Decreased venous thrombosis with an oral inhibitor of P selectin

BackgroundP-selectin inhibition with protein therapeutics such as antibodies or soluble ligands given intravenously can decrease thrombosis in a mouse ligation model of venous thrombosis. In this study, we hypothesized that oral inhibition of P selectin with a novel oral nonprotein inhibitor (PSI-697) would decrease thrombosis and circulating microparticle populations. This study evaluated the effects on thrombosis and circulating microparticle populations in this murine venous thrombosis model.MethodsMice underwent inferior vena cava ligation to induce thrombosis. Mice with high circulating level of P selectin, Delta Cytoplasmic Tail (^CT), mice gene-deleted for both E- and P-selectin knockout (EPKO), and wild-type C57BL/6 mice (WT) were studied without and with administration of PSI-697 in food (100 mg/kg daily) from 2 days before thrombosis until the end of the study. Animals were killed 2 and 6 days later. Evaluations included thrombus weight (TW), vein wall morphometrics, microparticle quantification by using fluorescence-activated cell sorter analysis, and vein wall enzyme-linked immunosorbent assays for interleukin (IL)-10, P selectin, and monocyte chemotactic protein 1.ResultsPSI-697 significantly decreased TW in WT and ^CT mice, with a treated vs nontreated TW of 132 ± 24 vs 228 ± 29 × 10−4 g (P = .014) and 166 ± 19 vs 281 ± 16 × 10−4 g (P = .001), respectively. At day 6, the effect was significant only in the ^CT group (P < .05). Drug therapy at day 2 significantly increased vein wall monocytes in WT mice and increased monocytes and total inflammatory cells in ^CT animals. A significant decrease in neutrophils and total inflammatory cells was seen in EPKO mice at day 2 with therapy. Therapy significantly increased platelet-derived microparticles and total microparticles in ^CT mice on day 2. Changes in treated WT and treated EPKO animals were not significant compared with respective vehicle treatments at day 2. On day 6, therapy significantly decreased total microparticles in EPKO animals. Vein wall expression of IL-10 increased in all groups with therapy at day 2 (n = 18) and was significantly increased in WT (2687.5 ± 903 pg/mL vs 636 ± 108 pg/mL total protein; P = .038) and ^CT (2078 ± 295 pg/mL vs 432 ± 62 pg/mL total protein; P = .001) mice. Therapy significantly decreased vein wall P selectin, monocyte chemotactic protein 1, and IL-10 levels at day 6.ConclusionsPSI-697 decreased thrombosis. P-selectin inhibition allowed vein wall inflammatory cell extravasation in this model of complete ligation. Circulating microparticles (platelet-derived microparticles and total microparticles) increased with P-selectin inhibition, possibly because of decreased consumption into the thrombus. In summary, the oral administration of an inhibitor to P selectin provides significant TW reduction.Clinical RelevanceDeep venous thrombosis is a significant national health problem in the general population. The average annual incidence of deep venous thrombosis is approximately 250,000 cases per year. The selectin family of adhesion molecules is thought to be largely responsible for the initial attachment and rolling of leukocytes on stimulated vascular endothelium. Recent studies have explored the possible therapeutic implications of P-selectin inhibition to modulate venous thrombosis. For example, prophylactic dosing of a recombinant P-selectin ligand decreases venous thrombosis in a dose-dependent fashion in both feline and nonhuman primate animal models. Additionally, treatment of 2-day iliac thrombi with a recombinant protein, P-selectin inhibitor, significantly improves vein reopening in nonhuman primates. It is interesting to note that P-selectin inhibition decreases thrombosis without adverse anticoagulation. On the basis of the results from these previous studies, the use of P-selectin antagonism is a logical therapeutic approach to treat venous thrombosis. All inhibitors developed to date are either proteins or small molecules with low oral bioavailability that require intravenous or subcutaneous injection. This study evaluates, for the first time, a novel orally bioavailable inhibitor of P-selectin (PSI-697)

