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Erythropoiesis occurs in specialized niches in the bone marrow consisting of a central macrophage, surrounded by differentiating erythroblasts. This central macrophage has been identified by several markers including, CD169 (Sialoadhesin or Siglec-1), F4/80, CD11b, VCAM-1, ER-HR3 and Ly-6G. These CD169+ macrophages support erythropoiesis both at steady state and during stress. Nuclear factor erythroid 2-related factor 2 (Nrf2) is the master regulator of the cellular oxidative defense system. It modulates hematopoietic stem cells but its loss produces no visible phenotype in steady state hematological parameters. However, the importance of Nrf2 and macrophage subsets has not been fully characterized during recovery from stress erythropoiesis. We examined specific subsets of CD169+ macrophage populations in Nrf2 knockout (Nrf2−/−) mice as well as the role of Nrf2 in recovery from stress erythropoiesis in vivo. We quantified the expression of CD169, F4/80 and CD11b, markers of central macrophages, in the BM and spleen of Nrf2+/+ and Nrf2-/- mice at steady state. Surprisingly, Nrf2-/- mice showed a phenotype characterized by lower percentages of cells expressing known macrophage markers. We observed a significant decrease of 47% (p≤0.01), 24% (p≤0.01) and 50% (p≤0.01) in BM macrophage subpopulations expressing F4/80hiCD169hi, F4/80hiCD11bhi and CD169hiCD11bhi respectively, in age-matched Nrf2-/- mice compared to Nrf2+/+ control mice (Fig. 1a). In the spleen, we also observed a similar significant deficiency in BM macrophages (p≤0.01). Further validating this phenotype, immunofluorescence staining of isolated spleen tissue showed that expression of CD169+ macrophages was dramatically lower in spleen sections of Nrf2-/- mice than in Nrf2+/+ control mice. We hypothesized that our macrophage-deficient mice would display a defect in recovery from blood loss. Five to seven days after acute blood loss, immature erythroid progenitors (CD71hiTer119hi) increased in marrow by about 5-fold in Nrf2+/+ mice (p≤0.001, Fig. 1b), and mature erythroid progenitors (CD71loTer119hi) increased in marrow by 12-fold (p≤0.05) but the erythroid marrow response was impaired significantly in the macrophage deficient Nrf2-/- mice. To extend our observations regarding macrophage deficiency and impaired erythroid response, we chose a more functional outcome of recovery from anemia after high-grade blood loss produced by daily phlebotomies over 3 consecutive days to induce stress erythropoiesis in Nrf2+/+ and Nrf2-/- mice. We found significantly lower packed cell volume values specifically on Days 2, 4 and 10, implying delayed erythroid recovery (p≤0.05, two-way ANOVA). The Nrf2-/- mice also showed a significant decline in total hemoglobin than the Nrf2+/+ mice (p≤0.05). Additionally, peripheral blood reticulocyte response to blood loss is delayed in Nrf2 deficient mice compared to age-matched controls (11.0 ± 0.6% vs. 14.8 ± 0.6%, p≤0.001). We analyzed expression of heme-oxygenase 1 (HO-1), a well-known Nrf2-regulated gene. HO-1 mRNA expression increased 3-fold and 23-fold in Nrf2+/+ mice animals subjected to phlebotomy and hemin treatment compared to 2-fold and 12-fold expression in Nrf2-deficient mice (p≤0.05). We demonstrate for the first time that Nrf2-deficient mice have a deficiency of macrophages that includes subsets considered erythroblastic island (EI) macrophages, and that this deficiency is associated with impaired erythroid response to induced stress. Secondly, our multiple phlebotomies data in aggregate demonstrate that Nrf2-/- mice deficient in BM macrophages have significant delay in functional erythroid response and recovery from experimentally-induced anemia. Thirdly, impaired inducibility of HO-1 is a known feature of Nrf2-/- mice, which we confirmed in our results, could be contributing to the impairment in erythroid response. However, it is not likely that restricted iron trafficking to erythroid progenitors occurs in Nrf2-/- mice, since there is no characteristic alteration of mean corpuscular volume and mean corpuscular hemoglobin in peripheral blood. This is an area worthy of additional investigation. We conclude that the Nrf2 gene plays a previously unappreciated role in erythroid biology that appears to be mediated through macrophage function. Disclosures Ofori-Acquah: Shire Human Genetic Therapies Inc: Other: Financial Relationship. Kato:Bayer: Research Funding; Novartis, Global Blood Therapeutics: Consultancy, Research Funding.

Background The hypercoagulable state associated with pancreatic adenocarcinoma (PDA) results in increased risk of venous thromboembolism, leading to substantial morbidity and mortality. Recently, neutrophil extracellular traps (NETs), whereby activated neutrophils release their intracellular contents containing DNA, histones, tissue factor, high mobility group box 1 (HMGB1) and other components have been implicated in PDA and in cancer-associated thrombosis. Methods Utilizing an orthotopic murine PDA model in C57/Bl6 mice and patient correlative samples, we studied the role of NETs in PDA hypercoagulability and targeted this pathway through treatment with the NET inhibitor chloroquine. PAD4 and RAGE knockout mice, deficient in NET formation, were used to study the role of NETs in platelet aggregation, release of tissue factor and hypercoagulability. Platelet aggregation was assessed using collagen-activated impedance aggregometry. Levels of circulating tissue factor, the initiator of extrinsic coagulation, were measured using ELISA. Thromboelastograms (TEGs) were performed to assess hypercoagulability and changes associated with treatment. Correlative data and samples from a randomized clinical trial of preoperative gemcitabine/nab-paclitaxel with and without hydroxychloroquine were studied and the impact of treatment on venous thromboembolism (VTE) rate was evaluated. Results The addition of NETs to whole blood stimulated platelet activation and aggregation. DNA and the receptor for advanced glycation end products (RAGE) were necessary for induction of NET associated platelet aggregation. PAD4 knockout tumor-burdened mice, unable to form NETs, had decreased aggregation and decreased circulating tissue factor. The NET inhibitor chloroquine reduces platelet aggregation, reduces circulating tissue factor and decreases hypercoagulability on TEG. Review of correlative data from patients treated on a randomized protocol of preoperative chemotherapy with and without hydroxychloroquine demonstrated a reduction in peri-operative VTE rate from 30 to 9.1% with hydroxychloroquine that neared statistical significance (p = 0.053) despite the trial not being designed to study VTE. Conclusion NETs promote hypercoagulability in murine PDA through stimulation of platelets and release of tissue factor. Chloroquine inhibits NETs and diminishes hypercoagulability. These findings support clinical study of chloroquine to lower rates of venous thromboembolism in patients with cancer. Trial registration This study reports correlative data from two clinical trials that registered with clinicaltrials.gov, NCT01128296 (May 21, 2010) and NCT01978184 (November 7, 2013). Electronic supplementary material The online version of this article (10.1186/s12885-018-4584-2) contains supplementary material, which is available to authorized users.

In this work we show preliminary results of an experimental and numerical investigation of a transonic axial compressor subjected to a 90 degree circumferential inlet total pressure distortion. The fundamental goal of the investigation is to establish an experimental dataset to be used in the validation of numerical simulation tools. The special interest of this investigation is the ability of the advanced blade row tools such as harmonic balance to reproduce the experimental results. Therefore, this report is account of the initial work of the ongoing project of the validation of advanced blade row tools in ANSYS CFX. Measurements of the compressor’s performance and efficiency were obtained by “traversing” the distortion around the test article inlet over 24 separate experiments. The resultant 2-D total pressure and total temperature contours at three measurement stations are shown. The experimental instrumentation package also included high sampling frequency rotor casing static pressure measurements and high sampling frequency rotor exit total pressure measurements. Phase locked averaged casing static pressure and rotor exit total pressure at four locations relative to the inlet distortion are shown. The initial set of computations presented in this paper concentrate on a full annulus transient simulation conducted in preparation for the harmonic analysis simulations.

