Evaluation of canine 2D cell cultures as models of myxomatous mitral valve degeneration.

The utility of cells cultured from the mitral valve as models of myxomatous diseases needs to be properly validated. In this study valve interstitial cells (VICs) and valve endothelial cells (VECs) were cultured from normal and diseased canine mitral valves in 2% (v/v) or 10% FBS media, in the presence of TGFβ1, 2 and 3, the TGFβ RI kinase inhibitor SB431542 and TGFβ neutralising antibodies, 5HT and the 5HT2RB antagonist LY272015. Cultures were examined by morphology, transcriptomic profiling, protein expression of the cell specific markers αSMA and SM22α (VICs), and CD31 (VECs), deposition of proteoglycans (PG), the PG versican, and the TGFβs themselves.

Characterisation of a novel Fc conjugate of macrophage colony-stimulating factor.

We have produced an Fc conjugate of colony-stimulating factor (CSF) 1 with an improved circulating half-life. CSF1-Fc retained its macrophage growth-promoting activity, and did not induce proinflammatory cytokines in vitro. Treatment with CSF1-Fc did not produce adverse effects in mice or pigs.

Oclacitinib (APOQUEL(®)) is a novel Janus kinase inhibitor with activity against cytokines involved in allergy.

Janus kinase (JAK) enzymes are involved in cell signaling pathways activated by various cytokines dysregulated in allergy. The objective of this study was to determine whether the novel JAK inhibitor oclacitinib could reduce the activity of cytokines implicated in canine allergic skin disease. Using isolated enzyme systems and in vitro human or canine cell models, potency and selectivity of oclacitinib was determined against JAK family members and cytokines that trigger JAK activation in cells.

Allergen-induced production of IL-31 by canine Th2 cells and identification of immune, skin, and neuronal target cells.

The canine cytokine IL-31 induces pruritus in dogs and can be detected in dogs with atopic dermatitis; however very little is understood around its interactions with specific canine cells. We hypothesize that IL-31 is involved in the progression of allergic skin disease by coordinating the interaction between the immune system with skin and neuronal systems. The goal of the following work was to identify cells that produce IL-31 as well as cells that may respond to this cytokine.

Interleukin-31: its role in canine pruritus and naturally occurring canine atopic dermatitis.

Background: Interleukin-31 (IL-31) is a member of the gp130/interleukin-6 cytokine family that is produced by cell types such as T helper 2 lymphocytes and cutaneous lymphocyte antigen positive skin homing T cells. When overexpressed in transgenic mice, IL-31 induces severe pruritus, alopecia and skin lesions. In humans, IL-31 serum levels correlate with the severity of atopic dermatitis in adults and children.

Cloning and expression of porcine Colony Stimulating Factor-1 (CSF-1) and Colony Stimulating Factor-1 Receptor (CSF-1R) and analysis of the species specificity of stimulation by CSF-1 and Interleukin 34.

Macrophage Colony Stimulating Factor (CSF-1) controls the survival, differentiation and proliferation of cells of the mononuclear phagocyte system. A second ligand for the CSF-1R, Interleukin 34 (IL-34), has been described, but its physiological role is not yet known. The domestic pig provides an alternative to traditional rodent models for evaluating potential therapeutic applications of CSF-1R agonists and antagonists.

Influence of various central moieties on the hypolipidemic properties of long hydrocarbon chain diols and diacids.

A series of long (11-15) hydrocarbon chain diols and diacids with various central functional groups and terminal gem-dimethyl or -methyl/aryl substituents was synthesized and evaluated in both in vivo and in vitro assays for its potential to favorably alter lipid disorders including metabolic syndrome. Compounds were assessed for their effects on the de novo incorporation of radiolabeled acetate into lipids in primary cultures of rat hepatocytes, as well as for their effects on lipid and glycemic variables in obese female Zucker fatty rats, Crl:(ZUC)-faBR. The most active compounds were hydroxyl-substituted symmetrical diacids and diols with a 13-atom chain and terminal gem-dimethyl substituents.

Lack of specific amyloid-beta(1-42) suppression by nonsteroidal anti-inflammatory drugs in young, plaque-free Tg2576 mice and in guinea pig neuronal cultures.