Basal Autophagy is altered in Lagotto Romagnolo Dogs with an ATG4D mutation

A missense variant in the autophagy-related ATG4D-gene has been associated with a progressive degenerative neurological disease in Lagotto Romagnolo (LR) dogs. In addition to neural lesions, affected dogs show an extraneural histopathological phenotype characterized by severe cytoplasmic vacuolization, a finding not previously linked with disturbed autophagy in animals. Here we aimed at testing the hypothesis that autophagy is altered in the affected dogs, at reporting the histopathology of extraneural tissues and at excluding lysosomal storage diseases. Basal and starvation-induced autophagy were monitored by Western blotting and immunofluorescence of microtubule associated protein 1A/B light chain3 (LC3) in fibroblasts from 2 affected dogs. The extraneural findings of 9 euthanized LRs and skin biopsies from 4 living affected LRs were examined by light microscopy, electron microscopy, and immunohistochemistry (IHC), using antibodies against autophagosomal membranes (LC3), autophagic cargo (p62), and lysosomal membranes (LAMP2). Biochemical screening of urine and fibroblasts of 2 affected dogs was performed. Under basal conditions, the affected fibroblasts contained significantly more LC3-II and LC3-positive vesicles than did the controls. Morphologically, several cells, including serous secretory epithelium, endothelial cells, pericytes, plasma cells, and macrophages, contained cytoplasmic vacuoles with an ultrastructure resembling enlarged amphisomes, endosomes, or multivesicular bodies. IHC showed strong membranous LAMP2 positivity only in sweat glands. The results show that basal but not induced autophagy is altered in affected fibroblasts. The ultrastructure of affected cells is compatible with altered autophagic and endo-lysosomal vesicular traffic. The findings in this spontaneous disease provide insight into possible tissue-specific roles of basal autophagy.Peer reviewe

Proteolytic activity, degradation, and dissolution of primary and permanent teeth

BackgroundPrimary and permanent teeth composition may influence dissolution and degradation rates.AimTo compare the dissolution and degradation of primary and permanent teeth.DesignEnamel and dentin powders were obtained from primary molars and premolars and incubated within different pH buffers. Calcium and inorganic phosphate release was quantified in the buffers by atomic absorption and light spectrophotometry. A colorimetric assay was used to assess the MMP activity of primary dentin (PrD) and permanent dentin (PeD). Collagen degradation was assessed by dry mass loss, change in elastic modulus (E), and ICTP and CTX release. Data were submitted to ANOVA and Tukey’s tests (α = 0.05).ResultsSimilar dissolution was found between PrD and PeD after 256 hours. At pH 4.5, enamel released more minerals than dentin whereas at pH 5.5 the inverse result was observed. MMP activity was similar for both substrates. PrD showed higher dry mass loss after 1 week. In general, greater reduction in E was recorded for PrD. Higher quantities of ICTP and CTX were released from PrD after 1 week.ConclusionsPrimary and permanent teeth presented similar demineralization rates. Collagen degradation, however, was faster and more substantial for PrD.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/156418/2/ipd12632_am.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/156418/1/ipd12632.pd

Tumor innate immunity primed by specific interferon-stimulated endogenous retroviruses.

Mesenchymal tumor subpopulations secrete pro-tumorigenic cytokines and promote treatment resistance1-4. This phenomenon has been implicated in chemorefractory small cell lung cancer and resistance to targeted therapies5-8, but remains incompletely defined. Here, we identify a subclass of endogenous retroviruses (ERVs) that engages innate immune signaling in these cells. Stimulated 3 prime antisense retroviral coding sequences (SPARCS) are oriented inversely in 3' untranslated regions of specific genes enriched for regulation by STAT1 and EZH2. Derepression of these loci results in double-stranded RNA generation following IFN-γ exposure due to bi-directional transcription from the STAT1-activated gene promoter and the 5' long terminal repeat of the antisense ERV. Engagement of MAVS and STING activates downstream TBK1, IRF3, and STAT1 signaling, sustaining a positive feedback loop. SPARCS induction in human tumors is tightly associated with major histocompatibility complex class 1 expression, mesenchymal markers, and downregulation of chromatin modifying enzymes, including EZH2. Analysis of cell lines with high inducible SPARCS expression reveals strong association with an AXL/MET-positive mesenchymal cell state. While SPARCS-high tumors are immune infiltrated, they also exhibit multiple features of an immune-suppressed microenviroment. Together, these data unveil a subclass of ERVs whose derepression triggers pathologic innate immune signaling in cancer, with important implications for cancer immunotherapy

A SINE Insertion in ATP1B2 in Belgian Shepherd Dogs Affected by Spongy Degeneration with Cerebellar Ataxia (SDCA2).

