Immunohistochemical and histomorphometric evaluation of vascular distribution in intact canine cranial cruciate ligament.

OBJECTIVES: To (1) describe vascular distribution in the grossly intact canine cranial cruciate ligament (CCL) using immunohistochemical techniques specific to 2 components of blood vessels (factor VIII for endothelial cells, laminin for basement membrane); and (2) compare the vascularity in different areas of interest (craniomedial versus caudolateral bands; core versus epiligamentous regions; and proximal versus middle versus distal portions) in the intact normal canine CCL. STUDY DESIGN: In vitro study. ANIMALS: Large, mature dogs (n=7) of breeds prone to CCL disease that were euthanatized for nonorthopedic conditions. METHODS: Intact CCL were collected from fresh canine cadavers free from stifle pathology. CCL tissue was processed for immunohistochemistry and stained for factor VIII and laminin. Vascular density was determined by histomorphometric analysis. RESULTS: Specific vascular staining was sparsely identified throughout the CCL; however, the proximal portion of the CCL appears to have a greater number of vessels than the middle or distal portion of the ligament. CONCLUSIONS: The CCL is a hypovascular tissue and its vascular distribution is not homogeneous.

Vascular distribution in ruptured canine cranial cruciate ligament.

OBJECTIVE: To (1) determine the microanatomic vascular distribution in ruptured canine cranial cruciate ligaments (CCL) using specific vascular immunohistochemical techniques, and (2) compare vessel density between ruptured and intact canine CCL and between different areas of interest in ruptured CCL using histomorphometric analysis. STUDY DESIGN: In vitro study. ANIMALS: Dogs (n=41) admitted for surgical treatment of ruptured CCL and 19 dogs euthanatized for nonorthopedic conditions. METHODS: Diseased (variable CCL rupture) and intact (normal control) CCL were processed for immunohistochemical staining specific to vessels (factor VIII, laminin). Mean vascular density was assessed and compared in areas of interest (torn end versus remaining core regions of CCL, proximal femoral versus distal tibial core CCL regions). RESULTS: Ruptured CCL was more vascular than intact CCL; however there was no difference in vascular density between the torn end and the remaining core area of the ruptured CCL. Ruptured CCL was vascularized to a greater degree at the proximal portion than the distal portion of the CCL. Partially ruptured CCLs had a higher vessel density than completely ruptured CCLs. CONCLUSIONS: Vascular density is increased in diseased CCL compared with intact CCL. It remains to be determined whether this finding is associated with the cause of CCL rupture or is a result of CCL degeneration and rupture.

Investigation of immune cell markers in feline oral squamous cell carcinoma.

Squamous cell carcinoma is the most common oral cancer in the cat and presents as a locally aggressive lesion for which an effective therapeutic protocol remains elusive. Feline oral squamous cell carcinoma (OSCC) shares many clinical characteristics with human head and neck squamous cell carcinoma (HNSCC). Accordingly, present studies were conducted to determine similarities for immune markers shared by feline OSCC and human HNSCC. Biopsies harvested from a feline patient cohort-1 (n = 12) were analyzed for lymphoid cell infiltrates by immunohistochemistry (IHC). Results revealed unique patterns of T cell infiltration involving both neoplastic epithelium and stroma that were detected in most patient tumor biopsies (92%) examined by IHC staining for CD3. Intratumoral B cell infiltrates were detected within tumor stroma only, based on IHC staining for CD79a and CD20 for all patients within the same cohort-1. Infiltration of tumors by a regulatory CD4 T cell subset (Tregs) defined by expression of the forkhead transcription factor FoxP3, was also detected in biopsies from 57% of patients and involved infiltration of neoplastic epithelium and stroma. Patient biopsies were also examined for expression of immunomodulator cyclooxygenase (COX)-2 and revealed positive but weak staining of neoplastic epithelium in a significant proportion of cases (75%). Interestingly, COX-2 expression was detected in both neoplastic epithelium and stroma. Blood collected from a second cohort of feline OSCC patients (n = 9) revealed an increased frequency of circulating CD4+FoxP3+ T cells when compared to healthy adult controls (n = 7) (P = 0.045), although frequencies of CD4+CD25+FoxP3+ T cells were comparable between patients and healthy pet cat controls. Lastly, biopsies from feline OSCC patients were characterized for histologic subtype using a classification scheme previously described for human HNSCC. This analysis revealed the conventional subtype as the predominant variant (75%) with conventional subtypes split evenly between well differentiated and moderately differentiated carcinomas. Two cases were classified as papillary and one case as basaloid subtypes. Correlations between subtype, immune marker scores or circulating Treg frequencies and clinical characteristics or outcome were not detected, most likely due to small patient numbers within patient cohorts. However, findings from these studies provide a preliminary step in the characterization of immune and histologic markers that will be critical to defining prognostic immune markers for feline OSCC and potential targets for testing of immunotherapeutics also relevant to human HNSCC in future studies.

