Immunohistochemical characteristics of normal canine eyes.

Immunohistochemistry is widely utilized in diagnostic laboratories to study neoplastic and nonneoplastic diseases. Knowledge of the immunohistochemical characteristics of normal tissue is essential for interpretation of immunoreactivity in pathologic conditions. In this study, immunohistochemistry was performed with a broad panel of diagnostically relevant antibodies on 4 normal canine globes--namely, vimentin, pan-cytokeratin (AE1/AE3), cytokeratin 7, cytokeratin 8/18, cytokeratin 20, α-smooth muscle actin, muscle specific actin, desmin, Melan-A, microphthalmia transcription factor, S-100, glial fibrillary acidic protein, triple neurofilaments, neuron-specific enolase, chromogranin A, synaptophysin, laminin and CD31. Results include cytokeratin immunoreactivity limited to the conjunctival epithelium, corneal epithelium, and retinal pigment epithelium; distinct patterns of immunopositivity of muscle markers; and widespread immunoreactivity for vimentin and most neural/neuroendocrine markers. These findings in normal eyes provide the basis for interpretation of ocular immunohistochemistry in dogs. Published immunophenotypes of primary ocular neoplasms are also reviewed.

Tumor morphology and immunohistochemical expression of estrogen receptor, progesterone receptor, p53, and Ki67 in urogenital carcinomas of California sea lions (Zalophus californianus).

Metastatic carcinoma of urogenital origin is a common cause of mortality in free-ranging California sea lions (Zalophus californianus). The etiology of this cancer is likely multifactorial, with viral infection, genetic factors, and exposure to environmental organochlorine contaminants possible contributing factors. In this study, expression of estrogen receptor alpha (ER alpha), progesterone receptor (PR), p53, and Ki67 were evaluated by immunohistochemistry in 12 sea lions with metastatic carcinoma, genital epithelial dysplasia, and intraepithelial neoplasia; 4 with genital epithelial dysplasia and intraepithelial neoplasia without metastases; and 6 control animals. Dysplastic and neoplastic lesions were identified in multiple areas of the cervix, vagina, penis, prepuce, and urethra in affected animals, suggesting multicentric development. Lesions were graded according to degree of epithelial dysplasia and infiltration and lesions of different grades were evaluated separately. Estrogen receptor expression was lower in intraepithelial lesions compared with normal genital epithelium, and expression in metastatic lesions was completely absent. There was progesterone receptor expression in neoplastic cells in intraepithelial lesions of all grades and in metastases, with no significant difference between lesion grades or between control and affected epithelium. Ki67 index and p53 expression increased with lesion grade and were higher in lesions than normal epithelium. Metastatic tumors exhibited highly variable morphology; however, proliferation index, ER alpha, PR, and p53 expression were similar in tumors with different patterns of growth. These results suggest that endogenous hormones, environmental contaminants that interact with steroid hormone receptors, and alterations in p53 may play a role in urogenital carcinogenesis in California sea lions.

Langerhans cell hyperplasia and IgE expression in canine atopic dermatitis.

Langerhans cells appear to be critical for IgE-mediated allergen capture and presentation in human atopic dermatitis. The present study sought to determine whether epidermal (i.e Langerhans cells) and dermal dendritic cells in the skin of dogs with atopic dermatitis are hyperplastic and expressed surface IgE. Frozen sections of lesional or non-lesional atopic and normal control canine skin were immunostained with CD1a-, CD1c-, and IgE-specific monoclonal antibodies. The enumeration of cells was performed by morphometry in both the epidermis and the dermis. Cell counts were compared with each individual's total serum IgE levels. Higher numbers of epidermal and dermal dendritic cells were present in atopic dogs than in normal control animals. Epidermal Langerhans cell counts were significantly higher in lesional than in non-lesional atopic specimens. IgE+ dendritic cells were observed in lesional atopic epidermis and dermis, and non-lesional atopic dermis, but not in normal control skin specimens. The percentages of IgE+ dendritic cells were correlated with each patient's total serum IgE levels. These results demonstrate dendritic cell hyperplasia and IgE expression in canine atopic dermatitis. Increased epidermal Langerhans cell counts in lesional specimens suggest an epidermal allergen contact in canine atopic dermatitis.

