Differences in second-intention wound healing between horses and ponies: histological aspects.

The histological aspects of second-intention healing were studied in 5 horses and 5 ponies. Biopsies were taken weekly from standardised wounds on the metatarsus and femoral biceps muscle of one horse and one pony. Sections were stained to enable cell counting and the detection of DNA synthesis, fibrin, smooth muscle actin (SMA), collagen, and bacteria. In the ponies, the number of polymorphonuclear leucocytes (PMNs) was high during the first 3 weeks and subsequently decreased rapidly. In the horses, the initial number of PMNs was lower, but remained persistently elevated during the evaluation period. PMNs were found mainly in the superficial zones. Significantly more fibrin was present in the wounds of the horses. No significant differences were observed in the number of fibroblasts, the amounts of SMA and collagen. However, myofibroblasts were significantly less regularly organised in the wounds of the horses, particularly in the metatarsal wounds. The mitotic activity of the epithelium was temporally reduced in week 3. The mitotic activity of the granulation tissue was initially high but declined rapidly from week 1 onwards, with the exception of the metatarsal wounds of the horses, in which mitotic activity remained significantly higher. Histology confirmed and explained the macroscopical differences in wound healing between horses and ponies by the strict organisation of the myofibroblasts and the more effective acute inflammation in the ponies. Stimulation of the organisation of myofibroblasts and improvement of the efficacy of the inflammatory response in horses may therefore result in better second-intention wound healing in horses in clinical practice.

Non-exfoliative canine cytology: the value of fine needle aspiration and scraping cytology.

The results of the cytological and histological examination of 348 canine lesions, localised in various organs, were compared with respect to the tumourous or non-tumour nature of the lesions and the malignancy or benignancy of tumours. The retrieval rate was 92.5%. Regarding the distinction between tumourous and non-tumourous lesions, the cytological examination showed a diagnostic accuracy of 83.9%, a sensitivity of 95.6%, a specificity of 65.4% and a predictive value for the presence of tumour of 93.5%. The diagnostic accuracy of cytology concerning the differentiation in malignancy and benignancy of the neoplasms was 83.7%, with a sensitivity of 86.8%, a specificity of 79.4% and a predictive value for the presence of malignant tumour of 85.6%. These results confirm the value of non-exfoliative cytology as a diagnostic method, providing rapid and valuable information with regard to diagnosis and prognosis and, consequently, for therapeutic handling. An eventual histological diagnosis remains indicated, especially in case of soft-tissue and mammary lesions.

Immunohistochemistry with keratin, vimentin, desmin, and alpha-smooth muscle actin monoclonal antibodies in canine mammary gland: normal mammary tissue.

Normal canine mammary gland tissue was studied immunohistochemically with monoclonal antibodies (MoAbs) directed against various human keratin types, vimentin, desmin, and alpha-smooth muscle actin. Both ductal and alveolar luminal cells were immunoreactive with MoAbs recognizing respectively human keratins no. 7, 8, 18 and 19. In addition, some ductal luminal cells were labelled with a keratin 4 and a keratin 10 MoAb. Basal/myoepithelial cells were immunoreactive only with MoAbs directed against keratin 14, keratins 14 and 17, and alpha-smooth muscle actin. The vimentin MoAb merely labelled solitary loose intraluminal cells representing macro-phages or sloughed epithelial cells. These findings correspond largely to observations made in human breast tissue.

Immunohistochemistry with keratin, vimentin, desmin, and alpha-smooth muscle actin monoclonal antibodies in canine mammary gland: benign mammary tumours and duct ectasias.

Duct ectasias (n = 2) and different types of benign canine mammary tumours (n = 19) were studied immunohistochemically with monoclonal antibodies (MoAbs) directed against various human keratin types (K), alpha-smooth muscle actin, vimentin, and desmin. In the duct ectasias and in most tumours the epithelial structures revealed an inner and outer cell layer. The inner cell layer was characterized by labelling with K 7, 8, 18, 19 and mostly also with K 4 and/or K 10 MoAbs. The outer cell layer was almost invariably labelled by K 14, K 14 and 17, and a-smooth muscle actin MoAbs. The labelling patterns of both duct ectasias and tumours corresponded largely to the patterns observed in normal mammary gland tissue, although a more distinct heterogeneity was seen. Tumours histomorphologically assumed to be of a myoepithelial origin did not show immunohistochemical features of myoepithelial cells. The myoepithelial nature of the vast majority of spindle-shaped cells present in the adenomas of the complex type and in the fibroadenomas of the benign mixed type could not be confirmed immunohistochemically. These cells, however, unequivocally expressed vimentin, suggesting proliferation of stromal cells in these tumours, which in the fibroadenomas of the benign mixed type may show metaplasia to bone or cartilage. In the duct ectasias and in some tumours, a fraction of elongated stromal cells, probably representing myofibroblasts, was labelled with the alpha-smooth muscle actin MoAb.