Microchip-associated leiomyosarcoma in an Egyptian fruit bat (Rousettus aegyptiacus).

Microchips are commonly used in companion, research, and zoo animal medicine for easy, reliable, and cost-effective identification with relatively low risk of side effects. This report describes development of a metastatic leiomyosarcoma associated with a microchip in an Egyptian fruit bat (Rousettus aegyptiacus).

Computer-assisted image analysis of neovascularization in thyroid neoplasms from dogs.

OBJECTIVE: To develop a computer-assisted image analysis procedure for quantitation of neovascularization in formalin-fixed paraffin-embedded specimens of thyroid gland tissue from dogs with and without thyroid gland neoplasia. SAMPLE POPULATION: 47 thyroid gland carcinomas, 8 thyroid gland adenomas, and 8 specimens of thyroid tissue from dogs without thyroid gland abnormalities (normal). PROCEDURE: Serial tissue sections were prepared and stained with antibodies against human CD31 or factor VIII-related antigen (factor VIII-rag). The areas of highest vascularity were identified in CD31-stained sections, and corresponding areas were then identified in factor VIII-rag-stained sections. Image analysis was used to calculate the total vascular density in each section, and neovascularization, expressed as a percentage, was determined as the absolute value of the total vascular density derived from factor VIII-rag-stained sections minus the vascular density derived from CD31-stained sections. RESULTS: Mean vascular density of thyroid gland carcinomas derived from CD31-stained sections was significantly greater than density derived from factor VII I-rag-stained sections. This incremental difference was presumed to represent degree of neovascularization. However, significant differences were not detected between vascular densities derived from CD31 and factor VIII-rag-stained sections for either normal thyroid gland tissue or thyroid gland adenomas. No significant correlations were found between vascular density in thyroid gland carcinomas and survival time following surgery. CONCLUSION AND CLINICAL RELEVANCE: A computer-assisted image analysis method was developed for quantifying neovascularization in thyroid gland tumors of dogs. This method may allow identification of dogs with tumors that are most likely to respond to treatment with novel antiangiogenesis agents.

Leukocyte differentiation antigens in canine cutaneous and oral plasmacytomas.

Seventeen cutaneous and oral tumours with light microscopic features of plasmacytomas from 16 dogs were studied. Clinically, most neoplasms were benign, although three recurred after excision and three were locally invasive. Tumours most often arose on the pinnae, digits, gingiva, and inguinal regions near areas of chronic inflammation and exhibited variable degrees of plasmacytic differentiation microscopically. Diagnosis of plasmacytoma was confirmed in paraffin-embedded tissues with a panel of leukocyte differentiation antigen markers that included cross-reactive antibodies for Mb-1 (CD79a), CD3, and vimentin and canine-specific antibodies for CD45RA and CD18. Immunoreactivity for Mb-1 and CD45RA, including staining of multinucleate cells and cells with karyomegaly, confirmed a B-cell origin of neoplasms, while staining for CD3 and CD18 revealed an extensive network of infiltrative T-cells and dendritic cells in tumours suggestive of a directed immune response. These findings (i) demonstrate the value of using a panel of antibodies for leukocyte antigens to differentiate plasmacytomas from other cutaneous and oral round cell tumours, and (ii) suggest that immune recognition and responsiveness within tumours may play a role in the behaviour of plasmacytomas in dogs by affecting tumour cell growth and differentiation.