Accumulation of senescent cells over time contributes to aging and age-related diseases. However, what drives senescence in vivo is not clear. Here we used a genetic approach to determine if spontaneous nuclear DNA damage is sufficient to initiate senescence in mammals. Ercc1-/∆ mice with reduced expression of ERCC1-XPF endonuclease have impaired capacity to repair the nuclear genome. Ercc1-/∆ mice accumulated spontaneous, oxidative DNA damage more rapidly than wild-type (WT) mice. As a consequence, senescent cells accumulated more rapidly in Ercc1-/∆ mice compared to repair-competent animals. However, the levels of DNA damage and senescent cells in Ercc1-/∆ mice never exceeded that observed in old WT mice. Surprisingly, levels of reactive oxygen species (ROS) were increased in tissues of Ercc1-/∆ mice to an extent identical to naturally-aged WT mice. Increased enzymatic production of ROS and decreased antioxidants contributed to the elevation in oxidative stress in both Ercc1-/∆ and aged WT mice. Chronic treatment of Ercc1-/∆ mice with the mitochondrial-targeted radical scavenger XJB-5–131 attenuated oxidative DNA damage, senescence and age-related pathology. Our findings indicate that nuclear genotoxic stress arises, at least in part, due to mitochondrial-derived ROS, and this spontaneous DNA damage is sufficient to drive increased levels of ROS, cellular senescence, and the consequent age-related physiological decline. Keywords: Reactive oxygen species, Free radicals, Genotoxic stress, Oxidative lesions, Endogenous DNA damage, Cellular senescence, Aging

Ischemia and reperfusion (I/R) injury following liver transplantation (LTx) is an important problem that significantly impacts clinical outcomes. IFN regulatory factor-1 (IRF-1) is a nuclear transcription factor that plays a critical role in liver injury. Our objective was to determine the immunomodulatory role of IRF-1 during I/R injury following allogeneic LTx. IRF-1 was induced in liver grafts immediately after reperfusion in both human and mouse LTx. IRF-1 contributed significantly to I/R injury because IRF-1–knockout (KO) grafts displayed much less damage as assessed by serum alanine aminotransferase and histology. In vitro, IRF-1 regulated both constitutive and induced expression of IL-15, as well as IL-15Rα mRNA expression in murine hepatocytes and liver dendritic cells. Specific knockdown of IRF-1 in human primary hepatocytes gave similar results. In addition, we identified hepatocytes as the major producer of soluble IL-15/IL-15Rα complexes in the liver. IRF-1–KO livers had significantly reduced NK, NKT, and CD8+ T cell numbers, whereas rIL-15/IL-15Rα restored these immune cells, augmented cytotoxic effector molecules, promoted systemic inflammatory responses, and exacerbated liver injury in IRF-1–KO graft recipients. These results indicate that IRF-1 promotes LTx I/R injury via hepatocyte IL-15/IL-15Rα production and suggest that targeting IRF-1 and IL-15/IL-15Rα may be effective in reducing I/R injury associated with LTx.

The effect of turbulent approach flow on the radiated sound from a circular cylinder was studied experimentally. The approach flow turbulence was provided by a single stream shear layer produced by an open jet anechoic tunnel facility. An instrumented cylinder was used to measure steady and unsteady surface pressure. The sound radiated from the cylinder placed in both an irrotational approach flow and the highly turbulent approach flow of the shear-layer was measured and compared. The cylinders located within the shear layer produced a less tonal sound with a higher broadband amplitude when compared to that of the free-stream approach flow. Secondly, the radiated sound from cylinders of different diameters was investigated to assess the effect of the ratio of cylinder diameter to approach flow length scale. It was found that as this ratio decreased, the broadband sound levels decreased as well. A simple theoretical model was then used to provide a prediction of the radiated sound for the cylinder within the shear layer. The theoretical model used statistics of the velocity field of the approach shear layer and the radiated sound spectrum the cylinder placed in a free stream. It was found that this method provided a similar spectral shape to the measured radiated sound.

An experimental and numerical investigation of the flow in a compressor duct with engine-realistic in-production features is presented in this paper. The experimental testing was conducted in the ND-FSCC test facility at University of Notre Dame, Indiana, USA. A baseline duct was also tested for back-to-back comparison. The ducts were heavily instrumented; duct inlet and exit flowfields were scanned using a five-hole pressure probe that provided total pressure, velocities and flow angles. Based on the five-hole probe total pressures, duct losses could be assessed. Furthermore the duct inlet boundary layers were traversed and turbulence intensity levels were assessed. For the CFD analysis of the production-like duct, a highly complex computational grid, resolving all the geometrical features present, was used. A previously validated surface roughness model was used to account for the cast aero-surfaces. Both experimental and numerical results show that there is a significant increase in loss for the production-like duct when compared to the baseline duct loss. The CFD results agree very well with experimental results for the baseline duct, which makes it possible to use the experimental data recorded for the production-like duct to validate CFD tools for real geometry effects, such as interface steps and surface roughness for example.

Although a key role of cross-dressing has been established in immunity to viral infection and more recently in the instigation of transplant rejection, its role in tolerance is unclear. We investigated the role of intragraft dendritic cells (DCs) and cross-dressing in mouse major histocompatibility complex (MHC)-mismatched liver transplant tolerance that occurs without therapeutic immunosuppression. Although donor interstitial DCs diminished rapidly after transplantation, they were replaced in the liver by host DCs that peaked on postoperative day (POD) 7 and persisted indefinitely. Approximately 60% of these recipient DCs displayed donor MHC class I, indicating cross-dressing. By contrast, only a very minor fraction (0%-2%) of cross-dressed DCs (CD-DCs) was evident in the spleen. CD-DCs sorted from liver grafts expressed much higher levels of T cell inhibitory programed death ligand 1 (PD-L1) and high levels of interleukin-10 compared with non-CD-DCs (nCD-DCs) isolated from the graft. Concomitantly, high incidences of programed death protein 1 (PD-1)These findings suggest that graft-infiltrating PD-L1

Liver interstitial dendritic cells (DC) have been implicated in immune regulation and tolerance induction. We found that the transmembrane immuno-adaptor DNAX-activating protein of 12 kDa (DAP12) negatively regulated conventional liver myeloid (m) DC maturation and their in vivo migratory and T cell allostimulatory ability. Livers were transplanted from C57BL/6(H2(b) ) (B6) WT or DAP12(-/-) mice into WT C3H (H2(k) ) recipients. Donor mDC (H2-K(b+) CD11c(+) ) were quantified in spleens by flow cytometry. Anti-donor T cell reactivity was evaluated by ex vivo carboxyfluorescein diacetate succinimidyl ester-mixed leukocyte reaction and delayed-type hypersensitivity responses, while T effector and regulatory T cells were determined by flow analysis. A threefold to fourfold increase in donor-derived DC was detected in spleens of DAP12(-/-) liver recipients compared with those given WT grafts. Moreover, pro-inflammatory cytokine gene expression in the graft, interferon gamma (IFNγ) production by graft-infiltrating CD8(+) T cells and systemic levels of IFNγ were all elevated significantly in DAP12(-/-) liver recipients. DAP12(-/-) grafts also exhibited reduced incidences of CD4(+) Foxp3(+) cells and enhanced CD8(+) T cell IFNγ secretion in response to donor antigen challenge. Unlike WT grafts, DAP12(-/-) livers failed to induce tolerance and were rejected acutely. Thus, DAP12 expression in liver grafts regulates donor mDC migration to host lymphoid tissue, alloreactive T cell responses and transplant tolerance.

Ischemia reperfusion injury (IRI) causes tissue and organ injury, in part, through alterations in tissue blood flow and the production of reactive oxygen species. The cell surface receptor signal-regulatory protein-α (SIRP-α) is expressed on inflammatory cells and suppresses phagocytosis, but the function of SIRP-α in IRI has not been determined. We reported previously that the matricellular protein thrombospondin-1 is upregulated in IRI. Here, we report a novel interaction between thrombospondin-1 and SIRP-α on nonphagocytic cells. In cell-free experiments, thrombospondin-1 bound SIRP-α. In vascular smooth muscle cells and renal tubular epithelial cells, treatment with thrombospondin-1 led to phosphorylation of SIRP-α and downstream activation of Src homology domain 2-containing phosphatase-1. Thrombospondin-1 also stimulated phosphorylation of p47(phox) (an organizer subunit for nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 1/2) and increased production of superoxide, both of which were abrogated by knockdown or antibody blockade of SIRP-α. In rodent aortic rings, treatment with thrombospondin-1 increased the production of superoxide and inhibited nitric oxide-mediated vasodilation in a SIRP-α-dependent manner. Renal IRI upregulated the thrombospondin-1-SIRP-α signaling axis and was associated with increased superoxide production and cell death. A SIRP-α antibody that blocks thrombospondin-1 activation of SIRP-α mitigated the effects of renal IRI, increasing blood flow, suppressing production of reactive oxygen species, and preserving cellular architecture. A role for CD47 in SIRP-α activation in these pathways is also described. Overall, these results suggest that thrombospondin-1 binding to SIRP-α on nonphagocytic cells activates NADPH oxidase, limits vasodilation, and promotes renal IRI.