Investigation of epidermotropism in canine mycosis fungoides: expression of intercellular adhesion molecule-1 (ICAM-1) and beta-2 integrins.

In human mycosis fungoides (MF), interactions between LFA-1 (CD11a/CD18) and ICAM-1 (CD54) are involved in lymphocyte adhesion to keratinocytes. The purpose of this study was to evaluate the expression of ICAM-1, beta-2 integrins and class II major histocompatibility complex molecules (MHC II) on keratinocytes and infiltrating lymphocytes in canine MF. Sections of frozen skin biopsy specimens from normal dogs (n = 3) and dogs with MF (n = 17) were evaluated by immunohistochemistry for expression of ICAM-1, beta-2 integrins, and class II MHC molecules. Our results demonstrated that in canine MF, ICAM-1 was expressed variably on epidermal and follicular keratinocytes. The extent of keratinocyte ICAM-1 expression did not correlate with the degree of lymphocyte epithelial infiltration, nor with lymphocyte LFA-1 expression. This was especially evident in cases of Pagetoid reticulosis-like disease in which prominent lymphocyte epidermotropism was not accompanied by keratinocyte ICAM-1 expression. Keratinocyte class II MHC molecule expression did not correlate with keratinocyte ICAM-1 expression. In conclusion, in canine MF, the lack of statistically significant correlations between epithelial lymphocyte infiltration and keratinocyte ICAM-1 expression, and between keratinocyte ICAM-1 and lymphocyte LFA-1 staining, suggests that the LFA-1/ICAM-1 pathway is not the major adhesion mechanism between lymphocytes and keratinocytes. It is suspected that different ligands of the LFA-1 integrin (e.g. ICAM-2) or other adhesion molecules (e.g. CD2/LFA-3, VLA-1) might be involved in the epitheliotropism phenomenon in canine MF. These hypothesis cannot be evaluated in the dog at this time owing to the lack of specific monoclonal antibodies.

A comparison of six monoclonal antibodies for detection of cytokeratins in normal and neoplastic canine tissues.

Twenty normal canine tissue specimens, both fetal and adult; 19 epithelial neoplasms; and 18 nonepithelial neoplasms were examined using 6 commercially available monoclonal antibodies differing in their recognition of various molecular weight cytokeratins in human tissues. Fresh tissue samples were fixed in 100% ethanol and paraffin embedded prior to sectioning. The intermediate filament proteins were identified by an avidin-biotin-immunoperoxidase method. Primary antisera used included AE1/AE3, CAM-5.2, 35BH11, 34BE12, PKK1, MAK-6 cytokeratins, and vimentin. Monoclonal antibodies detected cytokeratins in a wide variety of canine epithelial tissues and neoplasms. Normal mesenchymal tissues and neoplasms, and stromal elements of epithelial tissues, showed no reactivity with anti-cytokeratins, but reacted positively with vimentin. Although PKK1, CAM-5.2, and MAK-6 were the most consistently reactive anti-cytokeratins, the full panel of monoclonals was required to detect cytokeratins in all of the epithelia evaluated.

Evaluation of commercially available antibodies to cytokeratin intermediate filaments and laminin in normal cat pinna.

The pattern of distribution of cytokeratin (CK) intermediate filaments can be used to characterize subsets of epithelial tissues. The purpose of the study was to examine the CK expression of feline pinna skin. Six normal feline pinnae were routinely processed in formalin. An immunohistochemical method was used to stain the pinnae with 8 commercially available anti-human CK antibodies (Abs) (PKK1, CAM 5.2, UCD 10/11, 35BH11, 34BE12, AE1/AE3, MAK 6, A575) and an anti-human laminin Ab. All the CK Abs selectively localized to epithelium except 35BH11, which did not react with any part of the pinna. Some epithelial subsets were identified by their unique staining pattern with CK Abs. Basal cells but not suprabasal cells of the epidermis stained with PKK1; basal but not lumenal cells of apocrine glands stained with 34BE12. Apocrine glands stained with all CK Abs except 35BH11. All epithelial structures were stained with A575. Basal lamina of epithelial and mesenchymal tissues was clearly identified by the anti-laminin Ab. The results indicate that in cat pinna some commercially available anti-human CK Abs selectively stain subsets of epithelium and adnexa. PKK1, 34BE12, and A575 were the CK Abs with the most consistent staining patterns, the other Abs stained more variably from pinna to pinna. The pattern of epithelial and adnexal staining was similar but not identical to that reported for humans.

