Immunohistochemical evaluation of expression of heat shock proteins HSP70 and HSP90 in mammary gland neoplasms in bitches.

Heat shock proteins have essential roles in a number of pathophysiologic conditions including carcinogenesis and represent a group of novel molecular markers in cancer management. The aim of this study was to investigate heat shock protein expression in correlation with other neoplasm traits such as: histological type, differentiation grade, proliferative activity, estrogenic receptor expression, and cyclooxygenase-2 and p53 proteins. Material for the investigation comprised 133 tumors of the mammary gland collected from bitches. In total 14 adenomas, 66 complex carcinomas, 47 simple carcinomas and 6 solid carcinomas were collected. Evaluations were conducted with histopathological and immunohistochemical methods using suitable antibodies. Expression of heat shock protein 70 was observed in all types of evaluated neoplasms. A higher average number of cells undergoing expression of heat shock protein 70, which was statistically insignificant, was established in complex and simple cancers and in cancers with the 1st and the 2nd degree of histological malignancy. Expression of heat shock protein 90 was observed in all studied neoplasms; it was very insignificant in adenomas, compared to cancers, and the highest expression was established in the solid cancers, as well as in cancers with the 2nd degree of histological malignancy. This high expression of heat shock protein 90 was correlated with proliferative activity. The results suggest that heat shock protein 90 is involved in canine mammary gland carcinogenesis. The results also suggest that heat shock protein 90 may be a prognostic factor, but this requires detailed clinical confirmation.

Proliferation activity in canine lymphomas.

Forty five lymphomas, 14 of T cell origin, 28 of B cell and 3 with null-cell phenotype, were included in this study. Tumors were classified according to the updated Kiel classification system adapted to canine lymphomas. The percentage of Ki67+ cells and mitotic index (MI) were estimated in each specimen. Most of lymphomas (39 of 45) had high proliferation activity. Among them in 27 cases 50-70% of lymphoma cells expressed Ki67, the highest Ki67 expression (> 70% Ki67+ cells) was identified less frequently, in 12 cases. Moderate Ki67 expression (20-50% positive cells) was observed in 5 cases, only one tumor had low Ki67 expression (< 20% positive cells). Lower percentage of Ki67+ cells was usually accompanied with lower MI. The mean MI values in discussed groups differed significantly. Mean MI value was also significantly higher in T cell than in B cell lymphomas (4.30 vs. 3.33). Moreover, high positive correlation between the expression of Ki67 and MI was found (r = 0.668; P < or = 0.001). In T-cell tumors the correlation was very high (r = 0.83; P < or = 0.001) and in B-cell lymphomas the correlation was high (r = 0.61; P < or = 0.001). There were also differences between mean MI values in the lymphomas of different morphological subtypes, but in some of them high variations in the range of MI values were identified and wide overlaps of MI between individual cases from different subtypes were observed. Because of differences in the proliferation activity in single cases of the same subtype of lymphoma, the proliferation activity assessment may be helpful to chose appropriate scheme of treatment and should be commonly performed during routine histopathological diagnosis of canine lymphomas.

Histopathology in diagnosis of broiler chicken and layer diseases--review of cases 1999-2010.

The aim of this study was to estimate the prevalence of histopathological lesions in the different organs in relation to the commercial-type and the age of birds (i.e. broiler chickens and layers). During the period 1999-2010 a total of 189 cases was submitted to the Division of Animal Pathomorphology, Department of Pathology and Veterinary Diagnostics at WULS. Most cases were found in broiler chickens (66.7%). The majority of the histopathological lesions were detected in the liver and lymphoid organs. In of 29% cases of hepatic injury pathognomonic lesions associated with inclusion body hepatitis (IBH) were found. The mean age of birds was 23 days. Among IBH cases proventriculitis (58%) was more often found than gizzard lesions (25.8%). Interestingly, we noted some intranuclear inclusions in the epithelial cells within the proventriculus. A low percentage of histopathological evidence of infectious bursal disease (IBD) was reported in chickens. The gastrointestinal tract was the second most frequent predilection site for histopathological lesions. Histopathological findings within the heart and lungs were less common and were more often seen in the upper respiratory tract. Cases of infectious laryngotracheitis (ILT) were registered in broiler chickens (3.2%, mean age 37 days) and in layers (4.8%; mean age 196 days). Lesions associated with Marek's disease, avian leukosis and fowl pox were recognized only in layers, respectively in 3.2% (mean age 176 days), 1.6% (mean age 205 days) and 1.1% (mean age 196 days) of all cases. Avian encephalomyelitis (AE) was noted only in 0.5% of all cases.