Introduction When EMG studies are performed for evaluation of suspected ALS, the El Escorial diagnostic criteria require fibrillation potentials to serve as evidence of lower motor neuron disease. The Awaji Criteria allow fasciculation potentials together with neurogenic motor unit potentials to serve as EMG evidence of lower motor neuron disease. Although the Awaji Criteria have been considered to facilitate the diagnosis of ALS by not requiring fibrillation potentials on EMG, the El Escorial Criteria for the diagnosis of ALS continue to serve as the most commonly used criteria for ALS clinical trials. Methods A retrospective review of the Mayo Clinic Arizona EMG laboratory database was performed for patients with the diagnosis of motor neuron disease over a 2 year period. The clinical and EMG findings were reviewed in detail. Results The EMG database review revealed 125 patients with a diagnosis of motor neuron disease. Review of records revealed the final diagnosis was ALS in 112 of these and 13 were diagnosed with other forms of motor neuron disease. The clinical and EMG findings were reviewed in detail. In the ALS patients, a total of 1844 muscles were studied by EMG which represents an average of 16.5 muscles studied per patient. Of the 1,844 muscles studied, 649 were reported to show fasciculation potentials (34%) and 1067 showed fibrillation potentials (58%). None of the patients diagnosed with ALS had 0 muscles showing fibrillation potentials. However, 15 patients (12%) were reported to have 0 muscles showing fasciculation potentials. The number of ALS patients with 5 or fewer muscles showing fasciculation potentials was high at 54 (48%) compared to the number of ALS patients with 5 or fewer muscles showing fibrillation potentials, which was only 16 (14%). Conclusion This retrospective review of EMG findings in ALS patients suggests that fasciculation potentials are not identified as often as fibrillation potentials. All patients with a final diagnosis of ALS had fibrillation potentials on their EMG study whereas 12% of patients with a final diagnosis of ALS did not have fasciculation potentials identified on their EMG study. Thus, absence of fasciculation potentials should not be considered an unusual circumstance in ALS patients or an argument against the diagnosis of ALS. In contrast, fibrillation potentials are an expected finding in patients with ALS and the absence of fibrillation potentials does provide a strong argument against the diagnosis of ALS.

First described as essential to the phagocytic activity of leukocytes, Nox2-derived ROS have emerged as mediators of a range of cellular and tissue responses across species from salubrious to deleterious consequences. Knowledge of their role in inflammation is limited, however. We postulated that TNFα-induced endothelial reactive oxygen species (ROS) generation and pro-inflammatory signaling would be ameliorated by targeting Nox2. Herein, we in silico-modelled two first-in-class Nox2 inhibitors developed in our laboratory, explored their cellular mechanism of action and tested their efficacy in in vitro and mouse in vivo models of inflammation. Our data show that these inhibitors (CPP11G and CPP11H) disrupted canonical Nox2 organizing factor, p47phox, translocation to Nox2 in the plasma membrane; and abolished ROS production, markedly attenuated stress-responsive MAPK signaling and downstream AP-1 and NFκB nuclear translocation in human cells. Consequently, cell adhesion molecule expression and monocyte adherence were significantly inhibited by both inhibitors. In vivo, TNFα-induced ROS and inflammation were ameliorated by targeted Nox2 inhibition, which, in turn, improved hind-limb blood flow. These studies identify a proximal role for Nox2 in propagated inflammatory signaling and support therapeutic value of Nox2 inhibitors in inflammatory disease., Graphical abstract Image 1

Pulmonary hypertension is recognized as a leading cause of morbidity and mortality in patients with sickle cell disease (SCD). We now report benchtop phenotyping from the explanted lungs of the first successful lung transplant in SCD. Pulmonary artery smooth muscle cells (PASMCs) cultured from the explanted lungs were analyzed for proliferate capacity, superoxide (O2 (•-)) production, and changes in key pulmonary arterial hypertension (PAH)-associated molecules and compared with non-PAH PASMCs. Upregulation of several pathologic processes persisted in culture in SCD lung PASMCs in spite of cell passage. SCD lung PASMCs showed growth factor- and serum-independent proliferation, upregulation of matrix genes, and increased O2 (•-) production compared with control cells. Histologic analysis of SCD-associated PAH arteries demonstrated increased and ectopically located extracellular matrix deposition and degradation of elastin fibers. Biomechanical analysis of these vessels confirmed increased arterial stiffening and loss of elasticity. Functional analysis of distal fifth-order pulmonary arteries from these lungs demonstrated increased vasoconstriction to an α1-adrenergic receptor agonist and concurrent loss of both endothelial-dependent and endothelial-independent vasodilation compared with normal pulmonary arteries. This is the first study to evaluate the molecular, cellular, functional, and mechanical changes in end-stage SCD-associated PAH.

Background Chronic Lymphocytic Leukemia (CLL) is characterised by a highly heterogeneous natural history and treatment response. Indeed, 50% of immunoglobulin heavy chain variable region (IgHV) hypermutated patients have an excellent progression free survival (PFS) after chemoimmunotherapy. Conversely, 25% of FCR treated patients relapse within 24 months (high risk CLL). Recent studies have shown that complex karyotype with or without TP53 disruption predicts for relapse after BCL2 therapy and BTK inhibitors. However, TP53 is the only marker for which routine testing is available. Overall, nearly 80% of patients relapsing after frontline FCR do not present a known poor risk genomic marker. Additional candidate genomic predictors of poor outcome including mutations in coding regions of NOTCH1, SF3B1 and RPS15, non-coding regions of NOTCH1 and enhancer regions of PAX5, telomere length, IgHV status, and DNA Damage Repair (DDR) germline mutations including TP53 and ATM have been reported in CLL. Further, the role of mutational signatures and regions of kataegis also merit additional investigation in progressive CLL. Evaluating all candidate predictors requires complex time consuming, multi-modality testing outside the scope of routine clinical diagnostic practice, however, in isolation, each has low predictive value. Here, we show preliminary data on a novel patient stratification method based on whole genome sequencing (WGS) data incorporating multiple genomic features in a single test. Patients and Methods Tumor (peripheral blood) and germline (saliva) samples were collected from 321 patients from 6 UK trials via the Genomics England CLL pilot: ARCTIC (n=61), AdMIRe (n=64), CLL 210 (n=30), CLEAR (n=12), RIAltO (n=88) and FLAIR (n=66). We performed WGS on the HiSeqX (Illumina). After read alignment, we detected somatic variants using Strelka 2.4.7 for small variants detection (SNV and InDels), Manta 0.28.0 for structural variant (SV) detection, and Canvas 1.3.1 for copy number variant (CNV) detection (Illumina). Non-coding regions were annotated with information from primary CLL, CLL cell lines and B-cell ENCODE databases. Mutational signatures and putative regions of kataegis were calculated based on Alexandrov et al. (Nature, 2013) and Lawrence et al. (Nature, 2013). Telomere lengths were assessed using Telomerecat. Data aggregation was performed using contingency tables combined with non-negative matrix factorization. Results Mean coverage was 94.2X for tumor and 28.5X for germline samples. We found a median of 9172 SNPs/sample after filtering and 2348 indels/sample across 321 patients. High risk CLL was enriched for genomic complexity and poor prognostic mutations. The most frequently mutated genes were SF3B1 (17%), TP53 (13%), NOTCH1 (12%), IGLL5 (12%), and ATM (11%). Analysis of non-coding regions using DNA methylation markers, ATAC-seq and Hi-C revealed potential candidate regions associated with early relapse. Using CNA and SV data, we identified interesting patterns of genomic complexity and structural variants, including a trend towards enrichment of del8p in Relapse/Refractory and FCR non-responders. Additionally, we investigated mutation signatures and kataegis across coding and non-coding regions of the genome. We correlated exonic regions of DDR genes in germline data with clinical outcomes and extended this to genes mutated in both tumor and germline data, termed germline-tumor double-hits. We examined the relationship between the Alexandrov hypermutation signature, IgHV status (determined by % homology to the reference genome) and PFS, and combined mutational density at the Ig locus with mutation signature aiming to predict IgHV status. Finally, we produced a binary contingency matrix, using non-negative matrix factorization to cluster the samples. This method highlighted patient groups with shared genomic profiles. Conclusion We present preliminary data on a patient stratification method derived from WGS of 321 paired germline and CLL trial samples. Our predictive signature includes driver gene mutations, CNAs, IgHV status, genomic complexity, telomere length, overall mutation burden and genes with germline-tumor double-hits. Our comprehensive, NGS-based patient stratification attempts to predict patient outcome in a single sequencing run. Disclosures Becq: Illumina: Employment. He:Illumina: Employment. Ross:Illumina: Employment. Bentley:Illumina: Employment. Pettitt:Celgene: Research Funding; Gilead: Research Funding; Roche: Research Funding; GSK/Novartis: Research Funding; Napp: Research Funding; AstraZeneca: Research Funding; Chugai: Research Funding. Hillmen:Novartis: Research Funding; Gilead Sciences, Inc.: Honoraria, Research Funding; Alexion Pharmaceuticals, Inc: Consultancy, Honoraria; F. Hoffmann-La Roche Ltd: Research Funding; Celgene: Research Funding; Acerta: Membership on an entity's Board of Directors or advisory committees; Abbvie: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pharmacyclics: Research Funding; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Schuh:Giles, Roche, Janssen, AbbVie: Honoraria.

