Phosphoproteomics analysis of serum from dogs affected with pulmonary hypertension secondary to degenerative mitral valve disease.

Pulmonary hypertension (PH), a common complication in dogs affected by degenerative mitral valve disease (DMVD), is a progressive disorder characterized by increased pulmonary arterial pressure (PAP) and pulmonary vascular remodeling. Phosphorylation of proteins, impacting vascular function and cell proliferation, might play a role in the development and progression of PH. Unlike gene or protein studies, phosphoproteomic focuses on active proteins that function as end-target proteins within signaling cascades. Studying phosphorylated proteins can reveal active contributors to PH development. Early diagnosis of PH is crucial for effective management and improved clinical outcomes. This study aimed to identify potential serum biomarkers for diagnosing PH in dogs affected with DMVD using a phosphoproteomic approach. Serum samples were collected from healthy control dogs (n = 28), dogs with DMVD (n = 24), and dogs with DMVD and PH (n = 29). Phosphoproteins were enriched from the serum samples and analyzed using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Data analysis was performed to identify uniquely expressed phosphoproteins in each group and differentially expressed phosphoproteins among groups. Phosphoproteomic analysis revealed nine uniquely expressed phosphoproteins in the serum of dogs in the DMVD+PH group and 15 differentially upregulated phosphoproteins in the DMVD+PH group compared to the DMVD group. The phosphoproteins previously implicated in PH and associated with pulmonary arterial remodeling, including small nuclear ribonucleoprotein G (SNRPG), alpha-2-macroglobulin (A2M), zinc finger and BTB domain containing 42 (ZBTB42), hemopexin (HPX), serotransferrin (TRF) and complement C3 (C3), were focused on. Their unique expression and differential upregulation in the serum of DMVD dogs with PH suggest their potential as biomarkers for PH diagnosis. In conclusion, this phosphoproteomic study identified uniquely expressed and differentially upregulated phosphoproteins in the serum of DMVD dogs with PH. Further studies are warranted to validate the diagnostic utility of these phosphoproteins.

VDX-111 targets proliferative pathways in canine cancer cell lines.

VDX-111 (also identified as AMPI-109) is a vitamin D derivative which has shown anticancer activity. To further assess the function of this compound against multiple cancer types, we examined the efficacy of VDX-111 against a panel of 30 well characterized canine cancer cell lines. Across a variety of cancer types, VDX-111 induced widely variable growth inhibition, cell death, and migration inhibition, at concentrations ranging from 10 nM to 1 µM. Growth inhibition sensitivity did not correlate strongly with tumor cell histotype; however, it was significantly correlated with the expression of genes in multiple cell signaling pathways, including the MAPK and PI3K-AKT pathways. We confirmed inhibition of these signaling pathways as likely participants in the effects of VDX-111. These results suggest that a subset of canine tumors may be sensitive to treatment with VDX-111, and suggests possible predictive markers of drug sensitivity and pharmacodynamic biomarkers of drug exposure that could be employed in future clinical trials.

Feeding dogs a high-fat diet induces metabolic changes similar to natural aging, including dyslipidemia, hyperinsulinemia, and peripheral insulin resistance.

OBJECTIVE: The goal of this study was to characterize changes induced by a high-fat diet in body composition, insulin levels and sensitivity, blood lipids, and other key biomarkers also associated with the metabolic dysfunction that occurs with natural aging. ANIMALS: 24 male Beagle dogs, 3 to 7 years of age, of mixed castration status. METHODS: Dogs were randomly assigned to continue twice daily feeding of the commercial adult maintenance diet (n = 12, including 2 intact) that they were previously fed or to a high-fat diet (12, including 2 intact) for 17 weeks between December 1, 2021, and April 28, 2022. Assessments included body composition (weight, body condition score, and adipose mass determined by deuterium enrichment), clinical chemistries, plasma fatty acid quantification, oral glucose tolerance test, and histology of subcutaneous and visceral adipose biopsy samples. RESULTS: The high-fat diet led to increased body weight, body condition score, fat mass and adipocyte size, hyperinsulinemia and peripheral insulin resistance, and elevations in serum lipids, including cholesterol, triglycerides, and several species of free fatty acids. Leptin levels increased in dogs fed a high-fat diet but not in control dogs. There were no significant changes in routine clinical chemistry values in either group. CLINICAL RELEVANCE: Feeding a high-fat diet for 17 weeks led to potentially deleterious changes in metabolism similar to those seen in natural aging in dogs, including hyperinsulinemia, insulin resistance, and dyslipidemia. A high-fat diet model may provide insights into the similar metabolic dysfunction that occurs during natural aging.

