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.

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).

Immunohistochemical identification method of tumour cells in the S phase of mitotic cycle and its usefulness in diagnostics of mammary gland adenocarcinomas in bitches.

The studies aimed at identification of neoplastic cells at the S phase of mitotic cycle in mammary gland adenocarcinomas of bitches. The material was sampled from bitches of various races, aging 6 to 12 years, in which the mammary gland tumours developed spontaneously. The tumours were verified histopathologically and, then, immunohistochemical reactions were performed in order to detect cells which had incorporated BrdU (bromodeoxyuridine), contained Ki-67 or PCNA antigen. The histological preparations were photographed and obtained pictures were subjected to computer-assisted image analysis using Axiophot microscope (Carl Zeiss) coupled to a computer and the Multi-ScaneBase V 8.08 software, working under Windows. Fifty percent of sections from mammary gland adenocarcinomas demonstrated BrdU labelling index of 4-5%, 40% of 1-3%, while in the remaining 10% of examined tumours no BrdU incorporation could be demonstrated. No evident relationship could be detected between the presence of BrdU incorporation and Ki-67 or PCNA antigen presence but a significant correlation was demonstrated between the expression of Ki-67 and PCNA.

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.

Ultrastructural studies on malignant lymphoma in dogs, with the use of computer imaging.

Malignant lymphoma is one among the most often diagnosed malignant tumors in dogs. In the course of the disease lymphatic glands become enlarged and infiltrations form in internal organs or skin. Studies on the ultrastructure of neoplastic lymphocytes were carried out on a formalin-fixed and parafin-embedded material with a Hulquist and Karlsson's method. Numerous aplastic cells with large nuclei, abundan eu- or heterochromatin and unstabilised nucleolus were observed on TEM slides. The cells differed clearly from normal lymphocytes with highly condesed heterochromatin and stabilised nucleoli.

Therapeutic potential of biological response modifiers against transplantable mouse tumors of spontaneous origin. II. Local interleukin 2 treatment of tumors of different immunogenic strength.

This report describes tumor susceptibility to interleukin 2 (IL-2) therapy in relation to tumor immunogenicity. The following lines recently established from spontaneous tumors were used: X5, X6, and X9 mammary tumors of DBA/2, BALB/c and CBA origin, respectively, X7 carcinoma of BALB/c and X18 papilloma of CBA mice. Two spontaneous tumors of long transplantation history, SL2 lymphoma (SL2) of DBA/2 and M109 Madison lung carcinoma (M109) of BALB/c origin, were used as control systems. Mice were transplanted with different inocula of tumor cells at day 0; IL-2 treatment was initiated on day 1-3 or 10 and consisted of daily injections of 5000 or 20,000 IU/mouse given 5 times a week for 3 weeks. SL2 (i.p. - 2 x 10(4) cells) treatment consisted of i.p. injections of 5000 or 20,000 IU IL-2 given on days 10-14. IL-2 therapy of SL2 bearing DBA/2JIco mice resulted in the significant proportion of cures; however, no response to IL-2 treatment was achieved in SL2 bearing DBA/2Crliw mice. BALB/c mice with the i.p. transplant of M109 responded to IL-2 treatment with 40% increase in lifespan. The low-dose IL-2 therapy of the 5 recently established tumors resulted, in general, in transient growth inhibition of the i.m. transplants of line X5, X6, and X7 provided IL-2 was administered locally. The therapeutic effect depended on the number of transplanted tumor cells, and the best results being achieved at cell numbers close to the dose inducing tumor growth in 50% of animals (TD50). We found that the tumors responding to IL-2 treatment were all slowly growing and immunogenic (X6 and X7) or might have viral association (X5) and as such might express foreign antigens.

Ozonotherapy.

The medical use of ozone intraarterially, subcutaneously, intramuscularly and externally and in autohaemotransfusion or as a gas introduced into body cavities, as well as drops, compresses or liquids for rinsing etc. brings about very good therapeutic results. Ozone exerts a positive effect on oxygen and nutrient supply of cells, it enhances immunological processes, inhibits inflammatory processes, has a bacterio-, fungi- and virusostatic action when there is impaired resistance to microorganisms, improves rheological properties of the blood and exhibits no side effects in patients. Consequently ozonotherapy may be regarded asan effective method of treatment.

Ultrastructural studies on organs of cadmium-poisoned rats treated with oxygen-ozone mixture.

