Cytoplasmic Immunolabeling Research Articles

Dermal melanoma with plasmacytoid differentiation in a dog.

A 10-y-old spayed female Staffordshire Terrier dog was evaluated because of a cutaneous left ear base mass. Cytology revealed sheets of cells with anisocytosis and anisokaryosis, round-to-oval or plasmacytoid cytoplasm, and round, central, or eccentric nuclei; binucleate cells were present. Cytologic findings were consistent with a round cell tumor (plasmacytoma or agranular mast cell tumor), amelanotic melanoma, or anaplastic carcinoma. Histologically, neoplastic cells were polygonal to elongate, with round-to-oval nuclei and prominent nucleoli and arranged in sheets and nests on a fibrovascular stroma. Neoplastic cells with plasmacytoid morphology (round, glassy, eosinophilic cytoplasm with eccentric nuclei) were present in ~30% of the neoplasm. There were 18 mitoses in 2.37 mm2 (10 FN22/40× fields). Neoplastic cells had cytoplasmic immunolabeling for melan A and PNL2 and no immunolabeling for AE3/1 and MUM1, consistent with a dermal melanoma with plasmacytoid differentiation. The patient was re-evaluated ~1 mo after the first biopsy because of local recurrence of the original mass and new masses on the interscapular area and right elbow; these neoplasms were histologically identical to the original submission, plus scattered neoplastic cells in the new masses contained brown cytoplasmic pigment. The dog was euthanized because of swelling and hemorrhage of the tumors and right pelvic limb lameness. Our findings were consistent with a dermal melanoma with plasmacytoid features that were similar to human plasmacytoid melanoma, a rare variant of human melanoma that is diagnostically challenging as it may mimic a plasmacytoma.

Cytokeratin AE1/AE3 immunolabeling in epithelioid hemangiosarcoma.

Epithelioid hemangiosarcoma (EH), a rare histological variant of hemangiosarcoma, is reported in various animal species, including humans, dogs, cows, horses, and cats. Epithelioid hemangiosarcomas are composed of highly pleomorphic epithelioid cells arranged in cords, islands, nests, or solid cellular areas, similar to epithelial neoplasms. Moreover, in humans, approximately 50% of EHs have cytoplasmic immunolabeling for cytokeratin AE1/AE3 (CK AE1/AE3), making it challenging to distinguish them from carcinomas. This retrospective study assessed the CK AE1/AE3 immunolabeling in canine EH cases from 5 veterinary institutions. Immunohistochemistry for CD31 and CK AE1/AE3 was performed on 30 cases. CK AE1/AE3 immunolabeling was detected in 43% (13/30) of cases, with cytoplasmic labeling ranging from 5% to 100% of neoplastic cells. All tumors consistently had membranous immunolabeling for CD31. The CK AE1/AE3 immunolabeling pattern in canine EHs closely resembled those documented in humans, indicating a similar diagnostic challenge. Therefore, it is recommended to include a vascular immunohistochemistry marker, such as CD31, whenever EH is suspected, particularly in small incisional cutaneous and subcutaneous biopsies.

Development of a peptide-generated antibody to rabbit hemorrhagic disease virus 2 VP60 and its immunohistochemical application in natural cases.

Rabbit hemorrhagic disease virus 2 (RHDV2) has spread across the United States infecting and causing death in domestic and wild rabbits. Immunohistochemistry (IHC) would be a useful tool for the detection of RHDV2 antigen in tissues as it is inexpensive and readily achievable in most diagnostic laboratories. However, there is no readily available antibody for this purpose. To fill this void, we generated an RHDV2 capsid protein VP60-specific antibody in chicken eggs and validated the antibody using formalin-fixed tissues from 5 domestic rabbits naturally infected with RHDV2. Viral antigen was detected immunohistochemically in various tissues, most prominently in hepatocytes and macrophages in liver, and in macrophages in spleen and cecal lymphoid tissue. Intravascular mononuclear cells in lung and renal tubular and biliary epithelium also were immunolabeled. Both nuclear and cytoplasmic immunolabeling were observed. This peptide-generated antibody is a potentially useful tool as an adjunct to reverse-transcription PCR or in situ hybridization for detection of RHDV2 in tissues.

