Merit Review Research Articles

Platelet extravasation across inflamed venules depends on CD18, mast cells and neutrophils

Platelets and neutrophils are often recruited at sites of microvascular inflammation. While neutrophil extravasation in inflammation is well characterized, platelet extravasation in inflammation is poorly understood and has only been documented in a small number of studies. We showed previously that mice expressing low levels of the leukocyte integrin CD18 (CD18hypo) had reduced total recruitment of platelets (intravascular and extravascular) in the corneal limbus following corneal epithelial wound injury. In this study we sought to determine the role of CD18 in platelet extravasation. Following corneal wound injury, wild‐type (WT) mice had evidence of platelet extravasation across limbal venules; however, CD18hypo mice had markedly reduced platelet extravasation. In view of isolated reports that platelets express CD18, we compared blood platelet counts and ex vivo platelet function under shear stress between CD18hypo and WT mice. Blood platelet counts were measured with a Scil Vet ABC hematology analyzer (Scil animal care company). Flow chamber assays were conducted using a microfluidic BioFlux System (Fluxion Biosciences) with plates coated with type I collagen (25 μg/ml; Helena) and blocked with 5% Bovine serum albumin. The labeled blood was perfused over the collagen‐coated surfaces at two values of fixed shear stress (16 dyne/cm2 and 30 dyne/cm2). Platelet counts and platelet adhesion to collagen at both levels of shear stress were comparable between CD18hypo and WT mice. Since a variety of leukocytes express CD18, including mast cells and neutrophils, we performed experiments to assess the role of these leukocytes in platelet extravasation. We found that CD18hypo mice had a small but statistically significant decrease in mast cell number and a marked decrease in degranulated mast cells as compared to WT mice following wound injury. Further, corneal epithelial wound injury in mice deficient in mast cells (KitW‐sh/W‐sh) resulted in a marked decrease in platelet extravasation as compared to WT mice. Finally, antibody‐induced depletion of circulating neutrophils resulted in a marked decrease in platelet extravasation relative to control mice. Overall, these studies demonstrate that platelet extravasation in a corneal wound injury model of inflammation depends on CD18, mast cells, and neutrophils.Support or Funding InformationSupported by: NIH EY018239, EY17120, HL079368 and VA BLR&D Merit Review Award I01 BX002551. M.A.B. was supported by NHLBI T32 HL139425Extravascular platelets (green) are evident surrounding venules 24 hours postwounding in wild type mice.Figure 1Platelets from CD18 hypomorphic mice had similar adhesion under shear as those from Wild‐type mice; blood platelet counts were similar in both strainsFigure 2

Effects of combined oxytocin and beta‐3 receptor agonist (CL 316243) treatment on body weight and adiposity in high fat diet‐induced obese rats

Previous studies have indicated that oxytocin (OXT) reduces body weight in high fat diet (HFD)‐induced obese (DIO) rodents, nonhuman primates and obese humans through reductions in energy intake and increases in energy expenditure. Recent findings support a role for brown adipose tissue (BAT) thermogenesis in OXT‐elicited weight loss as chronic administration 1) increases interscapular brown adipose tissue (IBAT) temperature (TIBAT) in DIO rats during a period that coincides with the OXT’s initial weight‐lowering effect and 2) upregulates beta‐3 receptor mRNA expression in IBAT. Based on these findings, we hypothesized that OXT would enhance the effectiveness of the beta‐3 receptor agonist, CL 316243, to evoke weight loss in DIO rats. To test this hypothesis, rats were fed a HFD for approximately 7 months prior to being implanted with a fourth ventricular (4V) cannula (as a strategy to target hindbrain OXT receptors), 28‐day minipump that infused OXT (16 nmol/day) or vehicle (VEH; saline) and temperature transponder underneath an IBAT depot. DIO rats were subsequently matched for body weight and adiposity, as well as OXT‐elicited reductions in body weight during the first week of the infusion period, prior to receiving 1x daily injections of VEH (sterile water, IP) or CL 316243 at a dose (0.5 mg/kg, IP) found to stimulate TIBAT in a separate group of DIO rats (P<0.05; N=4–5/group). Daily energy intake, TIBAT and body weight were tracked for 28 days. Body composition was assessed immediately prior to VEH or OXT treatment and near treatment completion using Quantitative Magnetic Resonance (QMR; EchoMRI 4‐in‐1). Preliminary data indicate that OXT (16 nmol/day) alone and CL 316243 alone tended to reduce body weight by approximately 6.5±2.2% (0.05<P<0.1) and 4.7±0.9% (P<0.05), respectively (N=4–5/group), relative to pre‐infusion baseline. However, the combination of OXT and CL 316243 produced a more pronounced reduction in body weight (13.9±1.6%; P<0.05) compared with either treatment alone (N=4–5/group). Weight loss in response to the combination treatment (OXT and CL 316243) was the result of a sustained reduction of body fat adiposity stores (P<0.05) without any change in lean mass (P=NS). These effects were associated with reductions in energy intake (P<0.05) suggesting that decreased energy intake contributes, in part, to the effectiveness of OXT and CL 316243 to reduce body weight. Furthermore, repeated 1x daily administration of CL 316243 alone (0.05<P<0.1), and in combination with OXT (P=0.1), tended to elevate TIBAT at 0.5‐h post‐injection relative to vehicle treatment throughout the entire treatment period (N=4–5/group). Together, these findings support the hypothesis that the combination of OXT and a beta‐3 agonist produces an additive effect to evoke weight loss and body adiposity in DIO rats by reducing energy intake and increasing BAT thermogenesis.Support or Funding InformationNational Institutes of Health R01 DK115976 and VA Merit Review Award BX004102