Spongy degeneration with cerebellar ataxia (SDCA) is a genetically heterogeneous neurodegenerative disorder with autosomal recessive inheritance in Malinois dogs, one of the four varieties of the Belgian Shepherd breed. Using a combined linkage and homozygosity mapping approach we identified a ~10.6 Mb critical interval on chromosome 5 in a Malinois family with four puppies affected by cerebellar dysfunction. Visual inspection of the 10.6 Mb interval in whole genome sequencing data from one affected puppy revealed a 227 bp SINE insertion into the ATP1B2 gene encoding the β2 subunit of the Na(+)/K(+)-ATPase holoenzyme (ATP1B2:c.130_131insLT796559.1:g.50_276). The SINE insertion caused aberrant RNA splicing. Immunohistochemistry indicated a reduction of ATP1B2 protein expression in the central nervous system of affected puppies. Atp1b2 knock-out mice had previously been reported to show clinical and neurohistopathological findings similar to the affected Malinois puppies. Therefore, we consider ATP1B2:c.130_131ins227 the most likely candidate causative variant for a second subtype of SDCA in Malinois dogs, which we propose to term spongy degeneration with cerebellar ataxia subtype 2 (SDCA2). Our study further elucidates the genetic and phenotypic complexity underlying cerebellar dysfunction in Malinois dogs and provides the basis for a genetic test to eradicate one specific neurodegenerative disease from the breeding population in Malinois and the other varieties of the Belgian Shepherd breed. ATP1B2 thus represents another candidate gene for human inherited cerebellar ataxias, and SDCA2 affected Malinois puppies may serve as naturally occurring animal model for this disorder

Mutations in MITF and PAX3 Cause “Splashed White” and Other White Spotting Phenotypes in Horses

During fetal development neural-crest-derived melanoblasts migrate across the entire body surface and differentiate into melanocytes, the pigment-producing cells. Alterations in this precisely regulated process can lead to white spotting patterns. White spotting patterns in horses are a complex trait with a large phenotypic variance ranging from minimal white markings up to completely white horses. The “splashed white” pattern is primarily characterized by an extremely large blaze, often accompanied by extended white markings at the distal limbs and blue eyes. Some, but not all, splashed white horses are deaf. We analyzed a Quarter Horse family segregating for the splashed white coat color. Genome-wide linkage analysis in 31 horses gave a positive LOD score of 1.6 in a region on chromosome 6 containing the PAX3 gene. However, the linkage data were not in agreement with a monogenic inheritance of a single fully penetrant mutation. We sequenced the PAX3 gene and identified a missense mutation in some, but not all, splashed white Quarter Horses. Genome-wide association analysis indicated a potential second signal near MITF. We therefore sequenced the MITF gene and found a 10 bp insertion in the melanocyte-specific promoter. The MITF promoter variant was present in some splashed white Quarter Horses from the studied family, but also in splashed white horses from other horse breeds. Finally, we identified two additional non-synonymous mutations in the MITF gene in unrelated horses with white spotting phenotypes. Thus, several independent mutations in MITF and PAX3 together with known variants in the EDNRB and KIT genes explain a large proportion of horses with the more extreme white spotting phenotypes

A Missense Change in the ATG4D Gene Links Aberrant Autophagy to a Neurodegenerative Vacuolar Storage Disease

