Feline Facial Spindle Cell Tumors in 29 Cats: Histomorphological and Immunohistochemical Characterization.

Soft tissue tumors/sarcomas (STSs) in felines, encompassing a variety of mesenchymal tumors with similar histomorphological features, present diagnostic challenges due to their diverse cellular origins and the overlap with other tumor types such as feline sarcoid. This study aimed to delineate the clinical, histomorphological, and immunohistochemical characteristics of 34 feline facial spindle cell tumors affecting 29 cats, including testing for bovine papillomavirus type 14 (BPV14), the virus causing feline sarcoids. Only five out of 12 tumors previously diagnosed as feline sarcoids based on histomorphology were confirmed by PCR for BPV14, underscoring the importance of comprehensive diagnostic approaches to accurately distinguish between STSs and feline sarcoids. This study shows that most facial spindle cell tumors were compatible with peripheral nerve sheath tumors (PNSTs) based on positive immunohistochemical staining for Sox10 and other immunohistochemical markers such as GFAP, NSE, and S100. Some of these tumors displayed as multiple independent masses on the face or as erosive and ulcerative lesions without obvious mass formation, an atypical presentation and an important highlight for general practitioners, dermatologists, and oncologists. This study also describes periadnexal whorling of neoplastic cells as a novel histomorphologic finding in feline facial PNSTs and emphasizes Sox10 as a useful complementary immunohistochemical marker for the diagnosis of facial PNST in cats, providing valuable insights for veterinary pathologists.

Cross-species evaluation of fibroblast activation protein alpha as potential imaging target for soft tissue sarcoma: a comparative immunohistochemical study in humans, dogs, and cats.

Introduction: Complete surgical tumor resection is paramount in the management of soft tissue sarcoma (STS) in humans, dogs, and cats alike. Near-infrared targeted tracers for fluorescence-guided surgery (FGS) could facilitate intraoperative visualization of the tumor and improve resection accuracy. Target identification is complicated in STS due to the rarity and heterogeneity of the disease. This study aims to validate the expression of fibroblast activation protein alpha (FAP) in selected human, canine, and feline STS subtypes to assess the value of FAP as a target for FGS and to validate companion animals as a translational model. Methods: Formalin-fixed and paraffin-embedded tissue samples from 53 canine STSs (perivascular wall tumor (PWT), canine fibrosarcoma (cFS), and STS not further specified (NOS)), 24 feline fibrosarcomas, and 39 human STSs (myxofibrosarcoma, undifferentiated pleomorphic sarcoma, dermatofibrosarcoma protuberans, and malignant peripheral nerve sheath tumor) as well as six canine and seven feline healthy controls and 10 inflamed tissue samples were immunohistochemically stained for their FAP expression. FAP labeling in tumor, peritumoral, healthy skin, and inflamed tissue samples was quantified using a visually assessed semiquantitative expression score and digital image analysis. Target selection criteria (TASC) scoring was subsequently performed as previously described. Results: Eighty-five percent (85%) of human (33/39), 76% of canine (40/53), and 92% of feline (22/24) STSs showed FAP positivity in over 10% of the tumor cells. A high expression was determined in 53% canine (28/53), 67% feline (16/24), and 44% human STSs (17/39). The average FAP-labeled area of canine, feline, and human STSs was 31%, 33%, and 42%, respectively (p > 0.8990). The FAP-positive tumor area was larger in STS compared to healthy and peritumoral tissue samples (p < 0.0001). TASC scores were above 18 for all feline and human STS subtypes and canine PWTs but not for canine STS NOS and cFS. Conclusion: This study represents the first cross-species target evaluation of FAP for STS. Our results demonstrate that FAP expression is increased in various STS subtypes compared to non-cancerous tissues across species, thereby validating dogs and cats as suitable animal models. Based on a TASC score, FAP could be considered a target for FGS.

Ovarian cancer G protein-coupled receptor 1 deficiency exacerbates crystal deposition and kidney injury in oxalate nephropathy in female mice.