Recent studies indicating that some nonsteroidal anti-inflammatory drugs (NSAIDs) selectively modulate gamma-secretase cleavage of amyloid precursor protein (APP) while sparing Notch processing have generated interest in discovery of novel gamma-secretase modulators with the "NSAID-like" efficacy profile. The objective of the present studies was to compare the efficacy of a subset of NSAIDs with previously reported classical gamma-secretase inhibitors LY-411575 [N(2)-[(2S)-2-(3,5-difluorophenyl)-2-hydroxyethanoyl]-N(1)-[(7S)-5-methyl-6-oxo-6,7-dihydro-5H-dibenzo[b,d]azepin-7-yl]-L-alaninamide]and DAPT [N-[N- (3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester] in Tg2576 mice. Flurbiprofen (10 and 25 mg/kg/day) was overtly toxic and elicited significant (but nonselective) reductions in both Abeta(1-40) and Abeta(1-42) in the plasma in one of two studies.

Effects of a novel dual lipid synthesis inhibitor and its potential utility in treating dyslipidemia and metabolic syndrome.

We have identified a novel omega-hydroxy-alkanedicarboxylic acid, ESP 55016, that favorably alters serum lipid variables in obese female Zucker (fa/fa) rats. ESP 55016 reduced serum non-HDL-cholesterol (non-HDL-C), triglyceride, and nonesterified fatty acid levels while increasing serum HDL-C and beta-hydroxybutyrate levels in a dose-dependent manner. ESP 55016 reduced fasting serum insulin and glucose levels while also suppressing weight gain.

Positive and negative regulation of the gamma-secretase activity by nicastrin in a murine model.

Nicastrin is a component of the gamma-secretase complex that has been shown to adhere to presenilin-1 (PS1), Notch, and APP. Here we demonstrate that Nicastrin-deficient mice showed a phenotype that is indistinguishable from PS1/PS2 double knock-out mice, whereas heterozygotes were healthy and viable. Fibroblasts derived from Nicastrin-deficient embryos were unable to generate amyloid beta-peptide and failed to release the intracellular domain of APP- or Notch1-Gal4-VP16 fusion proteins.

Spontaneous aggregation and cytotoxicity of the beta-amyloid Abeta1-40: a kinetic model.

The time dependency of the spontaneous aggregation of the fibrillogenic beta-amyloid peptide, Abeta1-40, was measured by turbidity, circular dichroism, HPLC, and fluorescence polarization. The results by all methods were comparable and they were most consistent with a kinetic model where the peptide first slowly forms an activated monomeric derivative (AM), which is the only species able to initiate, by tetramerization, the formation of linear aggregates. The anti-Abeta antibody 6E10, raised against residues 1-17, at concentrations of 200-300 nM delayed significantly the aggregation of 50 microM amyloid peptide.

Short-term effects of the 21-aminosteroid lazaroid tirilazad mesylate (PNU-74006F) and the pyrrolopyrimidine lazaroid PNU-101033E on energy metabolism of human peripheral blood mononuclear cells.

Two groups of antioxidant compounds, the 21-aminosteroids and the pyrrolopyrimidines, have been found to act as neuroprotective drugs against lipid peroxidation in the injured CNS. Like glucocorticoids at high doses they are assumed to produce their effects at least in part by direct membrane stabilizing effects. In order to prove this hypothesis, we have investigated in this study the effects of these drugs on the energy metabolism of activated human peripheral blood mononuclear cells (PBMC) since these cells have been shown to serve as a suitable test system for substances affecting processes of ATP turnover.

Pharmacological approaches to counter the toxicity of Dopa.

Dopa and related catecholamines and their degradation products have been demonstrated to have neurotoxic potential in a number of cellular and in vivo experiments. Several mechanisms have been hypothesized to be involved including generation of prooxidant products that subsequently oxidize membrane lipids and exposed macromolecules. We have utilized a neuronal culture of cerebellar granule cells to study the toxicity of Dopa and the ability of various neuroprotective and antiparkinsonian compounds to offer protection therefrom.

D1 dopamine receptor activity of anti-parkinsonian drugs.

