Characterization of the plasma proteome from healthy adult dogs.

Introduction: Bloodwork is a widely used diagnostic tool in veterinary medicine, as diagnosis and therapeutic interventions often rely on blood biomarkers. However, biomarkers available in veterinary medicine often lack sensitivity or specificity. Mass spectrometry-based proteomics technology has been extensively used in the analysis of biological fluids. It offers excellent potential for a more comprehensive characterization of the plasma proteome in veterinary medicine. Methods: In this study, we aimed to identify and quantify plasma proteins in a cohort of healthy dogs and compare two techniques for depleting high-abundance plasma proteins to enable the detection of lower-abundance proteins via label-free quantification liquid chromatography-mass spectrometry. We utilized surplus lithium-heparin plasma from 30 healthy dogs, subdivided into five groups of pooled plasma from 6 randomly selected individuals each. Firstly, we used a commercial kit to deplete high-abundance plasma proteins. Secondly, we employed an in-house method to remove albumin using Blue-Sepharose. Results and discussion: Among all the samples, some of the most abundant proteins identified were apolipoprotein A and B, albumin, alpha-2-macroglobulin, fibrinogen beta chain, fibronectin, complement C3, serotransferrin, and coagulation factor V. However, neither of the depletion techniques achieved significant depletion of highly abundant proteins. Despite this limitation, we could detect and quantify many clinically relevant proteins. Determining the healthy canine proteome is a crucial first step in establishing a reference proteome for canine plasma. After enrichment, this reference proteome can later be utilized to identify protein markers associated with different diseases, thereby contributing to the diagnosis and prognosis of various pathologies.

Plasma proteome signature of canine acute haemorrhagic diarrhoea syndrome (AHDS).

Acute haemorrhagic diarrhoea is a common complaint in dogs. In addition to causes like intestinal parasites, dietary indiscretion, intestinal foreign bodies, canine parvovirus infection, or hypoadrenocorticism, acute haemorrhagic diarrhoea syndrome (AHDS) is an important and sometimes life-threatening differential diagnosis. There is some evidence supporting the link between Clostridium perfringens toxins and AHDS. These toxins may be partially responsible for the epithelial cell injury, but the pathogenesis of AHDS is still not fully understood. Recent studies have suggested that severe damage to the intestinal mucosa and associated barrier dysfunction can trigger chronic gastrointestinal illnesses. Besides bloodwork and classical markers for AHDS such as protein loss and intestinal bacterial dysbiosis, we focused mainly on the plasma-proteome to identify systemic pathological alterations during this disease and searched for potential biomarkers to improve the diagnosis. To accomplish the goals, we used liquid chromatography-mass spectrometry. We compared the proteomic profiles of 20 dogs with AHDS to 20 age-, breed-, and sex-matched control dogs. All dogs were examined, and several blood work parameters were determined and compared, including plasma biochemistry and cell counts. We identified and quantified (relative quantification) 207 plasmatic proteins, from which dozens showed significantly altered levels in AHDS. Serpina3, Lipopolysaccharide-binding protein, several Ig-like domain-containing proteins, Glyceraldehyde-3-phosphate dehydrogenase and Serum amyloid A were more abundant in plasma from AHDS affected dogs. In contrast, other proteins such as Paraoxonase, Selenoprotein, Amine oxidases, and Apolipoprotein C-IV were significantly less abundant. Many of the identified and quantified proteins are known to be associated with inflammation. Other proteins like Serpina3 and RPLP1 have a relevant role in oncogenesis. Some proteins and their roles have not yet been described in dogs with diarrhoea. Our study opens new avenues that could contribute to the understanding of the aetiology and pathophysiology of AHDS.

Traces of Canine Inflammatory Bowel Disease Reflected by Intestinal Organoids.

