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Characterization and expression of the equine chitotriosidase gene
Get PDFChitin, an essential structural carbohydrate polymer of some simple life forms, is degraded by chitinases. Mammals lack chitin, but express two chitinases: acidic mammalian chitinase and chitotriosidase. Horses are prone to develop recurrent airway obstruction from chronic exposure to organic dust. We sought to investigate whether chitotriosidase is expressed in the lung of horses and associated with inflammation. The entire equine chitotriosidase gene was amplified, and was determined to have similar structure as in other mammals consisting of 11 exons spanning 13 Kb of gDNA. Chitotriosidase was expressed in multiple tissues of horses, but not in muscle. Relative gene quantification in bronchoalveolar lavage cells showed a trend towards higher chitotriosidase gene expression in horses with active inflammation relative to horses in remission, but this was not statistically significant. In summary, it has been found that horses express a chitinase that it is present in many horse tissues including leukocytes in lung
Immunophenotypic Characteristics of Equine Monocytes and Alevolar Macrophages
Get PDFHematopoietic cells of the myelomonocytic lineage play a central role in orchestrating both innate and adaptive immunity. They are important in the control of infectious agents and in the pathogenesis of diseases characterized by dysregulated immune response. Like allergic asthma in human patients, recurrent airway obstruction (RAO) of horses is a disease exemplified by chronic airway inflammation in the absence of infectious agents. However, unlike allergic asthma, RAO is marked by preponderance of neutrophils rather than eosinophils in the airways. Attempts to understand the immunological basis of RAO by studying lymphocytes produced equivocal results. This thesis examined the possible role of alveolar macrophages (AM) recovered from bronchoalveolar lavage fluid (BALF) in RAO. Since macrophages are predominantly derived from circulating monocytes, the thesis investigated first the phenotypic characteristics of circulating monocytes, second those of macrophages in vitro derived from monocytes, and finally attributes of AM derived in vivo. Flow cytometric analysis following antibody staining of monocytes from 61 horses showed that the clustering pattern of human leukocytes may not always be extrapolated to horses when using this technique since clusters of granulocytes often spill over into the monocyte population. The study showed that DH24A, a monoclonal antibody directed against CD90, which recognizes T cells in other species, will specifically recognize granulocytes in horses and was therefore used to separate neutrophils from monocytes during analysis. In addition, investigation of circulating monocytes showed that expression of the hemoglobin-haptoglobin receptor CD163 on circulating monocytes is significantly increased in horses with systemic inflammation when compared with healthy horses. Evaluating cytokine and chemokine production by macrophages, it was demonstrated that CD163+ macrophages preferentially expressed IL10 while CD163- macrophages showed predominant expression of CCL17. It was, therefore, concluded that CD163+ IL10-producing macrophages of horses are homologues of the alternatively activated anti-inflammatory macrophage subset of humans. Finally, probing of alveolar macrophages for CD163 and CD206 expression showed a significant reduction in the proportion of CD163+ macrophages in horses with RAO. These findings suggest that RAO is associated with a reduction in anti-inflammatory macrophages, an observation that may in part explain the chronic airway inflammation associated with this disease
Optimal Procurement, Preparation and Analysis of Canine Bone Marrow for Histomorphology, Cell Composition, Gene Amplification and Immunohistochemistry
No full textBone marrow aspirates and biopsies are crucial for diagnosing hematologic changes. The processing of bone marrow samples, consisting of bone and hematopoietic tissue, involves fixation, demineralization, paraffin embedding, sectioning, and staining. This thesis focused on optimizing fixation and demineralization methods for canine bone marrow, ensuring suitability for tests like DNA amplification and immunohistochemistry. It also examined hematopoietic cellularity, granulocytic-to-erythrocytic (G:E) ratio, and the distribution of megakaryocytes and hemosiderin across four anatomic sites with trabecular bone: rib, ilium, humerus, and sternum. The first study evaluated two fixatives (acetic acid-zinc-formalin [AZF] and neutral buffered formalin [NBF]) and three demineralizing agents (EDTA, formic acid, and hydrochloric acid) for their effects on histomorphology and DNA amplification. Both fixatives provided comparable histomorphology, but EDTA outperformed acid demineralizers in preserving histomorphology and DNA for further amplification. DNA amplification was more successful in NBF-fixed samples than AZF-fixed samples and markedly better in EDTA-treated samples than those demineralized with acids. The second study assessed bone marrow cellular parameters across four different anatomic sites. The rib had higher cellularity, megakaryocyte counts, and hemosiderin content compared to the humerus, while the humerus had a higher G:E ratio. The rib and sternum had greater cellularity than the humerus and ilium. Individual animal variation rather than the site of collection or raters accounted for the majority of differences observed. The third study evaluated the effects of fixatives and demineralizers on immunohistochemical detection of six antigens (CD3, CD20, CD34, CD204, FLT3, and FVIII-rAG). AZF and NBF fixatives yielded similar signal quality, though AZF generated slightly more background in CD3 and CD34 staining. EDTA provided superior antigen preservation, especially for CD204, FLT3, and FVIII-rAG, compared to acid-based demineralizers, although it increased background in CD20 sections. Overall, formalin-based fixatives effectively preserve canine bone marrow, while EDTA outperforms acid demineralizers in maintaining DNA and antigen integrity. Differences in cellularity, G:E ratio, megakaryocyte number, and hemosiderin content across the evaluated sites were of relatively small magnitude and unlikely of biological significance
Induction and mechanisms of recurrent airway obstruction in horses
No full textStabled horses are susceptible to an inflammatory disease of the lower airways, recurrent airway obstruction (RAO). Recent consensus defines RAO as a complex disease that arises in mature horses in response to chronic exposure to environmental particulate, bacterial, and fungal components. The inflammation of RAO results in bronchoconstriction, and small airway obstruction from an accumulation of mucus and neutrophils--leading to the clinical signs of cough, tachypnea, nostril flare, and increased abdominal effort on exhalation. The disease has been studied extensively in an attempt to characterize the pathogenesis and to validate treatment modalities. In order to more fully characterize the mechanisms involved in development of the RAO phenotype, a standardized model for disease exacerbation was needed. Such a model would permit comparisons between studies; however, no model thus far offered has fully characterized components. Further, recent work has indicated that both Clara cells and their major product, Clara cell secretory protein (CCSP), are reduced in horses with RAO. Ultrastructural studies showed CCSP within the cytoplasm and nucleus of neutrophils of RAO-affected horses, which suggested that CCSP might function as a cytokine or chemokine. The present research project had three goals. First, molds consistently present in moldy hay were identified using sensitive techniques. Second, a nebulizable particulate suspension of accurately quantified materials was created to cause reliable and repeatable exacerbation of RAG. Third, the localization pattern of recombinant equine CCSP (reCCSP) within stimulated equine neutrophils was examined by immunoblot analysis. Results of the studies were as follows. Three fungi consistently identified in moldy hay were: 'Lichtheimia corymbifera, Aspergillus fumigatus,' and 'Eurotium amstelodami'. Further, nebulization of a mixture of fungal spores, lipopolysaccharide, and silica microspheres induced changes consistent with RAG exacerbation in susceptible horses. And finally, reCCSP may have been detected in stimulated neutrophils. These findings suggest that RAG can be reproduced in susceptible horses through inhalation of a defined mixture of challenge materials, and additional studies are required to assess the interactions of reCCSP and neutrophils
Prognostication of Canine T Cell Lymphoma
