Sertoli Cell Tumour and Uterine Leiomyoma in Miniature Schnauzer Dogs with Persistent Müllerian Duct Syndrome Caused by Mutation in the AMHR2 Gene.

Disorders of sex development (DSD) are a serious health problem in dogs. Different types of DSD have been described, including persistent Müllerian duct syndrome (PMDS), for which the molecular background has been identified in miniature schnauzers. Human patients with PMDS are at increased risk for cancers of the gonads (predominantly) or the Müllerian duct structures (rarely). This report describes two miniature schnauzer dogs with PMDS caused by a known nonsense mutation in the AMHR2 gene, with concurrent development of genital neoplasia. The first case (78,XY and SRY-positive) had unilateral cryptorchidism and a Sertoli cell tumour in the abdominal testicle. The second case (mosaic karyotype 77,XY,rob/78,XY and SRY-positive) had both gonads descended in the scrotum and developed an abdominal mass derived from the uterine wall, which showed histological features typical of leiomyoma.

An immunohistochemical analysis of lymphocytic infiltrations in canine skin cancers.

Lymphocytic infiltrations located in the extracellular matrix often accompany canine skin cancer. They can be characterised as an inflammatory infiltration and/or a second tumour - lymphoma. The aim of this study was an immunohistochemical analysis of a lymphocytic infiltration which accompanies spontaneous skin cancer. Twenty basal cell carcinoma, 20 non-keratinizing squamous cell carcinoma, 20 keratinizing squamous cell carcinoma and 8 sebaceous gland carcinoma samples which were accompanied by a lymphocytic infiltration and/or secondary lymphatic follicles were verified histopathologically. The expression of bcl-2, CD3, CD79α, Ki-67, MCM-3 and MCM-7 in the lymphocytic infiltration was evaluated. Four types of lymphocytic infiltrations were found: I - diffuse bcl-2+, II - diffuse bcl-2-, III - follicular bcl-2+/- where the centre was bcl-2-, and the marginal zone of the follicles and the extrafollicular area were bcl-2+ and IV - aggregated bcl-2+, where the centre and periphery were bcl-2+. The I and IV type corresponds to lymphoma, II type is non-neoplastic immune response and III type suggest reactive follicular hyperplasia. The proliferation of lymphocytes which demonstrated the expression of neoplastic markers (I and IV), suggests preneoplastic phase (pseudolymphoma) or lymphoma - the second independent tumour. A high proliferative index of the follicular blc-2+/- follicular infiltration indicates an increased immunological response of the host against skin cancer.

Morphological analysis of testicles in cats with disorders of sex development.

Disorders of sex development (DSD) are rare in cats. They can be caused by chromosomal aberrations, gene mutations or other undefined factors. The aim of the present study was to compare the histological structure and immunohistochemical reactivity of testes in cats with DSD and in healthy cats. The research material consisted of the gonads of four cats - phenotypic males with an incorrect structure of the reproductive system. The control group consisted of the testes of four healthy cats - routinely castrated phenotypical males. The material was fixed with formalin and embedded in paraffin; the sections were stained with hematoxylin and eosin. The immunohistochemical investigation were performed using monoclonal and polyclonal antibodies directed against desmin, vimentin, actin of smooth muscles, S100 protein and MCM3 protein. The results obtained allow concluding that the testes of cats with DSD differed in certain respects, mainly in the number of blood vessels, from the normal testes. Moreover, the results of immunohistochemical examination indicate that in the testes of cats with DSD the number of supporting cells is lower, the amount of interstitial cells is comparable and spermatogenesis is correct es compared to those determined in the control gonads. The number of blood vessels in cats with DSD is reduced by about 30%. It confirms the recommendations for castration of these animals in order to eliminate the potential inheritance of sex development disorders.

Disorder of sex development in a cat with chromosome mosaicism 37,X/38,X,r(Y).

An 18-month-old European shorthair cat was subjected to genetic studies due to ambiguous external genitalia (underdeveloped both penis and scrotum). Further anatomic and histopathological studies revealed the presence of abdominal, atrophic testes and uterus. Cytogenetic analysis showed two cell lines, one with X monosomy-37,X [90% of the analysed metaphase spreads], and other line had 38 chromosomes with normal X chromosome and abnormally small Y-derived chromosome-38,X,der(Y) [10%]. Further fluorescence in situ hybridization study with telomeric probe revealed a ring structure of the der(Y). Eight Y chromosome-specific genes, SRY, TETY1, TETY2, CUL4BY, CYORF15, HSFY, FLJ36031Y and ZFY, were detected. We conclude that the described abnormality of the reproductive system, leading to sterility, was caused by a very rare type of chromosomal mosaicism-37,X/38,X,r(Y).

