differentiation of Patients with Symptomatic Low von Willebrand Factor from Those with Asymptomatic Low von Willebrand Factor.

BACKGROUND: Accurate diagnosis of symptomatic low von Willebrand factor (VWF) remains a major challenge in von Willebrand disease (VWD). However, present tests do not adequately take into account flow forces that, at very high shear rates, reveal a weakness in the VWF-platelet glycoprotein glycoprotein Ib bond in normal subjects. The degree of this weakness is greater in symptomatic, but not asymptomatic, low VWF. OBJECTIVE: The aim of this study is to distinguish patients with symptomatic low VWF (levels in the 30-50 IU/dL range) from those with asymptomatic low VWF and normal subjects. METHODS: We measured platelet adhesion (PA)/aggregation in our novel microfluidic flow system that permits real-time assessment of PA (surface coverage) and PA/aggregation (V, aggregate volume) using epifluorescence digital videomicroscopy in flowing noncitrated whole blood at 4,000 second-1. Blood samples from 24 low VWF patients and 15 normal subjects were collected into plastic tubes containing 4 U/mL enoxaparin. MetaMorph software was used to quantify rates of PA and V increase. RESULTS: Rates of PA increase showed a bimodal distribution, with values for 16/24 patients (Group I) all below the 2.5th percentile of normal, and values for 8/24 patients (Group II) similar to controls. Bleeding scores (mean ± standard error) were 5.50 ± 0.45 versus 2.75 ± 0.45 (p = 0.00077), and 10 clinically significant bleeding events were observed in seven versus zero (p = 0.0295) Group I and Group II subjects, respectively. CONCLUSION: The present approach may offer a definitive means to distinguish symptomatic low VWF from either asymptomatic low VWF or normal controls.

Risk of Intracranial Hemorrhage Following Intravenous tPA (Tissue-Type Plasminogen Activator) for Acute Stroke Is Low in Children.

Background and Purpose- Data regarding the safety and efficacy of intravenous tPA (tissue-type plasminogen activator) in childhood acute arterial ischemic stroke are inadequate. The TIPS trial (Thrombolysis in Pediatric Stroke; National Institutes of Health grant R01NS065848)-a prospective safety and dose-finding trial of intravenous tPA in acute childhood stroke-was closed for lack of accrual. TIPS sites have subsequently treated children with acute stroke in accordance with established institutional protocols supporting data collection on outcomes. Methods- Data on children treated with intravenous tPA for neuroimaging-confirmed arterial ischemic stroke were collected retrospectively from 16 former TIPS sites to establish preliminary safety data. Participating sites were required to report all children who were treated with intravenous tPA to minimize reporting bias. Symptomatic intracranial hemorrhage (SICH) was defined as ECASS (European Cooperative Acute Stroke Study) II parenchymal hematoma type 2 or any intracranial hemorrhage associated with neurological deterioration within 36 following tPA administration. A Bayesian beta-binomial model for risk of SICH following intravenous tPA was fit using a prior distribution based on the risk level in young adults (1.7%); to test for robustness, the model was also fit with uninformative and conservative priors. Results- Twenty-six children (age range, 1.1-17 years; median, 14 years; 12 boys) with stroke and a median pediatric National Institutes of Health Stroke Scale score of 14 were treated with intravenous tPA within 2 to 4.5 hours (median, 3.0 hours) after stroke onset. No patient had SICH. Two children developed epistaxis. Conclusions- The estimated risk of SICH after tPA in children is 2.1% (95% highest posterior density interval, 0.0%-6.7%; mode, 0.9%). Regardless of prior assumption, there is at least a 98% chance that the risk is <15% and at least a 93% chance that the risk is <10%. These results suggest that the overall risk of SICH after intravenous tPA in children with acute arterial ischemic stroke, when given within 4.5 hours after symptom onset, is low.

Coupling carbon and energy fluxes in the North Pacific Subtropical Gyre.

The major biogeochemical cycles of marine ecosystems are driven by solar energy. Energy that is initially captured through photosynthesis is transformed and transported to great ocean depths via complex, yet poorly understood, energy flow networks. Herein we show that the chemical composition and specific energy (Joules per unit mass or organic carbon) of sinking particulate matter collected in the North Pacific Subtropical Gyre reveal dramatic changes in the upper 500 m of the water column as particles sink and age. In contrast to these upper water column processes, particles reaching the deep sea (4000 m) are energy-replete with organic carbon-specific energy values similar to surface phytoplankton. These enigmatic results suggest that the particles collected in the abyssal zone must be transported by rapid sinking processes. These fast-sinking particles control the pace of deep-sea benthic communities that live a feast-or-famine existence in an otherwise energy-depleted habitat.