No full textCanine lymphoma is a heterogeneous disease with many different subtypes. T cell lymphoma (TCL) is variable in outcome, and includes subtypes with non-progressive, indolent and aggressive disease course. Methods used to categorize lymphoma including cytopathology, flow cytometry, histopathology, and immunochemistry. Many different classification schemes have been used in the past, however, currently an adaptation of the human World Health Organization (WHO) classification is proposed. This classification categorizes lymphoma based on cytopathology, flow cytometry, histopathology, immunohistochemistry, and molecular techniques. Association of immunotype and subtype with disease course is incompletely defined. The goal of this study was to associate immunotype and different WHO subtypes with prognosis. Retrospectively, flow cytometric immunotyping for 127 canine TCL were analyzed in relation to survival and progression free interval. Samples originated from 101 multicentric, 8 mediastinal, 6 cutaneous, 5 hepatosplenic, 5 gastrointestinal and 2 other anatomic subtypes of TCL. Compared to patients with multicentric TCL, those with gastrointestinal lymphoma had shorter survival and progression free interval, and those with hepatosplenic lymphoma had shorter progression free interval. Among patients with multicentric TCL, immunotypes of CD4+/CD8-/MHCII+, CD4-/CD8+/MHCII+ and CD4-/CD8+/MHCII- were associated with longer survival than the CD4+/CD8-/MHCII- immunotype, and immunotypes of CD4+/CD8-/MHCII+, CD4-/CD8+/MHCII+, and CD4-/CD8-/MHCII+ were associated with longer progression free intervals. Prospectively, cytopathological, flow cytometric, histopathological, and immunohistochemical data was collected from 16 patients. Samples included 8 peripheral T cell Lymphoma – not otherwise specified (PTCL-NOS), 6 T zone lymphomas (TZL), and 2 T lymphoblastic lymphomas (T LBL). Both aggressive and indolent forms were identified in the PTCL-NOS and T LBL categories with unique immunotypes consistent with the retrospective study. TZL were always indolent and had consistent immunotypes. MHC class II expression was associated with favourable prognosis. Grading based on mitoses or Ki67 expression did not significantly affect outcome. Interobserver agreement was non-existent for cytopathology and flow cytometry, and moderate for histopathology and immunohistochemistry. There is support for the judicious use of single agent or no chemotherapy for TCL.Ontario Veterinary College Pet Trus
Kidney Injury Molecule 1: A potential biomarker of renal injury in the cat
No full textThe prevalence of both acute and chronic kidney disease in animals and people is increasing. Increased serum creatinine concentration indicates a decline in renal function but is insensitive for diagnosing acute kidney injury (AKI). As such, there is need to develop sensitive biomarkers of renal injury. At the fore in human medicine is a biomarker of acute renal tubular injury called kidney injury molecule-1 (KIM-1). KIM-1 is a cell membrane glycoprotein with an extracellular domain that is shed from proximal tubular kidney cells and detectable in urine after ischemic and toxic renal injury. KIM-1 was undetectable in normal human urine, but presence in urine correlated with immunohistochemical detection of KIM-1 in injured tubular epithelial cells. For these reasons, KIM-1 was considered a good candidate to investigate as a biomarker of kidney injury in cats. Alignment of KIM-1 sequences from human, rat, dog, and mice indicated a high degree of identity between species and conservation of a cytoplasmic tyrosine motif. Primers based on the conserved regions were used to amplify feline KIM-1 genomic and renal cDNA sequences. PCR assays used to amplify feline KIM-1 revealed the presence of three transcript variants derived by alternative splicing with exon-intron organization similar to KIM-1 orthologous sequences. KIM-1 was detected by IHC in tubules of the cortex and outer stripe of the outer medulla in cats with suspected naturally occurring AKI and cats with experimental unilateral ischemia/reperfusion. The same cells also expressed aquaporin 1, confirming expression of KIM-1 in the proximal convoluted tubules. Further, KIM-1 expression correlated positively with tubular injury scores and vimentin expression in the injured proximal tubules. Available urine immunoassay to rat Kim-1 yielded positive reactions with urine from 11 cats. In conclusion, feline KIM-1 is similar but not identical to human KIM-1. Expression is increased in cats with naturally occurring and experimental kidney injury, and KIM-1 may be detected in urine. KIM-1 is expressed predominantly in cells of the S3 segment of proximal tubules, and appears to persist during dedifferentiation and repair of injured cells. Hence, detection of KIM-1 in urine is a promising indicator of kidney injury in cats
Programmed Death Ligand 2 Gene Regulation in T-cells During Homeostasis and Lentiviral Infection
