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Veterinary Medicine and Science Jan 2021Canine mastocytomas (MCTs) are characterized by rapid proliferation of neoplastic mast cells (MCs) and clinical signs caused by MC-derived mediators. In dogs suffering...
Canine mastocytomas (MCTs) are characterized by rapid proliferation of neoplastic mast cells (MCs) and clinical signs caused by MC-derived mediators. In dogs suffering from MCT, histamine receptor 1 (HR1) antagonists are frequently used to control mediator-related clinical symptoms. Previous studies have shown that the HR1 antagonists loratadine and terfenadine exert some growth-inhibitory effects on neoplastic MCs. We examined whether other HR1 antagonists used in clinical practice (desloratadine, rupatadine, cyproheptadine, dimetindene, diphenhydramine) affect proliferation and survival of neoplastic MCs. Furthermore, we analysed whether these HR1 antagonists counteract IgE-dependent histamine release from a MC line harbouring a functional IgE-receptor. HR1 antagonists were applied on two canine MC lines, C2 and NI-1, and on primary MCs obtained from three MCT samples. The HR1 antagonists desloratadine, rupatadine and cyproheptadine were found to be more potent in decreasing proliferation of C2 and NI-1 cells when compared with dimetindene and diphenhydramine. Similar effects were seen in primary neoplastic MCs, except for diphenhydramine, which exerted more potent growth-inhibitory effects than the other HR1 antagonists. Drug-induced growth-inhibition in C2 and NI-1 cells was accompanied by apoptosis. Loratadine, desloratadine and rupatadine also suppressed IgE-dependent histamine release in NI-1 cells. However, drug concentrations required to elicit substantial effects on growth or histamine release were relatively high (>10 µM). Therefore, it remains unknown whether these drugs or similar, more potent, HR1-targeting drugs can suppress growth or activation of canine neoplastic MCs in vivo.
Topics: Animals; Cell Proliferation; Dogs; Histamine Antagonists; Histamine Release; Mast Cells
PubMed: 32924324
DOI: 10.1002/vms3.336 -
European Journal of Immunology Jun 2021The dimeric cytokine IL-12 is important in the control of various infections but also contributes to the pathology of certain diseases making it a potential target for...
The dimeric cytokine IL-12 is important in the control of various infections but also contributes to the pathology of certain diseases making it a potential target for therapy. However, its specific inhibition with antibodies is complicated by the fact that its two subunits are present in other cytokines: p40 in IL-23 and p35 in IL-35. This has led to erroneous conclusions like the alleged implication of IL-12 in experimental autoimmune encephalomyelitis (EAE). Here, we report the development of a mouse anti-mouse IL-12 vaccine and the production of monoclonal antibodies (mAbs) that do not react with p40 or p35 (in IL-35) but specifically recognize and functionally inhibit the IL-12 heterodimer. Using one of these mAbs, MM12A1.6, that strongly inhibited IFN-γ production and LPS-induced septic shock after viral infection, we demonstrate the critical role played by IL-12 in the rejection of male skin graft by female C57BL/6 syngeneic recipients and in the clearance of an immunogenic mastocytoma tumor variant by DBA/2 mice, but not in a parent to F1 immune aggression model nor in MOG-induced EAE, which was clearly prevented by anti-p40 mAb C17.8. Given this selective inhibition of IL-12, these mAbs provide new options for reassessing IL-12 function in vivo.
Topics: Animals; Antibodies, Monoclonal; Disease Models, Animal; Encephalomyelitis, Autoimmune, Experimental; Epitopes; Graft Rejection; Humans; Hybridomas; Interleukin-12; Mastocytoma; Mice; Mice, Inbred C57BL; Mice, Inbred DBA; Multiple Sclerosis; Neoplasms, Experimental; Nidovirales; Nidovirales Infections; Protein Subunits; Sepsis; Skin Transplantation
PubMed: 33788263
DOI: 10.1002/eji.202048936 -
Veterinary and Comparative Oncology Dec 2021Histopathologic surgical margin assessment in veterinary patients is an imprecise science with assessment limited to a small proportion of the surgical margin due to...