Rats poisoned with cadmium acetate during 12 weeks, at a dose of 50 mg/dm3 given in drinking water, were treated with oxygen-ozone mixture as intraperitoneal injection during the last 10 days of the experiment, at a daily dose of 1 cm3 and ozone concentration 40 micrograms/cm3. The mixture was made of medical oxygen with a Bioozon U type apparatus produced by B. Prochazka GmbH, Germany, Reutlingen. Control groups included animals treated with the above mixture with no cadmium, and rats poisoned with cadmium, with no oxygen-ozone treatment. Liver and cardiac muscle were examined in TEM Philips EM 301. Morphological traits of a protective of the mixture against cadmium-poisoning were observed in both those organs. This was expressed as weaker destructive changes within the endoplasmic reticulum, basal cytoplasm and lysosome of the hepatocytes, and additionally as a stabilization of contractile apparatus fibres in the heart myocytes.

Therapeutic potential of biological response modifiers against transplantable mouse tumors of spontaneous origin. I. Characterization of biological properties of tumor lines and preliminary data on response to cytostatic drugs and biomodulators.

Several transplantable lines were derived from spontaneous tumors of mouse strains of low and high cancer incidence (CBA, BALB/c and DBA/2), and eight of them were used for an evaluation of the therapeutic potential of BRM. The lowest number of cells inducing tumors was in most cases around 10(4) cells. In transplantation tests, tumors of CBA and DBA/2 mice were non-immunogenic, while tumors of BALB/c mice showed different level of immunogenicity. Transplantation of a small number of tumor cells in admixture with BCG resulted in no tumor growth of the immunogenic line X6 and no effect on take rate of non-immunogenic line X1. Sensitivity of studied tumor lines to BCNU, CY and ADR was moderate. Therapy of the tumors with postulan and CMA, two biomodulators already shown to be active against various experimental tumors, failed. The significant therapeutic response was observed in DBA/2 mice bearing i.p. transplant of lymphosarcoma X19 and treated with i.p. injections of CMA.

Histological and ultrastructural examination of muscles in broilers administered tiamulin together with ionophoric anticoccidials.

Histopathological and ultrastructural examinations of muscles in broilers, receiving tiamulin and ionophoric anticoccidials were carried out. Myo- and cardiomyopathies, originating from primary mitochondria lesions were followed by ATP deficiency, oedema, degeneration and necrosis of myocytes. This process is reversible and manifested by slow regeneration of muscles.

The influence of long-term administration of Tolpa Peat Preparation on immune reactivity in mice. I. Morphological changes in the thymus.

In experiment I BALB/c 200 mice were given in drinking water the TPP (Tolpa Peat Preparation) in daily doses of 1, 10 and 50 mg/kg over the period of 3, 5, 7, 9 and 12 weeks. In experiment II before the administration of TPP (as in experiment I) mice were immunized with a single dose i.p. administration of 0.2 cm3 of a 10% suspension of sheep erythrocytes (SRBC), i.e. 4 x 10(8) cells. Histopathological and ultrastructural studies have shown that TPP in all three doses causes morphological changes characteristic of thymus activity stimulation. However, the doses of 10 and 50 mg/kg administered longer than 7 weeks caused retrograde changes that did not occur after administration of 1 mg/kg of TPP. Morphological changes in the thymus of immunized and non-immunized mice simultaneously receiving TPP were similar.

The influence of long-term administration of Tolpa Peat Preparation on immune reactivity in mice. II. The effect of intermittent TPP administration on the morphological picture of lymphatic organs.

The studies were conducted on 200 Balb/c mice (inbred strain), aged 8 weeks. The mice were administered Tolpa Peat Preparation (TPP) in drinking water (10 or 50 mg/kg/day) for seven weeks (experiment I). Two lobes of the thymus, the spleen and the axillary and mesenteric lymph nodes of mice were taken for histological examinations after 3, 5 and 7 weeks of continuous TPP treatment and also 12 and 16 weeks from the beginning of the treatment. In experiment II, TPP was administered daily at the same doses for 3 weeks, and intermittently for 9 consecutive weeks; a week interval followed each seven-day cycle. The lymphatic organs of mice were taken for histological examinations after 3, 5, 7, 9 and 12 weeks of TPP treatment. It was found that daily administration of TPP (as described above) to mice for 7 weeks (10 mg/kg) induced a marked stimulation of the lymphatic organs (in the first weeks of TPP treatment) and resulted in retrograde changes (depletion of thymocytes and lymphocytes) in the lymphatic structures when TPP administration was prolonged (4-7 weeks). The morphological changes due to TPP administered at the dose of 50 mg/kg were less pronounced in the organs examined. The retrograde changes in the lymphatic structures disappeared 9 weeks after TPP treatment had been stopped. Similarly, the changes showing stimulation of the lymphatic organs were maintained throughout the entire period of 9 weeks.(ABSTRACT TRUNCATED AT 250 WORDS)

Further studies on the etiopathology of malabsorption syndrome in broiler chickens.