Clinical and preclinical investigations suggest that stimulation of D1 dopamine receptors may be responsible for dyskinesias induced by dopamine agonist treatment of Parkinson's Disease (PD), and that these dyskinesias may be decreased by treatment with a D1 antagonist (clozapine). Therefore, the effects of dopamine agonists and antagonists have been investigated in a primary cerebellar granule cell model of cAMP formation that seems to be highly responsive to the D1 receptors. SKF 38393, lisuride, apomorphine, pergolide, dopamine, bromocriptine and 7-OH-DPAT showed concentration-dependent increases in cAMP formation, with EC50s (in microM) of 0.

Effects of lazaroids and a peroxynitrite scavenger in a cell model of peroxynitrite toxicity.

We developed a cerebellar granule cell model of peroxynitrite toxicity and showed that certain sulfhydryl-containing compounds (e.g., penicillamine) present as concurrent treatments could inhibit this toxicity.

D2 and D1 dopaminergic activity of 7-OH-DPAT.

The D1 and D2 dopamine receptor agonist properties of 7-hydroxy-2-(N,N-di-n-propylamino) tetraline (7-OH-DPAT) was determined by investigating the effect of this compound on rat striatal acetylcholine (ACh) concentration and increase in cAMP formation in primary cerebellar granule cell cultures. 7-OH-DPAT at low doses (0.01 to 0.

Protective effects of tirilazad mesylate in a cellular model of peroxynitrite toxicity.

Following CNS trauma or ischemia, peroxynitrite may be a toxic intermediate which forms in vivo when nitric oxide condenses with superoxide. Alone, peroxynitrite appears to directly react with aromatic and sulfhydryl nucleophiles. However, at physiological pH, peroxynitrite is protonated and, in that form, will rapidly (within seconds) decompose to species with hydroxyl radical and nitrogen dioxide characteristics.

U-19451A: a selective inducible nitric oxide synthase inhibitor.

Drugs with high selectivity for iNOS inhibition may be useful for treatment of neurodegenerative disorders, chronic inflammatory diseases, and septic shock. Therefore, U-19451A (2-benzyl-2-thio-pseudourea hydrochloride), a potential NOS inhibitor, has been investigated for its selectivity for iNOS using tissues, primary cerebellar granule cell cultures and glial cell cultures. Lungs isolated from rats treated with intravenous injection of E coli lipopolysaccharide and glial cell cultures treated with the same bacterial toxin plus gamma-interferon were used for iNOS activity.

Antibody transformation by peroxynitrite as determined using capillary electrophoresis: a feasibility study.

Peroxynitrite may be a physiologically relevant endogenous neurotoxin that forms following CNS trauma when excessive levels of NO and .O2 accumulate. Recently, peroxynitrite was found to inactivate the polyclonal antibody to cAMP.

Structure activity relationships of peroxynitrite scavengers an approach to nitric oxide neurotoxicity.

Nitric oxide (NO) is made by NO synthase during the conversion of arginine to citrulline. Researchers have found that they can block the actions of excitotoxins by inhibiting NO synthase. Released from excitable cells during trauma, NO may react with superoxide to form peroxynitrite.

Development of venous occlusions in mice transgenic for the plasminogen activator inhibitor-1 gene.

The fibrinolytic potential of the vasculature is modulated primarily by the availability and activity of plasminogen activators, which convert the zymogen plasminogen into the active fibrin-degrading enzyme plasmin. The activities of these key regulatory enzymes are directly neutralized by their primary endogenous inhibitor, plasminogen activator inhibitor-1 (PAI-1). Although some individuals with a tendency to develop thrombotic disorders exhibit elevated levels of PAI-1 in their plasma, the cause-and-effect relationship between increased PAI-1 and thrombosis is still unclear.

Interleukin-1 promotes proliferation of vascular smooth muscle cells in coordination with PDGF or a monocyte derived growth factor.

We report that interleukin-1 (IL-1) potentiates the proliferation of vascular smooth muscle cells. Growth of early passage smooth muscle cells was not significantly affected by IL-1 alone. Treatment with IL-1 together with the platelet derived growth factor (PDGF) or another polypeptide growth factor derived from mitogen activated human monocytes (MDGF) resulted in a significant enhancement of cell growth over either PDGF or MDGF alone.