Apoptotic and proliferation indexes in canine lymphoma.

Proliferative and apoptotic fractions of tumors were evaluated in 41 dogs with lymphoma for prediction of response to chemotherapy. All dogs had advanced clinical stage tumors, were untreated prior to study, and received identical induction-remission chemotherapy. Tumor cell proliferation was determined in all pretreatment biopsy specimens and in 18 specimens collected at the time of clinical relapse from remission. Quantitative measures included mitotic index and immunoreactivities for proliferating cell nuclear antigen (PCNA) and Ki-67. Apoptotic index was evaluated from 40 dogs pretreatment and from 16 dogs at the time of first relapse. Pretreatment tumor values for Ki-67, PCNA, and apoptosis were compared with posttreatment values. The median first relapse-free interval (RFI) and overall survival (OS) time were 174 days and 445 days, respectively. Of the proliferation markers, only the results of the Ki-67 analysis were predictive for duration of the first RFI but not OS. Pretreatment apoptotic index was also predictive of the duration of first RFI but not OS. No significant predictive value for comparison of the pretreatment and postrelapse values was demonstrated. Ki-67 labeling index and apoptotic indexes were combined to form both a proliferation/apoptotic ratio (PAR) and a sum, or turnover index. Only the PAR was predictive for duration of first RFI on multivariate analysis. Other variables that were evaluated for their influence on treatment outcome included patient age, weight, gender, clinical stage, clinical substage, and tumor immunophenotype. Of these variables, only immunophenotype was found to be of value for predicting duration of first RFI and OS.

Meningoencephalitis due to a Sarcocystis neurona-like protozoan in Pacific harbor seals (Phoca vitulina richardsi).

Seven Pacific harbor seals with meningoencephalitis associated with Sarcocystis neurona-like protozoa are described. Six of the 7 seals were free-ranging and were found stranded over an 80-km stretch of central California coastline; the other was captive. All had marked to severe nonsuppurative meningoencephalitis, most severe in the cerebellar cortex. Immunohistochemistry for S. neurona antigens was positive on brain tissue in all cases, revealing numerous merozoites as well as developing and mature schizonts, including rosette forms. Electron microscopy performed on 3 animals revealed merozoites and schizonts consistent with Sarcocystis sp., with the absence of rhoptries in merozoites, lack of a parasitophorous vacuole around schizonts, and division by endopolygeny. Serology using western blotting revealed the presence of anti-S. neurona immunoglobulins in the sera of 4 of 5 seals tested. Four animals also had a concurrent mild to moderate nonsuppurative myocarditis; in 1 seal, rare sarcocysts of undetermined species were present within cardiomyocytes.

Canine transmissible venereal tumour: a morphological and immunohistochemical study of 11 tumours in growth phase and during regression after chemotherapy.

Eleven dogs with canine transmissible venereal tumour (CTVT) were given vincristine sulphate chemotherapy to induce tumour regression. Biopsy specimens were collected from tumours during the growth phase, before chemotherapy, and again from the same dogs during the regression induced by chemotherapy. Laboratory assessment included cytology, histology, the number of tumour cells in relation to the number of intratumoral leucocytes, proliferative and apoptotic fractions of tumour cells, intratumoral vessel density, and fibrosis. The results revealed that during regression, tumour cell proliferation ceased, apoptosis increased, leucocytes increased (with increased proportion of T lymphocytes), tumour parenchyma collapsed around intratumoral vessels, and fibrosis increased. These results, which were similar to findings in dogs with spontaneous regression of CTVT, suggest that tumour immunity plays a role in tumour regression after modest chemotherapy.