A novel canine B-cell leukaemia cell line. Establishment, characterisation and sensitivity to chemotherapeutics.

We established a new B-cell leukaemia cell line CLB70 from a dog with chronic lymphocytic leukaemia. This cell line is positive for CD20, CD45, CD79a, MHC class II, IgG, IgM; weakly positive for CD21; and negative for CD3, CD4, CD5, CD8, CD14, CD34, CD117. PCR for antigen receptor gene rearrangement (PARR) analysis revealed a biclonal immunoglobulin heavy chain (IgH) gene rearrangement and negative result for TCRγ. Western blot analysis of anti- and pro-apoptotic proteins showed increased expression of Bcl-2, Mcl-1, NF-kB, and Ras, and decreased expression of p53. CLB70 cells grow rapidly in vitro and are tumourigenic in nude mice. The CLB70 line is highly sensitive to doxorubicin, less sensitive to etoposide and imatinib, and resistant to piroxicam, celecoxib and dexamethasone. Our results indicate that CLB70 cells are derived from mature B-cells and they may be a useful tool for the development of new therapeutic strategies for both dogs and humans.

Immunophenotypic characterization of canine malignant lymphoma: a retrospective study of cases diagnosed in Poland Lower Silesia, over the period 2011-2013.

Lymphoma is the most frequently diagnosed cancer of the canine haematopoietic system. In this study, the flow cytometry and polymerase chain reaction (PCR) analysis were used to characterize a series of canine lymphomas in detail. The aim of this study was to determine the incidence of B- and T-cell high-grade lymphomas and their immunophenotypic characterization in Lower Silesia, Poland. The results show that the frequency of each type of lymphoma is 71% for B-cell and 17% for T-cell lymphomas. In two cases the PCR techniques confirmed the presence of simultaneous double gene rearrangements of the BCR and TCR receptors.

Comparative analysis of markers of cell proliferation in canine mast cell tumours according to current classifications.

The study aimed at immunohistochemical analysis of various markers of cell proliferation and comparison of the results with canine mast cell tumours grading systems according to the Patnaik and Kiupel. Tissue sections were stained using classical technique with haematoxylin and eosin, and immunohistochemical studies were performed with Ki-67, PCNA and MCM-3 antibodies. Additionally the mitotic index was assessed. Statistical analysis including rank correlation Spearman's and ANOVA Friedman analysis was performed. The significance was set at p<0.05. Expression of all examined antigens was detected. The results obtained allow concluding that there is a strong relationship between all the cell markers. However, due to the very strong response and positive reaction in the majority of tumours PCNA is not recommended as a prognostic indicator. Ki-67 and MCM-3 can be successfully used in the evaluation of canine mast cell tumours.

Immunohistochemical diagnostic of hibernoma in dog.

The diagnosis of hibernoma is uncommon in veterinary medicine. In this report, we present an attempt to confirm hibernoma diagnosed in dogs by applying immunohistochemical tests routinely used in human pathology i.e. antibodies specific to protein S100, protein CD31, or smooth muscle actin (SMA).

X monosomy in a virilized female cat.

An infertile Siamese female cat was subjected for clinical, histological, cytogenetic and molecular studies due to ambiguous external genitalia (vulva, vagina, rudimentary penis and scrotum-like structure) and masculine behaviour. An elevated oestrogen activity and a detectable level of testosterone were found. The cat underwent laparotomy. The gonads and the uterus were removed and subjected for histological studies, which showed ovaries with corpora lutea and a some primordial follicles. Chromosome studies of lymphocyte and fibroblast cultures, with the use of Giemsa staining, G-banding and whole X chromosome painting by fluorescence in situ hybridization, revealed pure X monosomy. Molecular analysis showed the absence of the SRY gene. Our study revealed for the first time that X monosomy in cats may be associated with virilization, in spite of the lack of the SRY gene.

