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PDF file - 198K, Figure 1. Characterization of the anti-OX40 mAb (9B12). Figure 2. Total peripheral lymphocyte counts. Figure 3. Pharmacokinetics of CD134 (anti-OX40) mAb in patients. Figure 4. Direct ex vivo detection of the murine anti-OX40 mAb bound to T cells in treated patients. Figure 5. Regression of a pulmonary metastasis in a patient with renal carcinoma enrolled in cohort 1. Figure 6. Changes in Ki-67 expression within CD4+ and CD8+ T cell subsets examined over time after anti-OX40 from patients in cohorts 1 and 2. Figure 7. Increased proliferation of CD4+ Foxp3- T cells and CD8+ T cells correlates with a decrease or stabilization of tumor burden. Figure 8. Ki-67 expression by monkey CD4+ and CD8+ memory T cells after the administration of anti-OX40 , mouse Immunoglobulin or monkey OX40L:Ig. Figure 9. Determination of the Endpoint Titer for anti-KLH Ab.

PDF file - 68K, Details clinical trial, flow cytometry and monkey experiments referred to in the manuscript.

Chronic kidney disease (CKD) imposes a strong and independent risk for peripheral artery disease (PAD). While solutes retained in CKD patients (uremic solutes) inflict vascular damage, their role in PAD remains elusive. Here, we show that the dietary tryptophan-derived uremic solutes including indoxyl sulfate (IS) and kynurenine (Kyn) at concentrations corresponding to those in CKD patients suppress β-catenin in several cell types, including microvascular endothelial cells (ECs), inhibiting Wnt activity and proangiogenic Wnt targets in ECs. Mechanistic probing revealed that these uremic solutes downregulated β-catenin in a manner dependent on serine 33 in its degron motif and through the aryl hydrocarbon receptor (AHR). Hindlimb ischemia in adenine-induced CKD and IS solute–specific mouse models showed diminished β-catenin and VEGF-A in the capillaries and reduced capillary density, which correlated inversely with blood levels of IS and Kyn and AHR activity in ECs. An AHR inhibitor treatment normalized postischemic angiogenic response in CKD mice to a non-CKD level. In a prospective cohort of PAD patients, plasma levels of tryptophan metabolites and plasma’s AHR-inducing activity in ECs significantly increased the risk of future adverse limb events. This work uncovers the tryptophan metabolite/AHR/β-catenin axis as a mediator of microvascular rarefaction in CKD patients and demonstrates its targetability for PAD in CKD models.

Background: Diarrhoea is a common complaint in dogs and may cause significant patient morbidity and owner distress. It can be a clinical manifestation of gastrointestinal disorders (primary enteropathy) or non-gastrointestinal disorders (secondary enteropathy) and may often be accompanied by other clinical signs such as vomiting, weight loss and changes in appetite. Diarrhoea is considered to be chronic if it lasts more than three weeks or is seen intermittently over a period of one month or more. Effective treatment of chronic diarrhoea benefits from the identification of the underlying disease process. Aim of the article: This article discusses the investigation, treatment and major causes of chronic diarrhoea in dogs, with a focus on primary enteropathies.

BACKGROUND: CKD, characterized by retained uremic solutes, is a strong and independent risk factor for thrombosis after vascular procedures . Urem ic solutes such as indoxyl sulfate (IS) and kynurenine (Kyn) mediate prothrombotic effect through tissue factor (TF). IS and Kyn biogenesis depends on multiple enzymes, with therapeutic implications unexplored. We examined the role of indoleamine 2,3-dioxygenase-1 (IDO-1), a rate-limiting enzyme of kynurenine biogenesis, in CKD-associated thrombosis after vascular injury. METHODS: IDO-1 expression in mice and human vessels was examined. IDO-1(−/−) mice, IDO-1 inhibitors, an adenine-induced CKD, and carotid artery injury models were used. RESULTS: Both global IDO-1(−/−) CKD mice and IDO-1 inhibitor in wild-type CKD mice showed reduced blood Kyn levels, TF expression in their arteries, and thrombogenicity compared with respective controls. Several advanced IDO-1 inhibitors downregulated TF expression in primary human aortic vascular smooth muscle cells specifically in response to uremic serum. Further mechanistic probing of arteries from an IS-specific mouse model, and CKD mice, showed upregulation of IDO-1 protein, which was due to inhibition of its polyubiquitination and degradation by IS in vascular smooth muscle cells. In two cohorts of patients with advanced CKD, blood IDO-1 activity was significantly higher in sera of study participants who subsequently developed thrombosis after endovascular interventions or vascular surgery. CONCLUSION: Leveraging genetic and pharmacologic manipulation in experimental models and data from human studies implicate IS as an inducer of IDO-1 and a perpetuator of the thrombotic milieu and supports IDO-1 as an antithrombotic target in CKD.

We present the case of an adolescent male who presented to the emergency department with headache and vomiting. We discuss the differential diagnosis and the need to maintain a high index of suspicion to avoid missing ominous causes of headache. In this case, the patient had a pineoblastoma, detected on a noncontrast CT scan. The CT scan was done as part of the emergency department workup to evaluate headache accompanied by vomiting in this otherwise healthy teenager.