Antifollicular cell-mediated and humoral immunity in canine alopecia areata.

Abstract A combination of cellular and humoral immunological assaults directed against follicular matrical cells is suspected to cause alopecia areata (AA) in humans. The specific aims of this study were to determine whether cytotoxic T lymphocytes (CTL) were increased in the blood and skin lesions of canine AA. Additionally, we wished to determine if circulating antifollicular antibodies were present in the dog. Finally, we aimed to investigate whether bulbar inflammation was associated with decreased matrical cell proliferation and keratinocyte apoptosis. Canine AA lesions were infiltrated by intrabulbar CTL, perifollicular helper T cells and dendritic cells. A higher percentage of CTL was present in canine AA peripheral blood compared with that of normal dogs. Tissue-fixed and circulating antifollicular IgG antibodies were detected. Reduced matrical proliferation and increased keratinocyte apoptosis was observed in inflamed hair bulbs. This study demonstrates the existence of antifollicular cell-mediated and humoral immunological responses in canine AA. Résumé- L'association d'agressions immunologiques cellulaires et humorales dirigées contre les cellules matricielles folliculaires est suspectée d'être à l'origine de l'Alopécie Areata chez l'homme. Le but de cette étude était de montrer si le nombre de lymphocytes T cytotoxiques est augmenté dans le sang et dans les lésions cutanées lors d'Alopécie Areata chez le chien. En plus, nous souhaitions montrer la présence d'anticorps circulants antifolliculaire chez le chien. Enfin, nous voulions voir si l'inflammation du bulbe pileux était associée à une diminution de la prolifération des cellules matricielles et une apoptose des kératinocytes. Lors d'Alopécie Areata chez le chien, existe une infiltration lésionnelle par des lymphocytes T cytotoxiques intrabulbaires, des lymphocytes T auxiliaires périfolliculaires et des cellules dentritiques. Le pourcentage de lymphocytes T cytotoxiques était plus important dans le sang périphérique provenant de sujets à Alopécie Areata que dans le sang d'animaux sains. Par ailleurs, des anticorps antifolliculaire (IgG) circulants et fixés dans les tissus sont détectés. Une réduction de la prolifération des cellules matricielles et une augmentation de l'apoptose des kératinocytes sont observées dans les bulbes pileux atteints. Cette étude démontre l'existence de réponses immunologiques cellulaires et humorales contre le follicule pileux dans l'Alopécie Areata du chien. [Olivry, T., Moore, P.F., Naydan, D.K., Puget, B.J., Affolter, V.K., Kline, A.E. Antifollicular cell-mediated and humoral immunity in canine alopecia areata (Immunité contre le follicule pileux dans l'Alopécie Areata du chien). Veterinary Dermatology 1996; 7: 67-79.] Resumen Se cree que la alopecia areata (AA) en la especie humana resulta de la combinación de la acción inmunológica celular y humoral contra células de la matriz folicular. Los objetivos especificos de este estudio fueron determinar si los linfocitos T citotóxicos (CTL) se encontraban aumentados en sangre y en las lesiones cutáneas de animales con AA. Además, descamos investigar la posible presencia de anticuerpos antifoliculares circulantes en el perro. Finalmente, nos propusimos investigar si la inflamación bulbar se encontraba asociada a una disminución en la proliferación de células de la matriz y a la apoptosis de queratinocitos. Las lesiones de la AA canina mostraban infiltración por CTL intrabulbares, células T colaboradoras y células dendríticas. Se encontró un porcentaje mayor de CTL en la sangre periférica de perros con AA respecto a perros normales. Se detectaron anticuerpos IgG antifoliculares circulantes y en tejido fijado. Se observó una disminución en la proliferación matrical y un aumento en queratinocitos apoptóticos en los bulbos foliculares inflamados. Este estudio demuestra la existencia de respuestas inmunitarias antifoliculares de tipo celular y humoral en la AA canina. [Olivry, T., Moore, P.F., Naydan, D.K., Puget, B.J., Affolter, V.K., Kline, A.E. Antifollicular cell-mediated and humoral immunity in canine alopecia areata (Immunidad antifolicular en la alopecia areata canina). Veterinary Dermatology 1996; 7: 67-79.] Zusammenfassung- Eine Kombinaton von zellulären und humoralen immunologischen Angriffen, die gegen die follikulären Matrixzellen gerichtet sind, wird als Ursache bei der Alopecia areata (AA) des Menschen vermutet. Die besondere Absicht dieser Studie war, festzustellen, ob die zytotoxischen T-Lymphozyten (CTL) im Blut und in den Hautveränderungen bei kaniner AA erhöht sind. Zusätzlich wollten wir feststellen, ob beim Hund zirkulierende antifollikuläre Antikörper vorkommen. Schließlich beabsichtigten wir, zu untersuchen, ob die bulbäre Entzündung mit verminderter Matrixzellproliferation und Keratinozytenapoptose einhergeht. Die Veränderungen bei kaniner AA wurden von intrabulbären CTL, perifollikulären T-Helferzellen und dendritischen Zellen infiltriert. Verglichen mit dem peripheren Blut gesunder Hunde kam bei kaniner AA eine höherer Prozentsatz an CTL vor. Es wurden gewebsfixierte und zirkulierende antifollikuläre IgG-Antikörper festgestellt. In entzündeten Haarbulbi wurde eine reduzierte Matrixproliferation und eine erhöhte Keratinozytenapoptose beobachtet. Diese Studie zeigt die Existenz von antifollikulären zellvermittelten und humoralen immunologischen Reaktionen bei kaniner AA. [Olivry, T., Moore, P. F., Naydan, D. K., Puget, B. J., Affolter, V. K., Kline, A. E. Antifollicular immunity in canine alopecia areata (Antifollikuläre Immunität bei der Alopecia areata des Hundes). Veterinary Dermatology 1996; 7: 67-79.].