SOX-10 and TRP-1 expression in feline ocular and nonocular melanomas.

In felines, ocular and nonocular melanomas are uncommon tumors that represent a diagnostic challenge for pathologists, especially when amelanotic. To date, the immunohistochemical diagnostic panel in cats is based on specific melanocytic markers (Melan-A and PNL2) and a nonspecific but sensitive marker (S100). In human medicine, SOX-10 is reported to be a sensitive antibody for the detection of melanoma micrometastasis in the lymph node. TRP-1, an enzyme involved in melanogenesis, has recently been used in humans and dogs as a specific melanocyte marker. The aim of this study was to evaluate the cross-reactivity and the expression of SOX-10 and TRP-1 antibodies in feline normal tissue and melanocytic tumors. Thirty-one cases of ocular, cutaneous, and oral melanomas were retrospectively evaluated and confirmed by histopathological examination and by immunolabeling with Melan-A and/or PNL2. SOX-10 nuclear expression in normal tissues was localized in epidermal, subepidermal, hair bulb, and iridal stromal melanocytes and dermal nerves. In melanomas, nuclear expression of SOX-10 was detected in ocular (11/12; 92%), oral (6/7; 86%), and cutaneous sites (12/12; 100%). TRP-1 cytoplasmic immunolabeling in normal tissue was observed in epidermal and bulbar melanocytes and in the lining pigmented epithelium of the iris and in its stroma. Its expression was positively correlated to the degree of pigmentation in the tumor and was observed in 75% of ocular (9/12), 43% of oral (3/7), and 33% of cutaneous melanomas (4/12). This study demonstrated the cross-reactivity of SOX-10 and TRP-1 antibodies in feline non-neoplastic melanocytes and their expression in ocular and nonocular melanomas.

Intoxicação por Nerium oleander em bovinos: uso de imuno-histoquímica para troponina C como método de diagnóstico auxiliar

ABSTRACT: Nerium oleander, also known as “espirradeira” in Brazil, is a shrub or small tree widely used as an ornamental plant in temperate and subtropical regions of the world. Ingestion of N. oleander can cause intoxication due to the presence of cardioactive glycosides, in both humans and animals. This study aims to report the epidemiological, clinical, postmortem, and immunohistochemical features of an outbreak of natural intoxication by N. oleander in cattle and the use of immunohistochemistry (IHC) for troponin C, for the first time, in this intoxication in the species. Twenty-eight cattle had access to pruned branches of N. oleander and 20 animals started presenting clinical signs of apathy, anorexia, ataxia, tachypnea, tremors, and recumbency ten hours after the consumption. Eight cattle progressed to death 48 hours after the clinical progression and one was submitted for necropsy. Macroscopic findings included petechiae and ecchymosis on the epicardium and endocardium. Histologically, there were multifocal areas of necrosis of individual or multiple cardiomyocytes, mainly in the left ventricular papillary muscle. Immunohistochemistry for troponin C revealed decrease or absence of cytoplasmic immunolabeling in necrotic cardiomyocytes. Although intoxication by N. oleander is uncommon in cattle, it should be considered as a differential diagnosis for other conditions that cause sudden or acute death in livestock and IHC for troponin C is an auxiliary tool in the identification of peracute and acute cardiac lesions.

Vulvo-vaginal epithelial tumors in mares: A preliminary investigation on epithelial-mesenchymal transition and tumor-immune microenvironment.