Intracerebral hemorrhage (ICH) is a relatively common condition seen throughout the world, with the vast majority of cases referring to primary ICH. However, secondary ICH from other underlying conditions is also possible. In the present case, the patient presented with severe headaches. An initial computed tomography (CT) was taken which showed hyperdense regions in both the occipital lobe and right lateral ventricle. The patient was hypertensive upon arrival, so medication was given to lower his blood pressure. Due to the patient's history of hypertension, it was believed to be a case of primary ICH caused by high blood pressure, but because of the odd positioning of the hemorrhaging, it was recommended for magnetic resonance imaging (MRI) and angiography (MRA) to be taken. Using the MRI and MRA, it was found out that growing nodes were responsible for the hypodense regions on the CT. Considering the patient's history of renal cell carcinoma metastasizing to the abdomen and lungs, the nodes were diagnosed as brain metastasis (BM) developed from the patient's past kidney cancer. Considering the hemorrhaging locations in the brain, it was concluded that the ICH was secondary to BM. After consulting neurosurgery and hematology, the patient was discharged to his family. Although not very prevalent in cases of ICH, BM is a cause that can not be overlooked. Sometimes initial imaging does not reveal such an underlying source. It is always important to pay close attention to the characteristics of the ICH so that it is possible to determine the true reason for the hemorrhage.

We present the case of a 44-year-old female who presented to the emergency department (ED) via emergency medical services with a chief concern of shortness of breath and was found to have bilateral pulmonary emboli (PE) while taking the direct oral anticoagulant dabigatran. The authors highlight the importance of considering PE even in patients who are on anticoagulation.

BackgroundStereotactic body radiotherapy (SBRT) demonstrates excellent local control in early stage lung cancer, however a quarter of patients develop recurrence or distant metastasis. Transforming growth factor-beta (TGF-β) supports metastasis and treatment resistance, and angiotensin receptor blockade (ARB) indirectly suppresses TGF-β signaling. This study investigates whether patients taking ARBs while undergoing SBRT for early stage lung cancer exhibited improved overall survival (OS) or recurrence free survival (RFS) compared to patients not taking ARBs. MethodsThis was a single institution retrospective analysis of 272 patients treated with SBRT for early stage lung cancer between 2009-2018. Patient health data was abstracted from the electronic medical record. OS and RFS were assessed using Kaplan-Meier method. Log-rank test was used to compare unadjusted survival between groups. Univariable and multivariable Cox proportional hazard regression models were used to estimate hazard ratios (HRs). ResultsOf 247 patients analyzed, 24 (10%) patients took ARBs for the duration of radiotherapy. There was no difference in mean age, median tumor diameter, or median biologic effective dose between patients taking ARBs or not. Patients taking ARBs exhibited increased OS (ARB=96.7 mo.; no ARB= 43.3 mo.; HR=0.25 [95% CI: 0.10 to 0.62, P = .003]) and increased RFS (median RFS, ARB=64.3 mo.; No ARB=35.1 mo.; HR=0.26 [95% CI: 0.10 to 0.63, P = .003]). These effects were not seen in patients taking angiotensin converting enzyme inhibitors (ACEIs) or statins. ConclusionsARB use while undergoing SBRT for early stage lung cancer may increase OS and RFS, but ACEI use does not show the same effect.

Colorectal cancer (CRC) is a leading non-familial cause of cancer mortality among men and women. Although various genetic and epigenetic mechanisms have been identified, the full molecular mechanisms deriving CRC tumorigenesis remains incompletely understood. In this study, we demonstrate that cell adhesion molecule transmembrane and immunoglobulin domain containing1 (TMIGD1) is highly expressed in mouse and human normal intestinal epithelial cells. We have developed TMIGD1 knockout mice and show that the loss of TMIGD1 in mice results in the development of adenomas in small intestine and colon. Additionally, the loss of TMIGD1 in mouse impaired intestinal epithelium brush border formation, junctional polarity and maturation. Mechanistically, TMIGD1 inhibits tumor cell proliferation, cell migration, arrests cell cycle at G2/M phase and induces expression of p21CIP1 (cyclin-dependent kinase inhibitor 1), and p27KIP1 (cyclin-dependent kinase inhibitor 1B) expression, key cell cycle inhibitor proteins involved in the regulation of the cell cycle. Moreover, we demonstrate that TMIGD1 is progressively downregulated in sporadic human CRC and correlates with poor overall survival. Our findings identify TMIGD1 as a novel tumor suppressor gene and provide insights into the pathogenesis of colorectal cancer and possibilities as a potential therapeutic target.