Inflammatory bowel disease (IBD) is a chronic inflammatory condition that affects humans and several domestic animal species, including cats and dogs. In this study, we have analyzed duodenal organoids derived from canine IBD patients using quantitative proteomics. Our objective was to investigate whether these organoids show phenotypic traits of the disease compared with control organoids obtained from healthy donors. To this aim, IBD and control organoids were subjected to quantitative proteomics analysis via liquid chromatography-mass spectrometry. The obtained data revealed notable differences between the two groups. The IBD organoids exhibited several alterations at the levels of multiple proteins that are consistent with some known IBD alterations. The observed phenotype in the IBD organoids to some degree mirrors the corresponding intestinal condition, rendering them a compelling approach for investigating the disease and advancing drug exploration. Additionally, our study revealed similarities to some human IBD biomarkers, further emphasizing the translational and comparative value of dogs for future investigations related to the causes and treatment of IBD. Relevant proteins such as CALU, FLNA, MSN and HMGA2, which are related to intestinal diseases, were all upregulated in the IBD duodenal organoids. At the same time, other proteins such as intestinal keratins and the mucosal immunity PIGR were depleted in these IBD organoids. Based on these findings, we propose that these organoids could serve as a valuable tool for evaluating the efficacy of therapeutic interventions against canine IBD.

Dysregulation of intestinal epithelial electrolyte transport in canine chronic inflammatory enteropathy and the role of the renin-angiotensin-aldosterone-system.

Chronic diarrhea is a hallmark sign of canine chronic inflammatory enteropathy (CIE), leading to fluid and electrolyte losses. Electrolyte homeostasis is regulated by the renin-angiotensin-aldosterone-system (RAAS), which might be involved in (counter-)regulating electrolyte losses in canine CIE. Whether and which electrolyte transporters are affected or if RAAS is activated in canine CIE is unknown. Thus, intestinal electrolyte transporters and components of the RAAS were investigated in dogs with CIE. Serum RAAS fingerprint analysis by mass spectrometry was performed in 5 CIE dogs and 5 healthy controls, and mRNA levels of intestinal electrolyte transporters and local RAAS pathway components were quantified by RT-qPCR in tissue biopsies from the ileum (7 CIE, 10 controls) and colon (6 CIE, 12 controls). Concentrations of RAAS components and mRNA expression of electrolyte transporters were compared between both groups of dogs and were tested for associations among each other. In dogs with CIE, associations with clinical variables were also tested. Components of traditional and alternative RAAS pathways were higher in dogs with CIE than in healthy controls, with statistical significance for Ang I, Ang II, and Ang 1-7 (all p < 0.05). Expression of ileal, but not colonic electrolyte transporters, such as Na+/K+-ATPase, Na+/H+-exchanger 3, Cl- channel 2, down-regulated in adenoma, and Na+-glucose-cotransporter (all p < 0.05) was increased in CIE. Our results suggest that the dys- or counter-regulation of intestinal electrolyte transporters in canine CIE might be associated with a local influence of RAAS. Activating colonic absorptive reserve capacities may be a promising therapeutic target in canine CIE.

Time to eRAASe chronic inflammation: current advances and future perspectives on renin-angiotensin-aldosterone-system and chronic intestinal inflammation in dogs and humans.

Chronic idiopathic intestinal inflammation is an increasing worldwide problem that affects companion animals, especially dogs, and human patients. Although these disease entities have been intensely investigated recently, many questions remain, and alternative therapeutic options are needed. Diarrhea caused by dysregulation of intestinal electrolyte transport and subsequent fluid and electrolyte losses often leads to secondary consequences for the patient. Currently, it is not exactly clear which mechanisms are involved in the dysregulation of intestinal fluid absorption, but differences in intestinal electrolyte shifts between human and canine patients suggest species-specific regulatory or counterregulatory mechanisms. Several intestinal electrolyte transporters are differentially expressed in human patients with inflammatory bowel disease (IBD), whereas there are virtually no studies on electrolyte transporters and their endocrine regulation in canine chronic inflammatory enteropathy. An important mechanism involved in regulating fluid and electrolyte homeostasis is the renin-angiotensin-aldosterone-system (RAAS), which may affect intestinal Na+ transport. While RAAS has previously been considered a systemic regulator of blood pressure, additional complex roles of RAAS in inflammatory processes have been unraveled. These alternative RAAS pathways may pose attractive therapeutic targets to address diarrhea and, thus, electrolyte shifts in human IBD and canine chronic inflammatory enteropathy. This article comparatively summarizes the current knowledge about electrolyte transport in human IBD and canine chronic inflammatory enteropathy and the role of RAAS and offers perspectives for novel therapeutic avenues.

