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.

Effect of in ovo-delivered prebiotics and synbiotics on the morphology and specific immune cell composition in the gut-associated lymphoid tissue.

The purpose of this study was to examine how pre- and synbiotic administration in ovo into the air chamber at d 12 of egg incubation influenced the specific immune cell composition and distribution in the ileum, cecal tonsils (CT) and bursa of Fabricius of broilers. The experiment was performed on 800 hatching eggs of the meat-type chickens (Ross 308). Hatching eggs were treated with: prebiotic, consisting of inulin (Pre1) or Bi(2)tos(®) (Pre2); symbiotic, composed of inulin and Lactococcus lactis subsp. lactis IBB SL1 (Syn1) or Bi(2)tos and Lactococcus lactis subsp. cremoris IBB SC1 (Syn2); or physiological saline as a control group. Seven chickens from each treatment group were randomly selected on , 1, 7, and 21 after hatch for tissue collection. Ileum, cecal tonsil and bursa of Fabricius samples were immunohistochemically stained and the proportions of Bu-1(+), CD3(+), CD4(+), CD8α(+) and TCRγδ(+) cells were estimated. It was indicated that the pre- and synbiotics do not adversely affect the development of the GALT of the chicken. The temporary decrease in B-cell number in bursa on d 7 after hatch suggested an increased colonization rate of the peripheral lymphoid organs by these cells after Pre1, Pre2, and Syn2 treatment. In CT at d 7 after hatch more potent colonization of the GALT by T cells was observed in all pre- and synbiotic treated groups and by B cells in both synbiotic-treated groups than those in respective controls. Then, on d 21 in both synbiotic-treated groups, an increase in T-cell number in ileum was also noticed with faster colonization of the CT by B cells. In 21-day-old chickens, both synbiotics exerted stronger stimulatory effect on the GALT colonization by T cells then prebiotics respectively. Similarly, the colonization by B cells was more pronounced in the Syn2 than in the Pre2 group. The data obtained in this study indicated that prebiotics and particularly synbiotics administrated in ovo stimulated GALT development after hatch.

Morphology of immune organs after very virulent plus strain of Marek's disease virus infection in vaccinated hens.

Marek's disease (MD) outbreaks in poultry flocks may be associated with overriding of vaccine immune protection by very virulent (vvMDV) or very virulent plus (vv+MDV) strains. This paper presents the study on lymphoid organ morphology in the latent phase of MD caused by vv+MDV which break post-vaccinal protection in hens. We also immunohistochemically examined B and T populations as well as B/T and CD4+/CD8+ ratio of lymphocytes in lymphatic organs and, as a background, in MD lymphomas from non-lymphatic organs. The number of antigen expressed cells was evaluated as a percentage of positive cells in the one power field. Organ samples were collected from 24 dead reproductive hens (Ross 308 line) in age between 35-56 weeks, infected with vv+MDV. The hens originated from farms with MD outbreaks, despite earlier routine vaccination with CVI988/Rispens + HVT. The control organ samples originated from 15 clinically healthy hens at the same age and line, subjected to the same vaccination schedule. The number of CD3+, CD8+ and TCRγδ+ cells was significantly lower in MDV infected thymus, spleen and cecal tonsils in comparison to that found in the control organs. The proportion of CD4+ was also distinctly reduced in the thymus and limited in the spleen of MDV infected hens. This study revealed that infection with field vv+MDV isolates might break post-vaccinal protection and influence the central and peripheral immune system. The decrease in CD8+ and TCRγδ+ cell number in the thymus, spleen and cecal tonsils suggests that primarily these cells are involved in cell-mediated cytotoxicity against MDV transformed cells during latency.

Effect of in ovo-delivered prebiotics and synbiotics on lymphoid-organs' morphology in chickens.

