Chymase Inhibition Resolves and Prevents Deep Vein Thrombosis Without Increasing Bleeding Time in the Mouse Model.

Background Deep vein thrombosis (DVT) is the primary cause of pulmonary embolism and the third most life-threatening cardiovascular disease in North America. Post-DVT anticoagulants, such as warfarin, heparin, and direct oral anticoagulants, reduce the incidence of subsequent venous thrombi. However, all currently used anticoagulants affect bleeding time at various degrees, and there is therefore a need for improved therapeutic regimens in DVT. It has recently been shown that mast cells play a crucial role in a DVT murine model. The underlying mechanism involved in the prothrombotic properties of mast cells, however, has yet to be identified. Methods and Results C57BL/6 mice and mouse mast cell protease-4 (mMCP-4) genetically depleted mice (mMCP-4 knockout) were used in 2 mouse models of DVT, partial ligation (stenosis) and ferric chloride-endothelial injury model of the inferior vena cava. Thrombus formation and impact of genetically repressed or pharmacologically (specific inhibitor TY-51469) inhibited mMCP-4 were evaluated by morphometric measurements of thrombi immunochemistry (mouse and human DVT), color Doppler ultrasound, bleeding times, and enzymatic activity assays ex vivo. Recombinant chymases, mMCP-4 (mouse) and CMA-1 (human), were used to characterize the interaction with murine and human plasmin, respectively, by mass spectrometry and enzymatic activity assays. Inhibiting mast cell-generated mMCP-4, genetically or pharmacologically, resolves and prevents venous thrombus formation in both DVT models. Inferior vena cava blood flow obstruction was observed in the stenosis model after 6 hours of ligation, in control- but not in TY-51469-treated mice. In addition, chymase inhibition had no impact on bleeding times of healthy or DVT mice. Furthermore, endogenous chymase limits plasmin activity in thrombi ex vivo. Recombinant mouse or human chymase degrades/inactivates purified plasmin in vitro. Finally, mast cell-containing immunoreactive chymase was identified in human DVT. Conclusions This study identified a major role for mMCP-4, a granule-localized protease of chymase type, in DVT formation. These findings support a novel pharmacological strategy to resolve or prevent DVT without affecting the coagulation cascade through the inhibition of chymase activity.

Mast Cell Degranulation Increases Mouse Mast Cell Protease 4-Dependent Vasopressor Responses to Big Endothelin-1 But Not Angiotensin I.

Mouse mast cell protease 4 (mMCP-4), the murine functional analog to the human chymase, is a serine protease synthesized and stored in mast cell secretory granules. Our previous studies reported physiologic and pathologic roles for mMCP-4 in the maturation and synthesis of the vasoactive peptide endothelin-1 (ET-1) from its precursor, big ET-1. The aim of this study was to investigate the impact of mast cell degranulation or stabilization on mMCP-4-dependent pressor responses after the administration of big ET-1 or angiotensin I (Ang I). In anesthetized mice, mast cell degranulation induced by compound 48/80 (C48/80) or stabilization by cromolyn enhanced or repressed, respectively, the dose-dependent vasopressor responses to big ET-1 in wild-type (WT) mice but not in mMCP-4 knockout mice in a chymase inhibitor (TY-51469)-sensitive fashion. In addition, mMCP-4-dependent hydrolysis of the fluorogenic substrate Suc-Leu-Leu-Val-Tyr-7-amino-4-methylcoumarin was depleted or enhanced in peritoneal mast cells isolated from mice pretreated with C48/80 or cromolyn, respectively. Furthermore, C48/80 or cromolyn markedly increased or abolished, respectively, ET-1 (1-31) conversion from exogenous big ET-1 in WT mice peritoneal fluid-isolated mast cells, in vitro. Finally, the vasopressor responses to Ang I were unaffected by mast cell activation or stabilization, whereas those induced by the angiotensin-converting enzyme-resistant Ang I analog, [Pro11, D-Ala12] Ang I, were potentiated by C48/80. Altogether, the present study shows that mast cell activation enhances the mMCP-4-dependent vasoactive properties of big ET-1 but not Ang I in the mouse model. SIGNIFICANCE STATEMENT: The current work demonstrates a significant role for mast cell stability in the cardiovascular pharmacology of big endothelin-1 but not angiotensin I in the murine systemic circulation.

Inter-dependent Centrosomal Co-localization of the cen and ik2 cis-Natural Antisense mRNAs in Drosophila.