Introduction: In response to a perceived high incidence of acute kidney injury following cardiopulmonary bypass at our institution, a quality improvement initiative consisting of a systematic change to a delivered oxygen (DO2) goal-directed perfusion practice was implemented. We sought to maintain DO2 > 270 mL/min/m2 to reduce the incidence of acute kidney injury. Methods: ’The study population included all patients receiving isolated, non-emergent, on-pump coronary artery bypass grafting from January 2015 through December 2018, excluding patients requiring preoperative hemodialysis. DO2 goal-directed perfusion was instituted in February 2017. Acute kidney injury was defined using Acute Kidney Injury Network criteria. Results: The pre–goal-directed perfusion cohort included 257 patients, and the post–goal-directed perfusion cohort included 226 patients. The DO2 was significantly higher in the post–goal-directed perfusion group (p Conclusion: This initiative confirms previous assertions that DO2 is a critical intraoperative parameter and should direct perfusion intervention accordingly.

The proto-oncogene β-catenin drives colorectal cancer (CRC) tumorigenesis. Casitas B-lineage lymphoma (c-Cbl) inhibits CRC tumor growth through targeting nuclear β-catenin by a poorly understood mechanism. In addition, the role of c-Cbl in human CRC remains largely underexplored. Using a novel quantitative histopathologic technique, we demonstrate that patients with high c-Cbl-expressing tumors had significantly better median survival (3.7 years) compared with low c-Cbl-expressing tumors (1.8 years; P = 0.0026) and were more than twice as likely to be alive at 3 years compared with low c-Cbl tumors (P = 0.0171). Our data further demonstrate that c-Cbl regulation of nuclear β-catenin requires phosphorylation of c-Cbl Tyr371 because its mutation compromises its ability to target β-catenin. The tyrosine 371 (Y371H) mutant interacted with but failed to ubiquitinate nuclear β-catenin. The nuclear localization of the c-Cbl-Y371H mutant contributed to its dominant negative effect on nuclear β-catenin. The biological importance of c-Cbl-Y371H was demonstrated in various systems, including a transgenic Wnt-8 zebrafish model. c-Cbl-Y371H mutant showed augmented Wnt/β-catenin signaling, increased Wnt target genes, angiogenesis, and CRC tumor growth. This study demonstrates a strong link between c-Cbl and overall survival of patients with CRC and provides new insights into a possible role of Tyr371 phosphorylation in Wnt/β-catenin regulation, which has important implications in tumor growth and angiogenesis in CRC.

Individuals with CKD are particularly predisposed to thrombosis after vascular injury. Using mouse models, we recently described indoxyl sulfate, a tryptophan metabolite retained in CKD and an activator of tissue factor (TF) through aryl hydrocarbon receptor (AHR) signaling, as an inducer of thrombosis across the CKD spectrum. However, the translation of findings from animal models to humans is often challenging. Here, we investigated the uremic solute-AHR-TF thrombosis axis in two human cohorts, using a targeted metabolomics approach to probe a set of tryptophan products and high-throughput assays to measure AHR and TF activity. Analysis of baseline serum samples was performed from 473 participants with advanced CKD from the Dialysis Access Consortium Clopidogrel Prevention of Early AV Fistula Thrombosis trial. Participants with subsequent arteriovenous thrombosis had significantly higher levels of indoxyl sulfate and kynurenine, another uremic solute, and greater activity of AHR and TF, than those without thrombosis. Pattern recognition analysis using the components of the thrombosis axis facilitated clustering of the thrombotic and nonthrombotic groups. We further validated these findings using 377 baseline samples from participants in the Thrombolysis in Myocardial Infarction II trial, many of whom had CKD stage 2-3. Mechanistic probing revealed that kynurenine enhances thrombosis after vascular injury in an animal model and regulates thrombosis in an AHR-dependent manner. This human validation of the solute-AHR-TF axis supports further studies probing its utility in risk stratification of patients with CKD and exploring its role in other diseases with heightened risk of thrombosis.

Casitas B-lineage lymphoma (c-Cbl) is a recently identified ubiquitin ligase of nuclear β-catenin and a suppressor of colorectal cancer (CRC) growth in cell culture and mouse tumor xenografts. We hypothesized that reduction in c-Cbl in colonic epithelium is likely to increase the levels of nuclear β-catenin in the intestinal crypt, augmenting CRC tumorigenesis in an adenomatous polyposis coli (APC(Δ14/+)) mouse model. Haploinsufficient c-Cbl mice (APC(Δ14/+) c-Cbl(+/−)) displayed a significant (threefold) increase in atypical hyperplasia and adenocarcinomas in the small and large intestines; however, no differences were noted in the adenoma frequency. In contrast to the APC(Δ14/+) c-Cbl(+/+) mice, APC(Δ14/+) c-Cbl(+/−) crypts showed nuclear β-catenin throughout the length of the crypts and up-regulation of Axin2, a canonical Wnt target gene, and SRY-box transcription factor 9, a marker of intestinal stem cells. In contrast, haploinsufficiency of c-Cbl(+/−) alone was insufficient to induce tumorigenesis regardless of an increase in the number of intestinal epithelial cells with nuclear β-catenin and SRY-box transcription factor 9 in APC(+/+) c-Cbl(+/−) mice. This study demonstrates that haploinsufficiency of c-Cbl results in Wnt hyperactivation in intestinal crypts and accelerates CRC progression to adenocarcinoma in the milieu of APC(Δ14/+), a phenomenon not found with wild-type APC. While emphasizing the role of APC as a gatekeeper in CRC, this study also demonstrates that combined partial loss of c-Cbl and inactivation of APC significantly contribute to CRC tumorigenesis.