Pleomorphic rhabdomyosarcoma with pulmonary metastasis in a stranded Steller (northern) sea lion (Eumetopias jubatus).

A Steller (northern) sea lion (SSL), stranded in northern California in July 2000 had an anaplastic pleomorphic rhabdomyosarcoma in the latissimus dorsi muscle, with pulmonary metastasis. Diagnosis was based on light and electron microscopy and immunohistochemistry. Death was attributed to multiple parasitic and bacterial lesions. The SSL is of special concern because, for unknown reasons, the global population has declined by 50% over the last decade. Published post-mortem data, however, are scarce. This case report highlights several disease conditions that affect this species and is the first report of a malignant neoplasm in a free-ranging SSL.

The expression of calretinin and cytokeratins in canine acanthomatous ameloblastoma and oral squamous cell carcinoma.

Oral squamous cell carcinoma (OSCC) and canine acanthomatous ameloblastoma (CAA) represent two epithelium-derived neoplasms that affect the oral cavity of dogs. The expression of cytokeratins (CKs) and calretinin has been previously established in the canine tooth bud and odontogenic tumours. The aim of this study was to characterize the CK and calretinin expression profile of OSCC in comparison to CAA and canine tooth bud tissues. Samples from 15 OSCC and 15 CAA cases, as well as 6 tooth buds and 2 normal gingival tissues were examined. OSCC CK expression was consistent with the CK expression profile of CAA and canine tooth bud tissue. Calretinin was positively expressed in 10 of 15 OSCC cases, with 5 cases demonstrating high staining intensity. Only 2 of 15 CAA cases demonstrated mild-moderate staining intensity. The statistically significant difference in staining pattern and intensity of calretinin in OSCC and CAA can help distinguish between these two tumour types.

Analysis of immune cells within the healthy oral mucosa of specific pathogen-free cats.

The oral mucosa is an important interface for host-environment interactions. Based on previous studies, it is generally accepted that the cellular compartments of the oral immune system comprise organized mucosal-associated lymphoid tissues as well as diffusely and focally distributed T- and to lesser extent B-lymphocytes, oral mucosal Langerhans cells (OMLC), macrophages and mast cells. However, a comprehensive quantification of the cellular elements in the oral mucous membranes of the cat has not been reported. The aim of this study was to provide a comprehensive analysis of the immune cell compartments in the oral mucous membranes and anatomically related tissues of healthy cats. Multiple biopsies of the oral mucous membranes and related tissues were obtained from four specific pathogen-free cats for histological and immunohistochemical assessment of lymphocyte subsets, OMLC, macrophages and mast cells. T-lymphocyte subsets, OMLC, mast cells and macrophages were present in varying frequencies among the tissue compartments of the feline oral cavity. B-lymphocytes were not identified in any of the examined tissues except the tonsils and mandibular lymph nodes. Lymphocytic aggregates (follicles) were found in the palatoglossal folds and the gingiva. We describe the topographical distribution of various leucocyte subsets in the normal healthy feline oral mucosa, and demonstrate regional differences in the distribution of these cells.

Expression of cytokeratins in the epithelium of canine odontogenic tumours.

Odontogenic tumours are considered to be relatively rare; however, several histologically distinct types have been identified in dogs. The more common canine odontogenic tumours are peripheral odontogenic fibroma and canine acanthomatous ameloblastoma. The expression of cytokeratins (CKs) has been established for the human dental germ and odontogenic tumours. The aim of the present study was to describe the immunohistochemical expression of a panel of CKs in the epithelium of the canine dental germ, normal gingiva and odontogenic tumours arising in this species. Samples from 20 odontogenic tumours, 12 tooth germs and three normal gingival tissues were obtained. Each sample was stained with haematoxylin and eosin and subjected to immunohistochemistry for CK expression. The typical expression pattern of CKs in the odontogenic epithelium and gingiva of dogs was CK14 and CK5/6. CKs 7, 8, 18 and 20 were generally absent from the canine dental germ, gingiva and odontogenic tumours. Dogs and man therefore exhibit similar CK expression in the odontogenic epithelium.

Pathological features of amyloidosis in stranded California sea lions (Zalophus californianus).