Inherited neurodegenerative disorders are debilitating diseases that occur across different species. We have performed clinical, pathological and genetic studies to characterize a novel canine neurodegenerative disease present in the Lagotto Romagnolo dog breed. Affected dogs suffer from progressive cerebellar ataxia, sometimes accompanied by episodic nystagmus and behavioral changes. Histological examination revealed unique pathological changes, including profound neuronal cytoplasmic vacuolization in the nervous system, as well as spheroid formation and cytoplasmic aggregation of vacuoles in secretory epithelial tissues and mesenchymal cells. Genetic analyses uncovered a missense change, c.1288G>A; p.A430T, in the autophagy-related ATG4D gene on canine chromosome 20 with a highly significant disease association (p = 3.8 x 10(-136)) in a cohort of more than 2300 Lagotto Romagnolo dogs. ATG4D encodes a poorly characterized cysteine protease belonging to themacroautophagy pathway. Accordingly, our histological analyses indicated altered autophagic flux in affected tissues. The knockdown of the zebrafish homologue atg4da resulted in a widespread developmental disturbance and neurodegeneration in the central nervous system. Our study describes a previously unknown canine neurological disease with particular pathological features and implicates the ATG4D protein as an important autophagy mediator in neuronal homeostasis. The canine phenotype serves as a model to delineate the disease-causing pathological mechanism(s) and ATG4D function, and can also be used to explore treatment options. Furthermore, our results reveal a novel candidate gene for human neurodegeneration and enable the development of a genetic test for veterinary diagnostic and breeding purposes.Peer reviewe

Toxicosis with grapes or raisins causing acute kidney injury and neurological signs in dogs

Background: The ingestion of grapes or raisins has been reported to cause acute kid- ney injury (AKI) in dogs, with a clinical picture dominated by early gastrointestinal signs and rapidly developing uremia. Ataxia is mentioned in a few reports, but not further characterized. Objectives: To evaluate the clinical, laboratory, and pathological features of dogs diagnosed with grape or raisin toxicosis (GRT) with emphasis on renal and neurologi- cal manifestations, compared to a control group of dogs with AKI from other causes. Animals: Fifteen client-owned dogs with GRT and 74 control dogs. Methods: Retrospective study over 17 months. Results: All dogs with GRT were presented with severe AKI (grade 4, n = 5; grade 5, n = 10). Eleven dogs (73%) had marked forebrain, cerebellar, or vestibular signs. These manifestations dominated the clinical picture in some dogs, but were not associated with the severity of azotemia or the presence of systemic hypertension. Eight dogs (53%) survived, and 5 dogs experienced a complete neurological recovery. Causes of death were unrelated to the neurological manifestations. Neuropathological examina- tion of 4 dogs did not identify any structural central nervous system abnormality. Only 2 control dogs (3%) displayed neurological signs with seizures unrelated to the AKI; 42 control dogs (57%) survived. Conclusions and Clinical Importance: Severe forebrain, cerebellar, or vestibular signs may be an important feature of GRT and dominate the early clinical picture. The described features suggest a reversible functional brain injury specific to GRT and unrelated to uremia

Correlative Magnetic Resonance Imaging and Histopathology in Small Ruminant Listeria Rhombencephalitis

Background: Listeria rhombencephalitis, infection of the brainstem with Listeria monocytogenes, occurs mainly in humans and farmed ruminants and is associated with high fatality rates. Small ruminants (goats and sheep) are a large animal model due to neuropathological similarities. The purpose of this study was to define magnetic resonance imaging (MRI) features of listeria rhombencephalitis in naturally infected small ruminants and correlate them with histopathology. Secondly, the purpose of this study was to compare the results with MRI findings reported in humans. Methods: Twenty small ruminants (13 sheep and 7 goats) with listeria rhombencephalitis were prospectively enrolled and underwent in vivo MRI of the brain, including T2-weighted, fluid attenuation inversion recovery, and T1-weighted sequences pre- and post-contrast administration and postmortem histopathology. In MRI, lesions were characterized by location, extent, border definition, signal intensity, and contrast enhancement. In histopathology, the location, cell type, severity, and chronicity of inflammatory infiltrates and signs of vascular damage were recorded. In addition, histopathologic slides were matched to MRIs, and histopathologic and MRI features were compared. Results: Asymmetric T2-hyperintense lesions in the brainstem were observed in all animals and corresponded to the location and pattern of inflammatory infiltrates in histopathology. Contrast enhancement in the brainstem was observed in 10 animals and was associated with vessel wall damage and perivascular fibrin accumulation in 8 of 10 animals. MRI underestimated the extension into rostral brain parts and the involvement of trigeminal ganglia and meninges. Conclusion: Asymmetric T2-hyperintense lesions in the brainstem with or without contrast enhancement can be established as criteria for the diagnosis of listeria rhombencephalitis in small ruminants. Brainstem lesions were similar to human listeria rhombencephalitis in terms of signal intensity and location. Different from humans, contrast enhancement was a rare finding, and abscessation was not observed