Ovarian cancer G protein-coupled receptor 1 (OGR1) (Gpr68) and G protein-coupled receptor 4 (GPR4) (Gpr4) are proton-activated G protein-coupled receptors that are stimulated upon increased extracellular acidity. These receptors have various physiological and pathophysiological roles in renal acid-base physiology, tissue inflammation, and fibrosis among others. Their function in injured renal tissue, however, remains mostly unclear. To address this, we investigated their role in crystalline nephropathy by increasing the oxalate intake of GPR4 KO and OGR1 KO mice. After 10 days of high-oxalate intake and 4 days of recovery, renal crystal content, histopathology, filtration function, and inflammation were assessed. While GPR4 deficiency did not show major alterations in disease progression, OGR1 KO mice had higher urinary calcium levels and exacerbated crystal accumulation accompanied by decreased creatinine clearance and urea excretion and a decreased presence of regulatory T (Treg) cells in kidney tissue. When lowering the severity of the kidney injury, OGR1 KO mice were more prone to develop crystalline nephropathy. In this setting, OGR1 KO mice displayed an increased activation of the immune system and a higher production of proinflammatory cytokines by T cells and macrophages. Taken together, in the acute setting of oxalate-induced nephropathy, the lack of the proton-activated G protein-coupled receptor (GPCR) GPR4 does not influence disease. OGR1 deficiency, however, increases crystal deposition leading to impaired kidney function. Thus, OGR1 may be important to limit kidney crystal deposition, which might subsequently be relevant for the pathophysiology of oxalate kidney stones or other crystallopathies.

Lymph node metastasis in feline cutaneous low-grade mast cell tumours.

OBJECTIVES: This retrospective study aimed to determine the incidence of nodal metastatic disease in cats affected by low-grade cutaneous mast cell tumours (MCTs) in our study population. METHODS: The clinical records of two centres were retrospectively searched for cats with cutaneous MCTs that had undergone lymphadenectomy of enlarged and non-enlarged lymph nodes. All primary tumours were histologically reviewed by two experienced pathologists and graded as high- or low-grade based on the grading system for feline cutaneous MCT. We graded the lymph nodes based on the grading scheme used for canine MCTs and considered HN2 and HN3 nodes to be metastatic. The number of patients with nodal metastasis was calculated. RESULTS: We identified 17 cats with cutaneous MCT resection and concurrent lymphadenectomy. All 21 MCTs were graded as low grade and 30 nodes were removed, with 12 being considered early or overtly metastatic (HN2 or HN3, respectively). Based on nodal status, 10/17 (59%) cats were affected by nodal metastasis in our population. CONCLUSIONS AND RELEVANCE: In contrast to previous reports, high percentage of cats with cutaneous MCTs in which lymphadenectomy was performed were presented with metastatic lymph nodes. The clinical relevance of this finding and a potential benefit of lymphadenectomy must be determined in future studies.

Phaeohyphomycosis caused by Phialophora americana in a dog.

Phaeohyphomycosis was diagnosed in a 6-year-old, male castrated Dachshund on immunosuppressive treatment. The fungus was identified by culture and PCR as Phialophora americana. This is the first reported case of infection with this pathogen in a dog. The infection was successfully managed medically, without surgical intervention.

Une phaéohyphomycose a été diagnostiquée chez un teckel mâle castré de 6 ans sous traitement immunosuppresseur. Le champignon a été identifié par culture et PCR comme Phialophora americana. Il s'agit du premier cas rapporté d'infection par cet agent pathogène chez un chien. L'infection a été prise en charge médicalement avec succès, sans intervention chirurgicale.

Se diagnosticó feohifomicosis en un macho de Teckel castrado de 6 años en tratamiento inmunosupresor. El hongo fue identificado por cultivo y PCR como Phialophora americana. Este es el primer caso reportado de infección por este patógeno en un perro. La infección se manejó con éxito médicamente, sin intervención quirúrgica.