Formerly bile-farmed bears as a model of accelerated ageing.

Bear bile-farming is common in East and Southeast Asia and this farming practice often results in irreversible health outcomes for the animals. We studied long-term effects of chronic bacterial and sterile hepatobiliary inflammation in 42 Asiatic black bears (Ursus thibetanus) rescued from Vietnamese bile farms. The bears were examined under anesthesia at least twice as part of essential medical interventions. All bears were diagnosed with chronic low-grade sterile or bacterial hepatobiliary inflammation along with pathologies from other systems. Our main finding was that the chronic low-grade inflammatory environment associated with bile extraction in conjunction with the suboptimal living conditions on the farms promoted and accelerated the development of age-related pathologies such as chronic kidney disease, obese sarcopenia, cardiovascular remodeling, and degenerative joint disease. Through a biomimetic approach, we identified similarities with inflammation related to premature aging in humans and found significant deviations from the healthy ursid phenotype. The pathological parallels with inflammageing and immuno-senescence induced conditions in humans suggest that bile-farmed bears may serve as animal models to investigate pathophysiology and deleterious effects of lifestyle-related diseases.

Gut microbiome signatures of Yorkshire Terrier enteropathy during disease and remission.

The role of the gut microbiome in developing Inflammatory Bowel Disease (IBD) in humans and dogs has received attention in recent years. Evidence suggests that IBD is associated with alterations in gut microbial composition, but further research is needed in veterinary medicine. The impact of IBD treatment on the gut microbiome needs to be better understood, especially in a breed-specific form of IBD in Yorkshire Terriers known as Yorkshire Terrier Enteropathy (YTE). This study aimed to investigate the difference in gut microbiome composition between YTE dogs during disease and remission and healthy Yorkshire Terriers. Our results showed a significant increase in specific taxa such as Clostridium sensu stricto 1, Escherichia-Shigella, and Streptococcus, and a decrease in Bacteroides, Prevotella, Alloprevotella, and Phascolarctobacterium in YTE dogs compared to healthy controls. No significant difference was found between the microbiome of dogs in remission and those with active disease, suggesting that the gut microbiome is affected beyond clinical recovery.

Microbial dysbiosis and fecal metabolomic perturbations in Yorkshire Terriers with chronic enteropathy.

Dysbiosis and perturbations of fecal metabolic profiles have been reported in dogs with inflammatory bowel disease. Currently the incidence of dysbiosis and the fecal metabolomic profile in Yorkshire Terriers with chronic enteropathy (YTE) and the effects of treatment are unknown. This prospective observational study analyzed the dysbiosis index (DI) and fecal bile acid, sterol and fatty acid profiles in 14 Yorkshire Terriers with active YTE, 11 dogs in clinical remission, and 26 healthy Yorkshire Terriers. YTE was associated with dysbiosis and a significant increase in fatty acids (docosanoate, p = 0.002; gondoate, p = 0.026; erucate, p < 0.001; nervonate, p < 0.001; linolenate, p < 0.001), and plant sterols (campesterol, p < 0.001; brassicasterol, p = 0.024). The abundances of Fusobacterium (p < 0.001) and Cl. hiranonis (p = 0.018) and the concentrations of the secondary bile acid ursodeoxycholic acid (p = 0.033) and the plant sterol sitostanol (p = 0.003) were significantly decreased compared to healthy dogs. Dysbiosis, abundances of Fusobacterium, Cl. hiranonis and fecal concentrations of bile acids and sterols did not recover after treatment, while fecal fatty acid concentrations decreased in treated dogs. YTE is associated with dysbiosis and changes in bile acid, fatty acid, and sterol metabolism. These changes only recovered partially despite clinical remission. They might be breed-specific and involved in the pathogenesis of YTE.

