Immunohistochemical Profiling of Canine Intestinal T-Cell Lymphomas.

Human enteropathy-associated T-cell lymphoma (EATL) is classified into 2 distinct subgroups based on their phenotypes (type I and type II). Canine intestinal T-cell lymphoma can be morphologically classified into large and small cell lymphomas (LCL and SCL, respectively). Their association with human EATL or immunohistochemical and biological features has not been well characterized. In this study, the immunohistochemical profiles of 17 cases of LCL and 33 cases of SCL were evaluated with markers used for human EATL classification. Morphologically, LCL was characterized by sheet-like proliferation of large to moderately sized neoplastic lymphocytes, with scant clear cytoplasm and pleomorphic, irregularly shaped nuclei containing distinctive nucleoli and scattered chromatin. In contrast, SCL was characterized by the proliferation of monomorphic small neoplastic lymphocytes, accompanied by infiltration of nonneoplastic plasma cells. Interestingly, 8 cases demonstrated mixed LCL and SCL morphologies. Granular cytoplasmic expression of granzyme B was observed in most LCL and SCL cases. Membranous expression of CD56 was demonstrated in only 2 of 17 LCL and 0 of 33 SCL cases. Coexpression of CD20 by neoplastic T cells was observed in more SCL cases (16/33; 48%) than LCL cases (1/17; 6%). The CD56-positive cells in 2 cases were negative for CD20. Although canine LCL shares common features with human EATL type I, canine SCL cells and human EATL type II differ in their immunophenotype. Canine intestinal T-cell lymphoma had a homogeneous immunophenotype regardless of cell morphology. The findings of this study may indicate large cell transformation of SCL rather than 2 distinct entities.

A case of Exophiala dermatitidis-induced phaeohyphomycosis in a cat with multiple intra-abdominal masses.

Pus discharge containing black granular materials (1-2 mm in diameter) was found in the abdominal skin of a 13-year-old sterilized female cat. Abdominal ultrasonography revealed a large intra-abdominal mass with abundant blood flow beneath the skin lesion. Laparotomy revealed a large mass that adhered to the spleen and left kidney. Similar small lesions were found in the abdominal wall and mesentery. The masses were surgically removed along with the spleen and kidney. Histopathologically, the mass lesions consisted of granulomas with lesional pigmented fungi, and the cat was diagnosed with phaeohyphomycosis. Uisng genetic analysis, the Exophiala dermatitidis was identified as the causative pathogen.

The Philippines stingless bee propolis promotes hair growth through activation of Wnt/ß-catenin signaling pathway.

Although hair loss is not a horrible disease, it sometimes reduces the patients' quality of life (QOL) and increases their mental stress. Currently, there is no effective treatment for hair loss. It is known that honeybee propolis has various biological activities, including stimulating the proliferation of hair matrix keratinocytes. However, little is known with the hair promoting activity of stingless bee propolis. Hence, this study investigates the hair growth-promoting activity of Philippines stingless bee propolis extract and the underlying a molecular mechanism of promoting hair growth. For the evaluation of hair growth stimulating activity, 99.5% ethanolic extract of Philippines stingless bee propolis is examined using the simple shaving model in C57BL/6N mice. Melaninization of dorsal skin and histological analysis of hair follicles (HFs) revealed that propolis promotes hair growth by stimulating HFs development. The expression of mRNA (Wnt3a, Ctnnb1/ß-catenin, Lef1, and Bmp2) and protein (WNT3A and ß-catenin) of selected Wnt/ß-catenin associated genes explains Philippines stingless bee propolis promoting HFs development by activating Wnt/ß-catenin signaling pathway. These results suggest that the treatment of propolis strongly promotes hair growth by stimulating the development of HFs via activation of Wnt/ß-catenin signaling pathway. This further indicates the potential of Philippines stingless bee propolis as a novel promising agricultural product for hair growth.

Aged vervet monkeys developing transthyretin amyloidosis with the human disease-causing Ile122 allele: a valid pathological model of the human disease.