Lymphoma with cutaneous involvement in three domestic rabbits (Oryctolagus cuniculus).

Three domestic rabbits (Oryctolagus cuniculus) with cutaneous lymphoma are described. Two rabbits were young (7 weeks and 1 years) and were euthanized within 1 week of showing clinical signs. Lymphoma was found in the skin and internal organs. The third rabbit was 9.5 years of age, and lived for more than 1 year after diagnosis. No response was seen to either 2 months of alpha-interferon administration or a 2.5-week course of isotretinoin treatment. After 1 year the rabbit died suddenly; the owner refused necropsy. Immunologic stains of the tumour in all three rabbits showed T cells to be the lymphoma cell type.

Expression of cyclooxygenase genes in the jejunum of horses during low-flow ischemia and reperfusion.

OBJECTIVE: To determine expression of cyclooxygenase (COX) genes 1 and 2 (also called prostaglandin-endoperoxide synthases 1 and 2) and stability of housekeeping gene expression during low-flow ischemia and reperfusion in the jejunum of horses. ANIMALS: 5 healthy adult horses. PROCEDURES: Horses were anesthetized, and two 30-cm segments of jejunum were surgically exteriorized. Blood flow was maintained at baseline (untreated) values in 1 (control) segment and was decreased to 20% of baseline (low-flow ischemia) for 75 minutes, followed by 75 minutes of reperfusion, in the other (experimental) segment. Biopsy samples were collected from experimental segments at baseline (T0), after 75 minutes of ischemia (T1), and after 75 minutes of reperfusion (T2); samples were collected from control segments at T0 and T2. Horses were euthanized 24 hours after induction of ischemia (T3), and additional samples were collected. Samples were evaluated histologically. Total RNA was extracted; expression of COX genes and stability of 8 housekeeping genes were determined via quantitative real-time PCR assays. RESULTS: COX-1 and COX-2 genes were constitutively expressed in baseline samples. Low-flow ischemia resulted in significant upregulation of COX-2 gene expression at each subsequent time point, compared with baseline values. The most stably expressed reference genes were ß-actin and hypoxanthine phosphoribosyltransferase, whereas glyceraldehyde 3-phosphate dehydrogenase and ß-2 microglobulin were the least stably expressed. CONCLUSIONS AND CLINICAL RELEVANCE: Low-flow ischemia resulted in upregulation of COX-2 gene expression in the jejunum of horses. Housekeeping genes traditionally used as internal standards may not be stable in this tissue during arterial low-flow ischemia and reperfusion.