Utility of a canine TSH assay for diagnosis and monitoring of feline hyperthyroidism.

OBJECTIVE: This retrospective study was initiated to evaluate the utility of TSH measurements using a common canine TSH assay to diagnose and monitor feline hyperthyroidism after radioiodine or thyreostatic drug treatment. MATERIAL AND METHODS: The electronic database of the University of Veterinary Medicine Vienna was searched for combined TSH and T4 measurements. 217 pairs of TSH and T4 from 136 cats with possible hyperthyroidism were assigned to group A (untreated; n = 24) and B (treated; n = 193). Measurements in group B were then subcategorized according to T4 concentrations (reference range 15-50 nmol/L): group B1 = elevated T4 (n = 46), group B2 = normal T4 (n = 84) and group B3 = decreased T4 (n = 63). Group B2 was further divided into cats with low normal (group B2a; n = 35), medium normal (group B2b; n = 29) and high normal (group B2c; n = 20) T4 concentrations. RESULTS: TSH was detectable in 4 (17 %) of the 24 untreated cats (group A) and did not return to normal despite seemingly successful therapy in two. Increased TSH concentrations were observed in 3.6 % of the treated cats in group B2 and 2.9 %, 6.9 %, and 0 % in subgroups B2a, B2b and B2c, respectively. Forty-four percent of the treated cats with a decreased T4 (group B3) had an increased TSH concentration. TSH correlated with treatment length (r = 0.358, p = 0.004) and was significantly higher in cats treated for more than 3 months (p = 0.008). CONCLUSION: TSH was detectable in a significant number of untreated hyperthyroid cats and thus this parameter should not be used to definitively rule out feline hyperthyroidism. Furthermore, the very low prevalence of increased TSH concentrations in treated hyperthyroid cats with a normal T4 and cost benefit calculations do not support the routine measurement of TSH in these cats. The fact that TSH correlated with time since treatment start and 56 % of the cats with a decreased T4 had TSH concentrations within the reference limits, suggests delayed recovery of the pituitary thyrotrophs which might explain the low prevalence of subclinical hypothyroidism in the present study. CLINICAL RELEVANCE: TSH measurement in cats with suspected or treated hyperthyroidism using a canine assay lacks diagnostic sensitivity and can only complement therapeutic decision-making.

A Preliminary Metabolomic Study of Yorkshire Terrier Enteropathy.

Perturbations of metabolite profiles in human and canine enteropathies have been reported before. However, data in dogs are scarce and inconsistent. Currently, the metabolite profile in Yorkshire Terrier enteropathy (YTE) and the impact of treatment is unknown. The objective of this study was to investigate the plasma metabolome of 13 Yorkshire Terriers with YTE and compare it to 20 healthy Yorkshire Terriers. Furthermore, we studied the impact of treatment on the metabolome. In this prospective observational study, plasma metabolite profiles were analyzed by flow injection analysis-tandem mass spectrometry (FIA-MS/MS) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) using a targeted metabolomics kit. Metabolite analysis revealed that YTE is accompanied by changes in lipid and bile acid metabolism. YTE was associated with a significant decrease of long-chain fatty acids (octadecenoic acid, eicosadienoic acid, eicosatrienoic acid) and lower levels of long-chain acylcarnitines (tetradecanoylcarnitine, hexadecanoylcarnitine, hexadecenoylcarnitine, octadecenoylcarnitine) compared with healthy controls. Furthermore, taurodeoxycholic acid, a secondary bile acid, was decreased in plasma from YTE patients. These changes might be breed-specific and might be involved in the pathogenesis of YTE. Interestingly, changes in metabolite levels were not recovered after treatment and differed considerably from healthy controls.