Immunohistochemistry with keratin, vimentin, desmin, and alpha-smooth muscle actin monoclonal antibodies in canine mammary gland: malignant mammary tumours.

Ten malignant canine mammary gland tumours and five metastases from three of these tumours were studied immunohistochemically with monoclonal antibodies (MoAbs) directed against different human keratin types (K), alpha-smooth muscle actin, vimentin, and desmin. In all tumours the neoplastic epithelium was rather homogeneously labelled with the keratin MoAbs RCK 102 (K 5 and 8) and CAM 5.2 (K 8). The adenocarcinomas (n = 5), the solid carcinomas (n = 2), and the carcinosarcoma (n = 1) showed heterogeneous labelling with the MoAbs specific for luminal cell antigens in the normal canine mammary gland, i.e., K 18, K 7 and K 19 MoAbs. These cells were also immunoreactive with K 4 and K 10 MoAbs. The spindle cell carcinomas (n = 2), however, did not react with these MoAbs. All tumours except one adenocarcinoma were characterized by the absence of immunoreactive labelling with the alpha-smooth muscle actin MoAb. In the solid carcinomas this was associated with the absence of labelling with one or both basal cell specific keratin MoAbs, i.e., 8.7 (K 14 and 17) and RCK 107 (K 14), respectively. In contrast, the other malignant tumours showed marked labelling of neoplastic epithelium with these MoAbs. Another remarkable finding was the labelling of a limited to moderate number of neoplastic epithelial cells with the vimentin MoAb. The presence of such labelling patterns in canine mammary gland tumours may be indicative of malignancy. Metastatic tumour tissues had a labelling pattern largely similar to that of the primary tumour, although also loss of reactivity for some keratin MoAbs was seen.

Distribution of the intermediate filament proteins vimentin, keratin, and desmin in the bovine ovary.

The distribution of the intermediate filament (IF) proteins desmin, keratin, and vimentin was studied immunohistochemically in bovine ovaries. Special attention was paid to granulosa cells to examine possible marked changes of IF distribution in relation to folliculogenesis during ovarian development. Therefore, ovaries were used from fetuses from 3 months of gestation onward, calves, heifers, and cows. In all ovaries, desmin immunoreactivity was restricted to smooth muscle cells in blood vessel walls. Keratin appeared a characteristic of the ovarian surface epithelium. Co-localization of keratin and vimentin was observed in the epithelium of rete ovarii tubules in fetuses and calves, and in cortical cord epithelium and pregranulosa cells of primordial follicles in fetuses at 3-7 months of gestation. Vimentin was demonstrated in endothelium and in fibroblasts. In addition, vimentin immunoreactivity was present in granulosa cells of primary, secondary, and antral follicles. In antral follicles, these granulosa cells mainly had an elongated appearance and either contained an oblong or a round nucleus. Those with an oblong nucleus were characteristic for atretic antral follicles. In nonatretic follicles, numerous vimentin immunoreactive, elongated granulosa cells with a round nucleus were observed, especially in the peripheral granulosa layer and in small ( < 3 mm in diameter) antral follicles. Additionally, in antral follicles, protrusions of vimentin-positive corona radiata cells were observed, that penetrated the zona pellucida to contact the oocyte. The data show that the distribution of vimentin containing IFs is associated with various aspects of granulosa cell activity, as mitosis, atresia, and intercellular transport.

Keratin and vimentin distribution patterns in the epithelial structures of the canine anal region.

The intermediate filament labeling pattern of the epithelial structures of the canine anal region was studied with different polypeptide specific keratin monoclonal antibodies (MoAbs) and with a monoclonal and polyclonal vimentin antibody. The epithelial structures in this region could be discriminated and characterized by differences in their keratin staining pattern. The basal cells in the different epithelial structures showed a similar staining pattern characterized by reactivity with MoAbs staining keratins 5, 8, 14, and 17. Columnar epithelial cells showed a completely different phenotype mostly characterized by reactivity with MoAbs staining keratins 7, 5, 8, 18, and 19. A restricted number of differentiated perianal gland cells showed perinuclear vimentin staining. Myoepithelial cells did not stain for vimentin, but, as other basal cells, were positive for MoAbs staining keratins 5, 8, 14, and 17.