Introduction: Viruses are commonly cited as trig- gers for autoimmune disease. It is unclear if West Nile virus (WNV) initiates autoimmunity. Methods: We describe 6 cases of myasthenia gravis (MG) that developed several months after WNV infection. All patients had serologically confirmed WNV neuroinvasive disease. None had evidence of MG before WNV. Results: All patients had stable neurological deficits when they developed new symptoms of MG 3 to 7 months after WNV infection. However, residual deficits from WNV confounded or delayed MG diagnosis. All patients had elevated acetylcholine receptor (AChR) antibodies, and 1 had thymoma. Treatment varied, but 4 patients required acetylcholinesterase inhibitors, multiple immunosuppressive drugs, and intravenous immune globulin or plasmapheresis for recurrent MG crises. Conclu- sions: The pathogenic mechanism of MG following WNV remains uncertain. We hypothesize that WNV-triggered autoim- munity breaks immunological self-tolerance to initiate MG, pos- sibly through molecular mimicry between virus antigens and AChR subunits or other autoimmune mechanisms. Muscle Nerve 49:26-29, 2014

Mononuclear phagocyte recognition of apoptotic cells triggering suppressive cytokine signaling is a key event in inflammation resolution from injury. Mice deficient in thrombospondin (TSP)-1 (thbs1⁻/⁻), an extracellular matrix glycoprotein that bridges cell-cell interactions, are prone to lipopolysaccharide-induced lung injury and show defective macrophage interleukin (IL)-10 production during the resolution phase of inflammation. Reconstitution of IL-10 rescues thbs1⁻/⁻ mice from persistent neutrophilic lung inflammation and injury and thbs1⁻/⁻ alveolar macrophages show defective IL-10 production following intratracheal instillation of apoptotic neutrophils despite intact efferocytosis. Following co-culture with apoptotic neutrophils, thbs1⁻/⁻ macrophages show a selective defect in IL-10 production, whereas prostaglandin E2 and transforming growth factor beta 1 responses remain intact. Full macrophage IL-10 responses require the engagement of TSP-1 structural repeat 2 domain and the macrophage scavenger receptor CD36 LIMP-II Emp sequence homology (CLESH) domain in vitro. Although TSP-1 is not essential for macrophage engulfment of apoptotic neutrophils in vivo, TSP-1 aids in the curtailment of inflammatory responses during the resolution phase of injury in the lungs by providing a means by which apoptotic cells are recognized and trigger optimal IL-10 production by macrophages.

The mechanisms involved in the progression from monoclonal gammopathy of undetermined significance (MGUS) and smoldering myeloma (SMM) to malignant multiple myeloma (MM) and plasma cell leukemia (PCL) are poorly understood but believed to involve the sequential acquisition of genetic hits. We performed exome and whole-genome sequencing on a series of MGUS (n=4), high-risk (HR)SMM (n=4), MM (n=26) and PCL (n=2) samples, including four cases who transformed from HR-SMM to MM, to determine the genetic factors that drive progression of disease. The pattern and number of non-synonymous mutations show that the MGUS disease stage is less genetically complex than MM, and HR-SMM is similar to presenting MM. Intraclonal heterogeneity is present at all stages and using cases of HR-SMM, which transformed to MM, we show that intraclonal heterogeneity is a typical feature of the disease. At the HR-SMM stage of disease, the majority of the genetic changes necessary to give rise to MM are already present. These data suggest that clonal progression is the key feature of transformation of HR-SMM to MM and as such the invasive clinically predominant clone typical of MM is already present at the SMM stage and would be amenable to therapeutic intervention at that stage.

This study evaluated two chemical approaches for treatment of commingled cadmium-cyanide (Cd-CN) and zinc-nickel (Zn-Ni) wastewaters. The first approach, which involved application of sodium hypochlorite (NaOCl), focused on elimination of chelating substances. The second approach evaluated the use of sodium dimethyldithiocarbamate (DMDTC) to specifically target and precipitate regulated heavy metals. Results demonstrated that by maintaining a pH of 10.0 and an oxidation-reduction potential (ORP) value of +600 mV, NaOCl treatment was effective in eliminating all chelating substances. Cadmium, chromium, nickel, and zinc solution concentrations were reduced from 0.27, 4.44, 0.06, and 0.10 ppm to 0.16, 0.17, 0.03, and 0.06 ppm, respectively. Similarly, a 1% DMDTC solution reduced these same metal concentrations in commingled wastewater to 0.009, 1.142, 0.036, and 0.320 ppm. Increasing the DMDTC concentration to 2% improved the removal of all regulated heavy metals except zinc, the removal of which at high pH values is limited by its amphotericity.

Peanuts (Arachis hypogaea) in addition to milk, eggs, fish, crustaceans, wheat, tree nuts, and soybean are commonly referred to as the "big eight" foods that contribute to the majority of food allergies worldwide. Despite the severity of allergic reactions and growing prevalence in children and adults, there is no cure for peanut allergy, leaving avoidance as the primary mode of treatment. To improve analytical methods for peanut allergen detection, researchers must overcome obstacles involved in handling complex food matrices while attempting to decipher the chemistry that underlies allergen protein interactions. To address such challenges, we conducted a global proteome characterization of raw peanuts using a sophisticated GELFrEE-PAGE-LC-MS/MS platform consisting of gel-based protein fractionation followed by mass spectrometric identification. The in-solution mass-selective protein fractionation: (1) enhances the number of unique peptide identifications, (2) provides a visual map of protein isoforms, and (3) aids in the identification of disulfide-linked protein complexes. GELFrEE-PAGE-LC-MS/MS not only overcomes many of the challenges involved in the study of plant proteomics, but enriches the understanding of peanut protein chemistry, which is typically unattainable in a traditional bottom-up proteomic analysis. A global understanding of protein chemistry in Arachis hypogaea ultimately will aid the development of improved methods for allergen detection in food.

The genomic landscape of breast cancer is complex, and inter- and intra-tumour heterogeneity are important challenges in treating the disease. In this study, we sequence 173 genes in 2,433 primary breast tumours that have copy number aberration (CNA), gene expression and long-term clinical follow-up data. We identify 40 mutation-driver (Mut-driver) genes, and determine associations between mutations, driver CNA profiles, clinical-pathological parameters and survival. We assess the clonal states of Mut-driver mutations, and estimate levels of intra-tumour heterogeneity using mutant-allele fractions. Associations between PIK3CA mutations and reduced survival are identified in three subgroups of ER-positive cancer (defined by amplification of 17q23, 11q13–14 or 8q24). High levels of intra-tumour heterogeneity are in general associated with a worse outcome, but highly aggressive tumours with 11q13–14 amplification have low levels of intra-tumour heterogeneity. These results emphasize the importance of genome-based stratification of breast cancer, and have important implications for designing therapeutic strategies., Much effort has recently been devoted to understanding the genomics of breast cancer. In this study, the authors integrate somatic mutation data with previously published copy number aberration and gene expression information for nearly 2,500 breast cancer samples.