Mutant forms of transthyretin (TTR) cause the most common type of autosomal-dominant hereditary systemic amyloidosis. In addition, wild-type TTR causes senile systemic amyloidosis, a sporadic disease seen in the elderly. Although spontaneous development of TTR amyloidosis had not been reported in animals other than humans, we recently determined that two aged vervet monkeys (Chlorocebus pygerythrus) spontaneously developed systemic TTR amyloidosis. In this study here, we first determined that aged vervet monkeys developed TTR amyloidosis and showed cardiac dysfunction but other primates did not. We also found that vervet monkeys had the TTR Ile122 allele, which is well known as a frequent mutation-causing human TTR amyloidosis. Furthermore, we generated recombinant monkey TTRs and determined that the vervet monkey TTR had lower tetrameric stability and formed more amyloid fibrils than did cynomolgus monkey TTR, which had the Val122 allele. We thus propose that the Ile122 allele has an important role in TTR amyloidosis in the aged vervet monkey and that this monkey can serve as a valid pathological model of the human disease. Finally, from the viewpoint of molecular evolution of TTR in primates, we determined that human TTR mutations causing the leptomeningeal phenotype of TTR amyloidosis tended to occur in amino acid residues that showed no diversity throughout primate evolution. Those findings may be valuable for understanding the genotype-phenotype correlation in this inherited human disease.

Histological and immunohistochemical features of cutaneous mast cell tumor in six captive four-toed hedgehogs (Atelerix albiventris).

This report described the histopathological and immunohistochemical features of cutaneous mast cell tumor (MCT) in six hedgehogs. The hedgehogs presented single cutaneous mass with ulcer and crusting. Histologically, the neoplastic lesions were characterized by the proliferation of well-differentiated mast cells (3 cases), and atypical mast cells (3 cases) with one atypical histiocytic morphology. Immunohistochemically, tumor cells were positive for KIT and mast cell tryptase, and were negative for Iba-1. In well-differentiated MCT, all patients were clinically improved and survived more than 365 days after surgical excision, whereas an atypical histiocytic MCT showed aggressive behavior with re-recurrence, and the animal died 115 days after surgery. These findings suggest that, compatible with other animals, well-differentiated MCT has a better prognosis in hedgehogs.

Granulosa cell tumor in 8 African pygmy hedgehogs (Atelerix albiventris).

A retrospective study involving eight African pygmy hedgehogs histopathologically diagnosed with granulosa cell tumors was conducted. The age at onset was 2.2-4.5 years, with a median age of 3.6 years. The most common clinical signs were hematuria and abdominal distension, which were observed in >50% cases. Exploratory laparotomy was performed in all cases, and ovariohysterectomy or excision of the abdominal mass was performed. Patients with only hematuria survived for >250 days after surgery, whereas those with initial ascites showed recurrence of ascites or tumor growth and survived for approximately 130 days after surgery. Intraperitoneal injection of carboplatin was performed in three recurrent cases. In one of these three cases, the tumor mass disappeared. Hence, carboplatin can be considered a potential antineoplastic drug for the treatment of granulosa cell tumors.

Senile systemic amyloidosis in an aged savannah monkey (Cercopithecus aethiops) with tenosynovial degeneration.

Senile systemic amyloidosis (SSA) is a rather common disease in elderly people, but it is very rare in animals, including nonhuman primates. Pathological examination of a 26-year-old male savannah monkey (Cercopithecus aethiops) revealed systemic amyloidosis with severe cardiac fibrosis, and tenosynovial degeneration of the elbow and knee joints. The amyloid deposits were observed predominantly in the heart, lung, intestine and tenosynovium, and were positive for transthyretin (TTR) in immunohistochemistry. Immunohistochemical results, together with the distribution of the amyloid deposited lesions and the age of the monkey, were equivalent to those of human SSA. This is the second case of animal SSA with unprecedented TTR amyloid deposited lesions of the tenosynovium resembling human SSA. There may be a genetic factor that makes this species susceptible to SSA, since SSA has been reported in no other mammal besides humans.

Tophaceous pseudogout of the femorotibial joint in a painted turtle (Chrysemys picta).

A 5-year-old painted turtle (Chrysemys picta) presented with a 1cm nodular enlargement of the right femorotibial joint. The right leg was amputated, because the nodule continued to grow and motor problems appeared. The cut surface of the nodule was yellowish white, and had a colloidal to chalky gross appearance. Microscopically, a multinodular foreign-body granuloma was observed around the joint capsule. Morphological and histochemical examination showed that the foreign body consisted of calcium pyrophosphate. Histological findings revealed that the articular cartilage was mainly involved in the pathological onset of the disease, and was therefore diagnosed as tophaceous pseudogout. This disease is generic in elderly people, but is rare in animals. Also, there are only three reports in reptiles.

Tumor-suppressing potential of stingless bee propolis in in vitro and in vivo models of differentiated-type gastric adenocarcinoma.