Sodium iodide symporter immunolabelling as a predictor of clinical iodide uptake in canine thyroid carcinoma: A preliminary study.

Thyroid follicular tumours may take up iodide via the sodium-iodide symporter. Knowledge of iodide uptake could then allow treatment with I-131 in dogs with high-risk tumours. The objective of this study was to determine the relationship between clinically detectable iodide uptake (as determined by scintigraphy and/or thyroxine concentrations) and sodium iodide symporter immunohistochemical labelling on histologically fixed thyroid tumours. Nineteen dogs were identified who were diagnosed with thyroid carcinoma and underwent surgery from November 2017 to July 2021. All had recorded thyroid hormone concentrations and were hyperthyroid and/or underwent preoperative nuclear imaging using planar scintigraphy (technetium-99m or I-123), or I-124 PET-CT. All dogs subsequently underwent surgery to remove the thyroid mass. Twenty-two tumours were submitted for histopathologic analysis immediately following surgery, which confirmed a diagnosis of thyroid carcinoma for each tumour. Images and/or thyroid hormone concentrations were reviewed for the included cases, and tumours were sorted into an avid/functional group (group 1) and a non-avid/functional group (group 2). The tumour tissues were re-examined histologically using sodium iodide symporter (NIS) immunohistochemistry (IHC). Group 1 contained 15 avid/functional tumours. Twelve of these tumours had membranous NIS IHC labelling. Group 2 contained 7 non-avid tumours. One of these tumours had membranous NIS IHC labelling. This resulted in an overall sensitivity and specificity for identification of avid/functional tumours with membranous NIS of 80.0% and 85.7%, respectively. NIS IHC may predict ion trapping in canine follicular thyroid tumours. Further studies using iodide-based imaging are warranted to better determine the clinical utility of this diagnostic modality.

Evaluation of Ki-67, goblet cell and MUC2 mucin RNA expression in dogs with lymphoplasmacytic and granulomatous colitis.

Intestinal mucus barrier disruption may occur with chronic inflammatory enteropathies. The lack of studies evaluating mucus health in dogs with chronic colitis arises from inherent challenges with assessment of the intestinal mucus layer. It is therefore unknown if reduced goblet cell (GBC) numbers and/or mucin 2 (MUC2) expression, which are responsible for mucus production and secretion, correlate with inflammation severity in dogs with granulomatous colitis (GC) or lymphocytic-plasmacytic colitis (LPC). It is undetermined if Ki-67 immunoreactivity, which has been evaluated in dogs with small intestinal inflammation, similarly correlates to histologic severity in GC and LPC. Study objectives included comparing Ki-67 immunoreactivity, GBC population and MUC2 expression in dogs with GC, LPC and non-inflamed colon; and exploring the use of ribonucleic acid (RNAscope®) in-situ hybridization (ISH) to evaluate MUC2 expression in canine colon. Formalin-fixed endoscopic colonic biopsies were obtained from 48 dogs over an eight-year period. A blinded pathologist reviewed all biopsies. Dogs were classified into the GC (n=19), LPC (n=19) or no colitis (NC) (n=10) group based on final histopathological diagnosis. Ki-67 immunohistochemistry, Alcian-Blue/PAS staining to highlight GBCs, and RNAscope® ISH using customized canine MUC2-targeted probes were performed. At least five microscopic fields per dog were selected to measure Ki-67 labelling index (KI67%), GBC staining percentage (GBC%) and MUC2 expression (MUC2%) using image analysis software. Spearman's correlation coefficients were used to determine associations between World Small Animal Veterinary Association histologic score (WHS) and measured variables. Linear regression models were used to compare relationships between WHS with KI67%, GBC%, and MUC2%; and between GBC% and MUC2%. Median WHS was highest in dogs with GC. Median KI67% normalised to WHS was highest in the NC group (6.69%; range, 1.70-23.60%). Median GBC% did not correlate with colonic inflammation overall. Median MUC2% normalised to WHS in the NC group (10.02%; range, 3.05-39.09%) was two- and three-fold higher than in the GC and LPC groups respectively. With increased colonic inflammation, despite minimal changes in GBC% overall, MUC2 expression markedly declined in the LPC group (-27.4%; 95%-CI, -49.8, 5.9%) and mildly declined in the GC and NC groups. Granulomatous colitis and LPC likely involve different pathways regulating MUC2 expression. Decreased MUC2 gene expression is observed in dogs with chronic colitis compared to dogs without colonic signs. Changes in MUC2 expression appear influenced by GBC activity rather than quantity in GC and LPC.