Effects of Individual or Combined Inhibition of Interleukin‐1 and Interleukin‐18 on the Resuscitation of Donation after Circulatory Death Murine Heart

BackgroundThere is a limited amount of donor hearts to be used for transplantation, which is mainly dependent on Donation after Brain Death (DBD) donors. This pool could be expanded with adoption of Donation after Circulatory Death (DCD) donor hearts. The transplantation of a heart that undergoes the DCD process would expose the organ to ischemia and reperfusion injury (IRI). IRI activates the NLRP3 inflammasome, which promotes the production of interleukin‐1 (IL‐1) and IL‐18.HypothesisCombined pharmacologic inhibition of IL‐1 and IL‐18 confers superior protection compared to individual blockade with IL‐1 or IL‐18 in the DCD heart.MethodsThe DCD mouse model was developed reflecting the clinical DCD protocol. The DCD was induced in C57 mice anesthetized, intubated, ventilated and paralyzed (vecuronium bromide 100 μg/kg) and by removing the ventilatory support to induce anoxia and circulatory failure. Total ischemic time was set at 40 min. Reanimation was achieved on a Langendorff system at 37°C using Krebs Henseleit (KH) buffer alone or supplemented with IL‐1 receptor antagonist (IL‐1Ra, 1 μg/ml), IL‐18 binding protein (IL‐18BP, 1 μg/ml) or their combination (n=8–10/group). Heart rate (HR), perfusion rate, developed pressure (DP) and +/−dP/dt were obtained at 15 min intervals. The rate pressure product (RPP) was calculated. After 90 min, the hearts were collected to measure DNA fragmentation (TUNEL assay). Cardiac troponin‐I (cTnI) levels were measured in the perfusate.ResultsHR and perfusion flow rates were comparable in all groups (table ). IL‐1Ra improved myocardial contractility, although it reached statistical significance only for DP and –dP/dt when compared to the DCD control (table ). IL‐18BP significantly improved all the parameters of myocardial contractility, compared to the control (all p<0.05). Markers of cell injury were significantly better after resuscitation with IL‐18BP, while IL‐1Ra did not reduce the release of cTnI or the DNA fragmentation (Table ). The combination of IL‐1Ra and IL‐18BP did not further improve the contractility nor further reduce damage of the DCD heart compared to the monotherapy treatment.ConclusionPharmacologic blockade of IL‐1 or IL‐18 protects the DCD mouse heart, with better protection with IL‐18BP compared to IL‐1Ra. Combined treatment with IL‐1Ra and IL‐18BP did not give additional benefits compared to the individual inhibitors.Support or Funding InformationThis work was supported by a Merit Review Award and and American Heart Association grant to Dr. Mohammed Quader. DCD DCD KH buffer KH buffer + inhibitors N=10 IL‐1Ra N=8 IL‐18BP N=9 IL‐1Ra+IL‐18BP r>N=5 Functional Data Heart Rate (beats/min) 396±14 401±15 440±10 408±20 Perfusion rate (ml/min) 1.8±0.1 2.2±0.3 2.4±0.1 1.9±0.2 Developed pressure (mmHg) 78.7±5.8 94.1±9.3 108.3±6.5 # 103.0±8.6 # Rate Pressure Product (mmHgxbeats/min) 29233±3293 34198±3634 43082±3191 # 31715±467 (+)dP/dt (mmHg/ms) 3256±266 3272±695 4744±204 # 5312±120 # (−)dP/dt (mmHg/ms) −2278±18 −2992±28 # −3632±29 # −3405±24 # Makers of myocyte damage cTnI (ng/ml) 7.79±0.98 7.68±0.85 4.21±0.67 # 2.63±0.71 # TUNEL+ nuclei (%) 2.09±0.53 2.02±0.76 0.99±0.23 1.01±0.23 Values are expressed as mean ± Standard Error of Mean. #P<0.05 vs DCD K‐H buffer.

Early Postnatal Gut Microbiota Determines SHR Hypertension

Hypertension is regulated by immunological components. We have shown that spontaneously hypertensive rats (SHR) display a genetic abnormality in a large population of proinflammatory CD161+ immune cells. Although early postnatal gut microbiota is important for development of the immune system, this relationship has not been studied in hypertension. Cross‐fostering SHR pups by normotensive Wistar‐Kyoto (WKY) dams lowers the blood pressure in adulthood when cross‐fostering is initiated at a very young age. In this study, we tested whether early alteration in postnatal gut microbiota affect s the immune system and blood pressure of SHR. We first examined the microbial populations in the fecal samples of SHR and normotensive control WKY using 16s rDNA sequencing. We found that the newborn SHR (1‐week old) have abnormal gut microbiota that is qualitatively and quantitatively different from the newborns of normotensive WKY. The representation of the predominant bacterial phylum Proteobacteria was significantly less in 1‐week old SHR pups than in WKY (94% Proteobacteria in WKY vs. 65% in SHR neonates). Even within the phylum Proteobacteria, the colonizing genera in WKY and SHR differed dramatically. Whereas WKY microbiota was predominantly comprised of Escherichia‐Shigella (92.5%), SHR microbiota was represented by Klebsiella (34.7%) and Haemophilus (27.8%). In contrast, the representation of phylum Fermicutes in the neonatal SHR gut was greater than WKY (5% Fermicutes in WKY vs. 31% in SHR). We cross‐fostered newborn SHR pups by lactating WKY dams and measured the resulting changes in the gut microbiota, immune system and blood pressure. Cross‐fostering caused a dramatic shift in 1‐week old cross‐fostered SHR gut microbiota. The two major bacterial genera of phylum Proteobacteria, Klebsiella and Haemophilus, present in self‐fostered SHR pups, were depleted after cross‐fostering, and were replaced by the predominant genera of WKY (Escherichia‐Shigella). A proinflammatory CD161+ immune cell population in the spleen of cross‐fostered SHR was also reduced (30.7% in self‐fostered SHR vs. 12.6% in cross‐fostered SHR at 30 weeks of age) as was the systolic blood pressure in adult cross‐fostered SHR (SHR 204 mmHg vs. cross‐fostered SHR 177 mmHg, p= 0.02, n= 4 each group). Thus, cross‐fostering of SHR pups by WKY dams shifted the composition of their gut microbiota toward WKY within one week. Additionally, reduction in proinflammatory immune cells and lowering of systolic blood pressure were observed in cross‐fostered SHR. In summary, SHR have a congenital defect in their immune system that results in greater proinflammatory response associated with hypertension. Gut microbiota of the SHR is remarkably different from that of normotensive WKY, and likely contributes to SHR blood pressure by modulating early development of the immune system.Support or Funding InformationNational Institutes of Health Program Project Grant to FMA (HL 14388) VA Merit Review Award to MWC (1 I01BX001414), American Heart Association Innovative Research Grant to MVS (16IRG27260323).