Aims Thrombospondin-1 (TSP1) is a ligand for CD47 and TSP1−/− mice are protected from pulmonary hypertension (PH). We hypothesized the TSP1–CD47 axis is upregulated in human PH and promotes pulmonary arterial vasculopathy. Methods and results We analyzed the molecular signature and functional response of lung tissue and distal pulmonary arteries (PAs) from individuals with ( n = 23) and without ( n = 16) PH. Compared with controls, lungs and distal PAs from PH patients showed induction of TSP1–CD47 and endothelin-1/endothelin A receptor (ET-1/ETA) protein and mRNA. In control PAs, treatment with exogenous TSP1 inhibited vasodilation and potentiated vasoconstriction to ET-1. Treatment of diseased PAs from PH patients with a CD47 blocking antibody improved sensitivity to vasodilators. Hypoxic wild type (WT) mice developed PH and displayed upregulation of pulmonary TSP1, CD47, and ET-1/ETA concurrent with down regulation of the transcription factor cell homolog of the v-myc oncogene (cMyc). In contrast, PH was attenuated in hypoxic CD47−/− mice while pulmonary TSP1 and ET-1/ETA were unchanged and cMyc was overexpressed. In CD47−/− pulmonary endothelial cells cMyc was increased and ET-1 decreased. In CD47+/+ cells, forced induction of cMyc suppressed ET-1 transcript, whereas suppression of cMyc increased ET-1 signaling. Furthermore, disrupting TSP1–CD47 signaling in pulmonary smooth muscle cells abrogated ET-1-stimulated hypertrophy. Finally, a CD47 antibody given 2 weeks after monocrotaline challenge in rats upregulated pulmonary cMyc and improved aberrations in PH-associated cardiopulmonary parameters. Conclusions In pre-clinical models of PH CD47 targets cMyc to increase ET-1 signaling. In clinical PH TSP1–CD47 is upregulated, and in both, contributes to pulmonary arterial vasculopathy and dysfunction.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder characterized by a progressive death of motor neurons for which there is no cure or effective treatment. The cause of ALS and the specific mechanisms of neuronal death remain unknown. However, considerable evidence supports the existence of autoimmune mechanisms contributing to pathogenesis in ALS, including biochemical, morphological, pharmacological, and physiological studies performed in animal models, cell culture, or with ex vivo preparations (1–6). Typical hallmarks of autoimmunity, such as circulating immune complexes, higher frequency of a particular histocompatibility type, or association with other autoimmune diseases, have also been reported (7, 8). An important marker of autoimmunity is the degree of T-lymphocytic infiltration in the anterior horn of the spinal cord from ALS patients (9, 10). Using monoclonal antibodies against T-cells, B-cells, and macrophages, almost 80% of the specimens show a cellular mononuclear infiltration. The cellular composition of the spinal cord inflammation consists of subsets of suppressor or cytotoxic T-cells and macrophages in the anterior and lateral corticospinal tracts and anterior horns (10). T-helper cells are also observed in proximity to corticospinal tract degeneration (11). Hence, inflammation in ALS spinal cord and brain appears to be primarily due to T-cells and macrophages (12), and aberrant macrophage activity is believed by many investigators to contribute to the pathology underlying ALS. This may explain the recent promising results of an ALS phase 2 clinical trial of NP001, a regulator of inflammatory macrophage activity (13). Although the predefined endpoints in this study did not reach statistical significance, administration of NP001 was associated with cessation in disease progression in 27% of patients, approximately 2.5 times greater than the percentage in patients on placebo. Two major plasma markers of inflammation, interleukin-18 (IL-18) and lipopolysaccharide (LPS), differentiated NP001 responders from non-responders, suggesting that the subgroup of patients with greater baseline biomarkers of neuroinflammation experienced the most benefit (13). Additional evidence pointing toward pathologic involvement of autoimmune processes has been the finding that immunoglobulins from ALS patients have been shown to cause apoptosis of motor neurons in primary spinal cord cultures (14) and that passive transfer of immunoglobulins to mice caused abnormalities at motor end-plates and degeneration of motor neurons (4, 15). These findings suggest that antibodies can contribute to disease pathogenesis. Increased levels of interleukins IL-17 and IL-23 have also been found in serum and cerebrospinal fluid of ALS patients (16). This increment is thought to be a sign of T-helper 17 (Th17) activation, a subset of T-cells suggested to be crucial in destructive autoimmunity. Astrocytes have also been shown to participate in the pathogenesis of ALS by producing a microenvironment toxic to motor neurons through increased neuroinflammation, oxidative damage, and glutamate excitotoxicity (17, 18). Overactivated astroglia produce high levels of protein S100B and other proinflammatory factors, which exacerbate neuroinflammation. The extracellular effects of S100B vary, depending on the concentration attained; at nanomolar concentrations, S100B is trophic to neurons, but at micromolar concentrations, S100B causes neuronal apoptosis (19, 20). Many of the effects of S100B on neurons are transduced by the receptor for advanced glycation end-products (RAGE), which participates in the pathophysiology of brain inflammatory disorders by regulating several inflammation-related events, including activation and migration of microglia and neutrophils to inflammatory sites (19–21). Extravasation of S100B into the systemic circulation can also trigger a pathologic autoimmune reaction with circulating antibodies that may re-enter the CNS to initiate an autoimmune response (22). Hence, S100B can be viewed as an astrocytic endokine that can act as an immunoregulator to participate in inflammation and autoimmunity. Additional support for the autoimmune pathogenesis hypothesis is the finding that ALS has recently been included in the spectrum of neurologic manifestations associated with voltage-gated potassium channel (VGKC) autoimmunity (23–25).

This study uses expectancy theory in an experimental setting to explain student motivation in adopting e-Learning technology. Data gathered from 173 students, having classroom exposure to eLearning technologies, suggest that expectancy theory is appropriate for evaluating and understanding a student’s motivation to adopt an e-Learning technology. On average, students considered information acquisition as the most attractive outcome of an e-Learning technology. Further, empirical evidence suggests that e-Learning technology adoption is more likely to succeed 1) when the technology is perceived by students to be in their best interest, and 2) when students perceive that reasonable efforts will result in successful adoption.

Electrodiagnostic studies are an important component of the evaluation of patients with suspected peripheral nerve disorders. The pattern of findings and the features that are seen on the motor and sensory nerve conduction studies and needle electromyography can help to identify the type of neuropathy, define the underlying pathophysiology (axonal or demyelinating), and ultimately help to narrow the list of possible causes. This article reviews the electrodiagnostic approach to and interpretation of findings in patients with peripheral neuropathies.

Ischemia/reperfusion injury associated with kidney transplantation induces profound acute injury, influences early graft function, and affects long-term graft outcomes. To determine whether renal dendritic cells play any role during initial innate ischemia/reperfusion injury and the subsequent development of adaptive immune responses, we studied the behavior and function of renal graft and host infiltrating dendritic cells during early and late phases of renal ischemia/reperfusion injury. Wild type to green fluorescent protein (GFP) transgenic rat kidney transplantation was performed with and without 24-h cold storage. Ischemia/reperfusion injury in cold-stored grafts resulted in histopathological changes of interstitial fibrosis and tubular atrophy by 10 weeks, accompanied by upregulation of mRNAs of mediators of interstitial fibrosis and inflammation. In normal rat kidneys, we identified two populations of renal dendritic cells, predominant CD103(-)CD11b/c(+) and minor CD103(+)CD11b/c(+) cells. After transplantation without cold storage, grafts maintained CD103(-) but not CD103(+) GFP-negative renal dendritic cells for 10 weeks. In contrast, both cell subsets disappeared from cold-stored grafts, which associated with a significant GFP-expressing host CD11b/c(+) cell infiltration that included CD103(+) dendritic cells with a TNF-α-producing phenotype. These changes in graft/host dendritic cell populations were associated with progressive infiltration of host CD4(+) T cells with effector/effector-memory phenotypes and IFN-γ secretion. Thus, renal graft ischemia/reperfusion injury caused graft dendritic cell loss and was associated with progressive host dendritic cell and T-cell recruitment. Renal-resident dendritic cells might function as a protective regulatory network.

Methodology for sequence analysis of ∼150 kDa monoclonal antibodies (mAb), including location of post-translational modifications and disulfide bonds, is described. Limited digestion of fully denatured (reduced and alkylated) antibody was accomplished in seconds by flowing a sample in 8 m urea at a controlled flow rate through a micro column reactor containing immobilized aspergillopepsin I. The resulting product mixture containing 3–9 kDa peptides was then fractionated by capillary column liquid chromatography and analyzed on-line by both electron-transfer dissociation and collisionally activated dissociation mass spectrometry (MS). This approach enabled identification of peptides that cover the complete sequence of a murine mAb. With customized tandem MS and ProSightPC Biomarker search, we verified 95% amino acid residues of this mAb and identified numerous post-translational modifications (oxidized methionine, pyroglutamylation, deamidation of Asn, and several forms of N-linked glycosylation). For disulfide bond location, native mAb is subjected to the same procedure but with longer digestion times controlled by sample flow rate through the micro column reactor. Release of disulfide containing peptides from accessible regions of the folded antibody occurs with short digestion times. Release of those in the interior of the molecule requires longer digestion times. The identity of two peptides connected by a disulfide bond is determined using a combination of electron-transfer dissociation and ion–ion proton transfer chemistry to read the two N-terminal and two C-terminal sequences of the connected peptides.

Damage-associated molecular patterns (DAMPs) initiate inflammatory pathways that are common to both sterile and infectious processes. The DAMP, high-mobility group box 1 (HMGB1), and the transcription factor, interferon regulatory factor 1 (IRF-1), have been independently associated as key players in ischemia-reperfusion (I/R) injury. Our study demonstrates that IRF-1 contributes to hepatocellular release of HMGB1 and further that IRF-1 is a necessary component of HMGB1 release in response to hypoxia or after liver I/R. We also link the nuclear upregulation of IRF-1 to the presence of functional Toll-like receptor 4 (TLR4), a pattern recognition receptor also important in sterile and infectious processes. Using IRF-1 chimeric mice, we show that IRF-1 upregulation in hepatic parenchymal cells, and not in the bone marrow-derived immune cells, is responsible for HMGB1 release during ischemic liver injury. Finally, our study also demonstrates a role for IRF-1 in modulating the acetylation status and subsequent release of HMGB1 through histone acetyltransferases. We found that serum HMGB1 is acetylated after liver I/R and that this process was dependent on IRF-1. Additionally, liver I/R induced a direct association of IRF-1 and the nuclear histone acetyltransferase enzyme p300. Together, these findings suggest that I/R-induced release of acetylated HMGB1 is a process that is dependent on TLR4-mediated upregulation of IRF-1.