NS1-mediated enhancement of MVC transcription and replication promoted by KAT5/H4K12ac.

Histone modifications function in both cellular and viral gene expression. However, the roles of acetyltransferases and histone acetylation in parvoviral infection remain poorly understood. In the current study, we found the histone deacetylase (HDAC) inhibitor, trichostatin A (TSA), promoted the replication and transcription of parvovirus minute virus of canines (MVC). Notably, the expression of host acetyltransferases KAT5, GTF3C4, and KAT2A was increased in MVC infection, as well as H4 acetylation (H4K12ac). KAT5 is not only responsible for H4K12ac but also crucial for viral replication and transcription. The viral nonstructural protein NS1 interacted with KAT5 and enhanced its expression. Further study showed that Y44 in KAT5, which may be tyrosine-phosphorylated, is indispensable for NS1-mediated enhancement of KAT5 and efficient MVC replication. The data demonstrated that NS1 interacted with KAT5, which resulted in an enhanced H4K12ac level to promote viral replication and transcription, implying the epigenetic addition of H4K12ac in viral chromatin-like structure by KAT5 is vital for MVC replication.IMPORTANCEParvoviral genomes are chromatinized with host histones. Therefore, histone acetylation and related acetyltransferases are required for the virus to modify histones and open densely packed chromatin structures. This study illustrated that histone acetylation status is important for MVC replication and transcription and revealed a novel mechanism that the viral nonstructural protein NS1 hijacks the host acetyltransferase KAT5 to enhance histone acetylation of H4K12ac, which relies on a potential tyrosine phosphorylation site, Y44 in KAT5. Other parvoviruses share a similar genome organization and coding potential and may adapt a similar strategy for efficient viral replication and transcription.

Myeloid derived suppressor cells in peripheral blood can be a prognostic factor in canine transitional cell carcinoma.