Feohifomicose foi diagnosticada em um cão da raça Dachshund, macho castrado, de seis anos de idade, em tratamento imunossupressivo. O fungo identificado por cultura e PCR foi Phialophora americana. Este é o primeiro relato de caso de infecção por este patógeno em um cão. A infecção foi bem conduzida com tratamento medicamentoso, sem intervenção cirúrgica.

Immunotherapy with Immunocytokines and PD-1 Blockade Enhances the Anticancer Activity of Small Molecule-Drug Conjugates Targeting Carbonic Anhydrase IX.

Small molecule-drug conjugates (SMDCs) represent an alternative to conventional antitumor chemotherapeutic agents, with the potential to improve the therapeutic window of cytotoxic payloads through active delivery at the site of the disease. In this article, we describe novel combination therapies consisting of anti-carbonic anhydrase IX SMDCs combined with different immunomodulatory products. The therapeutic effect of the SMDCs was potentiated by combination with PD-1 blockade and with tumor-homing antibody-cytokine fusions in mouse models of renal cell carcinoma and colorectal cancer. The combination with L19-IL12, a fusion protein specific to the alternatively spliced EDB domain of fibronectin containing the murine IL12 moiety, was also active against large established tumors. Analysis of the microscopic structures of healthy organs performed 3 months after tumor eradication confirmed absence of pathologic abnormalities in the healthy kidney, liver, lung, stomach, and intestine. Our findings may be of clinical significance as they provide motivation for the development of combinations based on SMDCs and immunotherapy for the treatment of renal cell carcinoma and hypoxic tumors.

L1 Cell Adhesion Molecule Confers Radioresistance to Ovarian Cancer and Defines a New Cancer Stem Cell Population.

Many solid tumors, including ovarian cancer, contain small populations of cancer stem cells (CSCs). These cells are usually resistant against conventional cancer therapies and play a role in disease recurrence. We demonstrated that the L1 cell adhesion molecule (L1CAM) is a new CSC target in ovarian cancer, triggering radioresistance. Using fluorescence-activated cell sorting, specific cell populations expressing L1CAM alone or in combination with the established CSC marker CD133 were isolated from three ovarian cancer cell lines. Double-positive L1CAM+/CD133+ cells displayed higher spherogenic and clonogenic properties in comparison to L1CAM-/CD133- cells. Furthermore, L1CAM+/CD133+ cells retained highest clonogenic capacity after irradiation and exhibited up-regulation of some CSC-specific genes, enhanced tumor-initiating capacity, self-renewal and higher tumor take rate in nude mice when compared with other cell populations. Superior radioresistance by L1CAM expression was confirmed by deletion of L1CAM using CRISPR-Cas9 technology. Moreover, we found expression signatures associated with epithelial-to-mesenchymal transition phenotype in L1CAM deleted cells. These results indicate that L1CAM in combination with CD133 defines a new cancer cell population of ovarian tumor-initiating cells with the implication of targeting L1CAM as a novel therapeutic approach for ovarian CSCs.

The elevation of circulating fibroblast growth factor 23 without kidney disease does not increase cardiovascular disease risk.

High circulating fibroblast growth factor 23 (FGF23) levels are probably a major risk factor for cardiovascular disease in chronic kidney disease. FGF23 interacts with the receptor FGFR4 in cardiomyocytes inducing left ventricular hypertrophy. Moreover, in the liver FGF23 via FGFR4 increases the risk of inflammation which is also found in chronic kidney disease. In contrast, X-linked hypophosphatemia is characterized by high FGF23 circulating levels due to loss of function mutations of the phosphate-regulating gene with homologies to an endopeptidase on the X chromosome (PHEX), but is not characterized by high cardiovascular morbidity. Here we used a novel murine X-linked hypophosphatemia model, the PhexC733RMhda mouse line, bearing an amino acid substitution (p.Cys733Arg) to test whether high circulating FGF23 in the absence of renal injury would trigger cardiovascular disease. As X-linked hypophosphatemia patient mimics, these mice show high FGF23 levels, hypophosphatemia, normocalcemia, and low/normal vitamin D levels. Moreover, these mice show hyperparathyroidism and low circulating soluble αKlotho levels. At the age of 27 weeks we found no left ventricular hypertrophy and no alteration of cardiac function as assessed by echocardiography. These mice also showed no activation of the calcineurin/NFAT pathway in heart and liver and no tissue and systemic signs of inflammation. Importantly, blood pressure, glomerular filtration rate and urea clearance were similar between genotypes. Thus, the presence of high circulating FGF23 levels alone in the absence of renal impairment and normal/high phosphate levels is not sufficient to cause cardiovascular disease.