Introduction Carpal tunnel syndrome (CTS) is a common diagnosis that requires a thorough clinical history and examination. Electrodiagnostic (EDX) evaluation with nerve conduction studies (NCS) is frequently performed to assist the ordering physician when considering additional therapies or surgical intervention. It is a sensitive tool to examine median nerve dysfunction and aids in distinguishing CTS from cervical radiculopathy (CR) by identifying selective slowing of the conduction of median nerve fibers across the carpal tunnel. Some argue that if the NCS shows evidence of CTS, then needle EMG is not necessary. Current practice guidelines consider needle EMG to be an optional test better suited to exclude concurrent causes (cervical radiculopathy) of the patient’s symptoms for improving diagnostic sensitivity. We set out to determine the number of suspected CTS patients with evidence of CTS on NCS who also have concurrent needle EMG evidence of cervical radiculopathy. We aimed to further explore whether clinical examination performed by physicians ordering EDX could distinguish between the diagnoses of CTS and CR before testing is performed. Methods A retrospective chart review of 606 cases of CTS and CR in the Mayo Clinic Arizona EMG Laboratory from January 2015 to January 2016 was performed. Clinical notes of referring physicians for EMG/NCS and final EMG/NCS reports were reviewed for referring diagnoses, clinical history, physical exam and EMG diagnoses. Results Out of 472 patients diagnosed with CTS, 19.1% also had a concurrent diagnosis of CR confirmed by needle EMG ( n = 90). 17.2% of suspected CTS based on clinical assessment were not confirmed after electrodiagnostic testing ( n = 104). 29.0% of suspected CR patients were not confirmed after electrodiagnostic testing ( n = 176). 12 clinical assessment data points (clinical history and physical exam findings) felt to represent a common evaluation of diagnoses of CTS were compiled. These included cervicogenic pain, radicular pain, hand paresthesias, nocturnal pain, symptoms worse with activity, symptoms relieved by shaking out hands (“flick” sign), myotomal weakness, dermatomal sensory abnormalities, hyporeflexia, loss of sensation in the median nerve distribution, thenar atrophy, and hand weakness. The only clinical finding found to have a clinically significant association with both CTS and CR was hand paresthesias ( p = 0.001). Conclusion This series demonstrated that 19% of patients seen with CTS have an associated cervical radiculopathy. Without needle electromyography, these concurrent diagnoses would be missed, which can affect subsequent management and therapy. Clinical assessment alone may not be enough to distinguish between CTS and CR, as has been demonstrated in previous studies. Even though practice guidelines allude to optional needle EMG, this data provides a strong argument in favor of performing both NCS and needle EMG in patients with suspected CTS.

To understand and interpret active-sonar returns and effectively employ active sonar systems for purposes such as antisubmarine warfare (ASW), operators must understand fundamental concepts of acoustical oceanography that govern how sonar interacts with the environment. Experts achieve high levels of performance in analysis and employment not by solving acoustic-propagation equations, but rather by employing simplified mental models that characterize the functional relationships governing how the environment affects acoustic propagation. Thus, a key goal in educating operators is helping them to develop and use such mental models. Here, we describe the development and employment of ESAIL, the Environment for Surface ASW Interactive Learning, a learning platform that combines interactive simulation with scaffolded visualization to facilitate instructors and students in conducting and assessing the outcome of “what if…?” experiments. In ESAIL, users are scaffolded in performing “what if…?” experimentation b...

Pulmonary hypertension (PH) is a chronic pulmonary complication of sickle cell disease (SCD) and a leading cause of death in adults. The exact molecular causes of SCD-associated PH are unknown although pathologic hemolysis related to chronic nitric oxide depletion and oxidative stress are recognized as contributing factors. We have reported that the protein thrombospondin-1 (TSP1) is elevated in the plasma of patients with SCD and by interacting with its receptor CD47 limits vasodilation of distal pulmonary arteries ex vivo . We hypothesized that the TSP1-CD47 interaction may promote PH in the BERK sickle mouse model. To test this hypothesis, we inquired whether TSP1 and CD47 are upregulated in the lungs of BERK mice and patients with SCD-associated PH. We found that lungs of age matched male 13-14 months old BERK sickle mice overexpressed TSP1 and CD47 as compared to both C57BL/6J and Hemi BERK mice and also confirmed our prior findings that sickle mice develop PH as they age. Immunohistochemistry of lung sections from 6 patients with SCD-associated PH revealed increased levels of CD47 as compared to sections from patients without PH or overt lung disease. We interrogated the TSP1-CD47 axis in vivo by generating chimeric animals with a sickle erythropoiesis on a CD47KO background by transplanting BERK sickle bone marrow into C57BL/6J (n=24) and CD47 knockout (CD47KO, n=27) mice. Fully engrafted chimeric mice underwent pulmonary hemodynamic assessment after 6 months. Right ventricular (RV) pressures were lower in Sickle-to-CD47KO chimeras as compared to Sickle-to-C57BL/6J chimeras as shown by the reduced maximum pressure of the RV (22.1 ± 3.6 vs. 28.1 ± 8.8 mmHg, p=0.013) and mean pulmonary artery pressure (15.3 ± 2.6 vs. 18.8 ± 5.7 mmHg, p=0.020). The afterload of the Sickle-to-CD47KO chimeras was lower compared to Sickle-to-C57BL/6J chimeras as shown by the diminished pulmonary vascular resistance (1.2 ± 0.7 vs. 2.4 ± 2.2 Wood units, p=0.024) and RV effective arterial elastance (1.5 ± 0.9 vs. 2.7 ± 2.6 mmHg/µL, p=0.052). The RV diastolic function as measured by the RV dP/dtmin also showed a trend towards improvement as compared to Sickle-to-C57BL/6J chimeras (-1297.0 ± 318.2 vs. -1604.0 ± 668.2 mmHg/s, p=0.059). Finally, the Sickle-to-CD47KO chimeras had a tendency for less RV hypertrophy as evidenced by lower RV free wall weight as compared to Sickle-to-C57BL/6J chimeras (21.45 ± 5.0 vs. 24.63 ± 6.0 mg, p=0.056). Interestingly, plasma levels of soluble TSP1 were reduced in Sickle-to-CD47KO chimeras (61.45 ± 10.11 vs. 76.89 ± 10.84 pg/mL, p=0.012). On myography, aortic segments from Sickle-to-CD47KO chimeras had improved relaxation to the endothelial activator acetylcholine. We hypothesized that in SCD TSP1-CD47 signaling promotes PH, in part, by increasing ROS generation. Treatment with exogenous TSP1 (2.2x10-9 M, a concentration found in the plasma of SCD patients) stimulated increased levels of superoxide and hydrogen peroxide in human pulmonary artery endothelial cells by cytochrome C and Amplex Red assays, respectively. Furthermore, lungs of CD47KO chimeras had decreased oxidative damage as measured by reduced 4-hydroxynonenal and 3-nitrotyrosine staining vs. Sickle-to-C57BL/6J chimeras. Finally, suppression of CD47 with a morpholino (10 mg/kg body weight i.p. weekly) for 8 weeks (n=8 animals) also reduced pulmonary pressures in BERK mice. Our results show that genetic deletion or suppression of CD47 ameliorates SCD-associated PH, which may be due, in part, to decreased ROS levels. Targeting TSP1-CD47 may provide a new molecular approach to the treatment of SCD-associated PH. Disclosures Isenberg: Tioma Therapeutics: Equity Ownership; Radiation Control Technologies, Inc.: Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

Spinal accessory neuropathy (SAN) causes impaired arm mobility and pain. The spinal accessory nerve is often injured during surgical procedures such as neck dissection for tumor resection or cervical lymph node biopsy. Other traumatic injuries may also occur. SAN causes weakness of the trapezius muscle and, less frequently, of the sternocleidomastoid muscle. The clinical consequence of trapezius muscle weakness includes impaired stability of the scapula leading to upper limb dysfunction and pain. We present a rare and illustrative case of SAN associated with deep tissue massage leading to scapular winging and droopy shoulder as a result of weakness of the trapezius muscle.

Hepatic ischemia/reperfusion (I/R) injury significantly influences short-term and long-term outcomes after liver transplantation (LTx). The critical step initiating the injury is known to include sinusoidal endothelial cell (SEC) alteration during the cold preservation period. As carbon monoxide (CO) has potent cytoprotective functions on vascular endothelial cells, this study examined if CO treatment of excised liver grafts during cold storage could protect SECs and ameliorate hepatic I/R injury. Rat liver grafts were preserved in University of Wisconsin (UW) solution containing 5% CO (CO-UW solution) for 18 to 24 hours and were transplanted into syngeneic Lewis rats. After 18 hours of cold preservation, SEC damage was evident with propidium iodide (PI) nuclear staining on SECs, and the frequency of PI(+) SECs was significantly lower in grafts stored in CO-UW solution versus those stored in control UW solution. SEC protection with CO was associated with decreased intercellular cell adhesion molecule translocation and less matrix metalloproteinase release during cold preservation. After LTx with 18 hours of cold preservation, serum alanine aminotransferase levels and hepatic necrosis were significantly less in the CO-UW group than in the control UW group. With 24 hours of cold storage, 35% (7/20) survived with control UW solution, whereas the survival with CO-UW solution improved to 80% (8/10). These beneficial effects of CO-UW solution were associated with a significant reduction of neutrophil extravasation, down-regulation of hepatic messenger RNA for tumor necrosis factor alpha and intercellular cell adhesion molecule 1, and less hepatic extracellular signal-regulated kinase activation. Liver grafts from Kupffer cell-depleted donors or pseudogerm-free donors showed less SEC death during cold preservation, and CO-UW solution further reduced SEC death. In conclusion, CO delivery to excised liver grafts during cold preservation efficiently ameliorates SEC damage and hepatic I/R injury.