Casitas B lymphoma (c-Cbl) is an E3 ubiquitin ligase and a negative regulator of colorectal cancer (CRC). Despite its high expression in immune cells, the effect of c-Cbl on the tumor microenvironment remains poorly understood. Here we demonstrate that c-Cbl alters the tumor microenvironment and suppresses Programmed cell death-1 (PD-1) protein, an immune checkpoint receptor. Using syngeneic CRC xenografts, we observed significantly higher growth of xenografts and infiltrating immune cells in c-Cbl+/− compared to c-Cbl+/+ mice. Tumor-associated CD8+ T-lymphocytes and macrophages of c-Cbl+/− mice showed 2–3-fold higher levels of PD-1. Functionally, macrophages from c-Cbl+/− mice showed a 4–5-fold reduction in tumor phagocytosis, which was restored with an anti-PD-1 neutralizing antibody suggesting regulation of PD-1 by c-Cbl. Further mechanistic probing revealed that C-terminus of c-Cbl interacted with the cytoplasmic tail of PD-1. c-Cbl destabilized PD-1 through ubiquitination- proteasomal degradation depending on c-Cbl’s RING finger function. This data demonstrates c-Cbl as an E3 ligase of PD-1 and a regulator of tumor microenvironment, both of which were unrecognized components of its tumor suppressive activity. Advancing immune checkpoint and c-Cbl biology, our study prompts for probing of PD-1 regulation by c-Cbl in conditions driven by immune checkpoint abnormalities such as cancers and autoimmune diseases.

Patients with malignancy are at 4- to 7-fold higher risk of venous thromboembolism (VTE), a potentially fatal, yet preventable complication. Although general mechanisms of thrombosis are enhanced in these patients, malignancy-specific triggers and their therapeutic implication remain poorly understood. Here we examined a colon cancer-specific VTE model and probed a set of metabolites with prothrombotic propensity in the inferior vena cava (IVC) ligation model. Athymic mice injected with human colon adenocarcinoma cells exhibited significantly higher IVC clot weights, a biological readout of venous thrombogenicity, compared with the control mice. Targeted metabolomics analysis of plasma of mice revealed an increase in the blood levels of kynurenine and indoxyl sulfate (tryptophan metabolites) in xenograft-bearing mice, which correlated positively with the increase in the IVC clot size. These metabolites are ligands of aryl hydrocarbon receptor (AHR) signaling. Accordingly, plasma from the xenograft-bearing mice activated the AHR pathway and augmented tissue factor (TF) and plasminogen activator inhibitor 1 (PAI-1) levels in venous endothelial cells in an AHR-dependent manner. Consistent with these findings, the endothelium from the IVC of xenograft-bearing animals revealed nuclear AHR and upregulated TF and PAI-1 expression, telltale signs of an activated AHR-TF/PAI-1 axis. Importantly, pharmacological inhibition of AHR activity suppressed TF and PAI-1 expression in endothelial cells of the IVC and reduced clot weights in both kynurenine-injected and xenograft-bearing mice. Together, these data show dysregulated tryptophan metabolites in a mouse cancer model, and they reveal a novel link between these metabolites and the control of the AHR-TF/PAI-1 axis and VTE in cancer.

Emerging evidence in animal models of chronic kidney disease (CKD) implicates Aryl Hydrocarbon Receptor (AHR) signaling as a mediator of uremic toxicity. However, details about its tissue-specific and time-dependent activation in response to various renal pathologies remain poorly defined. Here, a comprehensive analysis of AHR induction was conducted in response to discrete models of kidney diseases using a transgenic mouse line expressing the AHR responsive-promoter tethered to a β-galactosidase reporter gene. Following validation using a canonical AHR ligand (a dioxin derivative), the transgenic mice were subjected to adenine-induced and ischemia/reperfusion-induced injury models representing CKD and acute kidney injury (AKI), respectively, in humans. Indoxyl sulfate was artificially increased in mice through the drinking water and by inhibiting its excretion into the urine. Adenine-fed mice showed a distinct and significant increase in β-galactosidase in the proximal and distal renal tubules, cardiac myocytes, hepatocytes, and microvasculature in the cerebral cortex. The pattern of β-galactosidase increase coincided with the changes in serum indoxyl sulfate levels. Machine-learning–based image quantification revealed positive correlations between indoxyl sulfate levels and β-galactosidase expression in various tissues. This pattern of β-galactosidase expression was recapitulated in the indoxyl sulfate–specific model. The ischemia/reperfusion injury model showed increase in β-galactosidase in renal tubules that persisted despite reduction in serum indoxyl sulfate and blood urea nitrogen levels. Thus, our results demonstrate a relationship between AHR activation in various tissues of mice with CKD or AKI and the levels of indoxyl sulfate. This study demonstrates the use of a reporter gene mouse to probe tissue-specific manifestations of uremia in translationally relevant animal models and provide hypothesis-generating insights into the mechanism of uremic toxicity that warrant further investigation.