Nondestructive analysis of single crystals of selenide spinels by X-ray spectrometry techniques.

The paper presents possibilities and difficulties in nondestructive analysis of small multielement single crystals performed by means of X-ray spectrometry techniques: micro-X-ray fluorescence spectrometry (µ-XRF), energy-dispersive electron probe microanalysis (ED-EPMA), and X-ray photoelectron spectroscopy (XPS). The capability of the X-ray spectroscopy techniques in elemental analysis is demonstrated with the single crystals of selenide spinels of the general formula M(x)N(y)Cr(z)Se(4) (M(+2) and N(+3) are, for example, Zn(+2), V(+3), Ga(+3), Cd(+2), In(+3), and Sb(+3)). The results of the nondestructive analyses (µ-XRF, ED-EPMA, and XPS) are compared with those obtained by inductively coupled plasma optical emission spectrometry (ICP-OES) and wavelength-dispersive X-ray spectrometry (WDXRF) following sample digestion. The present study shows satisfactory agreement between the results of µ-XRF analysis performed using the standardless fundamental parameter method and the results obtained with the WDXRF and ICP-OES analyses. If the measured single crystal is precisely positioned, the difference between µ-XRF and wet analysis (WDXRF and ICP-OES) does not exceed 5% rel. The reliable results of ED-EPMA can be obtained only if the measured area is sufficiently large, i.e., of 200 × 300 µm. Even if this condition is fulfilled, the relative difference between the ED-EPMA and the wet analysis may reach 10% rel. In case of the XPS analysis, the accuracy of results depends on the proper preparation of the sample surface. It should be free of contamination that can be obtained by scraping in situ in ultrahigh vacuum. The ion etching, commonly used for cleaning the surface, leads to preferential sputtering; therefore, the reliable results cannot be obtained.

Expression of cyclooxygenase-2 in neoplasms of the mammary gland in bitches.

The aim of the study was to investigate the cyclooxygenase-2 expression in correlation with other neoplasm traits such as: histological type, the differentiation grade, proliferative activity, estrogenic receptor, as well as Hsp70 and p53 proteins expression. Material for the investigation comprised mammary gland tumours, collected from dogs, the patients of veterinary clinics, during surgical procedures. All together 14 adenomas, 66 complex carcinomas, 47 simple carcinomas and 6 solid carcinomas were studied. Evaluations were conducted with histopathological and immunohistochemical methods using suitable antibodies. Expression of COX-2 was observed in 95% of cancers, in case of which, the complex cancers constituted the highest percentage (48.4%). The highest expression of COX-2 was revealed in simple and complex cancers and in cancers with the 3rd degree of histological malignancy. The significant correlation between expression of COX-2 and high mean value of the mitotic index was found. The high expression of COX-2 was also correlated with the expression of protein p53 and expression of the protein Hsp 70. Obtained results suggest that cyclooxygenase-2 may be a prognostic factor, but it requires detailed clinical confirmation.

Evaluation of immunohistochemical expression of P-glycoprotein in neoplasms of the mammary gland in bitches.