Flow Devices to Assess Platelet Function: Historical Evolution and Current Choices.

Platelet function testing, which began more than a hundred years ago, is a time-consuming and uncertain process. Simulating hemostasis and the blood vessel microenvironment in vitro is challenging, which poses a difficulty for diagnosing platelet dysfunction and mild von Willebrand disease (VWD). In an effort to simulate the rheological microenvironment within blood vessels, several blood flow devices have been introduced since the 1980s. These devices are capable of reproducing the shear rates found in arterioles and venules, and of incorporating endothelial cell monolayers and surfaces with adsorbed platelet-adhesive proteins. The authors will describe and review here the presently most well-known blood flow devices. The technologies inherent in these devices offer a combination of physiologic accuracy and small blood volume requirements in the evaluation of platelet disorders and mild VWD (or "symptomatic low von Willebrand factor") in flowing whole blood, with the potential to individualize therapeutic options for and to achieve greater diagnostic accuracy in mild platelet disorders and VWD.

Naphtho[2,3- b]carbaporphyrins.

Acid-catalyzed condensation of a benzo[ f]indane dialdehyde with a tripyrrane, followed by an oxidation step, afforded the first example of a naphtho[2,3- b]-21-carbaporphyrin. This π-extended porphyrinoid system is strongly aromatic and gave a porphyrin-like UV-vis spectrum with a Soret band at 432 nm. Protonation with TFA gave a monocation, but under highly acidic conditions a C-protonated dication was generated. Reaction of the naphthoporphyrin with ferric chloride produced a 21-chloro derivative. Alkylation with methyl iodide and potassium carbonate gave a 22-methyl derivative, and this reacted with palladium(II) acetate to afford a palladium(II) complex in which the internal methyl group had migrated from a nitrogen to a carbon atom. Treatment of the naphthocarbaporphyrin with silver(I) acetate generated the corresponding silver(III) complex. In naphtho[2,3- b]-21-carbaporphyrin and many of its derivatives, the aromatic conjugation pathways appear to bypass the naphthalene unit, and for this reason the UV-vis spectra were little affected. However, the diprotonated dication and the palladium(II) complex have aromatic pathways that pass through the naphthalene moiety, and this leads to large bathochromic shifts for these species. The results provide insights on the influence of fused aromatic units on the reactivity, spectroscopic properties, and aromatic characteristics of carbaporphyrinoid systems.

Mortality After Pediatric Arterial Ischemic Stroke.

OBJECTIVES: Cerebrovascular disease is among the top 10 causes of death in US children, but risk factors for mortality are poorly understood. Within an international registry, we identify predictors of in-hospital mortality after pediatric arterial ischemic stroke (AIS). METHODS: Neonates (0-28 days) and children (29 days-<19 years) with AIS were enrolled from January 2003 to July 2014 in a multinational stroke registry. Death during hospitalization and cause of death were ascertained from medical records. Logistic regression was used to analyze associations between risk factors and in-hospital mortality. RESULTS: Fourteen of 915 neonates (1.5%) and 70 of 2273 children (3.1%) died during hospitalization. Of 48 cases with reported causes of death, 31 (64.6%) were stroke-related, with remaining deaths attributed to medical disease. In multivariable analysis, congenital heart disease (odds ratio [OR]: 3.88; 95% confidence interval [CI]: 1.23-12.29; P = .021), posterior plus anterior circulation stroke (OR: 5.36; 95% CI: 1.70-16.85; P = .004), and stroke presentation without seizures (OR: 3.95; 95% CI: 1.26-12.37; P = .019) were associated with in-hospital mortality for neonates. Hispanic ethnicity (OR: 3.12; 95% CI: 1.56-6.24; P = .001), congenital heart disease (OR: 3.14; 95% CI: 1.75-5.61; P < .001), and posterior plus anterior circulation stroke (OR: 2.71; 95% CI: 1.40-5.25; P = .003) were associated with in-hospital mortality for children. CONCLUSIONS: In-hospital mortality occurred in 2.6% of pediatric AIS cases. Most deaths were attributable to stroke. Risk factors for in-hospital mortality included congenital heart disease and posterior plus anterior circulation stroke. Presentation without seizures and Hispanic ethnicity were also associated with mortality for neonates and children, respectively. Awareness and study of risk factors for mortality represent opportunities to increase survival.