Myeloid-derived suppressor cells (MDSCs) are immature cells with immunosuppressive properties found in the tumor microenvironment. MDSCs are divided into two major subsets: polymorphonuclear MDSCs (PMN-MDSCs) and monocytic MDSCs (M-MDSCs). Both MDSC subsets contribute to the creation of an immunosuppressive environment for tumor progression. In humans, patients with high levels of MDSCs show worse outcomes for several types of cancers. However, the association between MDSCs and clinical features has rarely been investigated in canine studies. In the present study, we measured the proportion of PMN-MDSCs and M-MDSCs in the peripheral blood and tumor tissue of dogs with hepatocellular carcinoma (HCC), prostate cancer (PC), transitional cell carcinoma (TCC), lymphoma, and pulmonary adenocarcinoma. Additionally, we examined immunosuppressive ability of PMN-MDSCs and M-MDSCs in peripheral blood mononuclear cells of TCC case on CD4+, CD8+ and interferon-γ+ cells and investigated the relationships of MDSCs with clinical features and outcomes. PMN-MDSCs increased in HCC, PC, TCC, and lymphoma. In contrast, M-MDSCs increased in the TCC. Both PMN-MDSCs and M-MDSCs exhibited immunosuppressive effects on CD8+, CD4+ and interferon-γ+ cells. In dogs with TCC, lymph node metastasis was associated with high level of PMN-MDSCs but not with M-MDSCs. High levels of both PMN-MDSCs and M-MDSCs were related to advanced tumor stage. Kaplan-Meier analysis revealed that high levels of both PMN-MDSCs and M-MDSCs were significantly associated with shorter overall survival. In addition, the Cox proportional hazard regression model showed that M-MDSCs and the tumor stage were independent prognostic factors for TCC. These results suggest that PMN-MDSCs and M-MDSCs may be involved in tumor progression and could be prognostic factors and promising therapeutic targets in dogs with TCC.

PD-L1 mRNA and protein expression in canine mammary carcinomas: Correlation with histopathological grade and molecular markers.

Programmed death ligand 1 (PD-L1) is an immune checkpoint molecule that plays a crucial role in regulating antitumor immune responses. Canine mammary carcinomas (CMCs) are common tumors of dogs. Despite extensive studies on the heterogeneity of CMCs, there is still a lack of effective precision therapies for the treatment of CMCs. In this study, we aimed to investigate the correlation between PD-L1 mRNA and protein expression in CMCs and explore its association with histopathological grade and molecular markers, including the estrogen receptor, epidermal growth factor receptor 2, and cytokeratin 5/6 (CK5/6). Formalin-fixed paraffin-embedded samples were evaluated for PD-L1 mRNA expression using RNA in situ hybridization and PD-L1 protein expression using immunohistochemistry. We observed no substantial correlation between PD-L1 mRNA and protein expression in CMCs; however, PD-L1 mRNA levels were significantly higher in grade 3 than in grade 1 tumors (P = .001). In addition, we observed a positive correlation between PD-L1 protein expression and CK5/6 expression in CMCs (P = .032). These findings suggest that PD-L1 expression in CMCs is heterogeneous and may be regulated post-transcriptionally. Further studies are needed to explore the prognostic and therapeutic implications of PD-L1 expression in different molecular subtypes of CMCs and their potential as predictive biomarkers for immunotherapy.

Expression of sphingosine-1-phosphate receptor 1 in neuroinflammation of canine brains.

Sphingosine-1-phosphate (S1P) is a signaling lipid mediator that is involved in multiple biological processes. The S1P/S1P receptor (S1PR) signaling pathway has an important role in the central nervous system. It contributes to physiologic cellular homeostasis and is also associated with neuroinflammation. Therefore, this study was performed to evaluate the expression of S1PR in dogs with meningoencephalitis of unknown etiology (MUE) and experimental autoimmune encephalomyelitis (EAE). The analysis used 12 brain samples from three neurologically normal dogs, seven dogs with MUE, and two canine EAE models. Anti-S1PR1 antibody was used for immunohistochemistry. In normal brain tissues, S1PR1s were expressed on neurons, astrocytes, oligodendrocytes, and endothelial cells. In MUE and EAE lesions, there was positive staining of S1PR1 on leukocytes. Furthermore, the expression of S1PR1 on neurons, astrocytes, oligodendrocytes, and endothelial cells was upregulated compared to normal brains. This study shows that S1PR1s are expressed in normal brain tissues and leukocytes in inflammatory lesions, and demonstrates the upregulation of S1PR1 expression on nervous system cells in inflammatory lesions of MUE and EAE. These findings indicate that S1P/S1PR signaling pathway might involve physiologic homeostasis and neuroinflammation and represent potential targets for S1PR modulators to treat MUE.