Non‐Tuberculous Mycobacteria Infections Increase NOS2 While Decreasing NO Levels

Non‐tuberculous mycobacteria (NTM) are rod‐shaped bacilli that cause lung infections in patients with weakened immune system or underlying lung disease such as cystic fibrosis, COPD, or prior tuberculosis. While it is well‐established that NTM cause lung disease, the host response to infection with NTM has not been well‐characterized. The production of reactive oxygen species (ROS) and reactive nitrogen species (RNS) by innate immune cells is considered a major defense mechanism against mycobacteria. Nitric oxide (NO), a product of inducible nitric oxide synthase (NOS2) in macrophages, plays an essential role in M. tuberculosis (MTb) infection through its bactericidal and anti‐inflammatory characteristics. Therefore, we hypothesized that NTM infection may upregulate NOS2 expression, which in turn may contribute to the host response to NTM infection. RAW 264.7 and MH‐S cells, virus transformed murine macrophages and alveolar macrophages, respectively, were exposed to NTM (M. avium‐intracellulare, or MAI) at a MOI of 25 and NO levels in cell media were monitored 4, 8, 12, 24, and 48 hours after NTM infection using a colorimetric GSNO assay. mRNA expression of NOS isoforms (NOS1, NOS2, and NOS3) in MH‐S and RAW cells were analyzed 24‐ and 48‐hours post‐infection. MH‐S cells were also transfected with siRNA to NOS2 and infected with MAI for 0.5, 4, and 24 hours, and their intracellular NTM were quantified to assess macrophage function. NO levels decreased following 4 hours of NTM exposure and remained decreased for 48 hours (P<0.05). NTM infection increased the expression of NOS2 without affecting levels of other NOS isoforms (P<0.05). siNOS2 transfected cells infected with MAI for more than four hours had significantly greater levels of intracellular MAI compared to scrambled negative control (P<0.05). Our preliminary data are the first to demonstrate that NTM increase NOS2 expression while decreasing NO levels. Furthermore, decreasing NOS2 expression exacerbates NTM infections. Future studies will examine mechanisms whereby NTM decrease NO levels. Therefore, we propose that approaches that restore NO can enhance macrophage host defense against NTMs and improve infection clearance.Support or Funding InformationThis work was supported in part by NIH Grant R01HL102167 (RLS), Merit Review funding from the Department of Veteran’s Affairs, Office of Research and Development (RTS). NTM isolates are Isolates were provided by Emory University Hospital

Simulated Stroke in a Mouse Neuronal Cell Culture Model

INTRODUCTIONDespite many efforts, stroke remains a cause of debilitating illness and death worldwide. The fact that not only ischemia itself, but also the return of blood flow aggravates cellular dysfunction, illustrates a clinical barrier. Hence, catheter‐based interventions, the therapy of ischemic strokes, can add detrimental tissue damage, described as ischemia/reperfusion (I/R) injury. It is therefore crucial to develop potential pharmacological strategies that attenuate reperfusion injury and improve neurological function. Animal in‐vivo and in‐vitro models are vital to determine potential targets for pharmacological therapies. While in‐vivo models better mimic the complexity of the clinical context, in‐vitro studies allow to focus on the pathophysiology and can be performed on individual cell types. This study aimed to establish an in‐vitro stroke model by investigating the effect of simulated I/R injury on mouse brain isolated neurons.METHODSNeurons were plated and grown to confluency. To simulate stroke, cells were then randomized to either control/normoxic (21% O2, 5% CO2, 74% N2) or hypoxic conditions in a hypoxic chamber (0.01% O2, 5% CO2, 94.99% N2) in serum‐ and glucose‐free media. Durations of hypoxia for 2.5, 5 and 10h were compared. After 2h of reoxygenation in regular media and normoxic culture conditions following hypoxia/normoxia, samples were assayed for cell number/viability, cytotoxicity by lactate dehydrogenase (LDH) release and mitochondrial viability. Statistics: Mann‐Whitney Rank Sum Test and regression analysis; alpha=0.05.RESULTSHypoxia significantly impaired cell number/viability after 5 and 10h, whereas 2.5h of hypoxia did not. A large decrease of cell number/viability was observed after 10h of hypoxia compared to 5h and 2.5h. The same observations were made for mitochondrial viability, apart from a slightly lower decrease after 5h hypoxia compared to the cell number/viability. Inverse relations were found for LDH release, except for an already significant increase in cytotoxicity after 2.5h of hypoxia.CONCLUSIONSOur data demonstrate that hypoxic injury in this model is time‐dependent. While 2.5h did not result in sufficient damage and 10h injured neurons too excessively, 5h hypoxia led to a moderate enough damage, still allowing for potential attenuation through protective strategies. Therefore, 5h hypoxia, followed by 2h reperfusion, appears to be a suitable duration for an in‐vitro mouse neuron stroke model to test new treatments.Support or Funding InformationThis work was supported by institutional funds, NIH grant (5R01 HL123227), and a Merit Review Award (I01 BX003482) from the U.S. Department of Veterans Affairs Biomedical Laboratory R&D Service.