Evaluation of surface blood flow in intact and ruptured canine cruciate ligaments using laser Doppler flowmetry.

OBJECTIVE: To evaluate the usefulness of laser Doppler flowmetry (LDF) to measure surface blood flow in canine cruciate ligaments, compare measurements in different sites of intact and partially ruptured canine cranial cruciate ligaments (CrCL) and intact caudal cruciate ligaments (CaCL), and investigate any association between surface blood flow in partially ruptured CrCL and synovitis or duration of clinical signs. STUDY DESIGN: Case-controlled clinical study. ANIMALS: Sixteen dogs with partially ruptured CrCL and five dogs with intact CrCL. METHODS: Blood cell flux (BCF) readings during three measurement cycles using LDF at two sites in each ligament (mid-substance and the distal portion of the CrCL, and mid-substance and the proximal portion of the CaCL) were recorded. Synovial changes were graded grossly and histologically using the Osteoarthritis Research Society International histopathology scoring system. RESULTS: The within-run coefficients of variation (CV) for a single BCF measurement cycle were 12.2% and 12.7% in the ruptured and intact CrCL groups, respectively. The between-run CV for three measurement cycles was 20.8% and 14.8%, respectively. The intraclass correlation coefficient (ICC, absolute agreement) was 0.66 for a single measurement cycle and 0.86 for the average of three cycles. No difference in average BCF readings was found between any two sites in either group, but BCF readings in both CrCL sites were significantly higher in the ruptured CrCL group than the intact CrCL group. No associations between BCF and synovial grades or duration of lameness were identified. CONCLUSIONS: Laser Doppler flowmetry can be used to assess surface blood flow in intact and partially ruptured canine cruciate ligaments with acceptable precision. Using this method, surface blood flow appears greater in partially ruptured canine CrCL than intact CrCL. Further studies are required to determine if this is a sequela of trauma or synovitis.

Anaplasma phagocytophilum DNA amplified from lesional skin of seropositive dogs.

Canine granulocytic anaplasmosis (CGA) is caused by the rickettsial microorganism Anaplasma phagocytophilum. CGA is typically characterized by fever, thrombocytopenia, lethargy, anorexia, arthropy, and other nonspecific clinical signs. Skin lesions have been described in naturally infected lambs and humans. The pathophysiology of CGA is not entirely clear, and the persistence of the organism after the resolution of clinical signs has been described. The aim of the study was to investigate if A. phagocytophilum can be detected in canine lesional skin biopsies from A. phagocytophilum-seropositive dogs with etiologically unclear skin lesions that improved after the treatment with doxycycline. Paraffin-embedded lesional skin biopsies were allocated into separate groups: biopsies from A. phagocytophilum-seropositive dogs responsive to treatment with doxycycline (n=12), biopsies from A. phagocytophilum-seronegative dogs (n=2), and biopsies in which skin lesions histopathologically resembled a tick bite (n=10). The serological status of the latter group was unknown. Histology of the seropositive and seronegative dog skin lesions did not indicate an etiology. DNA was extracted, and a conventional PCR for partial 16S rRNA gene was performed. Anaplasma phagocytophilum DNA was amplified from 4/12 seropositive dogs' skin biopsies. All sequences were 100% identical to the prototype A. phagocytophilum human strain (GenBank accession number U02521). Anaplasma phagocytophilum was not amplified from the 2 seronegative and 10 suspected tick bite dogs. Serum antibody titers of the PCR-positive dogs ranged from 1:200 to 1:2048. Histopathologically, a mild-to-moderate perivascular to interstitial dermatitis composed of a mixed cellular infiltrate and mild-to-moderate edema was seen in all seropositive dogs. In 8/12 seropositive dogs, vascular changes as vasculopathy, fibrinoid necrosis of the vessel walls, and leukocytoclastic changes were observed. In summary, our results support the hypothesis that the persistence of A. phagocytophilum in the skin may be causative for otherwise unexplained skin lesions in seropositive dogs.