Immunohistochemistry with keratin and smooth muscle actin monoclonal antibodies in canine digestive tract and extramural glands.

The canine digestive system and its extramural glands (parotid gland, liver, pancreas) were immunohistochemically studied using a panel of twelve monoclonal antibodies (MoAbs) specific for human keratin proteins and for alpha-smooth muscle actin. Various epithelial tissues and cells were characterized by different keratin staining patterns. So, the epithelial lining of the upper alimentary tract was characterized by staining with the MoAb 6B10, specific for keratin-type (K) 4, and the absence of staining with the MoAbs directed against K 8 and 18 (CAM 5.2 and RGE 53, DE-K18 respectively), whereas the lower alimentary tract epithelium was not labeled by 6B10, but stained by the latter MoAbs. In the salivary glands the luminal and basal cells of the adenomeres as well as the different ductal structures could be immunohistochemically differentiated. The duct epithelium in liver and pancreas showed next to keratin staining characteristics in common with hepatocytes and exocrine pancreatic cells, additional staining by several keratin MoAbs. The keratin staining patterns in the canine tissues showed, in addition to similarities also distinct discrepancies when compared to the staining patterns in corresponding human tissues. Myoepithelial cells in salivary and oesophageal glands could be differentiated from other basally located epithelial cells by their exclusive immunoreactivity for alpha-smooth muscle actin. Canine pancreatic endocrine cells were not labeled by any of the keratin MoAbs. It is concluded that immunohistochemistry with polypeptide specific MoAbs specific for human keratin-types can be used to differentiate between different types of canine epithelial tissues and epithelial cells in the digestive tract. As a result such reagents may find their application in developmental biology and pathology of this species.

Immunohistochemistry with keratin monoclonal antibodies in canine tissues: urogenital tract, respiratory tract, (neuro-)endocrine tissues, choroid plexus and spinal cord.

Twelve oligo- or monospecific monoclonal antibodies (MoAbs) directed against human keratin types were used in an immunohistochemical study of the canine male and female urogenital tract, the respiratory tract, the adrenal gland, the (para-)thyroid gland, the choroid plexus and the spinal cord. The keratin MoAbs showed differences in staining patterns in the various epithelial tissues and the diverse epithelial cells. The kidney was characterized by a complex keratin staining pattern and the canine urothelium showed regional differences in keratin staining. Also in the female genital tract different keratin staining patterns were observed. Testicular and adrenal gland cells did not react with any of the keratin MoAbs. The keratin staining patterns in the various canine tissues showed, in addition to similarities, also distinct differences when compared to the staining patterns in corresponding tissues of other species, e.g. of man. These staining dissimilarities indicate that the reactivity patterns of the keratin MoAbs with restricted keratin immunoreactivity can not be always extrapolated from one species to another. Nevertheless, MoAbs directed against human keratin proteins can apparently be used to differentiate between various types of canine epithelia or epithelial compartments.

Keratin staining of canine epithelial tissues by a polyclonal antiserum.

The keratin distribution pattern in various canine epithelial tissues has been studied using a commercially available rabbit antiserum, raised against human skin keratins, in an immunoperoxidase staining method with the peroxidase-antiperoxidase complex (PAP). The staining results of two fixation methods were compared. Paraffin sections after fixation in Carnoy's solution showed optimal results, whereas paraffin sections after fixation in 10% buffered formalin resulted in a strongly reduced keratin staining reaction. Keratinizing and non-keratinizing stratified epithelial showed a strong staining reaction. Glandular epithelium and the non-stratified epithelia of internal organs e.g. liver, kidney and intestine did not react with the antiserum. However, glandular ductal epithelium, myoepithelial cells and basal cells in various epithelial tissues showed a positive staining reaction. The results indicate the presence of different keratin types in these canine epithelial tissues. The keratin distribution pattern is compared with the distribution pattern observed in tissues of other species.

An immunohistochemical study of canine tissues with vimentin, desmin, glial fibrillary acidic protein, and neurofilament antisera.