Histopathologic surgical margin assessment in veterinary patients is an imprecise science with assessment limited to a small proportion of the surgical margin due to time and finances. Incomplete excision of canine mast cell tumours (MCTs) alters treatment recommendations and prognosis. Optical coherence tomography (OCT) is a novel imaging modality that has been reported in a single veterinary study for surgical margin assessment. Twenty-five dogs with 34 MCTs were enrolled in a prospective pilot-study to assess the imaging characteristics of canine MCTs with OCT and to evaluate the feasibility and utility of OCT-guided histopathology. All dogs underwent routine surgical excision of MCTs. OCT imaging was used to assess the entire surgical margin prior to placement in formalin. Either normal areas or areas suspected of incomplete MCT excision were inked. Standard histopathologic sectioning and tangential sectioning of inked areas were performed and compared to OCT results. OCT identified MCT near the surgical margin in 10 of 26 specimens (38.4%). Four specimens suspicious for incomplete margins on OCT had incomplete MCT excision that was missed on standard histopathologic sectioning. Six specimens had OCT-guided sections taken as suspicious, which did not show MCT on histopathology. OCT-guided pathology sections were able to detect incompletely excised MCT near the surgical margin with a sensitivity of 90% and specificity of 56.2% in this preliminary study. OCT imaging shows promise for guiding pathologists to areas of interest to improve the diagnostic accuracy of surgical margin assessment in excised canine MCTs.
Topics: Animals; Dog Diseases; Dogs; Feasibility Studies; Margins of Excision; Mastocytoma; Pilot Projects; Prospective Studies; Tomography, Optical Coherence
PubMed: 32951309
DOI: 10.1111/vco.12654 -
BMC Veterinary Research Oct 2020The WW domain-containing oxidoreductase (WWOX) tumor suppressor gene is frequently lost in a variety of solid and hematopoietic malignancies in humans. Dysregulation of...
BACKGROUND
The WW domain-containing oxidoreductase (WWOX) tumor suppressor gene is frequently lost in a variety of solid and hematopoietic malignancies in humans. Dysregulation of WWOX has been implicated as playing a key role in tumor cell survival, DNA damage repair, and genomic stability. The purpose of this study was to characterize WWOX expression in spontaneous canine mast cell tumors (MCTs) and malignant cell lines and investigate the potential contribution of WWOX loss on malignant mast cell behavior.
METHODS/RESULTS
WWOX expression is decreased in primary canine MCTs and malignant mast cell lines compared to normal canine bone marrow-cultured mast cells. In transformed canine mastocytoma cell lines, overexpression of WWOX or WWOX knockdown had no effect on mast cell viability. Inhibition of WWOX enhanced clonogenic survival following treatment with ionizing radiation in the C2 mast cell line. Lastly, immunohistochemistry for WWOX was performed using a canine MCT tissue microarray, demonstrating that WWOX staining intensity and percent of cells staining for WWOX is decreased in high-grade MCTs compared to low-grade MCTs.
CONCLUSIONS
These data suggest that WWOX expression is attenuated or lost in primary canine MCTs and malignant mast cell lines. Given the observed increase in clonogenic survival in WWOX-deficient C2 mast cells treated with ionizing radiation, further investigation of WWOX and its role in mediating the DNA damage response in malignant mast cells is warranted.
Topics: Animals; Cell Line, Tumor; Dogs; Gene Expression Regulation, Neoplastic; Mast Cells; Mastocytoma; Skin Neoplasms; Tumor Suppressor Proteins; WW Domain-Containing Oxidoreductase
PubMed: 33129329
DOI: 10.1186/s12917-020-02638-3 -
Frontiers in Microbiology 2020Mast cells play an important role in the pathogenesis of highly pathogenic H5N1 avian influenza virus (H5N1-HPAIV) infection. Defective viral particles (DPs) can...