Breeah S. Carey received her degree in speech, language, and hearing sciences with minors in Spanish and psychology from Purdue University in May 2018. She is currently pursuing her master’s degree in speech-language pathology at Purdue University and wants to serve pediatric clients in school and clinic settings. She has volunteered in various schools in Newark, New Jersey, as a tutor in her mother’s after-school program and as a speaker for the Diamond’s in the Rough Program at Belmont Runyon Elementary School, where she encouraged minority girls to attend college in the future. Lauren Jankowski is a senior in interdisciplinary engineering who plans to pursue graduate school and focus her research on mental illness. She has volunteered with several organizations, such as St. Vincent de Paul, Hannah’s House, and Upward Basketball, since early high school. Madison Long received her degree in accounting from Purdue University in 2017 and began her career with Microsoft Corporation in Seattle, Washington, in their finance division. At Purdue, she frequently participated in local Lafayette community initiatives centered around child literacy and engagement, including United Way’s Read to Succeed Program and the Beck Lane Boys and Girls Club after-school volunteering program. Joshua Walker is a senior in mechanical engineering with a minor in computer science and plans to earn a master’s degree in mechanical engineering after a few years of work experience. He has provided one-on-one assistance with students at the free Math and Science Tutoring Center in his high school, refurbished bicycles intended for community use at Revolutions Bicycle Cooperative in Memphis, Tennessee, and worked with Engineers for a Sustainable World to keep tailgating parties at Purdue clean of trash.

The oxidation of carbon monoxide on oxygen-modified Au(111) surfaces is studied using a combination of reflection-absorption infrared spectroscopy (RAIRS) and temperature-programmed desorption (TPD). TPD reveals that CO desorbs in two states with the low-temperature state have a peak temperature between ~130 and 150 K, and the higher-temperature state having a peak temperature that varies from ~175 to ~220 K depending on the initial oxygen and CO coverages. Infrared spectroscopy indicates that the low-temperature CO desorption state is predominantly associated with CO adsorbed on Au δ+ sites, while the higher-temperature states are due to CO on Au0 sites. No additional vibrational features are detected indicating that CO reacts directly with adsorbed atomic oxygen on gold to form CO2. Estimates of the activation energy for CO2 formation suggest that they are in the same range and found for supported gold catalysts at reaction temperature below ~300 K. Fil: Thuening, Theodore. University Of Wisconsin; Estados Unidos Fil: Walker, Joshua. University Of Wisconsin; Estados Unidos Fil: Adams, Heather. University Of Wisconsin; Estados Unidos Fil: Furlong, Octavio Javier. Universidad Nacional de San Luis. Facultad de Ciencias Fisico- Matematicas y Naturales; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico San Luis. Instituto de Física Aplicada; Argentina; Argentina Fil: Tysoe, Wilfred T.. University Of Wisconsin; Estados Unidos

The adsorption and self-assembly of 1,3-phenylene diisocyanide (1,3-PDI) are studied on Au(111) using reflection–adsorption infrared spectroscopy (RAIRS), scanning tunneling microscopy (STM), and temperature-programmed desorption (TPD) supplemented by density functional theory (DFT) calculations and the results compared with the structures formed from 1,4-PDI where it assembled to form −(Au–PDI)– oligomer chains that incorporate gold adatoms. The infrared spectra display a single isocyanide feature consistent with the isocyanide binding to gold adatoms, while DFT calculations confirm that isocyanide binding to gold adatoms is more energetically favorable than binding to the surface. STM images show that 1,3-PDI forms zigzag chains containing hairpin bends that cause the chains to double back on each other, consistent with the 120° angle between the isocyanide groups. Hexagonal structural motifs are also observed that are proposed to be due to the self-assembly of three isocyanides as well as small structu...

Alterations in the efficacy of neuronal inhibition mediated by GABAA receptors (GABAARs) containing β3 subunits are continually implicated in autism spectrum disorders (ASDs). In vitro, the plasma membrane stability of GABAARs is potentiated via phosphorylation of serine residues 408 and 409 (S408/9) in the β3 subunit, an effect that is mimicked by their mutation to alanines. To assess if modifications in β3 subunit expression contribute to ASDs, we have created a mouse in which S408/9 have been mutated to alanines (S408/9A). S408/9A homozygotes exhibited increased phasic, but decreased tonic, inhibition, events that correlated with alterations in the membrane stability and synaptic accumulation of the receptor subtypes that mediate these distinct forms of inhibition. S408/9A mice exhibited alterations in dendritic spine structure, increased repetitive behavior, and decreased social interaction, hallmarks of ASDs. ASDs are frequently comorbid with epilepsy, and consistent with this comorbidity, S408/9A mice exhibited a marked increase in sensitivity to seizures induced by the convulsant kainic acid. To assess the relevance of our studies using S408/9A mice for the pathophysiology of ASDs, we measured S408/9 phosphorylation in Fmr1 KO mice, a model of fragile X syndrome, the most common monogenetic cause of ASDs. Phosphorylation of S408/9 was selectively and significantly enhanced in Fmr1 KO mice. Collectively, our results suggest that alterations in phosphorylation and/or activity of β3-containing GABAARs may directly contribute to the pathophysiology of ASDs.