Amyloidosis was diagnosed in 26 stranded adult California sea lions between 1983 and 2006 by retrospective case analysis. The kidneys (92.3% of animals), blood vessels (80.7%) and thyroid glands (65.4%) were most commonly affected. Macroscopically, affected kidneys were swollen, with pale tan cortices and loss of corticomedullary differentiation. Amyloid deposits in the kidney were located in the glomeruli, blood vessels, and peritubular interstitium, most prominently in the outer stripe of the medulla. The amyloid deposits were identified as type amyloid A (AA) by potassium permanganate staining and immunolabelling with antibodies against AA protein. Concurrent diseases, including inflammatory processes and genital carcinoma, were common in affected animals. Serum amyloid A concentrations were high (>1200 microg/ml) in six of seven affected sea lions, while the median value in clinically healthy animals was <10 microg/ml. These findings suggest that renal amyloidosis contributes to morbidity and mortality in stranded adult California sea lions.

Histologic features of mammary carcinomas in zoo felids treated with melengestrol acetate (MGA) contraceptives.

Melengestrol acetate (MGA), a potent synthetic progestin, has been used as a contraceptive in zoo felids since 1975. Mammary gland carcinomas have been linked to MGA treatment in zoo felids, but the histologic features of these tumors and steroid receptor expression have not been described. Zoo felid mammary tumors were requested from participating zoos from 1986 through 1998, and 31 mammary carcinomas from 28 MGA-treated and 3 untreated felids were received. The carcinomas were evaluated on the basis of histologic pattern, tumor grade, and occurrence of metastasis; then features of the tumors were compared to determine if carcinomas in MGA-treated felids differed from those that occur spontaneously. Estrogen- and progesterone-receptor expression was evaluated in 17 of the 31 carcinomas. Of the 31 tumors, 22 (70.9%) had multiple histologic patterns, 29 (93.5%) were high grade, and 28 (90.3%) had metastasized. Within tumors, the tubulopapillary pattern was most common (87.1%, n = 27); solid (61.3%, n = 19), cribriform (38.7%, n = 12), and comedone (25.8%, n = 8) patterns were less common; and the mucinous (3.2%, n = 1) pattern was rare. Both MGA-treated and untreated zoo felids had similar patterns and grades of mammary gland cancer as well as prevalence of metastasis. These results indicate that mammary carcinomas in zoo felids are high grade with a predominant tubulopapillary pattern and aggressive behavior. Five of 17 carcinomas expressed progesterone receptors, and 1 of 17 expressed estrogen receptors. Although more zoo felids with cancer had been exposed to MGA in this study, mammary carcinomas were similar in appearance and behavior in untreated and MGA-treated zoo felids. The association of MGA with the development of malignant mammary gland tumors should be considered when using this contraceptive in zoo felids.

Development of a new canine osteosarcoma cell line.

Establishing a canine osteosarcoma (OSA) cell line can be useful to develop in vivo and in vitro models of OSA. The goal of this study was to develop, characterize and authenticate a new canine OSA cell line and a clone. A cell line and a clone were developed with standard cell culture techniques from a naturally occurring OSA in a dog. The clonal cell line induced a tumour after injection in RAG 1-deficient mouse. Histology was consistent with OSA. The original tumour from the dog and the tumour induced in the mouse were both reactive with vimentin and osteonectin (ON). The parent cell line and clonal cell line were reactive with ON, osteocalcin and alkaline phosphatase. Loss of heterozygosity was found in the same three microsatellite markers in the parent and clonal cell lines, and the tumour tissue grown in the mouse.

Interstitial lung disease in West Highland White Terriers.

Progressive respiratory failure and pulmonary fibrosis in West Highland White Terriers (WHWT) is an apparently genetic disorder of unknown pathogenesis. This study characterizes the light microscopic, ultrastructural, and immunohistochemical features of affected WHWT in comparison with lesions in usual interstitial pneumonia (UIP) of humans. Lesions in WHWT were confined to the expansion of the interstitial space of alveolar septa by extracellular matrix (ECM) determined to be mixtures of type-I and -III collagens. Features of UIP such as intra-alveolar fibroblastic foci, subpleural distribution, and honeycombing were not observed in six WHWT. Comparison with normal dogs showed no apparent increase in septal myofibroblasts. Ultrastructually, the ECM in alveolar septa consisted of large aggregates of periodic collagen filaments underlying alveolar capillaries that were surrounded by thick bands of amorphous to fine fibrillar matrix. This study suggests that chronic pulmonary disease of WHWT is a result of aberrant collagen regulation.