Chromatin Regulation and Genome Maintenance by Mammalian SIRT6 and SIRT7

Members of the Sirtuin family of enzymes are important regulators of genomic stability, stress responses, and metabolic programs that impact on human physiology, aging, and age‐related disease processes. We previously showed that the mammalian Sirtuins SIRT6 and SIRT7 have high‐selectivity histone deacetylase activities at chromatin, and inactivation of SIRT6 or SIRT7 results in dysregulated histone acetylation states and gene expression programs, with pathological consequences at the cellular and whole organism levels. Recently, we have been exploring novel functions of SIRT6 and SIRT7 in silencing of heterochromatic regions of the genome, the deregulation of which has been linked to aging and cancer biology. We found that pericentric heterochromatin silencing by SIRT6 prevents acute cellular senescence that is triggered by pathologic pericentric transcripts. We also uncovered a second novel trigger of human cellular senescence, ribosomal DNA instability in nucleoli, and we showed that SIRT7 guards against senescence induced by this instability. In our studies, a long‐term focus has been identifying substrates of SIRT6 and SIRT7 and their roles in aging and disease pathways. In new work, we have identified a novel physiologic substrate of SIRT7 at chromatin, and we are characterizing the genomic landscape of this SIRT7‐dependent deacetylation target, and its downstream chromatin and nuclear signaling mechanisms. We will also discuss mechanistic insights into the functions of SIRT6 and SIRT7 from new proteomic, cellular, and mouse model studies.Support or Funding InformationVA Merit Review NIH/NIA

Quantification of Mitochondrial Membrane Potential in the Isolated Rat Lung Using Rhodamine 6G

Mitochondrial membrane potential (ΔΨm) plays a key role in vital mitochondrial functions, and its dissipation is a hallmark of mitochondrial dysfunction. The objective of this study was to develop an experimental and computational approach for estimating ΔΨm in intact rat lungs using the lipophilic fluorescent cationic dye rhodamine 6G (R6G). Rat lungs were excised and connected to a ventilation‐perfusion system. The experimental protocol consisted of three single‐pass phases: loading, wash, and uncoupling, in which the lungs were perfused with R6G‐containing perfusate, fresh R6G‐free perfusate, or R6G‐free perfusate containing the mitochondrial uncoupler FCCP, respectively. This protocol was carried out with lung perfusate containing verapamil vehicle or verapamil, an inhibitor of the multi‐drug efflux pump P‐glycoprotein (Pgp). Results show that the addition of FCCP resulted in an increase in R6G venous effluent concentration (Figure 1), and that this increase was larger in the presence of verapamil than in its absence. A physiologically‐based pharmacokinetic (PBPK) model for the pulmonary disposition of R6G was developed and used for quantitative interpretation of the kinetic data, including estimating ΔΨm. The estimated value of ΔΨm (−139 ± 21 (SD) mV) was not significantly altered by inhibiting Pgp with verapamil, and is consistent with that estimated previously in cultured pulmonary endothelial cells. These results demonstrate the utility of the proposed approach for quantifying ΔΨm in intact functioning lungs. This approach has potential to provide quantitative assessment of the effect of injurious conditions on lung mitochondrial function, and to evaluate the impact of therapies that target mitochondria.Support or Funding InformationSupported by NIH 2R15HL129209‐02 and VA Merit Review Award BX001681.Solid symbols: R6G venous effluent concentrations during the loading phase, wash phase, and uncoupling phase using FCCP (67 μM). Values are mean ± SE (n = 7 for loading and wash phases, and n = 5 for uncoupling phase). Solid line is PBPK model fit to data.Figure 1

Sodium Caprate Enhances Transcellular LPS Transport via Clathrin‐Mediated Endocytosis in Rat Jejunum

Lipopolysaccharides (LPS) entry from the intestinal lumen to the circulation is associated with systemic inflammation. We have found that LPS is transcellularly transported to portal vein during long‐chain fatty acid (LCFA) exposure via CD36‐ and lipid raft‐mediated pathways. We examined the effects of medium‐chain fatty acids (MCFA) on LPS transport in rat jejunum.FITC‐LPS was applied to the mucosal bath of Ussing chambered muscle‐stripped jejunal and distal colonic mucosa‐submucosa preparations, in which short‐circuit current (Isc) and tissue electrical resistance (TER) were measured. Serosal appearance of FITC‐LPS was measured with or without luminal application of sodium caprate (C10, 10 mM). FITC‐dextran 4kDa (FD4) m‐to‐s movement was also separately measured.Luminal application of caprate increased FITC‐LPS m‐to‐s transport without any changes in FD4 m‐to‐s movement and TER in the jejunum, whereas caprate remarkably increased FITC‐LPS transport with increased FD4 movement and decreased TER in the distal colon, suggesting that caprate enhances transcellular LPS transport in the jejunum, but increases paracellular permeability in the distal colon. In the jejunum, C10‐mediated FITC‐LPS m‐to‐s transport was inhibited by pretreatment with luminal Pitstop2 (30 μM), a selective inhibitor of clathrin‐mediated endocytosis (CME), whereas the CD36 inhibitor sulfosuccinimidyl oleate (SSO, 0.1 mM), the lipid raft inhibitor methyl‐β‐cyclodextrin (MβCD, 1 mM), or the dynamin inhibitor Dynasore (50 μM) had no effect. In contrast, luminal OA (30 mM) with taurocholate (TCA, 0.1 mM) also increased FITC‐LPS transport with SSO, MβCD and Dynasore‐sensitive, but Pitstop2‐insensitive manner. In vivo, jejunal luminal perfusion of C10 with FITC‐LPS increased portal venous FITC‐LPS appearance 15 min after perfusion, higher than by OA/TCA, which was then inhibited by co‐perfusion of Pitstop2.These results suggest that luminal MCFA and LCFA differentially induce transcellular LPS transport in the jejunum; MCFA via CME, but LCFA via the CD36/lipid raft/caveolin pathway. Dietary fatty acids may impact LPS‐associated diseases, including metabolic disease and multiple organ failure.Support or Funding InformationVA Merit Review and Shire Pharmaceuticals