Chronic cholecystitis: Diagnostic and therapeutic insights from formerly bile-farmed Asiatic black bears (Ursus thibetanus).

Across Southeast Asia and China, more than 17000 Asian bears are kept under suboptimal conditions and farmed for their bile to meet the consumer demand for traditional medicine products. Years of unsterile and repetitive bile extraction contribute to the development of chronic sterile or bacterial cholecystitis, a pathology commonly diagnosed in formerly bile-farmed bears. In both human and veterinary medicine, the diagnostic value of the macroscopic bile examination for assessing gallbladder disease is unclear. The objective of this study is to identify the role of gallbladder bile color, viscosity, and turbidity, while comparing them with established markers of cholecystitis. Moreover, it aims to define the optimal duration of oral antibiotic treatment for chronic bacterial cholecystitis in bears associated with bile farming. Thirty-nine adult, formerly bile-farmed Asiatic black bears (Ursus thibetanus) were examined under anesthesia and underwent percutaneous ultrasound guided cholecystocentesis. A total of 59 bile samples were collected with 20 animals sampled twice to evaluate the therapeutic success. All bile aspirates were assessed macroscopically and microscopically followed by submission for bacterial culture and antimicrobial sensitivity. In the majority of bears, samples with cytological evidence of bactibilia lacked inflammatory cells and did not always correlate with positive bacterial cultures. The most common bacterial isolates were Enterococcus spp, Streptococcus spp and Escherichia coli. Based on our findings, the optimal duration of antibiotic treatment for chronic bacterial cholecystitis is 30 days. Moreover, unlike Gamma-glutamyl Transferase (GGT) and gallbladder wall thickness, the organoleptic properties of bile were found to be reliable markers of chronic gallbladder inflammation with color and turbidity indicating cholestasis. The current study highlights the importance of cholecystocentesis for the management of gallbladder disease and provides initial results on the possible diagnostic value of macroscopic bile examination.

Proliferation Activity in Canine Gastrointestinal Lymphoma.

Gastrointestinal lymphomas are uncommon in dogs and little is known about their distinct subtypes or proliferation rate. The aim of this study was to stratify 33 canine gastrointestinal lymphoma samples according to the latest World Health Organization classification and to determine the Ki67 proliferation index by manual counting, digital image analysis and visual estimation. The Ki67 index was then correlated with subtype, immunophenotype, mitotic index, grade and tumour location. The mitotic index correlated positively with the Ki67 index. A significantly higher number of Ki67-positive cells was found in enteropathy-associated T-cell lymphoma type I and in diffuse large B-cell lymphoma compared with enteropathy-associated T-cell lymphoma type II. There was also a significant difference in Ki67 immunolabelled cells between grade 1 and grade 2 lymphomas. Moderate agreement was found between the Ki67 index as obtained by manual counting and visual estimation, but there was strong agreement between manual counting and digital image analysis. The user-friendly digital imaging system used in this study could have potential for future determination of the Ki67 index in lymphoid neoplasms.

A Recurrent STAT5BN642H Driver Mutation in Feline Alimentary T Cell Lymphoma.