Characterization of the cutaneous inflammatory infiltrate in canine atopic dermatitis.

Sections from lesional atopic, clinically normal atopic, and normal canine skin were investigated by light microscopy and an immunoperoxidase method using monoclonal antibodies specific for canine leukocyte antigens. We confirmed that skin-infiltrating cells of canine atopic dermatitis are constituted of mast cells, dendritic antigen-presenting cells, memory helper T-lymphocytes, low numbers of eosinophils and neutrophils, and rare B-lymphocytes. The presence of epidermal eosinophil microaggregates and clustered Langerhans' cells supports the hypothesis of epidermal allergen contact. The hyperplasia of epidermal T-cells expressing the gamma/delta T-cell receptor appears specific to canine atopic dermatitis compared with its human counterpart. This finding could be explained by an interspecies difference in skin immune systems or, alternatively, by an active participation of these epitheliotropic gamma/delta T-cells in the cutaneous allergic immune response in dogs. The paucity of dermal neutrophils in spontaneous lesions of canine atopic dermatitis is notably different from the neutrophil-rich late-phase reactions provoked by intradermal allergen injections in allergic dogs. This difference in the cellular infiltrate probably results from variations in the immune reaction between single and repeated allergen exposure as well as epidermal versus dermal antigen contact.

Cytokeratin-filament expression in epithelial and non-epithelial tissues of the common carp (Cyprinus carpio).

Cytokeratin expression in mammals is generally restricted to epithelial cells and has been utilized to differentiate epithelial from nonepithelial tissues in these species. Since cytokeratins have been shown to be highly conserved during vertebrate evolution, the objective of the present study has been to ascertain the expression pattern of cytokeratins in tissues of the common carp (Cyprinus carpio). A panel of 10 anti-human cytokeratin antibodies was evaluated using a streptavidin-biotin-peroxidase complex detection system. Tissues were fixed in 10% neutral-buffered formalin, 100% ethanol or methacarn. Only formalin-fixed tissues were pre-digested with trypsin prior to immunostaining. Formalin-fixed tissues generally resulted in a less intense, more diffuse staining pattern with considerable background compared with ethanol and methacarn and was therefore the least desirable fixative. The diverse staining pattern observed with the various antibodies used in this study was consistent with previous findings in other teleosts. The results confirm that cytokeratin expression in teleosts is fundamentally different from that in mammals and therefore should be used as a method to differentiate epithelial cell types in these species only with discretion.

Rapid identification of tissue micro-organisms in skin biopsy specimens from domestic animals using polyclonal BCG antibody.

Immunostaining with polyclonal anti-Mycobacterium bovis (BCG) was evaluated as a single screening method for the histological identification of micro-organisms in skin biopsy specimens from various veterinary species. Confirmed archival cases infected with Mycobacteria, Nocardia, Actinobacillus, Actinomyces, Streptococcus/Staphylococcus, Dermatophilus, spirochetes, Blastomyces, Coccidioides, Cryptococcus, Histoplasma, dermatophytes, Malassezia, Sporothrix, Leishmania, Pythium, phaeohyphomycetes and Prototheca organisms were selected. A total of 70 skin biopsy specimens from the dog, cat, horse, ox and llama were evaluated. The anti-BCG immunostain labelled bacteria and fungi with high sensitivity and minimal background staining but did not label spirochetes and protozoa (Leishmania). Differences were not noted between veterinary species. The results indicate that immunostaining with polyclonal anti-BCG is a suitable screening technique for the rapid identification of most common bacterial and fungal organisms in paraffin-embedded specimens. Also, mycobacterial and nocardial organisms were identified more readily with the anti-BCG immunostain in comparison to the histochemical stains.