A novel mass spectrometry (MS)-based approach to the identification of host-derived biomarkers (BMs) in the circulating low-molecular-mass (LMM) fraction (

Free radical accumulation and oxidative stress have been proposed as contributing to the progression of amyotrophic lateral sclerosis (motor neuron disease). A range of antioxidant medications is available, and has been studied. We aimed to examine the effects of antioxidant medication in the treatment of people with amyotrophic lateral sclerosis, and searched the Cochrane Neuromuscular Disease Group Trials register (August 2005), MEDLINE (January 1966 to August 2005), EMBASE (January 1980 to August 2005) and other sources. Selection criteria were all randomized or quasi-randomized controlled trials of antioxidant treatment for amyotrophic lateral sclerosis. The authors independently applied the selection criteria, assessed study quality and two authors performed independent data extraction. The search identified 23 studies for consideration but only nine studies met the inclusion criteria. Only two studies used our predetermined primary outcome measure as the primary outcome measure (survival at 12 months treatment). However, sufficient data were available from four studies to allow analysis of this outcome measure, and a meta-analysis was performed. In the individual studies no significant effect was observed for vitamin E 500 mg twice daily; vitamin E 1 g five times daily; acetylcysteine 50 mg/kg daily subcutaneous infusion; or a combination of L-methionine 2 g, vitamin E 400 International Units, and selenium 0.03 mg three times daily (Alsemet). No significant effect on the primary outcome measure was observed in a meta analysis of all antioxidants combined. No significant differences were demonstrated in any of the secondary outcome measures. In the opinion of the reviewers, there is insufficient evidence of efficacy of individual antioxidants, or antioxidants in general, in the treatment of people with amyotrophic lateral sclerosis. One study reported a mild positive effect, but this was not supported by the analysis we used. Generally, the studies were poorly designed, and underpowered, with low numbers of participants and of short duration. Further well-designed trials of medications such as vitamin C and E are unlikely to be performed. If future trials of antioxidant medications are performed, careful attention should be given to sample size, outcome measures, and duration of the trial. The high tolerance and safety, and relatively low cost of vitamins C and E, and other considerations related to the lack of other effective treatments for amyotrophic lateral sclerosis, explain the continuing use of these vitamins by physicians and people with amyotrophic lateral sclerosis. While there is no substantial clinical trial evidence to support their clinical use, there is no clear contraindication.

Disease-associated blood biomarkers exist in exceedingly low concentrations within complex mixtures of high-abundance proteins such as albumin. We have introduced an affinity bait molecule into N-isopropylacrylamide to produce a particle that will perform three independent functions within minutes, in one step, in solution: (a) molecular size sieving, (b) affinity capture of all solution-phase target molecules, and (c) complete protection of harvested proteins from enzymatic degradation. The captured analytes can be readily electroeluted for analysis.

We evaluated the kinetics by which rat liver sinusoidal endothelial cells (LSECs) are repopulated in the reperfused transplanted liver after 18 hours of cold ischemic storage. We found that the majority of LSECs in livers cold-stored for 18 hours in University of Wisconsin solution are seriously compromised and often are retracted before transplantation. Sinusoids rapidly re-endothelialize within 48 hours of transplantation, and repopulation is coincident with up-regulation of hepatocyte vascular endothelial growth factor expression and vascular endothelial growth factor receptor-2 expression on large vessel endothelial cells and repopulating LSECs. Although re-endothelialization occurs rapidly, we show here, using several high-resolution imaging techniques and 2 different rat liver transplantation models, that engraftment of bone marrow–derived cells into functioning LSECs is routinely between 1% and 5%. Conclusion: Bone marrow plays a measurable but surprisingly limited role in the rapid repopulation and functional engraftment of bone marrow–derived LSECs after cold ischemia and warm reperfusion. (HEPATOLOGY 2007.)

The placenta contains different populations of stem/progenitor cells such as mesenchymal, hematopoietic, trophoblastic and pluripotent stem cells. Although some tissue-specific stem cells are restricted to particular parts of the placenta, the localization of embryonic stem cell-like cells in term human placenta has not been determined. We have used immunofluorescence staining techniques with antibodies to pluripotent stem cell antigens, SSEA-3, SSEA-4, TRA 1-60 and TRA 1-81, and confocal microscopic analysis to identify and localize stem cells within the placenta. Stem cell marker-positive cells were found in amnion but not in choriodecidua, tissues known to contain hematopoietic and trophoblastic stem cells. Amniotic mesenchymal cells did not react with these pluripotent stem cell markers, while all amniotic epithelial cells reacted with at least one antibody. The TRA 1-60 and TRA 1-81 positive cells were solitary and present throughout the surface of amniotic membrane without a specific pattern of distribution, whereas SSEA-3 was negative and SSEA-4 was weakly positive on all amniotic epithelial cells. These data suggest that the human amnion contains stem cell-like cells at different states of differentiation. Human term amnion may be useful source of pluripotent stem cells for regenerative medicine.

Treatment of specific immune-mediated neuromuscular disorders involves consideration of many factors including severity of illness, concurrent medical problems, supportive therapies, and immune-modulating therapies. Many immune-modulating therapies are available, including steroids, an increasing number of immunosuppressive drugs, plasmapheresis, and intravenous immunoglobulin (IVIG). Deciding on which immune-modulating therapy involves selecting from those with proven efficacy for a specific disorder and global considerations of which therapies are most appropriate for an individual patient's circumstances. IVIG has become a commonly used therapy as it is well tolerated, easily administered, and is often efficacious with a relatively rapid action. IVIG has become a first-line therapy for several immune-mediated demyelinating polyneuropathies and may play a role in treating exacerbations of myasthenia gravis and selected chronic treatment-refractory cases of Lambert-Eaton myasthenic syndrome, dermatomyositis, and polymyositis.

The vascular effects of arsenic in drinking water are global health concerns contributing to human disease worldwide. Arsenic targets the endothelial cells lining blood vessels, and endothelial cell activation or dysfunction may underlie the pathogenesis of both arsenic-induced vascular diseases and arsenic-enhanced tumorigenesis. The purpose of the current studies was to demonstrate that exposing mice to drinking water containing environmentally relevant levels of arsenic promoted endothelial cell dysfunction and pathologic vascular remodeling. Increased angiogenesis, neovascularization, and inflammatory cell infiltration were observed in Matrigel plugs implanted in C57BL/6 mice following 5-week exposures to 5-500 ppb arsenic [Soucy, N.V., Mayka, D., Klei, L.R., Nemec, A.A., Bauer, J.A., Barchowsky, A., 2005. Neovascularization and angiogenic gene expression following chronic arsenic exposure in mice. Cardiovasc.Toxicol 5, 29-42]. Therefore, functional in vivo effects of arsenic on endothelial cell function and vessel remodeling in an endogenous vascular bed were investigated in the liver. Liver sinusoidal endothelial cells (LSEC) became progressively defenestrated and underwent capillarization to decrease vessel porosity following exposure to 250 ppb arsenic for 2 weeks. Sinusoidal expression of PECAM-1 and laminin-1 proteins, a hallmark of capillarization, was also increased by 2 weeks of exposure. LSEC caveolin-1 protein and caveolae expression were induced after 2 weeks of exposure indicating a compensatory change. Likewise, CD45/CD68-positive inflammatory cells did not accumulate in the livers until after LSEC porosity was decreased, indicating that inflammation is a consequence and not a cause of the arsenic-induced LSEC phenotype. The data demonstrate that the liver vasculature is an early target of pathogenic arsenic effects and that the mouse liver vasculature is a sensitive model for investigating vascular health effects of arsenic.

SummaryMicroparticles (MPs) are small membrane vesicles generated by essentially all cell types. In the plasma, most MPs are derived from platelets, but those from other sources, particularly leukocytes (macrophages, lymphocytes, and neutrophils), endothelial cells, and even smooth muscle cells can be detected and appear to play an important role in normal physiology and various diseases. In previous work we analyzed the proteome of MPs generated from isolated platelets (platelet MPs). Here, we report on a comparative analysis of microparticles isolated from plasma (plasma MPs) versus platelet MP using two complementary methods of comparative analysis. The first method, spectral count analysis, yielded 21 proteins detected in plasma MPs (with a total spectral count of 10 or greater) that were essentially absent in platelet MPs (with a total spectral count of 1 or 0). An additional two proteins (von Willebrand Factor, albumin) were present in both types of MPs but enriched in the plasma MPs. The second method, isotope-coded affinity tag (ICAT) labeling of proteins, supported the spectral count results for the more abundant proteins and provided better relative quantitation of differentially expressed proteins. Proteins present only in the plasma MPs include several associated with apoptosis (CD5-like antigen, galectin 3 binding protein, several complement components), iron transport (transferrin, transferrin receptor, haptoglobin), immune response (complement components, immunoglobulin J and kappa chains), and the coagulation process (protein S, coagulation factor VIII).