Alimentary lymphomas arising from T cells are rare and aggressive malignancies in humans. In comparison, they represent the most common anatomical form of lymphoma in cats. Due to the low prevalence in humans, the underlying pathomechanism for these diseases is poorly characterised, limiting experimental analysis and therapeutic exploration. To date, activating mutations of the JAK/STAT core cancer pathway and particularly the STAT5B oncoprotein have been identified in human enteropathy-associated T cell lymphoma. Here, we describe a high homology of human and feline STAT3 and STAT5B proteins and strong conservation at the genomic level. Analysis of 42 samples of feline T cell alimentary lymphoma reveals broad activation of STAT3 and STAT5B. Screening for known activating mutations in STAT3 or STAT5B identifies the presence of the STAT5BN642H driver mutation in feline enteropathy-associated T cell lymphoma in 7 out of 42 (16.67%) samples in total. Regarding lymphoma subtypes, the majority of mutations with 5 out of 17 (29.41%) cases were found in feline enteropathy-associated lymphoma type II (EATL II). This identification of an oncogenic STAT5B driver mutation in felines recapitulates the genetic situation in the corresponding human disease, thereby establishing the cat as a potential new model for a rare and incurable human T cell disease.

Upregulation of signal transducer and activator of transcription 3 in dogs with chronic inflammatory enteropathies.

BACKGROUND: In inflammatory bowel disease (IBD) in humans, phosphorylated signal transducer and activator of transcription 3 (pSTAT3) is upregulated in mucosal epithelial cells and correlates with clinical severity. HYPOTHESIS/OBJECTIVE: To investigate the expression pattern of pSTAT3 in the mucosa of dogs with chronic inflammatory enteropathy (CIE) and explore correlations between its expression and clinical and histopathological severity scoring. ANIMALS: Twenty-eight canine CIE patients grouped into food-responsive enteropathy (FRE; 9), steroid-responsive enteropathy (SRE; 10), and protein-losing enteropathy (PLE; 9). Ten healthy beagle dogs served as controls (CO). METHODS: Retrospective case control study. Immunohistochemistry was used to detect pSTAT3 in canine duodenal mucosa samples. RESULTS: Compared to CO, SRE (P < .001) and PLE (P < .001) dogs had significantly higher pSTAT3 expression in the villus epithelium. The SRE group had a significantly higher expression in the villus lamina propria (VLP) compared to controls (P = .009). In the crypt epithelium (CE), all CIE dogs had significantly higher pSTAT3 expression (FRE, P = .002; SRE, P = .003; PLE, P < .001) compared to CO. In the lamina propria crypt region (CLP), dogs with FRE (P = .04) and SRE (P = .03) had significantly upregulated pSTAT3 compared to controls. A positive correlation was found between canine chronic enteropathy clinical activity index (CCECAI) scoring and pSTAT3 expression for both epithelial (rho = .541; P < .001) and crypt regions (rho = .32; P = .02). CONCLUSIONS AND CLINICAL IMPORTANCE: pSTAT3 is upregulated in CIE in dogs, correlates with clinical severity, and may be helpful as a clinical marker in dogs with CIE.

Aldosterone Is Positively Associated With Circulating FGF23 Levels in Chronic Kidney Disease Across Four Species, and May Drive FGF23 Secretion Directly.

BACKGROUND: Chronic kidney disease (CKD) is accompanied by increases in circulating fibroblast growth factor 23 (FGF23) and aldosterone levels. Here, we tested the hypothesis that aldosterone may be one of the driving forces behind increased FGF23 secretion in CKD. METHODS: Using data from a prospective study in humans, a retrospective study in dogs and cats, and an experimental study in 5/6-nephrectomized mice, we analyzed the relationship between circulating FGF23 and serum aldosterone levels in CKD across four species. To assess the effects of acute inhibition of aldosterone signaling on circulating FGF23, we acutely treated mice with established CKD with the mineralocorticoid receptor blocker canrenone (50 mg/kg iv/sc), and measured intact FGF23 before and 24 h as well as 72 h after start of administration of the drug. RESULTS: We found a tight positive association between circulating intact FGF23 and serum aldosterone in human, canine, and feline CKD patients, as well as in experimental murine CKD (humans: r S = 0.57, p = 0.0368; dogs: r S = 0.66, p = 0.0019; cats: r S = 0.75, p = 0.0003; mice: r S = 0.49, p = 0.0004). Injection of canrenone in mice with established CKD did not lead to changes in FGF23 levels within 24 h, but reduced FGF23 in all mice at 72 h. CONCLUSION: Aldosterone may drive enhanced FGF23 secretion in CKD, possibly explaining the tight positive association between circulating intact FGF23 and aldosterone in human, canine, and feline CKD patients as well as in experimental CKD models.