Vulvo-vaginal epithelial tumors are uncommon in mares, and data on the epithelial-to-mesenchymal transition (EMT) and the tumor-immune microenvironment (TIME) are still lacking. This is a study investigating the equus caballus papillomavirus type 2 (EcPV2) infection state as well as the EMT process and the tumor microenvironment in vulvo-vaginal preneoplastic/ benign (8/22) or malignant (14/22) epithelial lesions in mares. To do this, histopathological, immunohistochemical, transcriptomic, in situ hybridization, and correlation analyses were carried out. Immunohistochemistry quantification showed that cytoplasmic E-cadherin and β-catenin expression as well as nuclear β-catenin expression were features of malignant lesions, while benign/preneoplastic lesions were mainly characterized by membranous E-cadherin and β-catenin expression. Despite this, there were no differences between benign and malignant equine vulvo-vaginal lesions in the expression of downstream genes involved in the canonical and noncanonical wnt/β-catenin pathways. In addition, malignant lesions were characterized by a lower number of cells with cytoplasmic cytokeratin expression as well as a slightly higher cytoplasmic vimentin immunolabeling. The TIME of malignant lesions was characterized by more numerous CD204+ M2-polarized macrophages. Altogether, our results support the hypothesis that some actors in TIME such as CD204+ M2-polarized macrophages may favor the EMT process in equine vulvo-vaginal malignant lesions providing new insights for future investigations in the field of equine EcPV2-induced genital neoplastic lesions.

Undifferentiated pleomorphic sarcoma in a white-eared opossum (Didelphisalbiventris)

We report a case of high-grade undifferentiated pleomorphic sarcoma in a free-living white-eared opossum (Didelphis albiventris) in Central Brazil. Grossly, it appeared as a soft yellow mass, approximately 8 cm in diameter, with an irregular, adherent and ulcerated surface, which extended from the proximal epiphysis of the femur to the proximal third of the tibia and fibula. The mass invaded the medullary cavity and had a necrotic centre. Multiple nodular metastases (0.5–1.5 cm) were present in the liver, lungs and spleen. Histologically, the neoplasm was characterized by pleomorphic cells with indistinct boundaries, many multinucleated cells, necrosis and tissue invasion. There was intense cytoplasmic immunolabelling of neoplastic cells for vimentin and moderate cytoplasmic and nuclear labelling for human muscle actin, human smooth muscle actin and lysozyme. Immunolabelling for Iba1, CD18, CD1A, cytokeratin AE1/AE3, glial fibrillary acidic protein and desmin was negative. To our knowledge, this is the first report of an undifferentiated pleomorphic sarcoma in this species.

Intracranial embryonal neoplasm in an alpaca.

An 11-y-old hembra alpaca was admitted because of cerebellar and vestibular signs, dysphagia, and aspiration pneumonia; without clinical improvement following empirical therapy, the patient was euthanized. On autopsy, a neoplasm was found incorporating the right vestibulocochlear nerve at the level of the acoustic meatus. Histologically, the mass was composed of a multiphasic primitive cell population associated with a dense fibrous stroma and enveloping a remnant ganglion and nerve bundles. Patterns included dense ribbons and cords of embryonal neuroepithelial cells admixed with loosely defined interlacing spindle cells. The embryonal cells had angular cell profiles with variable amounts of lightly basophilic cytoplasm, ovoid-to-irregular nuclei, and an open chromatin pattern with a typically inapparent nucleolus. Necrosis was not evident, and there was 1 mitotic figure per 2.37 mm2. The entire mass was infiltrated by small numbers of lymphocytes and plasma cells. Immunohistochemistry (IHC) revealed strong and diffuse cytoplasmic immunolabeling for vimentin, microtubule-associated protein-2, protein gene product 9.5, and synaptophysin; ~50% immunolabeling for cytokeratin AE1/3; sporadic OLIG2 and S100 immunolabeling; and absent glial fibrillary acidic protein immunolabeling. Based on the histologic pattern and the IHC results, our diagnosis was a poorly differentiated embryonal tumor with ependymal differentiation associated with the vestibulocochlear nerve.

Unilateral vascular hamartomas of the vaginal tunic in a dog.