Overlapping genes are prevalent in most genomes, but the extent to which this organization influences regulatory events operating at the post-transcriptional level remains unclear. Studying the cen and ik2 genes of Drosophila melanogaster, which are convergently transcribed as cis-natural antisense transcripts (cis-NATs) with overlapping 3' UTRs, we found that their encoded mRNAs strikingly co-localize to centrosomes. These transcripts physically interact in a 3' UTR-dependent manner, and the targeting of ik2 requires its 3' UTR sequence and the presence of cen mRNA, which serves as the main driver of centrosomal co-localization. The cen transcript undergoes localized translation in proximity to centrosomes, and its localization is perturbed by polysome-disrupting drugs. By interrogating global fractionation-sequencing datasets generated from Drosophila and human cellular models, we find that RNAs expressed as cis-NATs tend to co-localize to specific subcellular fractions. This work suggests that post-transcriptional interactions between RNAs with complementary sequences can dictate their localization fate in the cytoplasm.

Experimental Autoimmune Encephalomyelitis Potentiates Mouse Mast Cell Protease 4-Dependent Pressor Responses to Centrally or Systemically Administered Big Endothelin-1.

Multiple sclerosis is a neurodegenerative disease affecting predominantly female patients between 20 and 45 years of age. We previously reported the significant contribution of mouse mast cell protease 4 (mMCP-4) in the synthesis of endothelin-1 (ET-1) in healthy mice and in a murine model of experimental autoimmune encephalomyelitis (EAE). In the current study, the cardiovascular effects of ET-1 and big endothelin-1 (big-ET-1) administered systemically or intrathecally were assessed in the early preclinical phase of EAE in telemetry instrumented/conscious mice. Chymase-specific enzymatic activity was also measured in the lung, brain, and mast cell extracts in vitro. Finally, the impact of EAE immunization was studied on the pulmonary and brain mRNA expression of different genes of the endothelin pathway, interleukin-33 (IL-33), and monitoring of immunoreactive tumor necrosis factor-α (TNF-α). Systemically or intrathecally administered big-ET-1 triggered increases in blood pressure in conscious mice. One week post-EAE, the pressor responses to big-ET-1 were potentiated in wild-type (WT) mice but not in mMCP-4 knockout (KO) mice. EAE triggered mMCP-4-specific activity in cerebral homogenates and peritoneal mast cells. Enhanced pulmonary, but not cerebral preproendothelin-1 and IL-33 mRNA were found in KO mice and further increased 1 week post-EAE immunization, but not in WT animals. Finally, TNF-α levels were also increased in serum from mMCP-4 KO mice, but not WT, 1 week post-EAE. Our study suggests that mMCP-4 activity is enhanced both centrally and systemically in a mouse model of EAE.

Use of a novel surgical approach for treatment of complete bilateral membranous choanal atresia in an alpaca cria.

CASE DESCRIPTION A 4-hour-old 6.3-kg (13.9-lb) female alpaca cria was evaluated because of severe respiratory distress and difficulty nursing since birth. CLINICAL FINDINGS The cria had open-mouth breathing and cyanotic membranes, with no airflow evident from either nostril. Supplemental oxygen was delivered, and the patient was anesthetized and intubated orotracheally; a CT evaluation of the head confirmed bilateral membranous obstruction of the nasal cavities, consistent with complete bilateral choanal atresia. TREATMENT AND OUTCOME Choanal atresia was treated with an endoscopically assisted balloon-dilation technique, and temporary tracheostomy was performed. Stenosis recurred, requiring revision of the repair and intranasal stent placement 3 days after the first surgery. The tracheostomy tube was removed the next day. Complications during hospitalization included mucoid obstruction of the tracheostomy tube, granulation tissue development in the trachea near the tracheostomy site, mucoid stent obstruction, aspiration pneumonia, and presumed partial failure of passive transfer of immunity. The stents were removed 2 weeks after admission, and the cria was discharged 3 days later. The owner was advised that the animal should not be bred. At last follow-up 3 years later, the alpaca was doing well. CLINICAL RELEVANCE Surgical treatment with a balloon-dilation technique and placement of nasal stents with endoscopic guidance were curative in this neonatal alpaca with bilateral membranous choanal atresia. Computed tomography was useful to determine the nature of the atresia and aid surgical planning. Because a genetic component is likely, owners should be advised to prevent affected animals from breeding.

Significant Contribution of Mouse Mast Cell Protease 4 in Early Phases of Experimental Autoimmune Encephalomyelitis.