The aim of the study was to investigate the P-glycoprotein expression in correlation with other neoplasm traits such as: histological type, the differentiation grade, proliferative activity, expression of the cyclooxygenase-2. Material for the investigation comprised 50 tumours of the mammary gland collected from bitches during surgical procedures performed in Warsaw Veterinary Clinics and Small Animal Clinic of the Department of Clinical Sciences, Faculty of Veterinary Medicine, Warsaw University of Life Sciences - SGGW. All together 8 adenomas, 22 complex carcinomas, 15 simple carcinomas and 5 solid carcinomas. In case of cancers, the degree of histological malignancy was established: 1st degree of histological malignancy--18 neoplasms, 2nd degree of histological malignancy--14 neoplasms and 3rd degree of histological malignancy--10 neoplasms. Evaluations were conducted with histopathological and immunohistochemical methods using suitable antibodies. Proliferative activity was highly dependent on type of the neoplasm and the degree of histological malignancy. The highest value of the mitotic index was characteristic for solid and simple cancers and neoplasms with the highest degree of histological malignancy. Results of expression of the nuclear antigen Ki-67 were similar. Expression of P-glycoprotein was revealed in all types of neoplasms. The expression of P-glycoprotein was identified in cytoplasm and cell membranes of neoplastic cells. Positive expression of P-gp was observed in 76% of cancers. Complex carcinomas were the biggest group among the cancer types which demonstrated positive reaction of P-gp. High expression of P-gp was also established in cancers with the highest degree of malignancy. In bitches aged 9 through 12 years, the cancers featuring a positive reaction of P-gp constituted the most numerous group (63.2%); on the other hand, this cancer type barely appeared in the oldest bitches (10.5%).

Immunohistochemical study of expression of immunoglobulins in canine B-cell lymphomas.

Nineteen canine lymphomas were included in this study. Tumors were classified according to the updated Kiel classification adapted for canine lymphomas by Fournel-Fleury et al. Immunoglobulin light chains (kappa and lambda) and IgM and IgG expression were determined by immunohistochemical method. In all examined cases neoplastic cells were positive for one of the immunoglobulin light chains. Expression of lambda light chains and kappa light chains was observed in 18/19 and 1/19 tumors, respectively. In the majority of neoplastic cells in each examined specimen this reaction had a membranous pattern (skappa/slambda). In all examined cases the presence of immunoglobulin light chains was also observed in the cytoplasm of some neoplastic cells (ckappa/clambda). These cells were usally rare and never constituted a dominant population. The expression of immunoglobulin was found in 13/19 cases. Most lymphomas were sIgM positive (11/13 cases). In one case expression of IgG was found, and in another lymphoma two populations of neoplastic cells with different expression of examined immunoglobulins (cells with IgM+ and IgG+ phenotypes) were observed. The reaction also had a membranous pattern. The cells containing cytoplasmic immunoglobulins were rare, and in most cases were of the same type as the surface immunoglobulins. Our study has confirmed that canine lymphomas are a monoclonal proliferation of B-cells usually expressing immunoglobulin lambda light chains and that the vast majority of tumors deriving from B-cells express IgM. Our study also indicates a possibility of occurence of biclonal lymphomas in canine species.

Comparison of cellular and tissue transcriptional profiles in canine mammary tumor.

Tumor-derived cell lines are widely used as in vitro cancer models. Cell lines historically served as the primary experimental model systems for exploration of tumor cell biology and pharmacology. However, their ability to accurately reflect the phenotype and genotype of the parental histology remains questionable, given the prevalence of documented cell line-specific cytogenetic changes. Sometimes cell line studies are interpreted in the context of artifacts introduced by selection and establishment of cell lines in vitro. This complication has led to difficulties in the extrapolation of biology observed in cell lines to tumor biology in vivo. The aim of our study was to compare gene expression profiles in canine mammary tumor tissue and cell cultures derived from those tumors using cDNA microarrays. Tumors of two different origins were used; chondrosarcoma and adenocarcinoma and their primary cell cultures. It has been found that cell culture gene expression profiles closely resembled those of their corresponding in vivo tumor. In adenocarcinoma and chondrosarcoma only 6.0% and 2.7% of genes respectively, have shown significant difference in expression. In the most cases the difference concerned up-regulation of gene expression in cell lines, particularly genes involved in: protein metabolism and modification, signal transduction and nucleotide, nucleoside and nucleic acid metabolism. These experiments revealed that transcriptome of our primary cell culture corresponds to transcriptome of its parental tumor tissue and for this reason cell culture represents the reliable in vitro model for oncogenomic and pharmacogenomic studies.

Percolation limit and stability conditions for the spin glass state in the spinel families based on the two matrices CuCr(2)S(4) and CuCr(2)Se(4) doped by Sb ions.