Prevalence and characterization of hypoadrenocorticism in dogs with signs of chronic gastrointestinal disease: A multicenter study.

BACKGROUND: Dogs with hypoadrenocorticism (HA) frequently show signs of gastrointestinal disease (SGD). The prevalence of dogs presented for chronic SGD with HA is unknown. OBJECTIVES: The aims of this study were to determine the prevalence of HA in dogs with chronic SGD and to identify clinical and laboratory variables for HA in this population. ANIMALS: One hundred fifty-one dogs with chronic SGD. METHODS: In this multicentered prevalence study a standardized workup was performed in prospectively enrolled dogs with SGD > 3 weeks duration. Basal serum cortisol concentration was measured in every dog with ACTH stimulation test (ACTHST) if basal serum cortisol concentration was <3 µg/dL. RESULTS: Basal serum cortisol concentration was <3 µg/dL in 80/151 (53%) dogs, <2 µg/dL in 42/151 (28%) dogs, and < 1 µg/dL in 9/151 (6%) dogs. In 6/151 dogs HA was diagnosed based on ACTHST (stimulated serum cortisol concentration < 2 µg/dL), a prevalence of 4%. There was no difference in history, physical examination, and laboratory variables between dogs with HA and those with other causes of chronic SGD. In 4/6 dogs with HA, there was melena or hematochezia indicating gastrointestinal blood loss. Hyperkalemia, hyponatremia, or both was not observed in any dog. CONCLUSION AND CLINICAL IMPORTANCE: The prevalence of HA among dogs with chronic SGD is higher than in the general population. Based on these results, testing adrenal function should be performed as a standard screening test in dogs with chronic SGD to differentiate between HA and chronic enteropathies.

Generation of Differentiating and Long-Living Intestinal Organoids Reflecting the Cellular Diversity of Canine Intestine.

Functional intestinal disorders constitute major, potentially lethal health problems in humans. Consequently, research focuses on elucidating the underlying pathobiological mechanisms and establishing therapeutic strategies. In this context, intestinal organoids have emerged as a potent in vitro model as they faithfully recapitulate the structure and function of the intestinal segment they represent. Interestingly, human-like intestinal diseases also affect dogs, making canine intestinal organoids a promising tool for canine and comparative research. Therefore, we generated organoids from canine duodenum, jejunum and colon, and focused on simultaneous long-term expansion and cell differentiation to maximize applicability. Following their establishment, canine intestinal organoids were grown under various culture conditions and then analyzed with respect to cell viability/apoptosis and multi-lineage differentiation by transcription profiling, proliferation assay, cell staining, and transmission electron microscopy. Standard expansion medium supported long-term expansion of organoids irrespective of their origin, but inhibited cell differentiation. Conversely, transfer of organoids to differentiation medium promoted goblet cell and enteroendocrine cell development, but simultaneously induced apoptosis. Unimpeded stem cell renewal and concurrent differentiation was achieved by culturing organoids in the presence of tyrosine kinase ligands. Our findings unambiguously highlight the characteristic cellular diversity of canine duodenum, jejunum and colon as fundamental prerequisite for accurate in vitro modelling.

Ki-67/CD3 ratio in the diagnosis of chronic inflammatory enteropathy in dogs.