The protective effect of the Cornus mas fruits (cornelian cherry) on hypertriglyceridemia and atherosclerosis through PPARα activation in hypercholesterolemic rabbits.

Cornelian cherry (Cornus mas L.) fruits have been used in traditional cuisine and in folk medicine in various countries. This study was conducted to evaluate the constituents and impact of cornelian cherry (C. mas L.) fruits lyophilisate on lipid levels, PPARα protein expression, atheromatous changes in the aorta, oxido-redox state, and proinflammatory cytokines in hypercholesterolemic rabbits. The HPLC-MS method was used for determining active constituents in cornelian cherry. In a subsequent in vivo study the protective effect of the cornelian cherry on diet-induced hyperlipidemia was studied using a rabbit model fed 1% cholesterol. Cornelian cherry (100mg/kg b.w.) or simvastatin (5mg/kg b.w.) were administered orally for 60 days. Two iridoids - loganic acid and cornuside - and five anthocyanins were identified as the main constituents of the cornelian cherry. The administering of the cornelian cherry led to a 44% significant decrease in serum triglyceride levels, as well as prevented development of atheromatous changes in the thoracic aorta. Cornelian cherry significantly increased PPARα protein expression in the liver, indicating that its hypolipidemic effect may stem from enhanced fatty acid catabolism. Simvastatin treatment did not affect PPAR-α expression. Moreover, the cornelian cherry had a significant protective effect on diet-induced oxidative stress in the liver, as well as restored upregulated proinflammatory cytokines serum levels. In conclusion, we have shown loganic acid to be the main iridoid constituent in the European cultivar of the cornelian cherry, and proven that the cornelian cherry could have protective effects on diet-induced hypertriglicerydemia and atherosclerosis through enhanced PPARα protein expression and via regulating oxidative stress and inflammation.

Sonoelastography in differentiation of benign and malignant testicular lesion in dogs.

Present study aimed to evaluae the elasticity of tumorous testicular lesions and usefulness of the elastographic examination for diagnosing lesions in the testes. The study was carried out on nine dogs in which tumorous testicular lesions had been found in the ultrasonographic examination. In all the animals examined, the elastographic examination of the lesions was performed and then castration and the histopathological examination of specimens in order to determine the type of the changes. On the basis of the results of the histopathological examination the dogs were divided into two groups: group I--consisted of three dogs in which nonneoplastic testicular lesions were found and group II comprised six dogs in which neoplasic lesions that began in testicular interstitial cells (Leydigoma) were detected. The lesions observed in dogs of group I showed low stiffness (average 11.25 kPa, range 6.1 to 16.4 kPa), whereas the lesions found in dogs of group II were characterized by high stiffness (average 91.85 kPa, range 52.3 to 131.4 kPa). On the basis of a scale proposed by Goodie et al. (2012), the lesions in group I were in the range of SC1, and in turn, the lesions in group II were in the range of SC 3 inverted. Based on the results obtained, it can be stated that the sonoelastographic examination is useful method for the screening diagnostics of testicular lesions.

A case of leucocyte chimerism (78,XX/78,XY) in a dog with a disorder of sexual development.

A 1-year-old Shih Tzu dog was presented for examination because of abnormal external genitalia. A residual penis with a prepuce was located in a position typical of a male. The dog had no palpable testicles or scrotum. The ultrasound examination revealed the presence of the prostate, but the gonads remained undetectable. Cytogenetic analysis performed on chromosome preparations obtained from lymphocyte culture showed two cell lines - 78,XX and 78,XY. Molecular analysis of 14 polymorphic microsatellite markers allowed us to distinguish leucocyte chimerism from whole body chimerism. The presence of 3 or 4 alleles was confirmed in DNA isolated from blood, while in DNA isolated from hair follicles only 1 or 2 alleles were detected. The case was classified as leucocyte 78,XX/78,XY chimerism. Our study showed that XX/XY leucocyte chimerism might be associated with disorder of sexual development in dogs. Furthermore, it is emphasized that the use of cytogenetic study, in combination with analysis of polymorphic markers in DNA isolated from different somatic cells, facilitates distinguishing between leucocyte and whole body chimerism.

Application of shear wave elastography in the diagnosis of mammary gland neoplasm in dogs.