Get PDFProgrammed Death Ligand 2 (PD-L2) is a co-stimulatory molecule predominantly expressed on antigen-presenting cells. PD-L2 interacts with membrane-bound receptor PD-1 on activated T-cells. This interaction transmits a negative signal to T-cells inducing anergy or apoptosis. Viruses such as HIV induce PD-L2, which enables viral persistence. In this study, regulation of PD-L2 gene expression in feline lymphocytes in homeostasis and lentiviral infection was investigated. The upstream region of PD-L2 was amplified and sequenced to identify putative transcription factor binding sites, including NF-κB, STAT6, Oct-1/2, GATA-3 and NFAT. Assays to assess transcriptional activation from incremental segments upstream of PD-L2 identified the first 2 kb as the possible location of the proximal promoter. Transfection of NF-κB or NFAT into T-cells increased PD-L2 transcription, but FIV infection did not. Results suggest that transcriptional activation of PD-L2 in homeostatic T-cells differs from macrophages and DCs; and that acute FIV infection does not affect PD-L2 expression
Response of the Bronchial Epithelium to Challenge in Severe Equine Asthma
No full textSevere equine asthma (recurrent airway obstruction, heaves) is an inflammatory lung disease affecting horses chronically exposed to adverse environments. Genetic predisposition to the disease is reported to be heterogeneous across families, but clinical signs during exacerbation are remarkably similar. In order to identify commonly expressed genes and pathways in horses with asthma, we sequenced RNA from endobronchial biopsies collected from control and affected horses before and after an asthmatic stimulus. Differential expression analysis of the bronchial epithelium yielded 111 genes significantly different between asthmatic and non-asthmatic horses. Gene set and network analyses identified overrepresentation of upregulated genes involved in neutrophil migration and chemotaxis, immune and inflammatory responses, secretion, blood coagulation and apoptosis. Downregulated genes were overrepresented in rhythmic processes, referring to physiological rhythm. Network analysis identified MMP4, MMP1, IL8 and TLR4 as pivotal molecules, suggesting they have key roles in disease. Furthermore, in asthmatic horses, a large number of significantly upregulated genes contained E2F binding motifs, and were associated with cell cycle, neutrophilic response, hedgehog signaling, hemostasis and coagulation. These genes and motifs were not detected at similar frequencies in non-asthmatic horses. RNA-Seq was also used to identify genetic variants potentially linked to disease mechanisms. Single base mutations in parkin co-regulated (PACRG) and rotatin (RTTN) were identified as more prevalent in asthmatic compared to non-asthmatic horses, and confirmed with Sanger sequencing. PACRG and RTTN are essential proteins in motile and primary cilia development and function, befitting further investigation of cilia morphology in asthmatics. As such, cilia and microvilli were evaluated by electron microscopy, and ultrastructural abnormalities consisting of cilia of abnormal shape and size and branching microvilli of heterogeneous length were identified. These changes are hypothesized to be a consequence of chronic inflammation and also to contribute to progressive deterioration of bronchial epithelial function. Abnormal cilia are likely to reduce mucociliary clearance and hedgehog signaling, which in turn links gene expression and variants to physiological abnormalities in the bronchiolar epithelium. In summary, studies in this thesis have revealed multiple unique aspects of the bronchial epithelial response in asthmatic relative to non-asthmatic horses. The epithelial barrier is of paramount importance to modulate the host response to environmental agents, but other components of the lung not examined in this thesis also contribute to development of asthma.University of GuelphEquine GuelphNatural Sciences and Engineering Research Council of CanadaOntario Ministry of Agriculture and Rural Affair
Salivary Scavenger and Agglutinin in Horses With and Without Severe Equine Asthma
No full textSevere equine asthma (SEA) is a common, debilitating inflammatory airway disease of horses. Next-generation sequencing identified numerous differentially expressed epithelial genes in asthmatic compared to healthy horses, including downregulation of the gene for salivary scavenger and agglutinin (SALSA). In humans, SALSA has numerous functions in innate immunity and modulates inflammation. In horses, SALSA was poorly characterized. This thesis addressed the hypothesis that SALSA has anti-inflammatory effects on equine leukocytes as assessed in healthy horses and those with SEA. First, an antibody against SALSA was validated by western blotting, immunoprecipitation followed by mass spectrometry, and immunohistochemical analysis of 22 equine tissues in a tissue microarray. The equine SALSA gene was