Prebiotics and probiotics, either alone or together (synbiotics), can influence the intestinal microbiota and modulate the immune response. We aimed to investigate the effects of prebiotic and synbiotic administration during the early stage of development on the histological structures of central (bursa of Fabricius and thymus) and peripheral (spleen) lymphatic organs in broilers. We used 800 hatching eggs from meat-type hens (Ross 308). Prebiotics and synbiotics were administered in ovo into the air chamber of chicken eggs at d 12 incubation, as follows: prebiotic inulin (Pre1), Bi2tos (Pre2), a synbiotic composed of inulin and Lactococcus lactis subsp. lactis IBB SL1 (Syn1), a synbiotic composed of Bi2tos and L. lactis subsp. cremoris IBB SC1 (Syn2), or physiological saline (control group, C). In ovo delivery of prebiotics and synbiotics had no adverse effect on the development of the immune system in exposed chickens. Administration of Bi2tos with L. lactis subsp. cremoris (Syn2) decreased the cortex/medulla ratio in the thymus and slowed the development of the cortex in bursal follicles on d 21 posthatching, with consequent impacts on the primary lymphatic organs. The above treatment also stimulated germinal centers' formation in the spleens of 21- and 35-day-old chickens, indicating enhanced B-cell proliferation in secondary lymphatic organs. Syn2 also caused an age-dependent increase in the spleen/bursa of Fabricius ratio. In conclusion, the in ovo administration of pre- and synbiotics at d 12 incubation can modulate the central and peripheral lymphatic organ development in broilers. This effect is more pronounced after synbiotic treatment than in prebiotic-treated groups.

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).

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.

Inhibition of in vitro Histophilus somni biofilm production by recombinant Hsp60 antibodies.

Histophilus somni is an opportunistic pathogen causing respiratory, genitourinary and generalized infections in cattle. An important virulence factor is its ability to produce a biofilm. The aim of this work was to confirm that H. somni Hsp60 (Gro-EL) is a constituent of the biofilm produced by this bacterium in vitro and to check whether or not the presence of a specific antibody within the culture medium can inhibit biofilm production. Biofilm production by H. somni cultured in vitro was confirmed by crystalline violet staining. The presence of Hsp60 in the biofilm was confirmed by using specific antibodies produced in a mouse and goat hyperimmunized with H. somni recombinant Hsp60 (rHsp60). Large complexes of biofilm stained with Hsp60 antibodies were microscopically detected. This indicates that the Hsp60 protein is a common constituent of the biofilm produced by H. somni in vitro. In a second experiment, mouse serum containing anti-H. somni rHsp60 antibodies was added to an H. somni culture. It was found that the presence of anti-rHsp60 antibodies in the culture medium inhibited biofilm production in vitro. Only small biofilm particles were seen in the presence of the specific antibody, whereas in control cultures (without specific antiserum) large biofilm complexes were produced. The results indicate that antibodies specific to Hsp60 may be useful for preventing H. somni biofilm formation in vitro. If this also occurs in vivo, it may be helpful for eradicating H. somni infection in cattle through the elimination of carriers. Further in vivo studies are needed to confirm this idea.

The influence of antibiotics on B-cell number, percentage, and distribution in the bursa of Fabricius of newly hatched chicks.

Antibiotics are commonly used to prevent and treat poultry microbial infections, but certain antibiotic families depress humoral immunity, such as antibody production. Poultry humoral immunity depends on the normal functioning of the bursa of Fabricius and the B lymphocytes that mature in that gland. In this study, recommended therapeutic doses of enrofloxacin, florfenicol, or ceftiofur were administered to 2-d-old chicks. On d 7 post-hatch, bursae were sampled for histological, immunohistochemical, and flow cytometric determination of Bu-1-positive (Bu-1+) cell number, percentage, and distribution. The bursa of Fabricius from all treatment and control groups had normal morphology. The administration of antibiotics significantly decreased the number of Bu-1+ cells in the bursal medulla, with a simultaneous increase of these cells in the cortex. Flow cytometry revealed a significant decrease in the percentage of bursal Bu-1+ cells from all of the studied antibiotics: enrofloxacin (93.91 ± 3.27), florfenicol (87.84 ± 7.14), and ceftiofur (89.16 ± 5.68) compared with that of the control (96.48 ± 2.60). The combination of reduced percentages of Bu-1+ cells and a decrease in these cells in the medullary region suggests lower B cell maturation.

Genotype-dependent development of cellular and humoral immunity in the spleen and cecal tonsils of chickens stimulated in ovo with bioactive compounds.