The aim of the study was to evaluate the usefulness of elastography in the differential diagnosis of benign and malignant tumours of the mammary gland in dogs. The study was performed to 12 female dogs of different breeds aged 5-12 years in which tumoral lesions of the mammary gland were found in the clinical examination. In all the animals elastographic examination of the lesions was carried out and then the fine-needle aspiration biopsy was performed to determine the nature and degree of malignancy of the lesions. The examinations proved that benign neoplasms of the mammary gland showed low stiffness (average 22.42 kPa, range 19 to 42.4 kPa), whereas malignant neoplasms were characterized by high stiffness (average 235.44 kPa, range 171 to 300 kPa). On the basis of the results obtained we conclude that the elastography of proliferative lesions of the mammary gland is a useful diagnostic method for distinguishing benign neoplastic lesions from malignant neoplasms.

Fine needle biopsy of abdominal organs in dogs -- indications, contraindications and performance technique.

Recent years have seen in both human and veterinary medicine the development of numerous techniques allowing for evaluation and classification of changes in individual organs and tissues. Despite introduction of such techniques into diagnostics as among others, CT, MRI, CEUS or elastography, biopsy is still considered a "golden standard" and it is a procedure performed in order to obtain a final diagnosis. There are many biopsy techniques, such as fine needle aspiration biopsy, core biopsy as well as methods of performing a procedure, e.g. blind biopsy, biopsy under USG control and biopsy during laparotomy. In the article usefulness of biopsy techniques in relation to diagnostics of individual abdominal organ, as well as the procedure technique, contraindication and complications are discussed.

Adverse effects associated with high-dose acetylsalicylic acid and sodium salicylate treatment in broilers.

1. Acetylsalicylic acid (ASA) and sodium salicylate (SS) are considered safe for poultry and often used in avian medicine. However, information on tolerance and specific side effects of these drugs in birds is lacking. 2. The aim of this study was to determine the effects of 14 d administration of high doses (200 or 400 mg/kg) of either ASA or SS on body weight gain, blood biochemistry, white and red blood cell counts and pathology in broilers. In addition, minimal plasma salicylate concentrations were determined on the 1st, 5th, 10th and 14th d of treatment. 3. The results showed that the dose of 400 mg/kg of either ASA or SS decreased weight gain and induced gizzard ulceration. Kidney to body weight ratio was increased in a dose-dependent manner, but serum concentrations of creatinine and uric acid were not affected. A time-dependent decrease in the minimal plasma salicylate concentration was evident.

Robertsonian translocation in a sex reversal dog (XX, SRY negative) may indicate that the causative mutation for this intersexuality syndrome resides on canine chromosome 23 (CFA23).

A Bernese mountain dog was subjected for clinical evaluation due to the presence of ambiguous external genitalia (enlarged clitoris). Anatomical and histological studies revealed the presence of one testicle, one ovotestis and a uterus. This dog was classified as a female-to-male sex reversal, with 2 normal X chromosomes and a lack of the Y chromosome-linked genes SRY and ZFY. It is the first case of this syndrome in this breed. Apparently a Robertsonian translocation, rob(5;23), was also identified in this dog and it is again the first case of this type of chromosome abnormality in this breed, as well as the first case of co-occurrence of the sex reversal syndrome along with a centric fusion in the dog. Since on the canine chromosome 23 (CFA23) 3 genes (FOXL2,PISRT1 and CTNNB1) involved in the sex determination process are present, further cytogenetic FISH studies were carried out with the use of BAC probes specific for this chromosome. It was found that a pericentromeric fragment of CFA23 was deleted as a result of the centric fusion. We hypothesize that a cis regulatory sequence for the sex determination genes on CFA23 (e.g. proximally located CTNNB1) is present in the deleted fragment. Thus, a causative mutation responsible for this sex reversal syndrome may reside on CFA23.

Lack of the SOX9 gene polymorphism in sex reversal dogs (78,XX; SRY negative).