The paper presents two cases of malabsorption syndrome in broiler chickens. The results indicate that the etiological factor appears to be the virus agent which causes necrosis in extra-excretional parts of the pancreas. During the course of the disease disorders in the enzymatic activity of gammaglutamyltransferase, leucylaminopeptidase and alanylaminopeptidase were observed. It consequently leads to the deterioration of intestines digestion and resorption of feed components. Application of Selcewet-Polfa to the diseased birds improved the health of the birds, morphological picture of peripheral blood and mineralisation of thigh bones. No radical improvement in the compensation of the final body weight was recorded.

Amino acids and cholesterol levels of leukemic lymphocytes infected with bovine leukemia virus (BLV).

We performed qualitative and quantitative amino acids analysis of lymphocytes from normal and leukemic cattle (infected with bovine leukemia virus) to determine cellular cholesterol levels. Leukemic lymphocytes exhibited quantitative differences in the levels of some "glycogenic", "ketogenic" and "glyco-ketogenic" amino acids. These differences may reflect the disturbances of Krebs cycle. Evidence was produced that increased cholesterol content in the above lymphocytes was associated with disorders in beta-oxidation processes of leukemic cells.

[The content of Ca2+ and Mg2+ ions and membrane enzyme activity (AP, 5'-NT, ATPases) in the lymphocytes infected in vitro with bovine leukemia virus]. / Zawartosc jonów Ca2+ i Mg2+ oraz aktywnosc enzymów blony komórkowej (FZ, 5'-NT, ATP-az) w limfocytach bialaczkowych in vitro u bydla.

In the lymphocytes infected in vitro with BLV (bovine leukemia virus) the contents of Ca2+ and Mg2+ were determined using roentgen microanalyser JXA-5 A Joel-form (Japan). In the smears prepared from these cells the activity of enzyme markers of cell membranes i.e. alkaline phosphatase (AP - EC 3.1.3.1), 5'-nucleotidase (5'-NT - EC 3.1.3.5) and adenosine-triphosphatases - Ca2+ and Mg2+ dependent (ATP-ase - EC 3.6.1.3) was determined. The decrease in AP and ATP-assess activity and increase in 5'-NT in the membranes of leukemic lymphocytes were observed. During these changes the increase in Ca2+ and decrease in Mg2+ ions occurred. These processes lead to clear disturbances in the metabolism of cells transformed by the neoplasm. The effect of this phenomenon is probably the opening of calcium canals with the following cytoplasmatic hypercalcemia. It's very destructive for the change in permeability of the membrane of lymphocytes.

In vitro studies into some parameters of protein and carbohydrate metabolism in lymphocytes infected with bovine leucosis virus.

Several parameters of protein and carbohydrate metabolism were determined in normal and leukemic lymphocytes in vitro in cattle, including arylamidase activity toward beta-naphthylamides of L-amino acids. The homogenate of bovine leukemic lymphocytes, in comparison with the control revealed increase of gamma-glutamyltransferase, activity trypsin inhibitor and papain inhibitor concentration and aldolase activity. On the other hand, proteolytic activity toward casein and histomucoid content decreased. Out of the 7 substrates used in the study, only 2, alanyl-beta-naphthylamide and leucyl-beta-naphthylamide, demonstrated lower activity in the leukemic material. Disorders in carbohydrate and protein metabolism in the observed lymphocytes in vitro in cattle are presented in the paper.

The content of fatty acids in leukemic lymphocytes in vitro in cattle.

In leukemic lymphocytes in vitro obtained from cattle the fatty acids content has been determined employing the method of gas chromatography of methyl esters. Significant increase of linolic acid (C18 = 2) and of linolenic acid (C18 = 3) in leukemic lymphocytes have been noted, while reduced amounts of eicanoic acid (C20) and arachidonic acid (C20 = 4) have been observed. Fatty acids contribution in destabilization of biological membranes of leukemic cells has been discussed. It is very likely that the destabilization of biological membranes promotes the "induction" of various cancero- and cocancerogenetic changes, as well as enables the penetration of leukemovirus into the cell.