Prebiotics, probiotics, and synbiotics, delivered in ovo influence the colonization and development of the peripheral immune system in poultry. This study aimed to investigate the influence of the host genotype (broiler chickens [Ross 308] and old native Polish breed Green-legged Partridgelike [GP] chickens) on the number of B and T cells in the spleen and cecal tonsils (CT). The solution of a bioactive compound was injected in ovo on day 12 of egg incubation: prebiotics (galactooligosaccharides [GOS]), probiotics (Lactococcus lactis subsp. cremoris IBB477), and synbiotics (GOS + L. lactis). The samples were collected on day 7, day 21, and day 42 after hatching (n = 8). The number of Bu-1+ (B) cells, CD4+ cells, and CD8+ cells in the spleen and CT was estimated using immunohistochemistry. The number of germinal centers (GC) was determined in the spleen. In broilers, probiotics increased (P < 0.05) the number of CD4+ cells in the CT on day 7. On day 21, prebiotics raised (P < 0.01) the number of cells involved in cellular immunity in the CT (CD4+ and CD8+ cells) and spleen (CD8+ cells). On day 42, it was synbiotics that stimulated the colonization of both the CT and spleen by B cells, but colonization of the spleen only by CD4+ and CD8+ cells. In GP chickens, synbiotics enforced the cellular immunity (CD4+ or CD8+ cells) in the spleen at all time points. Synbiotics also stimulated the GC appearance on day 21 and day 42. In GP chickens, the influence of bioactive compounds on colonization of the CT was very limited. In broilers, we determined pronounced and age-dependent effects of prebiotics and synbiotics on the number of B and T cells in both the CT and spleen. In GP chickens, the most potent compound was synbiotics, which stimulated cellular immunity in the spleen but not in the CT. However, given the long-term effects on adaptive immune cells, synbiotics were the most potent compounds in both chicken genotypes.

The relationship between concentrations of vitamin D-binding protein (DBP) in serum and colostrum of mares and in serum of their foals in the neonatal period.

Vitamin D-binding protein (DBP) participates in the actin scavenger system, it is a carrier of vitamin D and its derivatives, it manifests the capacity to bind mainly monounsaturated and saturated fatty acids, it binds to the surface of several cells and enhances chemotactic activity of C5a of the complement. The present study was aimed at answering the question whether serum DBP level in mares is related to levels of this protein in colostrum and in serum of its progeny. For this purpose, sera from 77 mares, colostra from 72 mares and sera from 69 Thoroughbred foals were collected. Mother's age, number of deliveries experienced in the past, month of delivery, feeding of foals with colostra were recorded. Blood of the foals was sampled from the umbilical vein during delivery (0h) and 36-48 h after delivery from the external jugular vein, colostra of the mares were obtained after delivery and blood of the mares was sampled 36-48 h after delivery. Concentration of DBP was estimated by a self-designed ELISA. In the present study, DBP concentrations in newborn's serum were found independent of their concentrations in mother's serum, her age and number of parities experienced in the past. Colostrum DBP level was found to be lower than that in the mare's serum and was not correlated to the concentration of this protein in mare's serum. There was no effect of colostrum feeding on DBP level in the foal serum. These results indicate that serum DBP concentration in newborn foals depends on factors which act directly on the foal. Because of the lack of correlation between plasma and colostrum concentrations of DBP, it can be assumed that DBP is synthesised in the mammary gland and/or specific transport mechanisms exist in the mammary gland.

Angiotensin 1-7 formation in breast tissue is attenuated in breast cancer - a study on the metabolism of angiotensinogen in breast cancer cell lines.

The tissue renin-angiotensin system (RAS) plays an important role in the development and progression of many diseases. It has been confirmed that angiotensin II (ANG II) participates in the proliferation and angiogenesis of breast cancer. Moreover, some RAS dysregulations in cancer have been observed. Recent studies on the role of two opposite axes of angiotensinogen metabolism - ACE (angiotensin-converting enzyme)/ANGII/AT1R (angiotensin receptor type 1) and ACE-2/ANG 1-7/MAS (mitochondrial assembly) - indicate their importance in tumor growth and invasion, but studies describing the metabolic pathways in breast cancer and the role of newer angiotensins, such as ANG 1-12, remain lacking. In this study, the metabolism of angiotensinogen fragments in three breast cancer lines, namely, MDA-MB-231, MCF-7, and T-47D, compared with normal breast tissue cells (PCS-600) was estimated. Incubation of the cancer cells with angiotensinogen resulted in the prevalent formation of ANG 1-7. A difference in the ability to form ANG II was observed between cell lines. In normal breast cells, the strong predominance of the ACE-2/ANG 1-7/MAS pathway was detected. In cancer cells, differences in angiotensinogen metabolism depending on cancer line were observed; the prevalence of the ACE/ANG II/AT1R pathway was shown. Expressions of the RAS component were dysregulated in cancer cells and differed between cell lines. In conclusion, the ability of breast cancer cells to produce numerous angiotensin peptide metabolites was demonstrated. The metabolism of angiotensinogen differed between various types of breast cancer cells. The obtained results indicate the greater importance of the classical pathway - ACE/ANG II/AT1R - in breast cancer cells. The production of ANG 1-12 seems to be marginal in breast tissue, but a tendency for the higher formation of this peptide in cancer cells was observed. The production of ANG 1-7 was significantly lower in cancer cells, whereas the expression of MAS receptor was higher than that in the control. This finding suggests that substances with MAS receptor agonist activity could be useful in the treatment of breast cancer, but this requires further investigations.