The molecular background of the most frequent intersexuality syndrome in dogs (female-to-male sex reversal with the female karyotype and a lack of the SRY gene) is unknown. In this article, new cases of this syndrome are described in two unrelated American Staffordshire terrier dogs and one miniature pinscher dog subjected to cytogenetic and molecular analysis due to the presence of an enlarged clitoris. One dog was operated on and histological studies of the gonads revealed a testicular structure without signs of spermatogenesis, but the uterus wall appeared to be normal. All three dogs had female chromosome complements and lacked the Y-linked genes SRY and ZFY. Eight fragments, representing the vast majority of the coding sequence of the SOX9 gene, and two fragments of the 5' flanking region of this gene were analyzed. The studied fragments had identical DNA sequences when comparing the intersexual dogs with GenBank sequences (AY237827; NW139883). Thus a mutation in the coding sequence as well as the promoter region of the SOX9 gene might be excluded as a cause of this type of intersexuality. The importance of further studies of the 5' flanking region of this gene is discussed.

Studies on nucleolar organiser regions (AgNors) in selected spontaneous and transplantable animal tumors.

Behaviour of argyrophilic nucleolus organising regions (AgNOR) was estimated in various types of spontaneous and transplantable tumors in animals. The studies were performed on spontaneous epithelial and mesenchymal tumors, malignant and non-malignant, as well as transplantable tumors: Morris hepatoma, mammary gland carcinoma and Yoshid sarcoma. The examinations were made on paraffin sections, using silver-staining method according to Ploton et al. Quantitative assessment was made with computer-aided microscopic image analysis system Multi-Scan Base V.8 for Windows, coupled with Carl Zeiss microscope. It was demonstrated that AgNOR index reflects malignancy of the tumor, since it increases clearly in cancers and sarcomas, both spontaneous and transplantable. The highest AgNOR index--0.13--was noted in the group of spontaneous tumors in epithelial malignant tumors, and in the group of transplantable tumors in mesenchymal tumors (Yoshid sarcoma) it was 0.15. Classification of the studied spontaneous and transplantable tumors into groups of the same histogenesis, though phenotypically different, was aimed at demonstration of the increasing tendency of AgNOR index.

Immunocytochemical identification of neoplastic cell clone in phase S of cell cycle in transplantable rat tumors.

Wistar and Buffalo rats of both sexes, aged 4 months, were divided into three groups: I which was given an intramuscular injection of 3 x 10(6) cells of Morris hepatoma (Buffalo males), II--subcutaneous injection of 3 x 10(4) cells of mammary gland carcinoma (Wistar females), III--intraperitoneal injection of 3 x 10(4) cells of Yoshid sarcoma (Wistar males). The animals were killed: in group I--19, group II--13 and in group III--6 days after tumor transplantation. Twenty four hours before euthanasia the rats were given 5-brome-2'-deoxyuridine (BRd-U) at a dose of 50 mg/kg body mass. The control group consisted of animals with tumour. They were not treated with BRd-U. Immunocytochemical reaction was performed on the sections of tumors, using monoclonal anti-BRd-U clone BU-33, Sigma. Computer measurements of tumor cells were carried out. There was a high similarity in morphological parameters between two kinds of cancer, and clear differences between them and Yoshid sarcoma. The main difference was noted in a twofold increase in the quantity of synthesised DNA in the nuclei of sarcoma cells. Immunocytochemical identification of tumor cells in phase S of the cell cycle with the use of monoclonal anti-BRd-U antibody is a precise and quick method of estimation of their proliferative potential.

Changes in gene expression in the lungs of Mg-deficient mice are related to an inflammatory process.

It has been well documented that experimental hypomagnesemia in rodents evokes, as an early consequence, an inflammatory response. This also leads to the activation of cells producing reactive species of oxygen and, as a result, to the oxidative damage of tissues. Several studies have shown that lungs might be a specific target of Mg deficiency. Here, we report that 3 weeks of Mg deficiency in mice resulted in inflammatory processes in the lungs, including interstitial and perivascular pneumonia, manifested by the infiltration of leukocytes, plasmocytes and histiocytes, as well as the phenomenon of disseminated intravascular coagulation (DIC). These phenomena were accompanied by changes in gene expression assessed by cDNA array. In this study we identified 26 genes significantly changed by Mg deficiency, mostly involved in the anti-oxidative response, regulation of cell cycle and growth, apoptosis as well as cell-cell and cell-matrix interactions. We conclude that these changes are related to the phenomena of inflammatory and oxidative processes and consecutive remodeling occurring in the tissues as a result of Mg deficiency. This may have implications for at least several lung pathologies, including allergies, asthma, SIDS (Sudden Infant Death Syndrome) or facilitate formation of lung metastases.