The percolation limit of the appearance of the spin glass state in the compounds under study has been obtained experimentally. The conditions of stability of the spin glass state have been analyzed here on the basis of the de Almeida-Thouless theory for two spinels differing in the magnetic coupling constants. It turned out that for the higher value of the coupling constant the magnetic field influences the freezing temperature more strongly. Moreover, the greater the coupling constant the broader the range of the possible values of freezing temperatures, in other words the greater the temperature range of the appearance of the spin glass states. It was proved that for the stability of the spin glass state the existence of a small magnetic field is necessary. In our case the value of this field is equal to 3.46 × 10(-23)T(G). For the compounds under study the value of the magnetic coupling constant J cannot exceed 130 K for the spin glass state to appear.

Immunohistochemical expression of protein p53 in neoplasms of the mammary gland in bitches.

The aim of the study was to investigate the presence of protein p53 in correlation with other tumor traits: histological type, tumor grade and proliferative activity. Material for the investigation comprised mammary gland tumours collected from dogs, the patients of veterinary clinics, during surgical procedures, and archival samples. Alltogether 21 adenomas, 31 complex carcinomas, 35 simple carcinomas and 12 solid carcinomas were qualified for further investigation. No protein p53 expression was found in adenomas. Cancers show positive reaction in 32.5%. The highest percent of p53 positive neoplasms was observed in solid carcinomas and neoplasms with the highest degree of histological malignancy. The smallest number showing this expression was observed in adenomas and the highest was characteristic for solid carcinomas. Considering the tumour grading, it was found that an increase in neoplasm malignancy was positively correlated with the number of the cells showing the expression of protein p53. The differences were statistically significant. Statistically significant positive correlations were observed between the proliferative activity and protein p53 expression. Higher accumulation of protein p53 in more malignant neoplasms suggests that mutations of protein p53 can be responsible for higher proliferation in neoplasms with advanced progression of malignancy.

E-cadherin immunohistochemical expression in mammary gland neoplasms in bitches.

The aim of the study was to investigate E-cadherin expression in correlation with other neoplasm traits such as: histological type, the differentiation grade and proliferative activity. Material for the investigation comprised mammary gland tumours, collected from dogs, the patients of veterinary clinics, during surgical procedures and archival samples. All together 21 adenomas, 32 complex carcinomas, 35 simple carcinomas and 13 solid carcinomas were qualified for further investigation. E-cadherin expression was higher in adenomas as compared with carcinomas but lower in solid carcinomas as compared with simple and complex carcinomas. More over, the expression of E-cadherin decreased with the increase in the neoplasm malignancy and proliferative activity (value of the mitotic index and number of cells showing Ki67). The study has shown that the expression of E-cadherin can be used as a prognostic factor.

Some morphological, biochemical and immunological blood parameters in European bison with or without lesions in the digestive, respiratory, urinary and reproductive tracts.

Ninety-seven bison (39 males and 58 females) were culled out during three successive winter seasons. No infection with BHV-1 and BHV-5 or other viruses was detected by PCR and SN methods. Advanced pathological lesions observed in males exhibiting symptoms of posthitis/balanoposthitis are probably due to secondary infection by microorganisms present in the prepuce (Corynebacterium spp., Bacillus spp., Fusobacterium necrophorum, Staphylococcus spp. (coagulase negative) and Staphylococcus hyicus). Histopathological examinations of sections from prepuce and lips of pudendum showed the presence of Onchocercinae nematode (Onchocerca sp.). A comparison of biochemical and morphological blood parameters in the bison with or without lesions in the digestive (presence of parasites), respiratory, urinary and reproductive tracts revealed that the most variable parameter during reproductive organ disorder is the leukocyte count, while disorders in the urinary and digestive tracts coincided with the age of animals. In cases of digestive disorder most changes were observed in the number of neutrophils and concentration of urea. The number of red blood cells, monocytes, the level of alkaline phosphatase (AP) and hematocrit negatively correlated with age. Negative correlation was also observed between the number of neutrophils, level of creatinine and lymphocyte proliferation index. However, positive correlation was seen between erythrocyte sedimentation rate (Spearman's correlation coefficient), level of alanine aminotransferase (ALT) and protein, and age of the bison. Positive correlation was also observed between the lymphocyte count and lymphocyte proliferation index. The attempt to determine the reference values for blood biochemical and morphological parameters showed similarities to those of cattle.