Chronic kidney disease (CKD/uremia) remains vexing because it increases the risk of atherothrombosis and is also associated with bleeding complications on standard antithrombotic/antiplatelet therapies. Although the associations of indolic uremic solutes and vascular wall proteins [such as tissue factor (TF) and aryl hydrocarbon receptor (AHR)] are being defined, the specific mechanisms that drive the thrombotic and bleeding risks are not fully understood. We now present an indolic solute-specific animal model, which focuses on solute-protein interactions and shows that indolic solutes mediate the hyperthrombotic phenotype across all CKD stages in an AHR- and TF-dependent manner. We further demonstrate that AHR regulates TF through STIP1 homology and U-box-containing protein 1 (STUB1). As a ubiquitin ligase, STUB1 dynamically interacts with and degrades TF through ubiquitination in the uremic milieu. TF regulation by STUB1 is supported in humans by an inverse relationship of STUB1 and TF expression and reduced STUB1-TF interaction in uremic vessels. Genetic or pharmacological manipulation of STUB1 in vascular smooth muscle cells inhibited thrombosis in flow loops. STUB1 perturbations reverted the uremic hyperthrombotic phenotype without prolonging the bleeding time, in contrast to heparin, the standard-of-care antithrombotic in CKD patients. Our work refines the thrombosis axis (STUB1 is a mediator of indolic solute-AHR-TF axis) and expands the understanding of the interconnected relationships driving the fragile thrombotic state in CKD. It also establishes a means of minimizing the uremic hyperthrombotic phenotype without altering the hemostatic balance, a long-sought-after combination in CKD patients., National Institutes of Health (U.S.) (Grant R01HL132325), National Institutes of Health (U.S.) (Grant R01CA175382), National Institute of General Medical Sciences (U.S.) (Grant R01GM49039), American Heart Association (Grant 12FTF12080241)

Background Despite effective combination antiretroviral therapy, HIV-infected individuals develop comorbidities, including cardiovascular disease, where activated macrophages play a key role. To date, few therapies target activated monocytes and macrophages. Methods We evaluated a novel oral form of the polyamine biosynthesis inhibitor methylglyoxal-bis-guanylhydrazone (MGBG) on cardiovascular inflammation, carotid artery intima-media thickness (cIMT), and fibrosis in a simian immunodeficiency virus infection model of AIDS. Eleven simian immunodeficiency virus-infected animals received MGBG (30 mg/kg) once daily and 8 received a placebo control both beginning at 21 days postinfection (dpi). Animals were time sacrificed at 49 days post infection (dpi), when their matched placebo controls developed AIDS (63, 70, 77, 80), or at the study end-point (84 dpi). Aorta, carotid artery, and cardiac tissues were analyzed. Quantitative analyses of macrophage populations and T lymphocytes were done and correlated with cIMT and fibrosis. Results MGBG treatment resulted in 2.19-fold (CD163), 1.86-fold (CD68), 2.31-fold (CD206), and 2.12-fold (MAC387) decreases in macrophages in carotid arteries and significant 2.07-fold (CD163), 1.61-fold (CD68), 1.95-fold (MAC387), and 1.62-fold (CD206) decreases in macrophages in cardiac tissues. cIMT (1.49-fold) and fibrosis (2.05-fold) also were significantly decreased with MGBG treatment. Numbers of macrophage and the degree of fibrosis in treated animals were similar to uninfected animals. A positive correlation between decreased macrophage in the carotid artery and cIMT, and cardiac macrophages and fibrosis was found. Conclusions These data demonstrate that directly targeting macrophages with MGBG can reduce cardiovascular inflammation, cIMT, and fibrosis. They suggest that therapies targeting macrophages with HIV could be used in conjunction with combination antiretroviral therapy.

The role of macrophage activation, traffic, and accumulation on cardiac pathology was examined in 23 animals. Seventeen animals were simian immunodeficiency virus (SIV) infected, 12 were CD8 lymphocyte depleted, and the remaining six were uninfected controls (two CD8 lymphocyte depleted, four nondepleted). None of the uninfected controls had cardiac pathology. One of five (20%) SIV-infected, non-CD8 lymphocyte-depleted animals had minor cardiac pathology with increased numbers of macrophages in ventricular tissue compared to controls. Seven of the 12 (58%) SIV-infected, CD8 lymphocyte-depleted animals had cardiac pathology in ventricular tissues, including macrophage infiltration and myocardial degeneration. The extent of fibrosis (measured as the percentage of collagen per tissue area) was increased 41% in SIV-infected, CD8 lymphocyte-depleted animals with cardiac pathology compared to animals without pathological abnormalities. The number of CD163+ macrophages increased significantly in SIV-infected, CD8 lymphocyte-depleted animals with cardiac pathology compared to ones without pathology (1.66-fold) and controls (5.42-fold). The percent of collagen (percentage of collagen per total tissue area) positively correlated with macrophage numbers in ventricular tissue in SIV-infected animals. There was an increase of BrdU+ monocytes in the heart during late SIV infection, regardless of pathology. These data implicate monocyte/macrophage activation and accumulation in the development of cardiac pathology with SIV infection.