Advancing canine mammary tumor diagnostics: Unraveling the diagnostic potential of Cytokeratin 19 through droplet digital PCR analysis.

Cytokeratin 19 (CK19) is a complex intracytoplasmic cytoskeletal protein primarily localized in the ducts of the mammary gland and skin epithelial cells. In humans, the expression of CK19 gene within circulating tumor cells (CTCs) extracted from blood samples of breast cancer patients reflects tumor cell activity, offering valuable insights for predicting early metastatic relapse or monitoring treatment effectiveness. However, knowledge of serum tumor markers is limited in veterinary oncology. Recently, droplet digital PCR (ddPCR), has been employed to explore rare target genes due to its heightened sensitivity and accuracy as a novel molecular diagnostic tool. The objectives of this study were to investigate the expression of the CK19 mRNA in CTCs, non-neoplastic mammary tissues, and both benign and malignant canine mammary tumors (CMTs) through ddPCR analysis. In Study I, we optimized the discard volume for blood samples to reduce CK19 contamination from skin epithelial cells post-venipuncture. The results revealed that discarding the initial 3 mL of blood was adequate and effective in eliminating CK19 mRNA contamination. In Study II, after the removal of the initial 3 mL of blood, we investigated CK19 mRNA-positive CTCs in the peripheral blood of normal healthy dogs, including those with benign and malignant CMTs. Intriguingly, CK19 mRNA was undetectable in all blood samples. The expression of CK19 mRNA in mammary tissues was investigated in Study III. The copy number (CN) ratios of the CK19 gene in non-neoplastic mammary tissues (14.77 ± 14.65) were significantly higher (P < 0.05) than those in benign (4.23 ± 3.35) and malignant groups (6.56 ± 5.64). Notably, no difference was observed between the benign and malignant groups. In conclusion, CK19 mRNA appeared unlikely to be a suitable candidate as a biomarker in the peripheral blood of CMTs, while the CN ratio in mammary tissues could serve as a potential discriminator between non-neoplastic and CMT groups, complementing the gold standard of histopathological examination.

Relationships between haptoglobin and inter-alpha trypsin inhibitor heavy chain 4 levels in local and peripheral blood and systemic inflammatory response syndrome in bitches with pyometra.

We aimed to assess the usefulness of monitoring inter-alpha trypsin inhibitor heavy chain 4 (ITIH4) and haptoglobin (Hp) in peripheral and local blood in canine pyometra, and evaluation the relationships among acute phase proteins (APPs), systemic inflammatory response syndrome (SIRS) and the presence of bacteria. The material was collected from bitches with pyometra and from healthy ones. Blood was taken from the cephalic and uterine veins. APPs levels were quantified by ELISA. In the peripheral circulation, the Hp was higher in animals with open-cervix pyometra (OCP) than in the closed-cervix pyometra (CCP) and the control group. The Hp concentration was not correlated with age, with the presence of SIRS or with the type of bacteria (Gram-negative, Gram-positive or mixed flora). The ITIH4 concentrations in the peripheral blood did not differ significantly in the cases of pyometra. The Hp concentration in the local circulation increased in the OCP but not in the CCP groups, although the histopathological changes in the endometrium were similar. Peripheral Hp concentrations may be a useful tool in differentiating between the types of pyometra.

Profiling canine mammary tumors: A potential model for studying human breast cancer.

Despite all clinical progress recorded in the last decades, human breast cancer (HBC) remains a major challenge worldwide both in terms of its incidence and its management. Canine mammary tumors (CMTs) share similarities with HBC and represent an alternative model for HBC. The utility of the canine model in studying HBC relies on their common features, include spontaneous development, subtype classification, mutational profile, alterations in gene expression profile, and incidence/prevalence. This review describes the similarities between CMTs and HBC regarding genomic landscape, microRNA expression alteration, methylation, and metabolomic changes occurring during mammary gland carcinogenesis. The primary purpose of this review is to highlight the advantages of using the canine model as a translational animal model for HBC research and to investigate the challenges and limitations of this approach.