Hepatic stellate cells are critically involved in modulation of inflammatory microenvironment during acute liver injury

Background and AimsDepletion of hepatic stellate cells (HSCs) was shown to protect the liver from acute injury due to ischemia/reperfusion and concanavalin A. Depletion protocol involves 3 administrations of carbon tetrachloride (CCl4) (3 days apart) to mice transgenic for thymidine kinase under the glial fibrillary acidic protein (GFAP (expressed exclusively by HSCs in the liver) promoter (GFAP‐TK/TG). This causes activation of HSCs that become vulnerable to ganciclovir (GCV)‐induced death. After 10‐days GCV treatment, 70–75% HSCs are depleted and the liver completely recovers from prior CCl4‐induced injury. We aimed to determine CCl4‐induced acute liver injury in HSC‐depleted mice and the role of HSCs in regulating hepatic inflammatory microenvironment.MethodsGFAP‐TK/TG or wild type (WT) B6 mice (Control) were treated with 0.16 ml/kg CCl4 (3 injections) then 40 μg/g/d GCV for 10 days. Other controls were GFAP‐TK/TG mice treated with CCl4 but not GCV (does not deplete HSCs) or naïve B6 mice. Mice then received 0.16 ml/kg CCl4 and mechanisms of liver injury were determined at 24h.ResultsHSC‐depleted mice showed similar CCl4‐induced necrotic liver damage and oxidative stress as HSC‐sufficient mice. CCl4 administration to HSC‐sufficient mice and not HSC‐depleted mice caused reduction in F4/80+ macrophages, but CD68+ cells and inflammation as measured by expression of TNFα, IL6 and IFNβ showed greater increase in HSC‐depleted mice than HSC‐sufficient mice. Surprisingly, CCl4 administration to HSC‐sufficient mice caused rapid activation of HSCs and significant fibrosis accompanied by increased expression of collagen 1a1, TGFβ1 and TGFβR1. As expected, α‐SMA+ activated fibrogenic HSCs were found in HSC‐sufficient and not HSC‐depleted mice. CCl4‐treated HSC‐sufficient mice also increased hepatic IL17‐producing γδT cells and Ly6c+ monocytes. In contrast, MMP13 and MMP9 (collagenases) expression increased in CCl4‐treated HSC‐depleted mice indicating increase in restorative macrophages (found during fibrosis resolution). Finally, in co‐culture, HSCs were found to affect the expression of various inflammatory cytokines/cytokines by CD4+ T cells, regulatory T cells and Kupffer cells variably.ConclusionThese data suggest that CCl4 has direct damaging effect on hepatocytes but HSCs regulate inflammatory response of the resident and infiltrating cells. Rapid activation of HSCs and fibrosis after CCl4 administration to CCl4/GCV‐pre‐treated and recovered WT mice indicate that even transiently activated HSCs that revert to the quiescent physiological phenotype upon removal of injury stimulus remain primed to become quickly re‐activated to cause fibrosis. This should be an important consideration when targeting HSCs to treat fibrosis clinically.Support or Funding InformationSupport: VA Merit Review Award 1IO1BX001174 and DoD W81XWH‐14‐PRMRP‐IIRA to CRG and NIH P30 DK078392 to the Digestive Diseases Health Center at Cincinnati Children’s Hospital Medical Center.

Effect of High Glucose on Hypoxia/Reoxygenation Injury in Mouse Diabetic Coronary Artery Endothelial Cells