Background The ability to noninvasively assess arterial CD206+ macrophages may lead to improved understanding of human immunodeficiency virus (HIV)-associated cardiovascular disease. Methods We trialed a novel macrophage-specific arterial imaging technique. Results We demonstrated colocalization between technetium Tc 99m tilmanocept (99mTc-tilmanocept) and CD206+ macrophages ex vivo. In vivo application of 99mTc-tilmanocept single-photon emission computed tomography/computed tomography revealed high-level 99mTc-tilmanocept uptake across 20.4% of the aortic surface volume among HIV-infected subjects, compared with 4.3% among non-HIV-infected subjects (P = .009). Among all subjects, aortic high-level 99mTc-tilmanocept uptake was related to noncalcified aortic plaque volume (r = 0.87; P = .003) on computed tomographic angiography, and this relationship held when we controlled for HIV status. Conclusion These first-in-human data introduce a novel macrophage-specific arterial imaging technique in HIV. Clinical trials registration NCT02542371.

The distribution of gold atoms on the surface of a Au/Pd(100) alloy with various gold coverages was explored using density functional theory (DFT) calculations and measurements of the low-energy el...

Elimination of porosity and refinement of the normally coarse cast microstructure ofaluminium cast alloys by the intensive plastic deformation during friction stir processing (FSP) iswell known. However less is known about the mechanical behavior of the FS processed regionwhich contains zone/pass boundaries and macro/microstructure segregations. In the present study ofFS processed cast Al-7Si-0.3Mg alloy, microstructures featuring the deformed α-Al, fragmented Siparticles and their distribution in the processed region were related to the fracture paths duringtensile testing. It has been found that under the condition of a high rotation speed and minimum pinoverlap there is a strong upward flow of deformed cast material in thermomechanial affected zone.The arrays of Si particles in that flow have provided favorable paths for crack propagation duringtensile testing. As a result, tensile elongation and thus UTS values are low. The mechanism of thatupward flow and FSP conditions for reducing the flow and thus for improving properties of theprocessed region are discussed.

KCC2 comprises the major Cl(-) extruding mechanism in most adult neurons. Hyperpolarizing GABAergic transmission depends on KCC2 function. We recently demonstrated that glutamate reduces KCC2 function by a phosphorylation-dependent mechanism that leads to excitatory GABA responses. Here we investigated the methods by which to estimate changes in E(GABA), as well as the processes that lead to depolarizing GABA responses and their effects on neuronal excitability. We demonstrated that current-clamp recordings of membrane potential responses to GABA can determine upper and lower limits of E(GABA). We also further characterized depolarizing GABA responses, which both excited and inhibited neurons. Our analyses revealed that persistently active GABA(A) receptors contributed to loading Cl(-) during the glutamate exposure, indicating that tonic inhibition can facilitate the development of depolarizing GABA responses and increase excitability after pathophysiological insults. Finally, we demonstrated that hyperpolarizing GABA responses could temporarily switch to depolarizing responses when they coincided with an afterhyperpolarization.

A 20-year-old male was involved in a motor vehicle accident and computed tomography revealed a completely transected right mainstem bronchus. An Emergency Department (ED) right anterior thoracotomy was necessary soon after arrival at our institution secondary to acute desaturation that was unresponsive to ventilator and chest tube management. This allowed direct intubation and ventilation of the right middle and lower lobes directly through the thoracotomy incision, which stabilized the patient for transport to the operating room. Once there, percutaneous cardiopulmonary support (CPS) was initiated to allow primary surgical repair of the transected bronchus. Post surgery, the patient was transported to the surgical intensive care unit on CPS which he required for an additional two days. The patient eventually did well and was discharged home. To our knowledge this is the first successful reported case of using the Avalon Elite dual lumen veno-venous cannula for CPS in a patient with complete right main-stem bronchus transection and bilateral pulmonary contusions.

Objectives Cerebral ischemia can occur during cardiopulmonary bypass, especially during low flow. HBOC-201 (OPK Biotech, Cambridge, Mass) is a hemoglobin-based oxygen-carrying solution that enhances oxygen delivery. This project evaluated the benefits on total body and cerebral oxygen delivery and consumption using HBOC-201 during cardiopulmonary bypass. Methods Twelve immature swine were assigned to one of 2 groups. One group used HBOC-201 in pump prime, and the other used donor porcine blood. Cardiopulmonary bypass was initiated and then flow was serially decreased from 100% to 75%, to 50%, and then back to full flow. At each interval, 15 O positron emission tomographic analysis was performed, and blood was collected. Total body and cerebral oxygen delivery and consumption were calculated. Statistical analysis was performed with a Tukey–Kramer adjusted P value based on a repeated measures linear model on log-transformed data. Results Total and plasma hemoglobin levels were higher in the HBOC-201 group. Oxygen delivery and consumption were not statistically different but did tend to be higher in the HBOC-201 group. Mixed venous saturation was lower in the HBOC-201 group but not significant. Mild metabolic acidosis with increased lactate levels developed in the blood group. Mean cerebral blood flow decreased in both groups when total flow was 50%. In the HBOC-201 group cerebral oxygen metabolism was maintained. Conclusions The addition of HBOC-201 for cardiopulmonary bypass appears to improve oxygen use and minimize anaerobic metabolism. Cerebral oxygen use was preserved in the HBOC-201 group, even during decrease in blood flow. These findings support the reported improved oxygen-unloading properties of HBOC-201 and might provide a benefit during cardiopulmonary bypass.