Evaluation of Ki67, Bax, Bcl-2 and KIT in canine cutaneous mast cell tumours and their relationship with histopathology and prognosis.

Canine cutaneous mast cell tumours (CCMCTs) are common in dogs and exhibit many unpredictable behaviors. This study aimed to encourage pathology laboratories in developing countries to routinely assess prognosis by applying commonly used histopathological grading systems and immunohistochemistry (IHC) markers. We performed histological grading according to both the Patnaik and Kiupel systems, determined the mitotic count (MC) and carried out IHC for the detection of Ki67, Bax, Bcl-2 and KIT in 54 CCMCT cases. MC was associated with both grading systems in terms of survival following diagnosis and prognostic factors differed among cases categorized by the cut-off value of 5. KIT patterns were associated with grading systems and MC. The cohort with pattern II had a lower survival rate than those with patterns I and III. Ki67 was associated with survival when evaluated over the cut-off value of 0.018. Bax expression was associated with both grading systems. Median survival time was longer in patients with lower Bax expression level. Immunohistochemical detection of KIT, Ki67 and Bax improves histopathology in predicting the prognosis. If IHC is unavailable, reports regarding MC and values from both grading systems are the most effective, convenient and cost-effective way to provide the most reliable prognostic data and guidance for the clinicians.

G-protein-coupled estrogen receptor prevents nuclear factor-kappa B promoter activation by Helicobacter pylori cytotoxin-associated gene A in gastric cancer cells.

Helicobacter pylori is a well-known pathogen that causes chronic gastritis, leading to the development of gastric cancer. This bacterium has also been detected in dogs, and symptoms similar to those in humans have been reported. The cytotoxin-associated gene A (CagA) is involved in pathogenesis through aberrant activation of host signal transduction, including the nuclear factor-kappa B (NF-κB) pathway. We have previously shown the anti-inflammatory effect of the G-protein-coupled estrogen receptor (GPER) via inhibiting of NF-κB activation in several cells. Therefore, here, we investigated the effect of GPER on CagA-mediated NF-κB promoter activity and showed that CagA overexpression in gastric cancer cells activated the NF-κB reporter and induced interleukin 8 (il-8) expression, both of which were inhibited by the GPER agonist.

Research progress of good markers for canine mammary carcinoma.

PURPOSE: Mammary gland tumors are the most common neoplastic diseases in elderly female dogs, about 50% of which are considered to be malignant. Canine mammary tumors are similar to human breast cancers in many respects, so canine mammary tumors are frequently studied alongside human breast cancer. This article mentioned KI-67, HER-2, COX-2, BRCA1, BRCA2, P53, CA15-3, MicroRNA, Top2α and so on. All these markers are expected to have an important role in the clinic. METHODS: Existing markers of canine mammary carcinoma are reviewed, and the expression of each marker and its diagnostic role for this tumor are described in detail. RESULTS: This article introduced several effective markers of canine mammary tumors, among them, antigen KI-67 (KI-67), human epidermal growth factor receptor 2 (HER-2), cyclooxygenase 2 (COX-2) are promising and can be detected in both serum and tissue samples. Breast cancer caused by mutations in the breast cancer 1 gene (BRCA1) and breast cancer 2 gene (BRCA2) is also a hot topic of research. In addition to the above symbols, tumor protein p53 (p53), cancer antigen15-3 (CA15-3), MicroRNA (miRNA), topoisomerase πα (Top2α), proliferating cell nuclear antigen (PCNA), epidermal growth factor receptor (EGFR) and E-cadherin will also be involved in this paper. We will also mention Mammaglobin, which has been rarely reported so far.

Altered E-cadherin/ß-catenin expression in feline cutaneous squamous cell carcinomas.