Experimental autoimmune encephalomyelitis (EAE) is a mouse model that reproduces cardinal signs of clinical, histopathological, and immunological features found in Multiple Sclerosis (MS). Mast cells are suggested to be involved in the main inflammatory phases occurring during EAE development, possibly by secreting several autacoids and proteases. Among the latter, the chymase mouse mast cell protease 4 (mMCP-4) can contribute to the inflammatory response by producing endothelin-1 (ET-1). The aim of this study was to determine the impact of mMCP-4 on acute inflammatory stages in EAE. C57BL/6 wild type (WT) or mMCP-4 knockout (KO) mice were immunized with MOG35-55 plus complete Freund's adjuvant followed by pertussis toxin. Immunized WT mice presented an initial acute phase characterized by progressive increases in clinical score, which were significantly reduced in mMCP-4 KO mice. In addition, higher levels of spinal myelin were found in mMCP-4 KO as compared with WT mice. Finally, whereas EAE triggered significant increases in brain levels of mMCP-4 mRNA and immunoreactive ET-1 in WT mice, the latter peptide was reduced to basal levels in mMCP-4 KO congeners. Together, the present study supports a role for mMCP-4 in the early inflammatory phases of the disease in a mouse model of MS.

Effect of castration on the urinary protein-to-creatinine ratio of male dogs.

OBJECTIVE: To assess the urinary protein-to-creatinine ratio (UPCR) of healthy sexually intact male dogs and to compare the UPCR of these dogs before and after castration. ANIMALS: 19 client- or shelter-owned healthy adult sexually intact male dogs. PROCEDURES: Physical, hematologic, and biochemical examinations and urinalysis (including calculation of the UPCR) were performed on each dog. Dogs were then castrated, and physical examination and urinalysis (including calculation of the UPCR) were performed again at least 15 days after castration. RESULTS: A dipstick test yielded positive results for protein in the urine of 10 sexually intact male dogs, but the UPCR was < 0.5 for all sexually intact male dogs. Mean UPCR for sexually intact male dogs was 0.12 (range, 0.10 to 0.32). The UPCR was < 0.2 for all castrated dogs, except for 1. Mean UPCR for all castrated dogs was 0.08 (range, 0.05 to 0.69). There was a significant difference between mean UPCR before and after castration. CONCLUSIONS AND CLINICAL RELEVANCE: In this study, pathological proteinuria was not detected in sexually intact male dogs. Positive results for a urine dipstick test should be interpreted with caution in sexually intact male dogs and should be confirmed by assessment of the UPCR. An increased UPCR in sexually intact male dogs may be considered abnormal.

Ileocecocolic volvulus in a German shepherd dog.

This report describes an ileocecocolic volvulus in a German shepherd dog with risk factors of previous abdominal surgeries and concurrent chronic enteropathy. Contrast-enhanced computed tomography (CT) with multiplanar reformatting was more sensitive than abdominal radiographs or ultrasound to obtain a diagnosis, because of the presence of a "whirl-sign" on CT. A combination of colopexy and cecopexy was succesfully used to treat the patient's condition.

Volvulus iléo-caeco-colique chez un Berger Allemand. Ce rapport de cas décrit un volvulus iléo-caeco-colique chez un Berger allemand ayant différents facteurs de risque incluant des chirurgies abdominales préalables à sa présentation et une entéropathie chronique. L'observation d'un 'whirl-sign' lors des reconstructions de l'examen tomodensitométrique a permis d'obtenir le diagnostic de volvulus et s'est révélé dans ce cas être plus sensible que les examens d'imagerie usuels, soit la radiographie ou l'échographie abdominale. Le traitement chirurgical a été effectué avec succès grâce à la combinaison d'une cecopexie et d'une colopexie.(Traduit par les auteurs).

Feline leptospirosis serosurvey from a Quebec referral hospital.

Epidemiologic studies have linked interactions with cats as a risk factor for human leptospirosis, but serosurveys of feline Leptospira spp. infection are scarce in the veterinary literature. The objective of this study was to conduct a serosurvey of Leptospira spp. infection in cats presenting to an eastern Canadian veterinary teaching hospital (VTH). All serum samples collected from cats presented to the VTH were tested by the microscopic agglutination test (MAT) for the Leptospira serovars Canicola, Grippotyphosa, Icterohaemorrhagiae, Bratislava, Pomona, and Autumnalis. Ten of 40 cats [25%; 95% confidence interval (CI): 12.7% to 41.2%] tested had positive antibody titers (≥ 1:100). All 10 cats with positive titers were positive for Bratislava and 2 were also positive for Autumnalis. This high incidence of seropositivity for Leptospira spp. may suggest that the disease could be of more clinical importance than previously recognized.