The molecular genetic relationship between esophageal adenocarcinoma (EAC) and its precursor lesion, Barrett’s esophagus, is poorly understood. Using whole-genome sequencing on 23 paired Barrett’s esophagus and EAC samples, together with one in-depth Barrett’s esophagus case-study sampled over time and space, we have provided new insights on the following aspects: i) Barrett’s esophagus is polyclonal and highly mutated even in the absence of dysplasia; ii) when cancer develops, copy number increases and heterogeneity persists such that the spectrum of mutations often shows surprisingly little overlap between EAC and adjacent Barrett’s esophagus; and iii) despite differences in specific coding mutations the mutational context suggests a common causative insult underlying these two conditions. From a clinical perspective, the histopathological assessment of dysplasia appears to be a poor reflection of the molecular disarray within the Barrett’s epithelium and a molecular Cytosponge™ technique overcomes sampling bias and has capacity to reflect the entire clonal architecture.

The endogenous bronchodilator, S-nitrosoglutathione (GSNO), increases expression, maturation, and function of both the wild-type and the DeltaF508 mutant of the cystic fibrosis transmembrane conductance regulatory protein (CFTR). Though transcriptional mechanisms of action have been identified, GSNO seems also to have post-transcriptional effects on CFTR maturation. Here, we report that 1) GSNO is only one of a class of S-nitrosylating agents that, at low micromolar concentrations, increase DeltaF508 and wild-type CFTR expression and maturation; 2) NO itself (at these concentrations) and 8-bromocyclic GMP are minimally active on CFTR; 3) a novel agent, S-nitrosoglutathione diethyl ester, bypasses the need for GSNO bioactivation by gamma-glutamyl transpeptidase to increase CFTR maturation; 4) surprisingly, expression-but not S-nitrosylation-of cysteine string proteins (Csp) 1 and 2 is increased by GSNO; 5) the effect of GSNO to increase full maturation of wild-type CFTR is inhibited by Csp silencing (si)RNA; 6) proteins relevant to CFTR trafficking are SNO-modified, and SNO proteins traffic through the endoplasmic reticulum (ER) and Golgi after GSNO exposure; and 7) GSNO alters the interactions of DeltaF508 CFTR with Csp and Hsc70 in the ER and Golgi. These data suggest that GSNO is one of a class of S-nitrosylating agents that act independently of the classic NO radical/cyclic GMP pathway to increase CFTR expression and maturation. They also suggest that the effect of GSNO is dependent on Csp and on intracellular SNO trafficking. We speculate that these data will be of relevance to the development of NO donor-based therapies for CF.

Summary Background Non-invasive methods for diagnosis of urothelial carcinoma have reduced specificity in patients with non-malignant genitourinary disease or other disorders. We aimed to use mass spectrometry and bioinformatics to define and validate a cancer-specific proteomic pattern. Methods We used capillary-electrophoresis-coupled mass spectrometry to obtain polypeptide patterns from urine samples of 46 patients with urothelial carcinoma and 33 healthy volunteers. From signatures of polypeptide mass, we established a model for predicting the presence of cancer. The model was refined further by use of 366 urine samples obtained from other healthy volunteers and patients with malignant and non-malignant genitourinary disease. We estimated the proportion of correct classifications from the refined model by applying it to a masked group containing 31 patients with urothelial carcinoma, 11 healthy individuals, and 138 patients with non-malignant genitourinary disease. We also sequenced several diagnostic polypeptides for urothelial carcinoma. Findings We identified a diagnostic urothelial-carcinoma pattern of 22 polypeptide masses. On masked assessment, prediction models based on these polypeptides correctly classified all samples of urothelial carcinoma (sensitivity 100% [95% CI 87–100) and all healthy samples (specificity 100% [84–100]). Correct identification of patients with urothelial carcinoma from those with other malignant and non-malignant genitourinary disease ranged from 86% to 100%. A prominent polypeptide from the diagnostic pattern for urothelial carcinoma was identified as fibrinopeptide A—a known biomarker of ovarian cancer and gastric cancer. Interpretation Validation of a highly specific biomarker pattern for urothelial carcinoma in a large group of patients with various urological disorders could be used in the diagnosis of other diseases that are identified in urine samples or in other body fluids.

The vascular effects of arsenic are a global public health concern that contribute to disease in tens of millions of people worldwide.1 Whereas the role of environmental contaminants in the etiology of vascular diseases and in the vascular contributions to organ dysfunction remains poorly defined, epidemiological studies have associated As(III) exposures to increased risk of cardiovascular diseases1 and vascular contributions to liver disease.2 Liver effects associated with arsenic in drinking water include noncirrhotic portal fibrosis and, to a lesser extent, portal hypertension.2,3 These pathologic conditions involve increased vascular channels in the portal regions of the liver. Higher levels of chronic As(III) consumption increase urinary levels of porphyrins, a biomarker for liver injury, which are more pronounced in people under 20 years of age.4 In addition, cardiac and liver disorders are the major side effects of therapeutic As(III) regimes that treat leukemias.5 Despite epidemiological evidence that the liver vasculature is a pathogenic target of chronic As(III) ingestion,2 the direct effects of As(III) on liver vascular cells are unknown. In other vascular beds and isolated cell cultures, As(III) affects both endothelial and smooth muscle cell physiology. Arsenic(III) stimulates angiogenic processes in cultured endothelial cells and neovascularization in intact mouse and avian models.6–9 These angiogenic effects promote tumorigenesis in mice, as well as vascular remodeling.10,11 Stimulation of endothelial cell proliferation occurs at concentrations of As(III) that are not cytotoxic, whereas higher concentrations are cytotoxic and inhibit angiogenesis in tumors.10,11 Arsenic(III) stimulates cultured smooth muscle cells to proliferate and express vascular endothelial cell growth factor (VEGF), a primary mediator of angiogenesis.12 Although these effects of As(III) on cells from systemic blood vessels are well recognized, there is little data on the effects of As(III) exposures on liver vascular remodeling or specifically on the fenestrated sinusoidal endothelium. The liver sinusoidal endothelial cells (SECs) are highly specialized fenestrated cells that are unique in the extensive heterogeneity of vascular endothelium.13 Early in development, these cells differentiate to lose markers of a continuous endothelium, such as junctional expression of platelet endothelial cell adhesion molecule-1 (PECAM-1/CD31) and a basement membrane containing the matrix protein laminin-1.14 In the differentiation process, the SECs become fenestrated to allow sieving of circulating nutrients, lipids, and lipoproteins for normal liver metabolism.13 The SEC angiogenic process is different from angiogenesis in endothelial cells of systemic vessels, because there is no increase in sinusoidal vessel number or density. Instead, SEC angiogenesis is a dedifferentiation and maturation process called capillarization with diagnostic hallmarks of SEC defenestration and renewed surface expression of PECAM-1 and laminin-1 proteins.13,15–18 SEC defenestration and formation of tight intercellular junctions limits transendothelial cell transport.13,16,19,20 Capillarization precedes vascular remodeling of other liver vessels, such as hepatic arterioles and PBVP, causing blood flow shunting, vascular channel formation, and eventually liver fibrosis.15,16,21 Liver angiogenesis in general is recognized as an important pathogenic process not only in portal fibrosis, but also in portal hypertension and progression of HCCs.18,22–25 Finally, animal models have been used to demonstrate that liver capillarization affects the systemic vasculature by decreasing liver metabolism of lipids, lipoproteins, and glucose to promote atherogenesis in response to environmental stresses and aging.13,26,27 In mouse tumors or surrogate in vivo neovascularization assays, low to moderate levels of As(III) (5–250 ppb) in drinking water are angiogenic.7,28 However, As(III) effects on the liver endothelium or pathogenic angiogenic processes in the liver are unknown. The aim of this study was to investigate whether a chronic, human-relevant, environmental exposure to As(III) causes SEC dedifferentiation and dysfunctional capillarization in intact mice. The data indicate that prolonged As(III) exposure causes SEC maturation and sustained functional changes in SEC cell signaling.

Inherited genetic variation has a critical but as yet largely uncharacterized role in human disease. Here we report a public database of common variation in the human genome: more than one million single nucleotide polymorphisms (SNPs) for which accurate and complete genotypes have been obtained in 269 DNA samples from four populations, including ten 500-kilobase regions in which essentially all information about common DNA variation has been extracted. These data document the generality of recombination hotspots, a block-like structure of linkage disequilibrium and low haplotype diversity, leading to substantial correlations of SNPs with many of their neighbours. We show how the HapMap resource can guide the design and analysis of genetic association studies, shed light on structural variation and recombination, and identify loci that may have been subject to natural selection during human evolution. © 2005 Nature Publishing Group., link_to_OA_fulltext