Mast cells play an important role in the pathogenesis of highly pathogenic H5N1 avian influenza virus (H5N1-HPAIV) infection. Defective viral particles (DPs) can interfere with the replication of infectious viruses and stimulate the innate immune response of host cells. However, DPs arising from mast cells during HPAIV replication and their potent antiviral actions has not been reported. Here, we showed that the human mastocytoma cell line, HMC-1, allowed for the productive replication of the H5N1-HPAIV. Compared with alveolar cell line A549, DPs were propagated preferentially and abundantly in mast cells following IAV infection, which can be attributed to the wide existence of Argonaute 2 (AGO2) in HMC-1 cells. In addition, DPs generated in H5N1-infected cells could provide great therapeutic protection on mice to fight against various influenza A viruses, which included not only homologous H5N1-HPAIV, but also heterologous H1N1, H3N2, H7N2, and H9N2. Importantly, DPs generated in H5N1-infected HMC-1 cells could diminish viral virulence and by triggering a robust antiviral response through type II interferon signaling pathways. This study is the first to illustrate the arising of DPs in H5N1-HPAIV infected mast cells and explore their favorable ability to protect mice from influenza A viruses infection, which provides a novel insight and valuable information for the progress of new strategies to fight influenza A viruses infection, especially highly pathogenic avian influenza virus infection by focusing on the DPs generated in mast cells.
PubMed: 33250863
DOI: 10.3389/fmicb.2020.553274 -
Scientific Reports Nov 2020Cutaneous mast cell tumours (MCTs) are common skin neoplasms in dogs. MicroRNAs (miRNAs) are post-transcriptional regulators involved in several cellular processes, and...
Cutaneous mast cell tumours (MCTs) are common skin neoplasms in dogs. MicroRNAs (miRNAs) are post-transcriptional regulators involved in several cellular processes, and they can function as tumour promoters or suppressors. However, the role of miRNAs in canine MCTs has not yet been elucidated. Thus, the current study aimed to characterize miRNA profiles and to assess their value as biomarkers for MCTs. miRNA expression profiles were assessed in formalin-fixed, paraffin-embedded samples by next-generation sequencing. Ten samples were MCT tissues, and 7 were healthy adjacent tissues. Nine dysregulated miRNAs (DE-miRNAs) were then validated using RT-qPCR in a larger group of MCT samples, allowing the calculation of ROC curves and performance of multiple factor analysis (MFA). Pathway enrichment analysis was performed to investigate miRNA biological functions. The results showed that the expression of 63 miRNAs (18 up- and 45 downregulated) was significantly affected in MCTs. Five DE-miRNAs, namely, miR-21-5p, miR-92a-3p, miR-338, miR-379 and miR-885, were validated by RT-qPCR. The diagnostic accuracy of a panel of 3 DE-miRNAs-miR-21, miR-379 and miR-885-exhibited increased efficiency in discriminating animals with MCTs (AUC = 0.9854) and animals with lymph node metastasis (AUC = 0.8923). Multiple factor analysis revealed clusters based on nodal metastasis. Gene Ontology and KEGG analyses confirmed that the DE-miRNAs were involved in cell proliferation, survival and metastasis pathways. In conclusion, the present study demonstrated that the miRNA expression profile is changed in the MCT microenvironment, suggesting the involvement of the altered miRNAs in the epigenetic regulation of MCTs and identifying miR-21, miR-379 and miR-885 as promising biomarkers.
Topics: Animals; Biomarkers, Tumor; Dog Diseases; Dogs; Factor Analysis, Statistical; Female; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; High-Throughput Nucleotide Sequencing; Lymphatic Metastasis; Male; Mastocytoma; MicroRNAs; Oligonucleotide Array Sequence Analysis; Skin Neoplasms; Tumor Microenvironment
PubMed: 33144602
DOI: 10.1038/s41598-020-75877-x -
Molecular Biology Reports Jan 2023Expression changes for tryptophan hydroxylase 1 (TPH1), the rate-limiting enzyme in serotonin synthesis, by environmental glutamine (GLN) were examined in mouse...