The protective property of propolis across a wide spectrum of diseases has long been realized, yet the anti-tumor efficacy of this bioactive substance from Philippine stingless bees has remained poorly understood. Here, we showed the tumor-suppressing potential of crude ethanolic extract of Philippine stingless bee propolis (EEP) in in vitro models of gastric cancer highlighting the first indication of remarkable subtype specificity towards differentiated-type human gastric cancer cell lines but not the diffuse-type. Mechanistically, this involved the profound modulation of several cell cycle related gene transcripts, which correlated with the prominent cell cycle arrest at the G0/G1 phase. To reinforce our data, a unique differentiated-type gastric cancer model, A4gnt KO mice, together with age-matched 60 week-old C57BL/6 J mice were randomly assigned to treatment groups receiving distilled water or EEP for 30 consecutive days. EEP treatment induced significant regression of gross and histological lesions of gastric pyloric tumors that consistently corresponded with specific transcriptional regulation of cell cycle components. Also, the considerable p21 protein expression coupled with a marked reduction in rapidly dividing BrdU-labeled S-phase cells unequivocally supported our observation. Altogether, these findings support the role of Philippine stingless bee propolis as a promising adjunct treatment option in differentiated-type gastric cancer.

Detection of human epidermal growth factor receptor 2 overexpression in canine anal sac gland carcinoma.

Canine anal sac gland carcinoma (ASGC) frequently occurs in the apocrine glands of the canine anal sac and shows aggressive biological behavior. The expression of human epidermal growth factor receptor 2 (HER2) has been reported in various human and canine tumors. HER2 is a promising therapeutic target of these tumors, and HER2-targeted drugs, such as trastuzumab and lapatinib, have improved the outcome of these patients. In this study, HER2 expression in ASGC was evaluated to investigate its potential as a therapeutic target for canine ASGC. HER2 mRNA expression in surgically resected ASGC tissues was significantly higher than that in normal anal sac tissue. To evaluate the expression of HER2 protein, paraffin-embedded ASGC tissues were immunohistochemically evaluated. Strong and broad staining of HER2 was detected in ASGC tissues, while HER2 was weakly to moderately stained in normal anal sac apocrine glands and squamous epithelia. The degree of HER2 expression in ASGC tissues was scored based on its intensity and positivity (score: 0-3+). Scoring of HER2 expression revealed 6 samples (24%) scored 3+, 14 (56%) scored 2+, and 5 (20%) scored 1+, with no samples scoring 0. In all, 80% of canine ASGC tissues were positive for HER2 (scored ≥2+). Furthermore, putative HER2-overexpressed cells in ASGC were detected with trastuzumab by flow cytometry. These preliminary data may lead to further evaluation of the role of HER2 in canine ASGC as a mechanism of malignancy and as a therapeutic target for HER2-targeted therapy.

Proliferative changes in the adrenal medulla of aged Chinese native pigs.

Four aged retired Chinese native pigs, three females and one male, estimated as over 10-year-old, were subjected to autopsy because of infertility due to aging. Grossly, nodular lesions were found bilaterally in the adrenal medulla of all four pigs. Based on the gross and the histopathological findings, they were diagnosed as either medullary nodular hyperplasia or pheochromocytoma. Immunohistochemically, proliferating cells of all these lesions were immuno-positive for chromogranin-A, indicating adrenal medulla-derived. Ultrastructurally, cytoplasmic neurosecretory granules suggestive of secretion were observed in these proliferating cells. There have been only limited numbers of reports on adrenal medullar proliferative changes including pheochromocytoma in pigs. The present cases will provide a valuable information for the characterization of similar changes in animals and human.

Increased expression of the stromal fibroblast-secreted periostin in canine squamous cell carcinomas.

Canine squamous cell carcinoma (SCC) shows highly invasive and locally destructive growth. In animal models and human cancer cases, periostin plays a critical role in the enhancement of cancer growth; however, the mechanism of involvement in canine cancers remains unknown. The aim of this study was to examine the involvement of periostin in the pathophysiology of SCC in dogs. We examined the localization of periostin and periostin-producing cells in 20 SCC and three squamous papilloma specimens. Furthermore, we focused on transforming growth factor (TGF)-ß1, which was assumed to be an inducing factor of periostin, using culture cells. By immunohistochemistry, limited periostin expression in the stroma was observed in all squamous papillomas. In SCC, periostin protein diffusely expressed at the tumor invasion front of cancer growth. In situ hybridization revealed that periostin mRNA was expressed in the stromal fibroblasts in SCC. In vitro analysis determined that canine SCC cells expressed significantly higher levels of TGF-ß1 mRNA compared with canine keratinocytes. In addition, recombinant TGF-ß1 induced secretion of periostin from cultured dermal fibroblasts. These data suggest that periostin produced by stromal fibroblasts may be involved in the pathophysiology of canine SCC. TGF-ß1 derived from SCC cells may stimulate fibroblasts to produce periostin.