Measurement of angiotensin metabolites in organ bath and cell culture experiments by liquid chromatography - electrospray ionization - mass spectrometry (LC-ESI-MS).

The metabolism of renin-angiotensin system (RAS) is more complicated than previously expected and understanding the biological phenomena regulated by variety of angiotensin metabolites requires their precise and possibly comprehensive quantitation. Physiological concentrations of angiotensins (Ang) in biological fluids are low, therefore their accurate measurements require very sensitive and specific analytical methods. In this study we developed an accurate and reproducible method of quantitation of angiotensin metabolites through coupling of liquid chromatography and electrospray ionization - mass spectrometry (LC-ESI-MS). With this method main angiotensin metabolites (Ang I, II, III, IV, 1-9, 1-7, 1-5) can be reliably measured in organ bath of rat tissues (aorta, renal artery, periaortal adipose tissue) and in medium of cultured endothelial cells (EA.hy926), exposed to Ang I for 15 minutes, in the absence or in the presence of angiotensin converting enzyme inhibitor, perindoprilat. Presented LC-ESI-MS method proved to be a quick and reliable solution to comprehensive analysis of angiotensin metabolism in biological samples.

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.

Perindoprilat changes ANG (1-9) production in renal arteries isolated from young spontaneously hypertensive rats after ANG I incubation.

We used mass spectrometry to quantitate production of angiotensinogen metabolites in renal artery of 3- and 7-month-old Wistar-Kyoto (WKY) and Spontaneously Hypertensive Rats (SHR). Tissue fragments were incubated for 15 min in oxygenated buffer, with added angiotensin I. Concentrations of angiotensins I (ANG I), II (ANG II), III (ANG III), IV (ANG IV), angiotensin (1-9) [ANG (1-9)], angiotensin (1-7) [ANG (1-7)], and angiotensin (1-5) [ANG (1-5)], excreted into the buffer during experiment, were measured using liquid chromatography-mass spectrometry (LC/MS) and expressed per mg of dry tissue. Effects of pretreatment with 10 microM perindoprilat on the production of ANG I metabolites were quantitated. Background production of any of ANG I metabolites differed neither between WKY and SHR rats nor between 3- and 7-month-old rats. Perindoprilat pretreatment of renal arteries resulted, as expected, in decrease of ANG II production. However, renal arteries of 7-month-old SHR rats were resistant to ACE inhibitor and did not change ANG II production in response to perindoprilat. In renal arteries, taken from 3-month-old rats, pretreated with perindoprilat, incubation with ANG I, resulted in the level of ANG (1-9) significantly higher in SHR than WKY rats. Our conclusion is that in SHR rats, sensitivity of renal artery ACE to perindoprilat inhibition changes with age.

Exogenous Angiotensin I Metabolism in Aorta Isolated from Streptozotocin Treated Diabetic Rats.

Purpose. Products of angiotensin (ANG) I metabolism may predispose to vascular complications of diabetes mellitus. Methods. Diabetes was induced with streptozotocin (75 mg/kg i.p.). Rat aorta fragments, isolated 4 weeks later, were pretreated with perindoprilat (3 µM), thiorphan (3 µM), or vehicle and incubated for 15 minutes with ANG I (1 µM). Products of ANG I metabolism through classical (ANG II, ANG III, and ANG IV) and alternative (ANG (1-9), ANG (1-7), and ANG (1-5)) pathways were measured in the buffer, using liquid chromatography-mass spectrometry. Results. Incubation with ANG I resulted in higher concentration of ANG II (P = 0.02, vehicle pretreatment) and lower of ANG (1-9) (P = 0.048, perindoprilat pretreatment) in diabetes. Preference for the classical pathway is suggested by higher ANG III/ANG (1-7) ratios in vehicle (P = 0.03), perindoprilat (P = 0.02), and thiorphan pretreated (P = 0.02) diabetic rat. Within the classical pathway, ratios of ANG IV/ANG II (P = 0.01) and of ANG IV/ANG III (P = 0.049), but not of ANG III/ANG II are lower in diabetes. Conclusions. Diabetes in rats led to preference toward deleterious (ANG II, ANG III) over protective (ANG IV, ANG (1-9), and ANG (1-7)) ANG I metabolites.