Cutaneous squamous cell carcinoma (CSCC) is the most common malignant skin tumour in cats and the nature of the molecular mechanisms involved is poorly defined. Included among the molecular mechanisms in human and canine CSCCs is altered expression of E-cadherin/ß-catenin. This study aimed to explore the immunohistochemical expression pattern of E-cadherin and ß-catenin in 43 samples of feline CSCC by using a tissue microarray to elucidate whether expression of these molecules is dysregulated. Membrane expression of E-cadherin and membrane and cytoplasmic expression of ß-catenin were significantly reduced in the CSCCs. Cytoplasmic expression of E-cadherin and nuclear expression of ß-catenin were also found in some CSCCs. These findings indicate that altered expression of E-cadherin and ß-catenin is a frequent event in feline CSCCs, suggesting that these molecules play an important role in acquisition of the malignant phenotype in feline patients with CSCC. The results also suggest the existence of a subpopulation of feline patients with CSCC in which the Wnt pathway may contribute to epidermal carcinogenesis.

Single-Nuclei Multiome (ATAC + Gene Expression) Sequencing of a Primary Canine Osteosarcoma Elucidates Intra-Tumoral Heterogeneity and Characterizes the Tumor Microenvironment.

Osteosarcoma (OSA) is a highly aggressive bone tumor primarily affecting pediatric or adolescent humans and large-breed dogs. Canine OSA shares striking similarities with its human counterpart, making it an invaluable translational model for uncovering the disease's complexities and developing novel therapeutic strategies. Tumor heterogeneity, a hallmark of OSA, poses significant challenges to effective treatment due to the evolution of diverse cell populations that influence tumor growth, metastasis, and resistance to therapies. In this study, we apply single-nuclei multiome sequencing, encompassing ATAC (Assay for Transposase-Accessible Chromatin) and GEX (Gene Expression, or RNA) sequencing, to a treatment-naïve primary canine osteosarcoma. This comprehensive approach reveals the complexity of the tumor microenvironment by simultaneously capturing the transcriptomic and epigenomic profiles within the same nucleus. Furthermore, these results are analyzed in conjunction with bulk RNA sequencing and differential analysis of the same tumor and patient-matched normal bone. By delving into the intricacies of OSA at this unprecedented level of detail, we aim to unravel the underlying mechanisms driving intra-tumoral heterogeneity, opening new avenues for therapeutic interventions in both human and canine patients. This study pioneers an approach that is broadly applicable, while demonstrating significant heterogeneity in the context of a single individual's tumor.

MUC5AC and MUC5B expression in canine gallbladder mucocele epithelial cells.

Gallbladder mucocele (GBM) is one of the most common gallbladder diseases in dogs. Its pathogenesis has not yet been clarified, but excessive accumulation of a secretory gel-forming mucin, MUC5AC in the gallbladder has been reported. This study aimed to ascertain if MUC5AC overproduction resulted in mucus accumulation in the gallbladder during GBM development. Eleven dogs undergoing cholecystectomy who were pathologically diagnosed with GBM were included, and the expression level of mucins, particularly MUC5AC and MUC5B, in their gallbladder epithelial cells was compared with those in normal gallbladder epithelial cells. On reverse transcription-quantitative polymerase chain reaction screening, there was a significant difference (P<0.05) in the mRNA expression level of MUC1, but not of other mucins including MUC5AC and MUC5B, between mucocele and normal gallbladder epithelial cells. Protein expression levels were also evaluated for MUC5AC and MUC5B using immunohistochemistry. There was little immunoreactivity for MUC5AC, whereas MUC5B showed definitive staining in gallbladder epithelial cells. There was no difference in MUC5AC and MUC5B protein expression levels between mucocele and normal gallbladder epithelial cells. These data suggest that excessive production of mucin, especially MUC5AC and MUC5B, does not occur in canine GBM, and that abnormal mucus excretion, rather than excessive mucus production, may be the cause of GBM development.

Significant increase in MIC-A and MIC-B and soluble MIC-A and MIC-B in canine lymphomas.