The normal genital tract of the female California sea lion (Zalophus californianus): cyclic changes in histomorphology and hormone receptor distribution.

Changes in reproductive tract histomorphology, and estrogen (ERalpha) and progesterone receptor (PR) expression throughout the breeding cycle were evaluated in free-ranging stranded female California sea lions (Zalophus californianus). Hormone receptor expression in the ovaries, uterus, cervix, and vagina was evaluated using an immunohistochemical technique with monoclonal antibodies. During a large portion of the cycle, ovaries contained both a corpora lutea (CL) and follicles in varying stages of development. In the periods of pupping and estrus during June and July, and in the spring morphologic features of the endometrium suggested estrogen influence. There were areas of squamous differentiation in the pseudostratified columnar epithelium of the cervix and vagina in some animals during estrus and in the spring. Estrogen receptor immunohistochemical scores were highest during pupping and estrus and in the spring and lowest during embryonic diapause. Cyclic changes in uterine PR expression throughout the cycle were minimal. Both ERalpha and PR were expressed in epithelial and stromal cells throughout the cervix and vagina, however, receptor expression was typically higher in the stroma. Stromal cell hormone receptors may play an important role in epithelial responses to circulating sex hormones. The results of this investigation add to the general knowledge of California sea lion reproduction and establish baseline information on reproductive tract hormone receptors that will aid in determining the factors involved in urogenital cancer development in sea lions.

Normal morphology and hormone receptor expression in the male California sea lion (Zalophus californianus) genital tract.

Histomorphology and estrogen alpha (ER alpha), and progesterone receptor (PR) expression were evaluated in free-ranging stranded male California sea lions (Zalophus californianus). Hormone receptor expression was evaluated using an immunohistochemical technique with monoclonal antibodies. Estrogen and PRs were identified in the efferent ductules, prostate gland, corpus cavernosa, corpus spongiosium, penile urethra, and in the epithelium and stroma of both the penis and prepuce. In some tissues, ER alpha expression was more intense in the stroma, emphasizing the importance of the stroma in hormone-mediated growth and differentiation of reproductive organs. To our knowledge, this is the first study to localize ER alpha and PR to the epithelium of the glans penis. The results of this investigation add to the general knowledge of male California sea lion reproduction and suggest that estrogens could have a role in the function of the male reproductive tract.

Hereditary equine regional dermal asthenia ("hyperelastosis cutis") in 50 horses: clinical, histological, immunohistological and ultrastructural findings.

Data on fifty horses with hereditary equine regional dermal asthenia (HERDA; "hyperelastosis cutis") were collected on clinical, histopathological, ultrastructural and immunohistological findings. All horses were Quarter horses or of Quarter horse ancestry. Pedigree evaluation strongly supported an autosomal recessive mode of inheritance. The most common lesions were seromas/haematomas, open wounds, sloughing skin, and loose, easily tented skin that did not return to its initial position. Definitive diagnosis could not be made via histopathology, although the presence of tightly grouped thin and shortened collagen fibres arranged in clusters in the deep dermis was suggestive of the disease. Trichrome, acid orcein-Giemsa and immunohistochemical stains for collagens I and III showed no consistent abnormalities compared to control horses; an increase in elastic fibres was not a consistent finding. Electron microscopy showed no abnormalities in the periodicity of the collagen bundles; neither orientation nor variation of cross-section diameter of the collagen fibrils differentiated control from affected horses. The diagnosis of HERDA relies on clinical presentation, but may be supported by suggestive (although not pathognomonic) histopathological lesions.