In Turkey, a politically autonomous and secular military is pitted against a popularly elected, Islamic-rooted government, which has acquired new energy for domestic and foreign policy agendas and political reform. The election of Barack Obama and the Democrats in the US, as well as reverberations from the ongoing Ergenekon revelations and attempts to address the Kurdish question have brought new influences and players into the game of domestic Turkish politics. This article analyses the possible impact of these changes domestically, as well as from the EU and US perspectives.

Recombinant Factor VIII (FVIII) therapies have been created to provide treatment for Hemophilia A, an inherited bleeding disorder caused by mutation in the FVIII gene. A major challenge in the purification of recombinant FVIII molecules is the development of an affinity chromatography step. Such a step must be highly specific and selective for the FVIII molecule, but also must be designed appropriately to ensure the FVIII molecule can be effectively recovered without resorting to harsh elution conditions which may be harmful to the product. Additionally, it is desirable to have affinity adsorbents designed to be reusable over a large number of column cycles while maintaining consistent purification performance. In this work, we describe the use of VIIISelect, a commercially available affinity adsorbent designed specifically for the purification of FVIII compounds. The VIIISelect adsorbent consists of a 13 kDa recombinant protein ligand attached to a cross-linked agarose base matrix. The structure of the recombinant ligand is based upon Camelid-derived single domain antibody fragments. The VIIISelect adsorbent is produced using a process free of animal-derived raw materials, which is a highly desirable attribute for adsorbents used in the purification processes of recombinant protein therapeutics. The VIIISelect adsorbent was used as the initial capture column to purify a FVIII compound directly from clarified cell culture fluid prior to further downstream purification. The purification performance of the VIIISelect was evaluated, which included measurement of the static binding capacity, dynamic binding capacity, product recovery, impurity clearance, and adsorbent reuse. Following laboratory-scale process development, the VIIISelect adsorbent was scaled up and used in the large scale manufacturing of a FVIII compound.

Introduction: Recirculation (R), the shunting of arterial blood back into to the venous lumen, commonly occurs during veno-venous extracorporeal membrane oxygenation (VV-ECMO) and renders the monitoring of the venous line oxygen saturation no longer reflective of patient mixed venous oxygen saturation (SVO2). Previously, we failed to prove the hypothesis that, once R is known, it is possible to calculate the SVO2 of a patient on VV-ECMO. We hypothesize that we can calculate SVO2 during VV-ECMO if we account for and add an additional correction factor to our model for dissolved oxygen content. Therefore, the purpose of this study is to derive a more accurate model that will allow clinicians to determine SVO2 during VV-ECMO when ultrasound dilution is being used to quantify R. Methods : Using an extracorporeal circuit primed with fresh porcine blood, two stocks of blood were produced; (1) arterial blood (AB), and (2) venous blood (VB). To mimic recirculation, the AB and VB were mixed together in precise ratios using syringes and a stopcock manifold. Six paired stock AB/VB sets were prepared. Two sets were mixed at 20% R increments and 4 sets were mixed at 10% R increments. The partial pressure of oxygen (pO2 ) and oxygen (O2) saturation of the stock blood and resultant mixed blood was determined. The original model was modified by modeling the residual errors with linear regression. Results: When using the original model, as the partial pressure of arterial oxygen (PaO 2) of the stock AB increased, the calculated SVO2 was higher than actual, especially at higher R levels. An iteration of the original model incorporating the PaO2 level (low, medium, high) and R was derived to fit the data. Conclusions : The original model using R and circuit saturations for the calculation of SVO 2 in VV-ECMO patients is an oversimplification that fails to consider the influence of the high pO2 of arterial blood during therapy. In the future, further improvements in this model will allow clinicians accurately to calculate SVO2 in conjunction with recirculation measurements.

The nanoscience revolution that sprouted throughout the 1990s is having great impact in current and future DNA detection technology around the world. In this review, we report our recent progress on gold nanoparticle based fluorescence resonance energy transfer assay to monitor DNA hybridization as well as the cleavage of DNA by nucleases. We tried to discuss a reasonable account of the science and the important fundamental work carried out in this area. We also report the development of a compact, highly specific, inexpensive and user-friendly optical fiber laser-induced fluorescence sensor based on fluorescence quenching by nanoparticles to detect single-strand DNA hybridization at femtomolar level.