Analysis of patient outcomes following proton radiation therapy for retinoblastoma.

PURPOSE: Proton radiotherapy (PRT) is used in the treatment of retinoblastoma (RB) and has the potential to minimize exposure of normal tissue to radiation and thus decrease risk of toxicity and second malignancies. However, comprehensive analyses of long-term patient outcomes are not available. METHODS: RB patients treated with PRT at our institution between 1986 and 2012 were invited to return for participation in a study designed to assess long-term outcomes. Enrolled patients underwent comprehensive analysis including oncologic, ophthalmic, endocrine, cephalometric, and quality of life (QOL) assessments. RESULTS: A total of 12 patients were enrolled in this study, and the average length of follow-up among enrolled patients was 12.9 years (range 4.8-22.2 years). All enrolled patients had bilateral disease, and the disease and visual outcomes for enrolled patients were similar to outcomes for all RB patients treated with PRT over the same time period at our institution. Endocrine evaluation revealed no growth abnormalities or hormonal deficiencies across the cohort. Based on MRI and external cephalometry, PRT was associated with less facial hypoplasia than enucleation. Patient and parent-proxy QOL assessments revealed that RB treatment did not appear to severely impact long-term QOL. CONCLUSIONS: In addition to providing an opportunity for long-term disease control and functional eye preservation, PRT does not appear to be associated with unexpected late visual, endocrine, or QOL effects in this cohort.

The Importance of H in Particulate Organic Matter Stoichiometry, Export and Energy Flow.

The discipline of marine ecological stoichiometry has progressed rapidly over the past two decades, and continues to be at the forefront of microbial oceanography. Most of this effort has been focused on the elements carbon (C) and nitrogen (N), and to a lesser extent phosphorus (P), with little consideration of hydrogen (H), or the redox state of the organic matter pools despite the fact that H is the most abundant, and possibly the most important, element in biogeochemistry. Obtaining accurate estimates of the H content of organic matter, either in suspended or sinking particles, is a major analytical challenge. While many aquatic science laboratories have access to commercial "C-H-N elemental analyzers," few investigators report H values due to analytical difficulties in obtaining accurate estimates of H. Because organic compounds vary considerably in their H:C ratio and therefore in their energy content, measurements of H combined with C-specific caloric estimates will ultimately be required for a more comprehensive understanding of ecosystem dynamics.

Human mannose-binding lectin inhibitor prevents Shiga toxin-induced renal injury.

Hemolytic uremic syndrome caused by Shiga toxin-producing Escherichia coli (STEC HUS) is a worldwide endemic problem, and its pathophysiology is not fully elucidated. Here we tested whether the mannose-binding lectin (MBL2), an initiating factor of lectin complement pathway activation, plays a crucial role in STEC HUS. Using novel human MBL2-expressing mice (MBL2 KI) that lack murine Mbls (MBL2(+/+)Mbl1(-/-)Mbl2(-/-)), a novel STEC HUS model consisted of an intraperitoneal injection with Shiga toxin-2 (Stx-2) with or without anti-MBL2 antibody (3F8, intraperitoneal). Stx-2 induced weight loss, anemia, and thrombocytopenia and increased serum creatinine, free serum hemoglobin, and cystatin C levels, but a significantly decreased glomerular filtration rate compared with control/sham mice. Immunohistochemical staining revealed renal C3d deposition and fibrin deposition in glomeruli in Stx-2-injected mice. Treatment with 3F8 completely inhibited serum MBL2 levels and significantly attenuated Stx-2 induced-renal injury, free serum hemoglobin levels, renal C3d, and fibrin deposition and preserved the glomerular filtration rate. Thus, MBL2 inhibition significantly protected against complement activation and renal injury induced by Stx-2. This novel mouse model can be used to study the role of complement, particularly lectin pathway-mediated complement activation, in Stx-2-induced renal injury.

Brain endothelial dysfunction in cerebral adrenoleukodystrophy.