Tumors of the urogenital system in dogs and cats. Retrospective review of 138 cases.

The aim of the study was to estimate the prevalence and localization of different tumors in the urogenital system in dogs and cats in relation to sex, age and breed of animals. The study was performed on tumors or tissue specimens from tumors of the urinary and genital system obtained during surgery from dogs and cats submitted to the Division of Pathological Anatomy, Department of Clinical Sciences Agricultural University of Warsaw from 1998 to 2005. Most tumors of the urogenital system recognized in the present study derived from dogs (94.20%, 130 cases), and only a few cases were obtained from cats (5.79%). Occurrence and localization of urogenital system tumors in present review is similar to findings reported by other authors. Testicular tumors in males, ovarian lesions in females and urinary bladder tumors in both sexes were most commonly recognized. Older dogs were most often affected, animals with nonmalignant tumors were a bit younger than those with malignant lesions. Any obvious breed predilections were found, but terriers were at increased risk for development of transitional cell carcinoma of the urinary bladder and mixed breed and German shepherd for development of testicular neoplasms.

The diagnosis of gastritis and helicobacter-like organisms infection in endoscopic biopsies of the canine gastric mucosa.

The aim of this study was to evaluate the prevalence of gastric Helicobacter-like organisms (GHLO) and gastritis in the gastric mucosa of dogs with gastric disorders. Tissue samples of the gastric mucosa were obtained from 30 dogs with gastrointestinal symptoms (vomiting, abdominal pain or discomfort, loss of appetite) during endoscopy. Histopathological examinations were performed and occurrence of GHLO infection, gastritis and other mucosal changes were estimated. The GHLO infection and gastritis were identified in 63.3 and 36.6% of dogs respectively; other mucosal changes included fibrosis in the lamina propria, degenerative changes of the gastric glands and hyperplasia of the parietal cells. The present study has revealed that microscopically found gastritis is not frequent in dogs examined by endoscopy. GHLO infection can be responsible for some cases of gastritis and hyperplasia of parietal cells in dogs.

p53 expression in canine lymphoma.

The tumour suppressor p53 plays a key role in DNA damage and repair. It is the most frequently altered gene in human cancers and these mutations may implicate the genesis and/or progression of tumours. Mutations of the p53 gene were also found in a number of canine cancers, although it is poorly estimated in canine lymphomas. Thus, the aim of this study was to investigate the p53 status in these types of tumours. We have shown that the expression of p53 in canine lymphomas is rare, however significantly differs between lymphomas of T- and B-cell origin.

Effect of gastric Helicobacter-like organisms on gastric epithelial cell proliferation rate in dogs.

The aim of this study was the evaluation of gastric epithelial cell proliferation rate in connection with the gastric Helicobacter-like organisms (GHLO) infection in dogs. The study was performed on samples of the gastric mucosa obtained during necropsies from 60 dogs euthanasized for various reasons. Microscopic preparations were stained with the hematoxylin-eosin method, Giemsa method, Warthin-Starry silver method and immunohistochemical method (MIB-1 antibody) to investigate the expression Ki67 antigen. In glands of the gastric body and antrum different proliferation patterns were observed. The gastric epithelial cell proliferation rate in the gastric antrum was significantly higher in dogs with GHLO colonization in relation to dogs without GHLO, and in the gastric body was significantly higher in dogs with mild GHLO colonization as compared to dogs with significant GHLO colonization and without GHLO colonization. The gastric epithelial cell proliferation rate in the gastric body was significantly higher in dogs with gastritis in comparision with the dogs without gastritis irrespectively from the presence or absence of the GHLO colonization. It seems that the presence of the GHLO colonization, as well as the inflammatory state in the gastric mucosae, can alter the rate of the gastric epithelial cells proliferation in dogs.

The presence of Helicobacter-like microorganisms in the gastric mucosa in dogs.