BackgroundType 2 diabetes mellitus (DM2) remains an increasing cause of morbidity and mortality in the Western world, including coronary artery disease and myocardial infarction. In order to eventually test mechanisms of cardioprotection by different agents, we sought to develop an in‐vitro model of mouse DM2 coronary artery endothelial cells (ECs) undergoing hypoxia/reoxygenation (HR) injury under normal and high glucose concentrations. We hypothesized that high glucose exacerbates HR injury.MethodsDM2‐ECs, grown to confluency in complete media (5% fetal bovine serum and 5.5 mM glucose), were exposed to normal (5.5 mM) or high (20.5 mM) glucose concentrations for 72 hrs. They were then were subjected to normoxic (21% O2, complete media) or hypoxic (0.0125% O2, FBS‐ and glucose‐free media) conditions for 8 hrs, followed by reoxygenation in complete media for 16 hrs. To assess the extent of injury, cell (CyQUANT GR dye) and mitochondrial viability (3‐(4,5‐dimethylthiazol‐2‐yl)‐5‐(3‐carboxymethoxyphenyl)‐2‐(4‐sulfophenyl)‐2H‐tetrazolium; MTS) as well as cellular injury (lactate dehydrogenase release; LDH) were measured. Statistics: Two‐Way ANOVA followed by Tukey post‐hoc testing; alpha = 0.05 (two‐tailed).ResultsUnder both normal and high glucose conditions, HR caused a significant decrease in cell number and increase in LDH release, while MTS remained unchanged. The injury by HR under high compared to under normal glucose conditions, was not different at either outcome parameter.ConclusionsIn contrast to our hypothesis, 20.5 mM glucose did not significantly exacerbate HR‐induced cellular or mitochondrial injury and also did not lead to significantly increased injury under normoxic conditions. It is conceivable that the DM2‐ECs in our in‐vitro model perceive a concentration of 20.5 mM as closer to normal and a concentration of 5.5 mM lower than normal than would be anticipated in‐vivo. Another explanation could be that the hypoxia we chose (0.0125% O2 for 8 hrs) was not strong and/or long enough to cause a significant enough damage to be exacerbated by an additional stressor. We also did not control for differences in osmolarity between normal and high glucose experiments that might have skewed our results. More research under different conditions will be necessary to elucidate the interplay of high glucose and HR injury in DM2.Support or Funding InformationThis work was supported, in part, by institutional funds; by a Merit Review Award (I01 BX003482) from the U.S. Department of Veterans Affairs Biomedical Laboratory R&D Service; by the American Heart Association’s PAECER Program (1R25HL145330); and by the National Institute of General Medical Sciences of the National Institutes of Health (R25GM113740).

Qualitative Assessment of Carbon Nanotube Toxicity to Mouse Liver using Dark‐field Microscopy

Broad use of nanomaterials may eventually lead to environmental poisoning, including the contamination of food. However, methods for the assessment of nanomaterial toxicity are not well developed. The goal of the current study was to develop a techniques to measure toxicity of multiwall carbon nanotubes (MWCNTs) in vivo. For this, male CD‐1 mice were fed by oral gavage with a water suspension of MWCNTs at varying doses up to 10 mg/kg/day. Twenty‐four hours later, blood chemistry testing showed elevation of aspartate aminotransferase (AST), a liver injury marker, at the highest used dose. The liver damage was confirmed by the terminal deoxynucleotidyl transferase dUTP nick‐end labeling (TUNEL) assay. Our further analysis included the application of dark‐field microscopy (DFM) for (a) the colocalization between MWCNTs and TUNEL‐positive cells and (b) development of a method to quantify MWCNTs burden in liver tissue. DFM clearly showed some elevation of particulate material in the liver with occasional association with TUNEL‐positive cells. DFM did not seem to allow precise quantification of nanoparticle aggregates due to low sensitivity of DFM at the concentration observed in the liver. However, we discovered that CNT gavage increased the percentage of nuclei colocalized with DF objects by systematically counting the number of liver cell nuclei in contact with dark‐field (DF) objects. TUNEL‐positive nuclei were photographed, analyzed at 100‐fold magnification and sorted by CNT treatment category. The process was repeated for DAPI nuclei to serve as a control. The percentage of nuclei with DF colocalization was recorded per group. Our results indicate that the rate of colocalization between dark‐field objects and nuclei appears to correlate directly with CNT‐gavage treatment. Another important observation, confirmed by an electron microscopy, was that there were many alive TUNEL‐negative cells containing MWCNTs. Overall, our data indicate that the DFM method designed for particle toxicology has only limited potential for measurement of nanoparticle toxicity, and that some mouse liver cells are capable of accumulating MWCNTs without inducing immediate cell death.Support or Funding InformationUAMS/ASPET Pharmacology/Toxicology Summer SURF Program USDA subcontract (Basnakian, PI) VA Merit Review grant (Basnakian, PI) COBRE grant (Hauer‐Jensen, PI)To study the effect of carbon nanotube (CNT) ingestion on mouse liver, we systematically counted the number of liver cell nuclei in contact with dark‐field (DF) objects. We found that CNT gavage increased the percentage of nuclei colocalized with DF objects at 100x. Of a total of 38 TUNEL cells recorded in gavage tissue, 22 were touching a DF‐positive object. Livers were extracted from male CD‐1 mice 72 hours after receiving a CNT gavage of 10mg CNT/kg mouse weight.Figure 1

MicroRNA‐34a Knockout Attenuates Endothelial Progenitor Dysfunction in Cholestatic Liver Injury