Expression changes for tryptophan hydroxylase 1 (TPH1), the rate-limiting enzyme in serotonin synthesis, by environmental glutamine (GLN) were examined in mouse mastocytoma-derived P815-HTR cells. GLN-treated cells exhibited a robust increase in TPH1 mRNA after a 6 h exposure to GLN. 6-Diazo-5-oxo-L-norleucine (DON), a glutamine-utilizing glutaminase inhibitor, significantly inhibited the GLN-induction of TPH1 mRNA. Nuclear run-on assays and mRNA decay experiments demonstrated that the primary mechanism leading to increased TPH1 mRNA levels was not due to transcriptional changes, but rather due to increased TPH1 RNA stability induced by GLN. Treatment with GLN also led to activation of p38 MAP kinase, but not p42/44 MAPK. In addition, SB203580, a p38 MAP kinase specific inhibitor, completely abolished the GLN-mediated increase of TPH1 mRNA levels, suggesting the pathway stabilizing TPH1 mRNA might be mediated by the activated p38 MAP kinase pathway. Additionally, SB203580 significantly reduced the stability of TPH1 mRNA, and this reduction of the stability was not affected by GLN in the culture medium, implying a sequential signaling from GLN being mediated by p38 MAP kinase, resulting in alteration of TPH1 mRNA stability. TPH1 mRNA stability loss was also dependent on de novo protein synthesis as shown by treatment of cells with a transcriptional/translational blocker. We provide evidence that TPH1 mRNA levels are increased in response to increased exogenous GLN in mouse mastocytoma cells via a stabilization of TPH1 mRNA due to the activity of the p38 MAP kinase.
Topics: Mice; Animals; Mitogens; Glutamine; RNA, Messenger; p38 Mitogen-Activated Protein Kinases; Mastocytoma; Enzyme Inhibitors; Tryptophan Hydroxylase
PubMed: 36331742
DOI: 10.1007/s11033-022-07693-7 -
Journal of Equine Veterinary Science Apr 2020Mast cell tumor (MCT) has long been considered as an uncommon neoplasm in horses. Cytological and behavioral evidence of its malignancy is usually lacking, and only a...
Mast cell tumor (MCT) has long been considered as an uncommon neoplasm in horses. Cytological and behavioral evidence of its malignancy is usually lacking, and only a few reports have described MCT displaying malignant behavior. An 18-year-old Friesian stallion presented with a one-year history of intermittent and progressive skin lesions on the left forelimb associated with intense, generalized pruritus and apathy temporarily responsive to glucocorticoids and antibiotics. The horse was alert and responsive with poor body condition and marked generalized pruritus. The left forelimb was markedly enlarged and surrounded by numerous firm 2- to 20-cm masses that were ulcerated and focally necrotic. A 7-cm round firm mass was observed on the left dorsal neck. Dermatological examination revealed generalized moth-eaten alopecia and scaling with erosions and ulcers secondary to pruritus. A direct skin smear from the affected leg showed severe eosinophilic inflammation and neutrophilic inflammation with pleomorphic bacteria. Histopathology of the skin and biopsies of the underlying tissues revealed an abundant population of atypical mast cells consistent with a malignant MCT. The horse was euthanized and necropsy revealed a marked fibrous reaction on longitudinal sections of the affected limb, and the tumor could be detected on only a few histological slides. Diagnosis of equine MCT can be challenging because of the massive accompanying fibrous reaction. Mast cell tumor should be suspected in the presence of eosinophilic infiltration of the affected tissue and in cases of generalized pruritus not attributable to other causes.