A 12-y-old, male Dachshund was presented for elective orchiectomy. The testes were of normal size. The left testis had numerous dark-red, blood clot-like foci within the vaginal tunic over the pampiniform plexus, epididymis, and testis. Histologically, the red foci were limited to the vaginal tunic and consisted of disorderly growing, variably sized, thin-walled blood vessels lined by a single layer of endothelial cells without mitoses and supported by a thin layer of pericytes. The blood vessels were distended by erythrocytes without thrombus formation. Endothelial cells had cytoplasmic immunolabeling for CD31; pericytes had strong cytoplasmic immunolabeling for α-smooth muscle actin. Our case of subclinical unilateral vascular hamartomas of the vaginal tunic in a dog has not been reported previously in domestic animals or humans, to our knowledge.

Immunohistochemical characterization of clear cell variant of hepatocellular carcinoma in a sheep

The cadaver and viscera of a mature female sheep (Ovis aries) underwent routine abattoir meat inspection. The liver was expanded by an infiltrative neoplastic mass comprising multifocal to coalescing, well-demarcated, pink to white–yellow nodules, up to 25 mm in diameter. An unencapsulated, moderately densely cellular, infiltrative neoplasm was present within the hepatic parenchyma. The neoplastic cells were arranged in solid sheets and acini supported by a moderately fine collagenous vascularized stroma. The neoplastic cells were moderately sized and polygonal, with clearly delineated cell borders and a moderate amount of cytoplasm that was clear or exhibited either globular eosinophilic deposits or fine fibrillar eosinophilic strands. The neoplastic cell nuclei were round and centrally located. The chromatin was lightly stippled and there was frequently a single, prominent, basophilic nucleolus. There were eight mitoses in 10 high-power fields (2.37 mm2). Most of the neoplastic cells had intense cytoplasmic immunolabelling for arginase 1, with frequent concurrent nuclear positivity, and mild to moderately intense punctate cytoplasmic labelling for hepatocyte specific antigen (Hep Par-1). The neoplastic cells did not label with anti-cytokeratin 19 antibody. Based on the histological appearance and the immunolabelling pattern, the neoplasm was diagnosed as the clear cell variant of a hepatocellular carcinoma.

Mesotelioma epitelial maligno em vacas senis Sindi do Brasil

ABSTRACT: Mesotheliomas in cattle are often described as isolated case reports, and investigations of multiple cases within the same bovine herd are lacking. A series of cases of malignant epithelial mesothelioma, tubulopapilary type, is described in five 15 to 21-year-old Red Sindhi cows from the same herd. Clinical signs included three to eight months of progressive emaciation, dehydration, subcutaneous edema of the lower extremities, and abdominal distension. Grossly, severe subcutaneous edema and hydroperitoneum were noted. Multiple organs’ parietal and visceral serosal surfaces had multifocal to coalescing yellow, firm, sessile nodules ranging from 0.1 to 29.0cm. Similar free nodules floated in the peritoneal fluid. Histologically, the masses comprised a layer of cubic to columnar neoplastic cells forming papillary or cystic proliferation supported by a dense fibrovascular stroma. Neoplastic cells had strong and diffuse cytoplasmic immunolabeling for pan-cytokeratin but were negative for cytokeratin 7 and vimentin. Ultrastructurally, neoplastic cells had delicate microvilli and tight and anchoring junctions. Within the cytoplasm, a moderate amount of loose aggregate of intermediary filament with small mitochondria was observed. Epidemiological investigation evidenced endogamy in this herd. Asbestos exposure was not detected. The diagnosis was based on clinical, gross, histological, and immunohistochemical findings and confirmed by transmission electron microscopy features. A definitive underlying etiology remains unknown.

Doublecortin immunolabeling in canine gliomas with distinct degrees of tumor infiltration.