BackgroundThe noncoding miRNA‐34a (miR‐34a) is involved in the pathogenesis of chronic liver disease and shows potential for application as a biomarker for early diagnosis and intervention. CD34+ endothelial progenitor cells (EPCs) have angiogenic potentials contributing to neovascularization within ischemic sites or to vascular repair after vessel wall injury. However, a clear mechanism of endothelial progenitor dysfunction in chronic liver disorders still remains elusive. The objective of current study is to define the microRNA regulated endothelial progenitor dysfunction during cholestatic liver injury.MethodsBile duct ligation (BDL) and miR‐34a knockout mice were used as the animal models. CD34+ cells were isolated from mouse liver using laser capture microdissection (LCM). A capture probe covalently bound to an oligonucleotide containing biotin and a color‐coded reporter probe were designed for 84 endothelial function‐related genes and analyzed with the nCounter Single Cell Gene Expression Assay. The mediators of endothelial injury were defined in BDL/miR‐34a knockout mice in vivo and cultured human liver sinusoidal endothelial cells (HLSECs) with miR‐34a modifications in vitro by real‐time PCR assay, immunohistochemistry and Western blot analysis.ResultsUsing BDL mouse model of cholestatic liver injury, results showed the significant increase in serum ALT, hepatic CD34 and miR‐34a, severe inflammation, pericellular fibrosis, and intensified nitrosative stress induced by a 16‐fold induction of nitric oxide synthase (NOS3). In CD34+ cells isolated from BDL mice liver by LCM, nCounter single cell analysis demonstrated the enhanced expressions of sinusoidal endothelial dysfunction (SED) markers ICAM1, endothelial marker vWF as well as the inflammatory cytokines TNFα, CCl2, IL‐1β, IFNβ and IL‐7. The upregulation of miR‐34a in HLSECs led to a time‐dependent repression of its target protein Sirt1 levels as shown by western blot analysis, and a significant increase of NOS3. SED marker ICAM‐1 and endothelial marker vWF were significantly up‐regulated in the progressive phases of CLI. Lack of miR‐34a in vivo reversed the serum ALT level, and restored the levels of Sirt1 coupled with decreased NOS3 expression as well as the reduced levels of TNFα, CCl2, IL‐1β, IFNβ and IL‐7 in LCM isolated CD34+ cells analyzed by nCounter single cell gene expression assay. Depletion of miR‐34a in vivo also induced a significant down‐regulation of profibrogenic genes such as α‐SMA, collagen A1 and MMPs in total liver tissues and LCM isolated CD34+ cells by single cell gene assay from BDL mice liver, along with significantly reduced Sirius red staining and liver angiogenesis.ConclusionOur discovery that CD34 associated endothelial progenitor dysfunction is regulated by miR‐34a during cholestatic liver injury implicates an exciting field in which the epigenomic microRNAs of endothelial progenitor cells may be manipulated with potential therapeutic benefits for the patients with chronic liver diseases.Support or Funding InformationNIH/NIDDK R01 award and VA Merit Review Award

Automation in Social Security: Implications for Merits Review?

This paper reviews illustrative aspects of the introduction of artificial intelligence into Australian social security's (Centrelink) original decision making and merits review. The review highlights the complexity and nuanced character of evaluating greater reliance on artificial intelligence over human decision‐making systems. It argues that technological change is inevitable, and is not necessarily either an unadulterated boon or bane, but calls for careful planning and a comparative assessment of the benefits and limitations of artificial intelligence versus human systems. In social security, it is concluded that particular attention should be given to avoiding discrimination against citizens who are technology poor or otherwise vulnerable.

A Reform Too Few or a Reform Too Many: Judicial Review, Appeals or a Prosecutorial System under the UK Competition Act 1998?

This article discusses the CMA’s proposals to lower the standard of review of certain antitrust decisions in the CAT from a merits appeal to judicial review principles or some other limited basis, while retaining the CAT’s ‘full jurisdiction’ over fines, and to amend the CAT’s rules of procedure to restrict the admissibility of new evidence on appeal and the use of oral testimony. The argument developed in this article is that such proposals are: (a) in conflict with the constitutional principle of the rule of law; (b) incompatible with the HRA98; (c) a step back even from the often criticised standard of review applied by the Union Courts in respect of European Commission’s decisions; (d) inappropriate as a matter of policy. If the current regime needs improving to reduce the cost and length of the proceedings, then three options should be considered: (a) moving from a merits appeal to a merits review (Option 1); (b) strengthening the independence of the decision-making panel within the CMA (Option 2), while lowering the standard of review to judicial review principles; (c) establishing a prosecutorial model (Option 3). Option 3 is the most radical but should be given serious consideration as it is likely to be the best suited to achieving the policy objective of reducing the cost and length of competition proceedings while at the same time retaining rigorous scrutiny of the facts and economic evidence, which is key to ensuring not only the fairness, and therefore the legitimacy, of the system, but also its effectiveness.

RELATIONSHIP BETWEEN CHOLINERGIC RECEPTOR BINDING AND ATTENTION IN COGNITIVE AGING USING POSITRON EMISSION TOMOGRAPHY AND THE STROOP

Introduction Cholinergic (Ach) neurotransmission has been associated with aspects of brain attention networks. Reductions in Ach are also thought to underlie cognitive symptoms in Alzheimer's Disease (AD). In a previous study using positron emission tomography (PET) imaging to measure nicotinic Ach receptor binding in vivo, our group found that Ach receptor binding was reduced in patients with AD relative to healthy controls. Reductions were observed in areas of the brain thought to mediate attention, including the anterior cingulate cortex/medial prefrontal cortex (ACC/mPFC) and insula. The Stroop is a neuropsychological measure used in research and clinical settings to assess attention and executive function. In a different study of AD patients, we found that performance on the Stroop was associated with metabolic activity in brain networks in the right hemisphere; the Stroop COLOR-WORD condition was associated with metabolic rate in the intraparietal sulcus (IPS), ACC/mPFC, and insula. In the present study, we sought to extend these findings by examining whether Ach binding was associated with performance on the Stroop prior to the onset of dementia. Methods The study included a combined group of healthy older adults or older adults with amnestic mild cognitive impairment (MCI). All participants completed the Stroop. The Stroop included three conditions: two simpler tasks requiring participants to name colors (COLOR) and read words (WORD) and a more complex task in which participants are required to name the color of ink used for a word that names a different color from the ink color (COLOR-WORD). Cholinergic (Ach) receptor binding was quantified using PET imaging, specifically 18F-2-FA PET (2FA), a radiotracer that binds to alpha4beta2 nicotinic receptor subunits. Scanning utilized a bolus plus infusion technique and measurement of nonmetabolized 2FA from plasma to calculate distribution volume (VT/fp) in PET Ach image voxels. Ach images were pre-processed using SPM12, which included motion correction, normalization to Montreal Neurological Institute (MNI) space using a co-registered T1 MRI image, and spatial smoothing of 8?mm full width at half maximum. First, a voxel-based analysis across the entire brain was used to measure the association between voxel VT/fp and performance on the Stroop COLOR-WORD test using a liberal statistical threshold (p Results Seventy-six participants were included in the sample, comprised of 43 healthy older adults and 33 patients with amnestic MCI (age: mean=73.1 (SD=7.7), MMSE: mean=28.3 (SD=2.1), 57 males and 19 females, education: mean=15.7?years (SD=2.5)). The combined group included 19 African-Americans, 48 Caucasians, 6 Hispanics, and 3?Asian/?Pacific Islanders. Performance on the COLOR task was associated with mean VT/fp in the ACC/mPFC (r=.23 p=.04), insula (r=.28, p=.02), and IPS (r=.23 p Conclusions Across the spectrum of cognitive aging, performance on the Stroop was associated with Ach binding in key brain regions involved in attention—the IPS, ACC/mPFC, and insula. These findings suggest that Ach binding, in part, mediates attentional processing. This appears true regardless of whether the task is simple, as in the COLOR and WORD conditions, or more complex, as in the COLOR-WORD condition. Thus, changes in Ach receptor binding may be an important factor in attentional deficits resulting from cognitive aging prior to onset of AD, and Ach neurotransmission could be a potential molecular target for novel pharmaceuticals to address these symptoms. This research was funded by: This work was supported by a VA Career Development grant awarded to Brandon C. Yarns, MD, MS, and VA Merit Review grants awarded to David L. Sultzer, MD, and Rebecca J. Melrose, PhD.