In a wide range of canine tissues the immunoreactivity with commercially available antisera against intermediate filament antigens viz. vimentin, desmin, glial fibrillary acidic protein (GFAP) and neurofilament proteins, was studied. In addition, the results of formalin and Carnoy fixation were compared. Carnoy fixation appeared to result in optimal reactivity for all antisera. Epithelial cells did not react with any of the antisera, with exception of ovarian surface epithelium, which showed staining with the vimentin and desmin antisera. The vimentin antiserum induced staining of several cell types viz. fibroblasts, endothelial cells, chondrocytes, Schwann cells, ependymal cells, astrocytes, Leydig cells, synovial cells, podocytes and some parietal cells of Bowman's capsule. Sertoli cells showed a faint staining reaction. Muscle cells in various tissues reacted with the desmin antiserum. In the kidney a varying number of parietal cells appeared to react as did a restricted number of epithelial cells of proximal tubules and loops of Henle. GFAP reactivity was confined to glial cells, predominantly fibrous astrocytes, Schwann cells and axons. Additionally, some neuronal cell bodies in peripheral ganglia showed staining of varying intensity. Neurofilament staining was restricted to axons and some neurons. The immunoreactivity of canine tissues with these antisera is compared to findings in other species. The results confirm a broad interspecies cross-reactivity of these antisera. They can be used in studying the nature of canine tissues.

The expression of keratins, vimentin, neurofilament proteins, smooth muscle actin, neuron-specific enolase, and synaptophysin in tumors of the specific glands in the canine anal region.

Eight canine tumors originating from specific glandular structures in the anal region, as well as metastatic tumor tissue of two of these cases (case Nos. 7, 8), were immunohistochemically analyzed using various monoclonal antibodies (MoAbs) directed against human keratin types, vimentin, neurofilament proteins, and alpha-smooth muscle actin. These tumors also were stained for the broad-spectrum neuroendocrine markers neuron-specific enolase (NSE) and synaptophysin. In histologically normal canine anal structures, alpha-smooth muscle actin and NSE antibodies stained basally localized (probably myoepithelial) cells in the anal glands and the anal sac glands. NSE staining also was present in a limited number of luminal cells in both anal glands and anal sac glands. Synaptophysin labeling was not observed in any of these glandular structures. Histologically, the tumors were differentiated into well- and moderately differentiated perianal gland tumors (n = 5) and carcinomas without perianal gland differentiation (n = 3), corresponding to the so-called apocrine carcinomas of the anal region. Immunohistochemically, the perianal gland tumors could be differentiated from the carcinomas by marked differences in staining pattern with the various keratin MoAbs, particularly MoAbs directed against human keratin types 7 and 18. The keratin-staining characteristics of the carcinomas suggest a glandular luminal cell origin. Metastases of the carcinomas showed loss of some keratin-staining characteristics as compared with the primary tumor. Staining for NSE was only observed in solitary cells and small cell clusters in the carcinomas and their metastases, whereas the alpha-smooth muscle actin antibody did not react with the carcinoma cells. None of the tumors stained for neurofilament proteins or synaptophysin. An unequivocal neuroendocrine nature of the carcinomas could not be substantiated by our immunohistochemical study, although the presence of a population of neuroendocrine cells within these neoplasms seems likely. Because the immunohistochemical features of the carcinomas with respect to various keratin MoAbs and NSE are similar to those of the anal glands and the anal sac glands, both these glands might be considered as site of origin of these carcinomas.

Rhabdomyosarcomas in young pigs in a swine breeding farm: a morphologic and immunohistochemical study.

Within a 6-month-period, solitary or multiple tumors were observed in 25 young pigs in their first weeks of life in a swine breeding farm. The herd comprised approximately 100 animals, and affected pigs were observed in several litters. The number of affected littermates varied from one to three. Five animals, all from different litters and with a total of 11 tumors, were studied. Histologically the tumors were classified as undifferentiated sarcomas. Electron microscopic examination of the tumors (n = 3) revealed myogenic differentiation, characterized by the presence of numerous cytoplasmic filaments with longitudinal densities and cytoplasmic dense bodies. Immunohistochemically, all 11 tumors were labeled by vimentin and desmin antibodies. Two tumors from which frozen material was available were additionally labeled by a titin antibody but did not show immunoreactivity with antibodies directed against myosin and alpha-sarcomeric actin. The tumors were finally diagnosed as undifferentiated rhabdomyosarcomas. The high incidence of these tumors within a short period of time in multiple young animals in different litters indicates a common causative event. The clinical history suggests a genetic cause.