Increased doublecortin (DCX) immunolabeling at the tumor margins has been associated with tumor infiltration in human glioma and canine anaplastic meningioma. No association between DCX immunolabeling and glioma infiltration has been reported in dogs, to our knowledge. Here we compare the DCX immunolabeling in 14 diffusely infiltrating gliomas (gliomatosis cerebri) and 14 nodular gliomas with distinct degrees of tumor infiltration. Cytoplasmic DCX immunolabeling was classified according to intensity (weak, moderate, strong), distribution (1 = <30% immunolabeling, 2 = 30-70% immunolabeling, 3 = >70% immunolabeling), and location within the neoplasm (random or at tumor margins). Immunolabeling was detected in 6 of 14 (43%) diffusely infiltrating gliomas and 8 of 14 (57%) nodular gliomas. Diffusely infiltrating gliomas had moderate and random immunolabeling, with distribution scores of 1 (4 cases) or 2 (2 cases). Nodular gliomas had strong (6 cases) or moderate (2 cases) immunolabeling, with distribution scores of 1 (3 cases), 2 (3 cases), and 3 (2 cases), and random (6 cases) and/or marginal (3 cases) immunolabeling. Increased DCX immunolabeling within neoplastic cells palisading around necrosis occurred in 4 nodular gliomas. DCX immunolabeling was not increased at the margins of diffusely infiltrating gliomas, indicating that DCX should not be used as an immunomarker for glioma infiltration in dogs.

Cell adhesion molecules E-cadherin and CADM1 are differently expressed in canine inflammatory mammary cancer

Human inflammatory breast cancer (IBC) and canine inflammatory mammary cancer (IMC) are the most aggressive and lethal types of mammary tumors with specific characteristics such as exacerbated angiogenesis, lymphangiogenesis and lymphangiotropism. E-cadherin expression is another specific feature of IBC not previously studied in canine IMC. In this study, the expression of E-cadherin and CADM1 (Cell Adhesion molecule 1) and their possible role as key molecules involved in the pathogenesis of IMC were immunohistochemically analyzed in 19 canine IMC and 15 grade III non-IMC cases. E-cadherin and CADM1 expression was higher in IMC cases (p = 0.002, p = 0.008, respectively). In the IMC group, E-cadherin cytoplasmic immunolabeling was more frequent (p = 0.035) and it was associated to the expression of the angiogenic and lymphangiogenic factors COX-2 (p = 0.009), VEGF-A (p = 0.031) and VEGF-D (p = 0.008). The differential mRNA expression between IMC and non-IMC was studied by microarray analysis in 6 cases. E-cadherin gene (CDH1) was not up-regulated in IMC cases at a transcriptional level; interestingly CADM1 was 7-fold upregulated. The differential expression of E-cadherin protein in IMC suggests a possible role of E-cadherin in the characteristic exacerbated angiogenesis and lymphangiogenesis and further support IMC as a natural model for the study of human IBC. Future studies in IBC and IMC including a broad panel of adhesion molecules are necessary to elucidate their role in the metastatic process and angiogenesis.