Orally administered brown seaweed-derived ß-glucan effectively restrained development of gastric dysplasia in A4gnt KO mice that spontaneously develop gastric adenocarcinoma.

ß-Glucan refers to a heterogeneous group of chemically defined storage polysaccharides containing ß-(1,3)-d-linked glucose polymers with branches connected by either ß-(1,4) or ß-(1,6) glycosidic linkage. To date, an extensive amount of scientific evidence supports their multifunctional biological activities, but their potential involvement in the progression of premalignant lesions remains to be clarified. A4gnt KO mice that lack α1,4-N-acetylglucosamine-capped O-glycans in gastric gland mucin are a unique animal model for gastric cancer because the mutant mice spontaneously develop gastric cancer through hyperplasia-dysplasia-adenocarcinoma sequence. In particular, A4gnt KO mice show gastric dysplasia during 10-20 weeks of age. Here we investigated the putative gastro-protective activity of brown seaweed-derived ß-glucan (Laminaran) against development of gastric dysplasia, precancerous lesion for gastric cancer in A4gnt KO mice. The mutant mice at 12 weeks of age were randomly assigned into three treatment groups namely, wildtype control + distilled water (normal control), A4gnt KO mice + distilled water (untreated control), and A4gnt KO mice + 100 mg/kg Laminaran. After 3 weeks, the stomach was removed and examined for morphology and gene expression patterns. In contrast to the untreated control group, administration of Laminaran substantially attenuated gastric dysplasia development and counterbalanced the increased induction in cell proliferation and angiogenesis. Furthermore, Laminaran treatment effectively overcame the A4gnt KO-induced alteration in the gene expression profile of selected cytokines as revealed by real-time PCR analysis. Collectively, our present findings indicate that ß-glucan can potentially restrain the development of gastric dysplasia to mediate their tissue-preserving activity.

Anaplastic large T-cell lymphoma in three black-tailed prairie dogs (Cynomys ludovicianus).

Anaplastic large T-cell lymphoma (ALTCL) is rarely reported in domestic animals. Accordingly, the histopathological and immunohistochemical characteristics of ALTCL have not been well established in prairie dogs. The present report documents three cases in which prairie dogs were diagnosed with ALTCL arising in the subcutaneous tissue, oral mucosa or the mesenteric lymph nodes. Of the three cases, one was available for necropsy and the others were biopsy cases. Microscopically, moderate to large, pleomorphic neoplastic lymphocytes with ovoid to polygonal, bizarre-shaped nuclei, abundant cytoplasm and eosinophilic granules were seen in all cases. Immunohistochemical staining revealed membranous or cytoplasmic CD3 expression of the neoplastic lymphocytes. The neoplastic cells often had granzyme B-positive cytoplasmic granules. One of the prairie dogs with nodal ALTCL suffered systemic dissemination of the tumor and died suddenly. In the two biopsy cases, one animal died on the day of the biopsy examination and the other died six weeks after chemotherapy. ALTCL in prairie dogs displays a cytotoxic T cell phenotype and presumably carries a poor prognosis regardless of the anatomical type.

Hodgkin's-like lymphoma in a ferret (Mustela putorius furo).

A 7-year-old castrated male ferret developed unilateral cervical lymphadenomegaly over a 1-month period. Histological examination revealed proliferation of tumor cells in a diffuse and partially nodular pattern. The tumor cells were predominantly Hodgkin cells and binucleated Reed-Sternberg cells, characterized by abundant, clear, vacuolated cytoplasm, pleomorphic, ovoid nuclei with thick nuclear membranes and distinct nucleoli. Multinucleated cells, resembling lymphocytic and histiocytic (L&H) cells, were also observed. Immunohistochemically, the tumor cells expressed Pax-5, BLA-36 and vimentin. A small population of the tumor cells expressed CD20. This case showed proliferation of Hodgkin/Reed-Sternberg cells in conjunction with L&H cells that were histologically analogous to feline Hodgkin's-like lymphoma. However, Pax-5 and BLA-36 expression along with rare CD20 expression were consistent with classical Hodgkin's lymphoma in humans.

Identification of the PLA2G6 c.1579G>A Missense Mutation in Papillon Dog Neuroaxonal Dystrophy Using Whole Exome Sequencing Analysis.