amplified and functional motifs predicted. Equine SALSA was found to have three to five scavenger receptor cysteine-rich domains, two complement C1r/C1s, Uegf, Bmp1 (CUB) domains, and one zona pellucida domain. There was interindividual variability in domain frequency. Immunohistochemical labeling was most prominent at mucosal surfaces of the duodenum and sections of the airways. Labeling was cytoplasmic and apical, consistent with a secreted protein. Second, gene and protein expression of SALSA in horses with and without SEA was evaluated, and bronchial brushing and biopsy were compared to bronchoalveolar lavage as in vivo diagnostic modalities. Quantitative PCR confirmed lower SALSA gene expression in horses with SEA while immunohistochemically SALSA did not significantly differ between affected and unaffected horses. Bronchial brushing but not bronchial biopsy was a good method for distinguishing affected from unaffected horses. In the third study, the effect of purified equine SALSA on neutrophil bacterial phagocytosis and alveolar macrophage cytokine production was determined. Neutrophils incubated with SALSA had a dose-dependent decrease in the phagocytosis of fluorescently-labeled bacteria. Alveolar macrophages incubated with SALSA had reduced production of CXCL1, IL-8, IL-10 and TNF-α and increased production of G-CSF, as measured with an equine-specific multiplex bead immunoassay. Overall, the findings from this research indicate that equine SALSA is secreted by mucosal epithelial cells, reduces neutrophil phagocytosis, and suppresses pro-inflammatory cytokine production. These features define characteristics of SALSA that are likely influential in dampening the host response to microbial components at mucosal surfaces.University of GuelphEquine GuelphNatural Sciences and Engineering Research Council of Canad
Expression profile and role of sterile alpha motif domain-and histidine domain-containing protein 1 (SAMHD1) in restriction of feline immunodeficiency virus (FIV)
No full textSAMHD1 is one of the type-I interferon-induced proteins known collectively as “restriction factors”. The triphosphohydrolase activity of SAMHD1 reduces the cellular dNTP pool in non-dividing myeloid cells and resting CD4+ T cells to levels below that required for efficient HIV-1 cDNA synthesis. SAMHD1 also has putative exonuclease activity that degrades ssRNA and ssDNA. The feline genome encodes a SAMHD1 orthologue, but neither expression nor physiologic or antiviral functions are known. Using a feline whole-body tissue microarray with 24 tissues for comprehensive immunohistochemical analysis, SAMHD1 protein was identified in a wide range of tissues with most concentrated detection at sites of viral entry and replication. Nuclear and cytoplasmic immunoreactivity were variable among tissues. Among six commonly used feline cell lines, SAMHD1 protein and mRNA were most abundant in the lymphocyte cell line (FeTJ) with the highest rate of replication.Subsequently, the response of SAMHD1 to different types of interferon was examined through several experimental approaches. SAMHD1 mRNA in FeTJ cells increased in a dose-related manner in response to IFNγ treatment concurrent with increased nuclear localization and phosphorylation. In contrast, IFNα treatment induced SAMHD1 mRNA but did not significantly alter SAMHD1 protein detection, phosphorylation or nuclear translocation. In purified primary feline CD4+ lymphocytes, interleukin-2 supplementation increased SAMHD1 expression, but addition of IFNγ did not further alter SAMHD1 protein expression or nuclear localization. These findings imply that while SAMH1 was inducible by IFNγ, overall activity was cell type- and compartment-specific. Promoter methylation regulates SAMHD1 transcription. When the promoter is non-methylated and accessible to transcription factor binding and recruitment of RNA polymerases, gene transcription is enabled. However, the methylated promoter is inaccessible to transcription factors. Azacytidine reduced DNA methylation in feline lymphocytes, resulting in increased cellular and nuclear SAMHD1 protein without an increase in phosphorylated SAMHD1. These findings suggest that the antiviral activity of SAMHD1 may be augmented by promoter demethylation with compounds such as Azacytidine.In summary, tissue-, cell- and subcellular compartment-specific expression of SAMHD1, responsiveness to IFNγ stimulation, and transcriptional regulation by demethylation, suggest that SAMHD1 has unique functions in viral restriction and regulation of nucleotide availability.Natural Sciences and Engineering Research Council of CanadaUniversity of Guelp
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