Hemoperitoneum in a Cow with Lumbar Muscle Hemangiosarcoma

Background: Hemangiosarcoma is a malignant neoplasm of endothelial cells with an infiltrative growth pattern. Hemangiosarcomas are frequently reported in canines and rare in felines, sheep, goats, swine, horses and cattle. Few cases of hemangiosarcoma were reported in cattle. In the present report, we describe the clinicopathological findings of a bovine muscle hemangiosarcoma.Case: A 6-year-old, Girolando cow from the Dairy Cattle Sector of the Federal Rural University of Rio de Janeiro (UFRRJ), Seropédica, presented sternal decubitus. Clinical signs were markedly pale mucous membranes, moderate dehydration, respiratory distress, and increased heart rate. The hematological examination revealed intense regenerative anemia. Due to the worsening of the clinical condition, the cow was submitted to euthanasia. The necropsy and collection of various fragments of organs were performed, which were sent to the “Setor de Anatomia Patológica” (SAP-UFRRJ). Tissues were fixed in 10 % buffered formalin, routinely processed for histology and stained with Hematoxylin and Eosin (HE). The external mucous membranes were markedly pale. Multifocal areas of 1.5 x 1.0 cm, irregular and dark red were observed dissecting the quadratus lumborum muscle (hemangiosarcoma) fibers. These neoplasms were associated with an extensive cruoric clot adhered to the muscle fibers. The extensive, red, friable mass measured approximately 76 x 55 x 20 cm on the serous surfaces of the organs of the peritoneal cavity (hemoperitoneum). The spleen was moderately reduced. The bone marrow was markedly pale. Histologically, it was observed that there was an extensive proliferation of endothelial cells in the quadratus lumbar muscle mass dissecting the epimysium and perimysium. Endothelial cells had moderate pleomorphism, organized in vascular channels and forming multifocally solid areas with a significant amount of eosinophilic fibrillar material (fibrin). Sections of muscle neoplasm were subjected to immunohistochemistry with anti-von Willebrand factor primary antibody, which showed a multifocal moderate cytoplasmic immunolabeling of neoplastic endothelial cells.Discussion: There are few reports of striated muscle hemangiosarcoma in cattle. Muscular hemangiosarcomas were reported in a 4-month-old calf in the left cervical trapezius muscle and a 6-year-old Holstein cow with left pelvic limb mass lateral and distal to the knee. Some reports presented hemangiosarcoma in the iliopsoas muscle, left cervical trapezius muscle, pelvic limb muscles and right cervical muscle of the bovine. In the presented report, hemoperitoneum occurred as a result of hemorrhages from muscle hemangiosarcoma. Other studies have demonstrated cavity hemorrhages in joint, pelvic, pleural and cranial cavities associated with hemangiosarcoma. Hemangiosarcoma with regenerative anemia must be distinguished from other diseases that cause anemia. The main differential diagnoses of bovine with anemia are vena cava syndrome, coumarin derivatives poisoning, acute poisoning by Pteridium spp., tick fever, anaplasmosis, babesiosis and trypanosomiasis. Hemangiosarcoma should be differentiated from other lesions as hemangioma, vascular tumor of lymphatic endothelium and perivascular wall tumors. Cases with poorly differentiated morphology should be submitted for immunohistochemistry. In the present hemangiosarcoma case, we have used the von Willebrand factor for immunohistochemistry diagnosis. Expression of angiogenic growth factors such as CD31, vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF) and angiopoietin-1 (Ang-1) have also been used in the diagnosis of vascular proliferation lesions. Hemangiosarcoma in cattle should be included mainly in the differential diagnosis of diseases that cause acute anemia in cattle.Keywords: cattle, neoplasia, hemangiosarcoma, hemoperitoneum, anemia.

Feline sarcomatoid renal cell carcinoma with peritoneal carcinomatosis and effusion.

A 9-y-old, castrated male, domestic medium-hair cat diagnosed previously with chronic kidney disease developed anorexia and vomiting. Ultrasonography revealed abdominal effusion and a left renal perihilar mass. Cytologic evaluation of the peritoneal fluid and mass identified atypical epithelioid cells suspected to be of renal epithelial or possible mesothelial origin. Immunohistochemical (IHC) evaluation of a formalin-fixed, paraffin-embedded peritoneal fluid cell block indicated both pancytokeratin and vimentin expression in the atypical epithelioid cell population. With scanning electron microscopic evaluation, similar epithelioid cells lacked the cell-surface microvilli expected of mesothelium, supporting an antemortem diagnosis of probable carcinoma. On postmortem examination, the left kidney was effaced by an infiltrative neoplasm with myriad similar nodules throughout the peritoneum. The neoplasm was composed primarily of polygonal-to-spindle-shaped cells with strong vimentin and weak pancytokeratin cytoplasmic immunolabeling. Further IHC characterization with PAX8, CK18, KIT, napsin A, SMA, desmin, CD18, and claudin 5 was performed. Histologic and IHC findings supported a diagnosis of sarcomatoid renal cell carcinoma with peritoneal carcinomatosis. An in vitro cell culture line of neoplastic cells harvested from the primary tumor was successfully established for future research endeavors.

ING3 and ING4 immunoexpression and their relation to the development of benign odontogenic lesions.