Topics: Animals; Fibrosis; Horse Diseases; Horses; Male; Mast Cells; Mastocytoma, Skin; Neoplasms; Pruritus
PubMed: 32172911
DOI: 10.1016/j.jevs.2020.102921 -
FEBS Open Bio May 2020The tumour microenvironment comprises a diverse range of cells, including fibroblasts, immune cells and endothelial cells, along with extracellular matrix. In...
The tumour microenvironment comprises a diverse range of cells, including fibroblasts, immune cells and endothelial cells, along with extracellular matrix. In particular, fibroblasts are of significant interest as these cells are reprogrammed during tumorigenesis to become cancer-associated fibroblasts (CAFs), which in turn support cancer cell growth. MicroRNAs (miRNAs) have been shown to be involved in this intercellular crosstalk in humans. To assess whether miRNAs are also involved in the activation of fibroblasts in dogs, we cocultured primary canine skin fibroblasts with the canine mast cell tumour cell line C2 directly or with C2-derived exosomes, and measured differential abundance of selected miRNAs. Expression of the CAF markers alpha-smooth muscle actin (ACTA2) and stanniocalcin 1 confirmed the activation of our fibroblasts after coculture. We show that fibroblasts displayed significant downregulation of miR-27a and let-7 family members. These changes correlated with significant upregulation of predicted target mRNAs. Furthermore, RNA interference knockdown of miR-27a revealed that cyclin G1 (CCNG1) exhibited negative correlation at the mRNA and protein level, suggesting that CCNG1 is a target of miR-27a in canine fibroblasts and involved in their activation. Importantly, miR-27a knockdown itself resulted in fibroblast activation, as demonstrated by the formation of ACTA2 filaments. In addition, interleukin-6 (IL-6) was strongly induced in our fibroblasts when cocultured, indicating potential reciprocal signalling. Taken together, our findings are consistent with canine fibroblasts being reprogrammed into CAFs to further support cancer development and that downregulation of miR-27a may play an important role in the tumour-microenvironment crosstalk.
Topics: Animals; Cancer-Associated Fibroblasts; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cell Transformation, Neoplastic; Coculture Techniques; Dog Diseases; Dogs; Endothelial Cells; Exosomes; Fibroblasts; Mast Cells; Mastocytoma, Skin; MicroRNAs; Signal Transduction; Tumor Microenvironment
PubMed: 32133790
DOI: 10.1002/2211-5463.12831 -
Journal of Veterinary Diagnostic... Sep 2019DNA amplification by PCR detects exon 11 internal tandem duplications in canine mast cell tumors (MCTs). Tissue-specific inhibitors often contaminate DNA extracted from...
DNA amplification by PCR detects exon 11 internal tandem duplications in canine mast cell tumors (MCTs). Tissue-specific inhibitors often contaminate DNA extracted from formalin-fixed, paraffin-embedded (FFPE) canine MCTs, blocking PCR amplification and, consequently, preventing mutation detection. We used a commercial kit to extract DNA from FFPE canine MCTs. Two independent PCR assays, each with one primer set, were used to amplify target genes ( and ) directly after FFPE DNA extraction. PCR amplification failed with at least one primer set in 153 of 280 samples (54.6%, 95% CI: 48.8-60.5%). One or 2 DNA washing steps were required to remove PCR inhibitors in 130 of 280 (46.4%) and 23 of 280 (8.2%) of these cases, respectively. DNA concentration and quality (A/A and A/A) either pre- or post-washing were not associated with ability of the samples to be amplified by PCR using both and primer sets. Low-grade and subcutaneous MCTs were less likely to amplify directly after DNA extraction and without any washing steps compared to high-grade MCTs using gene primers.
Topics: Animals; Circulating Tumor DNA; Dog Diseases; Dogs; Formaldehyde; Mast Cells; Mastocytoma; Mutation; Nucleic Acid Amplification Techniques; Paraffin Embedding; Polymerase Chain Reaction; Proto-Oncogene Proteins c-kit; Skin Neoplasms
PubMed: 31378162
DOI: 10.1177/1040638719867743