Whole exome sequencing (WES) has become a common tool for identifying genetic causes of human inherited disorders, and it has also recently been applied to canine genome research. We conducted WES analysis of neuroaxonal dystrophy (NAD), a neurodegenerative disease that sporadically occurs worldwide in Papillon dogs. The disease is considered an autosomal recessive monogenic disease, which is histopathologically characterized by severe axonal swelling, known as "spheroids," throughout the nervous system. By sequencing all eleven DNA samples from one NAD-affected Papillon dog and her parents, two unrelated NAD-affected Papillon dogs, and six unaffected control Papillon dogs, we identified 10 candidate mutations. Among them, three candidates were determined to be "deleterious" by in silico pathogenesis evaluation. By subsequent massive screening by TaqMan genotyping analysis, only the PLA2G6 c.1579G>A mutation had an association with the presence or absence of the disease, suggesting that it may be a causal mutation of canine NAD. As a human homologue of this gene is a causative gene for infantile neuroaxonal dystrophy, this canine phenotype may serve as a good animal model for human disease. The results of this study also indicate that WES analysis is a powerful tool for exploring canine hereditary diseases, especially in rare monogenic hereditary diseases.

Histological and immunohistochemical studies on primary intracranial canine histiocytic sarcomas.

Histiocytic sarcoma is a progressive and fatal malignant neoplasm that mainly occurs in middle- to old-aged dogs. This study describes clinicopathological, histological and immunohistochemical characteristics of intracranial histiocytic sarcomas in 23 dogs. Magnetic resonance imaging and/or computed tomography of the brains revealed that the tumors mainly located in the cerebrum, particularly the frontal lobe. Seizure was a predominant clinical sign in most of the cases. Histologically, the tumor cells were morphologically classified into round/polygonal- and spindle-shaped cell types. There was a significant association between tumor cell types and hemophagocytic activity (P<0.05). However, there was no significant difference in other clinicopathological parameters and mitotic index between the 2 types. Immunohistochemically, tumor cells were strongly positive for HLA-DR, Iba-1 and CD204 in all the 23 cases, for iNOS in 20, for CD163 in 17, for CD208 (DC-LAMP) in 9, for lysozyme in 8 and for S100 in 5 cases. In addition, the Ki67-proliferative index showed range of 0.50-64.33% (Average 26.60 ± 3.81%). These observations suggest that canine primary intracranial histiocytic sarcomas tend to exhibit both dendritic cell and macrophage phenotypes of histiocytic differentiation.

Expression of cell adhesion molecules in canine choroid plexus tumors.

Choroid plexus tumor (CPT) is a primary intracranial neoplasm of the choroid plexus epithelium in the central nervous system. In the current World Health Organization classification, CPT is classified into two categories; choroid plexus papilloma (CPP) and carcinoma (CPC). In the present study, we investigated immunohistochemical expressions of N-cadherin, E-cadherin and ß-catenin in 5 canine CPT cases (1 disseminated CPC, 2 CPCs and 2 CPPs). One CPP case was positive for N-cadherin and ß-catenin, but negative for E-cadherin. The disseminated CPC case was positive for E-cadherin and ß-catenin, but negative for N-cadherin. The other cases were positive for the three molecules examined. These results suggest that loss of the N-cadherin expression might associate with the spreading of CPC cells.

Common variable immune deficiency in a Pomeranian with Pneumocystis carinii pneumonia.

A Pomeranian dog, 1 year- and 8 month-old neutered female, was presented with persistent respiratory distress and recurrent generalized demodicosis. Physical examination revealed cyanosis, rough respiratory sounds, multifocal alopecia and dermal erosions on the dorsal side of the forelimbs, perineal area and skin around the eyes. A severe diffuse interstitial lung pattern was observed on thoracic radiographs. The blood examination revealed neutrophilia and hypoglobulinemia. Serum immunoglobulin concentrations of IgG and IgA were low. Histopathological examination revealed severe diffuse interstitial pneumonia with Pneumocystis carinii infection. Severe lymphoid depletion was observed in the spleen and other organs with lymphoid follicles consisted mainly of CD3-positive T cells and few cells of B-cell lineage. B-cell hypoplasia with subsequent antibody deficiency was suspected.

Mycotic aneurysm caused by Graphium species in a dog.

A 10-year-old castrated male mixed-breed dog exhibited vomiting, lethargy and anorexia. An abdominal ultrasound examination revealed a focal dilation of the abdominal aorta. The dog died 2 days after examination, and necropsy revealed a ruptured aneurysm at the abdominal aorta between the kidneys. Histological examination revealed severe granulomatous and necrotizing pan-arteritis with fungal organisms. Graphium species was identified through DNA sequence analysis of the PCR product from formalin-fixed paraffin-embedded samples. To our knowledge, this is the first report of aortic aneurysm caused by Graphium species in a dog. Sequence was submitted to the DNA Data Bank of Japan with the accession number LC007972.