The sample comprised of 20 odontogenic keratocysts (OKC), 20 ameloblastomas (AM), and 15 adenomatoid odontogenic tumors (AOT) specimens. Nuclear and cytoplasmic immunolabeling of ING3 and ING4 were semi-quantitatively evaluated in epithelial cells of the odontogenic lesions, according to the percentage of immunolabelled cells in each case. Descriptive and statistics analysis were computed, and the p-value was set at 0.05. No statistically significant differences were found in cytoplasmic and nuclear ING3 immunolabeling among the studied lesions. In contrast, AOTs presented higher cytoplasmic and nuclear ING4 labeling compared to AMs (cytoplasmic p-value = 0.01; nuclear p-value < 0.001) and OKCs (nuclear p-value = 0.007). ING3 and ING4 protein downregulation may play an important role in the initiation and progression of more aggressive odontogenic lesions, such as AMs and OKCs.

Tilapia lake virus immunoglobulin G (TiLV IgG) antibody: Immunohistochemistry application reveals cellular tropism of TiLV infection

Tilapia lake virus (TiLV) is a notable contagious agent that causes massive economic losses in the tilapia industry globally. Evaluations of the histological changes associated with TiLV infection are not only crucial for diagnosis, but also to gain an understanding of the disease. We therefore synthesized a rabbit polyclonal immunoglobulin G antibody against TiLV and developed an immunohistochemical (IHC) procedure to detect TiLV localization in the tissues of infected fish for comparison with in situ hybridization (ISH) testing. A total of four different sample cohorts derived from TiLV-infected fish was used to validate the IHC procedure. The TiLV IHC application was successfully developed and facilitated nuclear and cytoplasmic immunolabelling in the intestines, gills, brain, liver, pancreas, spleen, and kidneys that corresponded with the ISH results. Apart from the ISH results, TiLV-IHC signals were clearly evident in the endothelial cells of various organs, the circulating leukocytes in the blood vessels, and the areas of tissue inflammation. Among the tested sample cohorts, the intestines, gills, and brain had IHC-positive signals, highlighting the possibility of these organs as common TiLV targets. Immunological staining pattern and distribution corresponded with the TiLV viral load but not the inoculation route. The TiLV IHC was also capable of detecting TiLV infection in the experimentally challenged ornamental cichlids, Mozambique tilapia, giant gourami, and naturally infected tilapia, indicating the dynamic range of IHC for TiLV detection. Overall, our study delivers the first IHC platform to detect TiLV infection and provides novel evidence of cellular tropism during TiLV infection. Our findings also reveal the TiLV distribution pattern of infected fish and propose the endotheliotropism and lymphotropism of this virus, which requires further elaboration. Importantly, this new IHC procedure could be applied to study the pathogenesis and interaction of TiLV in future research.

Cathepsin K (Clone EPR19992) Demonstrates Uniformly Positive Immunoreactivity in Renal Oncocytoma, Chromophobe Renal Cell Carcinoma, and Distal Tubules.

Introduction. Cathepsin K is overexpressed in several tumors associated with microphthalmia transcription factor (MiTF) family or mechanistic target of rapamycin (mTOR) upregulation. Among renal neoplasms, MiTF translocation renal cell carcinoma (RCC), perivascular epithelioid cell neoplasms (PEComa), and eosinophilic solid and cystic RCC have demonstrated Cathepsin K immunoreactivity. In this study, we demonstrate a uniform Cathepsin K expression in oncocytoma, chromophobe RCC (CHRCC), and distal tubules. Design. We stained 13 oncocytomas, 13 CHRCC, 14 clear cell RCC (CCRCC), 9 papillary RCC (PRCC), 9 PEComas, and 5 MiTF RCC. Additionally, we assessed immunoreactivity for Cathepsin K in non-neoplastic renal parenchyma. Immunolabeling was performed on regularly charged slides from formalin-fixed paraffin-embedded tissue with monoclonal anti-rabbit antibodies to human Cathepsin K (clone EPR19992, Abcam). Results. All oncocytomas demonstrated diffuse strong cytoplasmic immunolabeling. CHRCC demonstrated uniform less intense immunolabeling in all cases with membranous accentuation. The assessment of the non-neoplastic renal parenchyma in all cases showed strong cytoplasmic immunoreaction in distal tubules and proximal tubules stained faintly. Mesangial cells were not immunoreactive. All MiTF RCC and PEComas were immunoreactive for Cathepsin K, whereas CCRCC and PRCC were negative in all cases. Conclusions. In this study, we expand the spectrum of renal neoplasms reactive with a particular clone of Cathepsin K (EPR19992). Distal tubules are strongly immunoreactive for Cathepsin K. Our conclusions need to be taken into consideration when differential diagnosis includes MiTF RCC or PEComa and this Cathepsin K clone is included in the immunohistochemical panel. This newer antibody clone was not tested in prior publications, potentially explaining the difference in conclusions.