BACKGROUND: T cells play a key role in the pathogenesis of chronic inflammatory enteropathy (CIE) in dogs. Cluster of differentiation 3 (CD3) antigen serves as a marker for T cells. In human medicine, Ki-67 is an indicator for cell growth but there are only a few studies in dogs with CIE. OBJECTIVE: To investigate Ki-67 in relation to T cells as a marker for CIE in dogs. ANIMALS: Eleven dogs with CIE and 6 healthy beagle controls (CO). METHODS: Retrospective case-control study. Dogs were clinically assessed by the Canine Chronic Enteropathy Clinical Activity Index (CCECAI). Duodenal mucosal biopsy samples were endoscopically obtained for histopathologic examination by means of the World Small Animal Veterinary Association score. Double-labeled immunofluorescence was used to investigate colocalization of Ki-67 and CD3 in epithelium and lamina propria (LP) of villi and crypts. RESULTS: Dogs with CIE had significantly higher clinical score (median, 5.0; interquartile range [IQR], 3-7) compared to CO (all 0; P < .001). The Ki-67/CD3 double-positive cells were significantly increased in the LP of the crypt region of CIE dogs (0.63 cells/mm2 ; IQR, 0-0.54) versus CO (0.08 cells/mm2 ; IQR, 0-0.26; P = .044). A significant correlation was found between CCECAI and the Ki-67/CD3 ratio in the LP of the crypt region (r = 0.670; P = .012) in dogs with CIE. CONCLUSIONS AND CLINICAL IMPORTANCE: The Ki-67/CD3 ratio is upregulated in the LP crypt region of dogs with CIE and it correlates with clinical severity. Therefore, Ki-67/CD3 could be a useful tool for detection of CIE.

Companion Animals as Models for Inhibition of STAT3 and STAT5.

The use of transgenic mouse models has revolutionized the study of many human diseases. However, murine models are limited in their representation of spontaneously arising tumors and often lack key clinical signs and pathological changes. Thus, a closer representation of complex human diseases is of high therapeutic relevance. Given the high failure rate of drugs at the clinical trial phase (i.e., around 90%), there is a critical need for additional clinically relevant animal models. Companion animals like cats and dogs display chronic inflammatory or neoplastic diseases that closely resemble the human counterpart. Cat and dog patients can also be treated with clinically approved inhibitors or, if ethics and drug safety studies allow, pilot studies can be conducted using, e.g., inhibitors of the evolutionary conserved JAK-STAT pathway. The incidence by which different types of cancers occur in companion animals as well as mechanisms of disease are unique between humans and companion animals, where one can learn from each other. Taking advantage of this situation, existing inhibitors of known oncogenic STAT3/5 or JAK kinase signaling pathways can be studied in the context of rare human diseases, benefitting both, the development of drugs for human use and their application in veterinary medicine.

Clonality testing as complementary tool in the assessment of different patient groups with canine chronic enteropathy.

Differentiation between canine chronic enteropathy (CCE) and intestinal lymphoma is a diagnostic challenge as histopathology might fail to yield unequivocal results. Detection of clonal rearrangements of the T-cell-receptor gamma (TCRG) chain and IG heavy chain (IGH) V-J genes offer a useful solution. In this retrospective study, histopathology samples of 35 CCE patients and 7 healthy Beagle dogs underwent clonality testing. Patients suffered either from inflammatory bowel disease (IBD), food responsive diarrhea (FRD) or protein loosing enteropathy secondary to IBD (PLE/IBD). Healthy Beagles served as controls (CO). Canine IBD activity index (CIBDAI) and histopathological WSAVA-grading differed significantly (p<0.001) between groups. CIBDAI improved significantly after appropriate therapy (p < 0.0001). Intestinal biopsies of all CO showed polyclonal patterns for B- and T-cell primers. All samples from CCE patients showed polyclonal patterns for the B-cell primers. Targeting TCRG, 4 patients showed a monoclonal or oligoclonal pattern of the lymphocytic infiltrates in the duodenum and/or colon. Clinical improvement was observed in all dogs. Although a small cell lymphoma cannot be excluded in view of the short follow up duration, a false positive result, in the sense of a canonical rearrangement or unspecific amplification due to a antigenic stimulation in a non-neoplastic inflammatory process is possible.