Pulmonary aspergillosis in an African elephant (Loxodonta africana).

A 26-year-old female African elephant (Loxodonta africana) with a history of purulent pododermatitis, recurrent abdominal pain, and severe weight loss died spontaneously after a period of deteriorating disease. The main pathological finding was a severe bilateral pyogranulomatous, partially necrotizing pneumonia with numerous intralesional fungal hyphae. At microbiological examination Aspergillus spp. were isolated. The present case indicates that mycotic pneumonia should to be considered as a differential diagnosis of pulmonary disorders in elephants.

Influence of persistent canine distemper virus infection on expression of RECK, matrix-metalloproteinases and their inhibitors in a canine macrophage/monocytic tumour cell line (DH82).

A morbillivirus infection of tumour cells is known to exert oncolytic activity, but the mechanism of this inhibitory action has not been well defined. Matrix metalloproteinases (MMPs) are important enzymes degrading the extracellular matrix and are often upregulated in malignant neoplasms. Recent studies have demonstrated that RECK may potently suppress MMP-2 and -9 activity, thus inhibiting angiogenesis and metastasis. In this study, real time quantitative polymerase chain reaction (RT-qPCR) was used to determine the effect of persistent infection with canine distemper virus (CDV) infection on the expression of MMPs and their inhibitors (TIMPS) in a canine macrophage/monocytic tumour cell line (DH82). The activity of proMMP-2 and proMMP-9 was also verified zymographically. Following CDV infection, MMP-2, TIMP-1 and TIMP-2 were down-regulated, while RECK was upregulated. These findings suggest that CDV infection restores RECK expression in tumour cells and may interfere with the intracellular processing of MMPs and TIMPs, thus possibly influencing tumour cell behaviour beneficially for the host. However, this needs to be verified in in vivo studies.

Purification and in vitro characterization of adult canine olfactory ensheathing cells.

Olfactory ensheathing cells (OECs) are known to promote neural repair under experimental conditions. The experimental focus has so far been almost entirely on rodent OECs (rOECs), and hence whether human OECs (humOECs) display similar properties is unclear. Studies on larger mammals as an "intermediate" model may be helpful for translating the experimental evidence gathered so far into novel therapeutic strategies. In the present study, we purified adult canine OECs (caOECs) from the olfactory bulb and analyzed their in vitro properties with respect to antigen expression, proliferation, and differentiation. Secondary caOECs shared the expression of marker molecules and the reactivity toward growth factors, with rOECs and humOECs. CaOECs were positively immunostained for the low affinity neurotrophin receptor p75, GFAP, and O4 and proliferated in response to fibroblast growth factor-2 and heregulin-1beta. No decline in proliferation was noted at higher passages (>8). The effects of forskolin, which neither increased proliferation nor stimulated the expression of O4, were clearly different from those on rOECs. Moreover, caOECs displayed their typical spindle-shaped morphology only upon growth factor/forskolin addition, whereas mitotically quiescent caOECs had a flattened morphology. Thus, caOECs can readily be purified from adult canine olfactory bulb and expanded by using established OEC mitogens. The behavior of caOECs toward forskolin suggests that caOECs and humOECs share a number of properties amd implies the presence of common intracellular signalling pathways. CaOECs therefore represent a suitable model system relevant for humOECs in neural repair studies.