Who Files Amicus Curiae Briefs in the Texas Supreme Court? – 2020 Edition

The ANNUAL STATISTICAL REPORT FOR THE TEXAS JUDICIARY provides detailed information on caseload and dispositions for the Texas Supreme Court (SCOTX), but does not shed any light on amicus curiae participation in such cases. This paper endeavors to fill the void with quantitative data and identifying information on amicus curiae filings and filers. The friend-of-the-court activity data was extracted from the electronic docket management system of the Texas appellate court system (TAMES) and was processed into more user-friendly formats (tables and lists). Over the 12-month observation period, a total of 236 amicus submissions were received and docketed in a total of 134 state supreme court cases, consisting of two major categories: amicus briefs and amicus curiae letters. Relative to the entire SCOTX caseload of about 1,200 per year, amicus filings are relatively rare, occurring in about 10% of cases. The most common pattern is the submission of a single amicus brief (N=89 in 2019). In cases that the supreme court agrees to hear, however, friend-of-court activity is higher. Like the U.S. Supreme Court, the Texas high court exercises discretionary review. The legal disputes that receive a final merits review in the state’s court of last resort for civil matters are atypical: they are either important to the jurisprudence of the state, involve challenges to a high-dollar trial court judgment, or are otherwise salient. The greatest amici magnet in 2019 was No. 17-0862, styled Energy Transfer Partners, L.P. v. Enterprise Products Partners, L.P., a high-stakes dispute over a failed pipeline development project. It received 13 amicus curiae submissions that year, in addition to two already e-filed in 2018. The Supreme Court issues its opinion on January 31, 2020. The case with the second highest number of amicus filings was Barrow-Shaver v. Carrizo Oil & Gas, No. 17-0332, but ten (10) of them were submitted in 2018 and thus fall outside the study period. With three additional amicus filings in 2019, however, this oil & gas contract case still ranked in the top 20 in the dataset for the calendar year on which this report is based.

Automation in social security: Implications for merits review?

AbstractThis paper reviews illustrative aspects of the introduction of artificial intelligence into Australian social security's (Centrelink) original decision making and merits review. The review highlights the complexity and nuanced character of evaluating greater reliance on artifical intellignce over human decision‐making systems. It argues that technological change is inevitable, and is not necessarily either an unadulterated boon or bane, but calls for careful planning and a comparative assessment of the benefits and limitations of artificial intelligence versus human systems. In social security, it is concluded that particular attention should be given to avoiding discrimination against citizens who are technology poor or otherwise vulnerable.

INTEREST GROUP SESSION—GERIATRIC EDUCATION: AGHE PROGRAM OF MERIT: PRACTICAL AND TECHNICAL APPLICATION INFORMATION AND ASSISTANCE

At a time in education when it is important for programs at colleges and universities to be productive in teaching, student recruitment, and income generation, the AGHE Program of Merit review process can aid in advancing gerontology/geriatrics programs. The Program of Merit has expanded its review now Colleges of Osteopathic Medicine and any Health Professions program may apply for Program of Merit status – your institution need not be an AGHE member to apply. The POM designation provides gerontology programs and those health disciplines that include geriatrics/gerontology content in the curriculum with an AGHE “stamp of approval” which can be used to verify program quality to administrators, to lobby for additional resources, to maintain a quality program, to market the program, and to recruit prospective students into the program. This session will begin with the “Why,” “What” and “How” of applying for Program of Merit and then provide time for small group consultation regarding the Program of Merit application. Preparation of the self-study document will be detailed by the experienced faculty members followed by the review process and timelines associated with application process. Free consultation available!

Innovations in functional and rehabilitative knee bracing

New knee brace designs are available that have the potential to improve patient outcomes relative to traditional bracing. For the indications of post-knee injury/surgery recovery, conservative management of knee osteoarthritis (OA), total knee arthroplasty (TKA) pre-habilitation, and the treatment of post-surgical extension deficits/flexion contractures, innovative new bracing designs merit review and discussion. The researchers requested information from industry brace manufacturers, and from the information received, have selected those products considered significant improvements over traditional functional brace designs for review in this article. Clinical research supporting the benefits of the innovative products listed in the article have been cited when available. The authors are both Certified Orthotists with over 50 years of combined knee bracing experience.