See Aubourg (doi:10.1093/awv271) for a scientific commentary on this article.X-linked adrenoleukodystrophy is caused by mutations in the ABCD1 gene leading to accumulation of very long chain fatty acids. Its most severe neurological manifestation is cerebral adrenoleukodystrophy. Here we demonstrate that progressive inflammatory demyelination in cerebral adrenoleukodystrophy coincides with blood-brain barrier dysfunction, increased MMP9 expression, and changes in endothelial tight junction proteins as well as adhesion molecules. ABCD1, but not its closest homologue ABCD2, is highly expressed in human brain microvascular endothelial cells, far exceeding its expression in the systemic vasculature. Silencing of ABCD1 in human brain microvascular endothelial cells causes accumulation of very long chain fatty acids, but much later than the immediate upregulation of adhesion molecules and decrease in tight junction proteins. This results in greater adhesion and transmigration of monocytes across the endothelium. PCR-array screening of human brain microvascular endothelial cells after ABCD1 silencing revealed downregulation of both mRNA and protein levels of the transcription factor c-MYC (encoded by MYC). Interestingly, MYC silencing mimicked the effects of ABCD1 silencing on CLDN5 and ICAM1 without decreasing the levels of ABCD1 protein itself. Together, these data demonstrate that ABCD1 deficiency induces significant alterations in brain endothelium via c-MYC and may thereby contribute to the increased trafficking of leucocytes across the blood-brain barrier as seen in cerebral adrenouleukodystrophy.

Proton radiation therapy for the treatment of retinoblastoma.

PURPOSE: To investigate long-term disease and toxicity outcomes for pediatric retinoblastoma patients treated with proton radiation therapy (PRT). METHODS AND MATERIALS: This is a retrospective analysis of 49 retinoblastoma patients (60 eyes) treated with PRT between 1986 and 2012. RESULTS: The majority (84%) of patients had bilateral disease, and nearly half (45%) had received prior chemotherapy. At a median follow-up of 8 years (range, 1-24 years), no patients died of retinoblastoma or developed metastatic disease. The post-PRT enucleation rate was low (18%), especially in patients with early-stage disease (11% for patients with International Classification for Intraocular Retinoblastoma [ICIR] stage A-B disease vs 23% for patients with ICIR stage C-D disease). Post-PRT ophthalmologic follow-up was available for 61% of the preserved eyes (30 of 49): 14 of 30 eyes (47%) had 20/40 visual acuity or better, 7 of 30 (23%) had moderate visual acuity (20/40-20/600), and 9 of 30 (30%) had little or no useful vision (worse than 20/600). Twelve of 60 treated eyes (20%) experienced a post-PRT event requiring intervention, with cataracts the most common (4 eyes). No patients developed an in-field second malignancy. CONCLUSIONS: Long-term follow-up of retinoblastoma patients treated with PRT demonstrates that PRT can achieve high local control rates, even in advanced cases, and many patients retain useful vision in the treated eye. Treatment-related ocular side effects were uncommon, and no radiation-associated malignancies were observed.

Emergence of the primary pediatric stroke center: impact of the thrombolysis in pediatric stroke trial.

BACKGROUND AND PURPOSE: In adult stroke, the advent of thrombolytic therapy led to the development of primary stroke centers capable to diagnose and treat patients with acute stroke rapidly. We describe the development of primary pediatric stroke centers through preparation of participating centers in the Thrombolysis in Pediatric Stroke (TIPS) trial. METHODS: We collected data from the 17 enrolling TIPS centers regarding the process of becoming an acute pediatric stroke center with capability to diagnose, evaluate, and treat pediatric stroke rapidly, including use of thrombolytic therapy. RESULTS: Before 2004, <25% of TIPS sites had continuous 24-hour availability of acute stroke teams, MRI capability, or stroke order sets, despite significant pediatric stroke expertise. After TIPS preparation, >80% of sites now have these systems in place, and all sites reported increased readiness to treat a child with acute stroke. Use of a 1- to 10-Likert scale on which 10 represented complete readiness, median center readiness increased from 6.2 before site preparation to 8.7 at the time of site activation (P≤0.001). CONCLUSIONS: Before preparing for TIPS, centers interested in pediatric stroke had not developed systematic strategies to diagnose and treat acute pediatric stroke. TIPS trial preparation has resulted in establishment of pediatric acute stroke centers with clinical and system preparedness for evaluation and care of children with acute stroke, including use of a standardized protocol for evaluation and treatment of acute arterial stroke in children that includes use of intravenous tissue-type plasminogen activator. CLINICAL TRIAL REGISTRATION URL: http://www.clinicaltrials.gov. Unique identifier: NCT01591096.