Non-Hodkin's lymphoma (NHL) is the most frequent hematologic malignancy in humans and dogs. NKG2D is one of the most critical receptors on NK cells, recognizing their natural ligands on malignant cells such as A and B major histocompatibility complex-related proteins (MIC-A and MIC-B). Soluble molecules (sMIC-A and sMIC-B) can interfere with immune synapsis between NK cells and tumor cells, impeding NK cytotoxicity. The main objectives of this study were to analyze, in dogs with diffuse large B cell lymphoma, NK cell lymphoma, and reactive lymphadenopathies, the role of NK cells, their activating receptors NKG2D and NKp46, and their ligands MIC-A and MIC-B, as well as soluble molecules sMIC-A and sMIC-B. Thirty-six dogs with a possible diagnosis of NHL and eight healthy dogs were studied. NHL was diagnosed in 28 (78 %) dogs; in the other 8 (22 %), reactive lymphadenopathies were present. Most of the lymphomas corresponded to B cell NHL (82 %). The most predominant subtype was diffuse large B cell lymphoma (21, 71.5 %), followed by five cases (18 %) that were Non-B Non-T lymphomas (presumably NK cell lymphomas) and other B cell lymphomas (3, 10.5%). There were no cases of T cell NHL. MIC-A was positive in 7 of 27 (26 %) cases of NHL, and MIC-B in 20 of 27 (74 %) NHL. In non-malignant lymphadenopathies, three (37.5 %) dogs were positive for MIC-A, and five (62.5 %) expressed MIC-B. Dogs with lymphoma had higher numbers of NK cells than eight healthy dogs. In 15 dogs (12 cases with NHL and three cases with reactive adenopathies) and eight controls, there were no differences in the number of NK cells expressing NKP46 and NKG2D. NHL dogs had higher values of sMIC-A and sMIC-B. B-cell and NK cell lymphomas correspond to 86 % and 14 % of all canine lymphomas. MIC-A, MIC-B, and sMIC-A and sMIC-B were increased in canine lymphomas.

Serum/Faecal S100A12, CRP and lactoferrin can be used to distinguish infectious and non-infectious canine diarrhoea.

OBJECTIVES: This study aimed to evaluate the diagnostic efficacies of selected inflammatory and intestinal biomarkers in cases of infectious and non-infectious diarrhoea in dogs. METHODS: A total of 60 dogs, 12 healthy (Control Group) and 48 with diarrhoea were used. Viral, Bacterial, Parasitic (infectious) and Nutritional diarrhoea (non-infectious) subgroups (n: 12) were formed according to the aetiology, on the basis of clinical and laboratory examinations. Selected inflammatory and intestinal biomarkers (Calgranulin, S100A12; Lactoferrin, LCTF; C-reactive protein, CRP) were measured both in serum and faecal samples. RESULTS: Compared to the Control and Nutritional Diarrhoea groups, the infectious diarrhoea groups had higher serum S100A12, LCTF, CRP, blood urea nitrogen, creatinine (CR), alanine transaminase and alkaline phosphatase, and lower glucose (GLU), sodium (Na) and potassium (K) concentrations (p < 0.05); Viral and Parasitic Diarrhoea groups had lower serum albumin (ALB) and total protein (TP) concentrations (p < 0.05). Faecal S100A12, LCTF and CRP concentrations were higher in infectious diarrhoea groups compared to the Control and Nutritional Diarrhoea groups (p < 0.05). Faecal LCTF and CRP concentrations were higher in the Bacterial Diarrhoea group than in the Viral and Parasitic Diarrhoea groups (p < 0.05). CLINICAL SIGNIFICANCE: It was determined that serum (area under curve, AUC: 0.842 and 0.956) and faecal (AUC: 0.975 and 0.786) S100A12 and CRP concentrations in viral diarrhoea; serum (AUC: 0.956) and faecal (AUC: 0.992) LCTF concentrations in bacterial diarrhoea have diagnostic values in the diagnosis of the presence of intestinal inflammation and damage and can be used in the differential diagnosis of infectious and non-infectious diarrhoea.