Cimetidine-induced androgenic failure causes cell death and changes in actin, EGF and V-ATPase immunoexpression in rat submandibular glands.

Submandibular gland (SMG) is responsive to androgens via androgen receptor (AR). We verified whether cimetidine induces androgenic dysfunction in SMG, and evaluated the structural integrity, cell death and immunoexpression of actin, EGF and V-ATPase in androgen-deficient SMG. Male rats received cimetidine (CMTG) and control animals (CG) received saline. Granular convoluted tubules (GCTs) diameter and number of acinar cell nuclei were evaluated. TUNEL and immunofluorescence reactions for detection of AR, testosterone, actin, EGF and V-ATPase were quantitatively analysed. In CG, testosterone immunolabelling was detected in acinar and ductal cells cytoplasm. AR-immunolabelled nuclei were observed in acinar cells whereas ductal cells showed AR-immunostained cytoplasm, indicating a non-genomic AR action. In CMTG, the weak testosterone and AR immunoexpression confirmed cimetidine-induced androgenic failure. A high cell death index was correlated with decreased number of acinar cells, GCTs diameter and EGF immunoexpression under androgenic dysfunction. Actin immunofluorescence decreased in the SMG cells, but an increased and diffuse cytoplasmic V-ATPase immunolabelling was observed in striated ducts, suggesting a disruption in the actin-dependent V-ATPase recycling due to androgenic failure. Our findings reinforce the androgenic role in the maintenance of SMG histophysiology, and point to a potential clinical use of cimetidine against androgen-dependent glandular tumour cells.

Overexpression of GDNF and FGF-1 in Canine Benign Prostatic Hyperplasia: Evidence for a Pathogenetic Role of Neural Growth Factor

Benign prostatic hyperplasia (BPH) is common in aged dogs, but the pathogenesis has not been clearly elucidated. A total of 33 male Iranian dogs of mixed breed and in three age groups (under 3 years [n=10]; 3-6 years [n=15]; over 6 years [n=8]), were investigated. BPH was confirmed by ultrasonography and histopathology in 13 cases. The highest prevalence of BPH was in the 3-6 years age group (8/15; 53.3%). Examination of sections of prostate that had been stained with Masson's trichrome revealed that the intensity of stromal smooth muscle cell staining (P<0.05) and the number of fibroblasts (P=0.002) were significantly increased in BPH compared with normal prostate glands. Prostate cells from dogs with BPH (n=13) had a significantly higher intensity of cytoplasmic immunolabelling with antibodies against glial cell line-derived neurotrophic factor (GDNF), cytokeratin (CK) AE1/AE3, vimentin, fibroblast growth factor-1 (FGF-1) and prostate-specific antigen (PSA), compared with normal prostate glands (n=20) (P=0.001), except for PSA, which was negative in both normal and BPH affected prostates. The overexpression of GDNF and FGF-1 in stromal and epithelial cells of prostate glands of dogs with BPH suggests that GDNF has a paracrine or autocrine role in stimulating cellular proliferation. GDNF overexpression may also play a pathogenetic role in promoting chronic prostatitis and increasing fibrosis and the smooth muscle component of the prostate gland in BPH.