Enquête sérologique sur la leptospirose féline dans un hôpital de référence du Québec. Les études épidémiologiques ont associé les interactions avec les chats comme un facteur de risque pour la leptospirose humaine, mais les enquêtes sérologiques sur l'infection à la leptospirose féline sont rares dans les publications vétérinaires. La présente étude avait pour objectif de réaliser une enquête sérologique de l'infection à Leptospira chez les chats présentés dans un hôpital d'enseignement vétérinaire (HEV) de l'Est du Canada. Tous les échantillons de sérum prélevés auprès des chats présentés à l'HEV ont été testés à l'aide du sérodiagnostic d'agglutination microscopique pour les sérotypes de Leptospira Canicola, Grippotyphosa, Icterohaemorrhagiæ, Bratislava, Pomona et Autumnalis. Dix des 40 chats [25 %; intervalle de confiance (IC) de 95 % : 12,7 % à 41,2 %] ont obtenu des titres d'anticorps positifs (≥ 1:100). Les 10 chats avec des titres positifs étaient positifs pour Bratislava et 2 étaient aussi positifs pour Autumnalis. Cette incidence élevée de séropositivité pour Leptospira peut suggérer que la maladie pourrait être d'une importance clinique supérieure à ce qui était reconnu auparavant.(Traduit par Isabelle Vallières).

Elevation of neutrophil gelatinase-associated lipocalin (NGAL) in non-azotemic dogs with urinary tract infection.

Neutrophil gelatinase-associated lipocalin (NGAL) is a promising biomarker in humans and dogs with kidney disease. This protein is expressed by many cells including renal tubular cells and neutrophils. The aim of this study was to evaluate the effect of urinary tract infection (UTI) on urinary NGAL (uNGAL) concentration in dogs. Urine culture and measurement of uNGAL level were performed in 80 non-azotemic dogs suspected of UTI and 19 healthy dogs. Dogs were divided in three groups: 19 healthy dogs, 25 dogs with positive culture and 55 dogs suspected of UTI but with negative culture. uNGAL and uNGAL/Creatinine was significantly higher (P < 0.0001) in dogs with UTI (14.22 ng/mL;19.74 µg/g) compared to Healthy (0.24 ng/mL;0.11 µg/g) and Negative (1.13 ng/mL;1.28 µg/g) dogs. A uNGAL value <3.38 ng/mL had a negative predictive value for UTI of 87%. Presence of UTI has to be considered when uNGAL is used to detect kidney disease.

An out-of-frame overlapping reading frame in the ataxin-1 coding sequence encodes a novel ataxin-1 interacting protein.

Spinocerebellar ataxia type 1 is an autosomal dominant cerebellar ataxia associated with the expansion of a polyglutamine tract within the ataxin-1 (ATXN1) protein. Recent studies suggest that understanding the normal function of ATXN1 in cellular processes is essential to decipher the pathogenesis mechanisms in spinocerebellar ataxia type 1. We found an alternative translation initiation ATG codon in the +3 reading frame of human ATXN1 starting 30 nucleotides downstream of the initiation codon for ATXN1 and ending at nucleotide 587. This novel overlapping open reading frame (ORF) encodes a 21-kDa polypeptide termed Alt-ATXN1 (Alternative ATXN1) with a completely different amino acid sequence from ATXN1. We introduced a hemagglutinin tag in-frame with Alt-ATXN1 in ATXN1 cDNA and showed in cell culture the co-expression of both ATXN1 and Alt-ATXN1. Remarkably, Alt-ATXN1 colocalized and interacted with ATXN1 in nuclear inclusions. In contrast, in the absence of ATXN1 expression, Alt-ATXN1 displays a homogenous nucleoplasmic distribution. Alt-ATXN1 interacts with poly(A)(+) RNA, and its nuclear localization is dependent on RNA transcription. Polyclonal antibodies raised against Alt-ATXN1 confirmed the expression of Alt-ATXN1 in human cerebellum expressing ATXN1. These results demonstrate that human ATXN1 gene is a dual coding sequence and that ATXN1 interacts with and controls the subcellular distribution of Alt-ATXN1.

Computed tomographic evaluation of a bronchogenic cyst in a German shepherd dog.

A German shepherd dog was referred for further evaluation of a cavitary pulmonary lesion. Computed tomography identified a well-defined rounded radiolucent area in the left cranial lung lobe in continuity with the bronchial lumen. These findings were consistent with a bronchogenic cyst.