The aim of this study was the evaluation of the occurrence of Helicobacter-like organisms in the gastric mucosa of dogs. The study was performed on samples of gastric mucosa obtained during necropsies from 94 dogs euthanasized for various reasons. Histopathological examination and also urease test was performed. Helicobacter sp. was found in 85.10% of examined dogs. In 36% cases the presence of bacteria was accompanied by chronic inflammation of gastric mucosa localized mainly in pyloric region. In 97.59% samples positive results of urease test were associated with histopathological confirmation of gastric Helicobacter-like organisms.

Relationships among ecology, demography and diseases of European bison (Bison bonasus).

The bison population in the Bialowieza Forest in Poland has now grown to approximately 300, while the herds in the Belarusian part of the forest total about 240 bison. The first signs of a health problem in these herds appeared in 1980, when two cases of balanoposthitis were detected in two bulls (2 and 5 years of age). Since 1980 research has been conducted to explain the cause of diseases, particularly balanoposthitis, and to monitor the health of bison in Bialowieza Forest. A total number of 614 bison (294 male and 320 female) of different ages was eliminated between 1980 and 2000. Not all the culled bison were examined (postmortem analysis, histopathological, bacteriological, virological and toxicological examinations, serological tests, molecular research). Based on the increase in numbers, reproduction in this population for the past 21 years is generally considered successful. Among 182 male bison eliminated during 1990-2000, only 85, or 47%, of the animals had balanoposthitis. Thus, the percentage of balanoposthitis cases went from 100% during the 1980s down to 47% in the past decade. It appears that the culling process has been a major factor leading to this decrease. It can be assumed that a set of factors is involved in the appearance of the disease (Corynebacterium spp., Bacillus sp., Pseudomonas aeruginosa, Escherichia coli, Ureoplasma spp, Fusobacterium necrophorum, Streptoccocus spp., Staphyloccocus spp.) while opportunistic infections including nematodes (Onchocerca spp.) are responsible for the occurrence of secondary lesions.

Estrogen receptors in canine mammary gland tumours.

The aim of the present study was to establish the relationship between estrogen receptor expression in neoplastic parenchymal cells and the type of canine mammary gland tumour. The research material included mammary gland tumours obtained from 66 dogs. Paraffin sections were stained with hematoxylin-eosin and immunohistochemically with monoclonal antibodies and LSAB/Peroxidase/Universal Kit. The estrogen receptor expression was observed in nuclei of neoplastic cells in about 59% of the cases and in the cytoplasm in about 89% of the cases. In about 20% of the cases the expression in the nuclei and in the cytoplasm was extremely weak. No correlation was found between the expression of estrogen receptors and the value of mitotic indexes in the neoplasms investigated.

Homing studies on distribution of ectromelia (mousepox) virus-specific T cells adoptively transferred into syngeneic H-2d mice: paradigm of lymphocyte migration.

Mousepox (infectious ectromelia) may be used as a model for studies on the cellular immune response and pathogenesis of generalized viral infections. Ectromelia virus (EV) initially replicates in the footpad (f.p.) skin at the site of infection, next in draining lymph nodes, and then in the spleen and liver where the virus may induce extensive necrotic process with inflammatory reaction. We show in this study that after recipient BALB/c mice (H-2d) f.p. infection with EV prior to the adoptive transfer of syngeneic donor EV-specific cytotoxic T lymphocytes interferon-gamma-positive (IFN-gamma-+), interleukin-2-positive (IL-2+), and IL-4+ of both phenotypes, CD8+ approximately 70%, and CD4+ approximately 30%) preferentially migrated to the inguinal and auxiliary lymph nodes, spleen, liver, and skin at the site of infection (f.p.). Many particles of EV with the morphology characteristic for orthopoxviruses and virus-specific immunofluorescence within the cells of inguinal and auxiliary lymph nodes, liver, spleen, and skin have been observed using high-resolution transmission electron microscopy and fluorescence antibody technique, respectively. Results presented in this article support the concept that immune T cells adoptively transferred into infected recipient mice are able not only to specific migration in the host and homing in the sites of virus replication, but also to develop immunoprotection in the transferred animals.