The renin-angiotensin system and its vasoactive metabolite angiotensin-(1-7) in the mechanism of the healing of preexisting gastric ulcers. The involvement of Mas receptors, nitric oxide, prostaglandins and proinflammatory cytokines.

The inhibition of angiotensin-converting enzyme (ACE) or the blockade of angiotensin (Ang) AT-1 receptors affords protection against acute gastric mucosal injury, but whether the major metabolite of renin-angiotensin system (RAS), Ang-(1-7), accelerates the healing process of preexisting gastric ulcers remains unknown. Previous studies documented that Ang-(1-7) acting via its own Mas receptor exerts vascular responses opposing those of Ang II. We studied the effects of the Ang-(1-7)/Mas receptor axis on the healing rate of acetic-acid-induced gastric ulcers with or without the blockade of Mas receptors by A 779 and compared it with the effects of activation and blockade of the AT-1 receptor by the treatment with Ang II and losartan, respectively, the inhibition of ACE by lisinopril, the NO/cNOS inhibition by L-NAME and inhibition of prostaglandin/COX system by indomethacin in the presence of Ang-(1-7). Additionally, ex vivo metabolism of Ang I in gastric tissue was assessed by LC/MS method. At day 9 after ulcer induction, the area of these ulcers and the accompanying changes in total gastric blood flow (GBF) were determined as were gastric mucosal blood flow (GMBF) at ulcer margin and gastric oxygen uptake (GVO2). The gastric mucosal expression of mRNAs for constitutive nitric oxide synthase (cNOS), superoxide dismutase (SOD), and pro-inflammatory cytokines interleukin 1ß (IL-1ß) and tumor necrosis factor alpha (TNF-α) and plasma level of both cytokines were determined by RT-PCR and ELISA. The 9 days treatment with Ang II dose-dependently increased the area of gastric ulcers and this effect was accompanied by a significant fall in the GBF, GVO2 and GMBF at ulcer margin. In contrast, treatment with Ang-(1-7) which produced a significant rise in the luminal content of NO significantly reduced the area of gastric ulcer and significantly increased the GBF, GVO2 and the GMBF at ulcer margin. Similar GMBF changes and significant reduction the area of gastric ulcer was observed in rats with gastric ulcers treated with the agonist of Mas receptor, AVE 0991. These effects of Ang-(1-7) and AVE 0991 were eliminated by blockade of the Mas receptor with A779. Similarly to Ang-(1-7), treatment with losartan or lisinopril significantly reduced the area of gastric ulcers and the accompanying increase in the GMBF at ulcer margin and these effects were significantly attenuated by a concomitant administration of L-NAME and indomethacin. The rate of healing of ulcers was associated with a decrease in ex vivo Ang-(1-7) formation and this effect was attenuated by lisinopril. The treatment with Ang-(1- 7) or AVE 0991 increased the expression of mRNA for cNOS and SOD and downregulated that of IL-1ß and TNF-α followed by the decrease in the plasma IL-1ß and TNF-α levels. We conclude that the Ang-(1-7)/Mas receptor system accelerates the healing of preexisting gastric ulcers via an increase in the gastric macro- and microcirculations, and an increase in gastric tissue oxygenation. These effects are mediated by PG and NO derived from overexpression of cNOS, an increase in the expression of antioxidizing enzyme SOD 2 and an anti-inflammatory action involving the inhibition of expression and release of pro-inflammatory cytokines IL-1ß and TNF-α. Our results seem to underlie the importance of the Ang-(1-7), AT-1 and Mas receptors in the regulation of local vascular and metabolic effects associated with mechanism of gastric ulcer healing.

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.

Metastatic potential of human CX-1 colon adenocarcinoma cells is dependent on the expression of sialosyl Le(a) antigen.

Several lines of evidence indicate that sialosyl Le(a), tumor-associated carbohydrate antigen present on human colon carcinoma cells, is involved in formation of metastases. To study the role of this carbohydrate structure in development of metastases, we have used the clone of human colon carcinoma CX-1 cells transfected with antisense expression vector containing fragment of cDNA for alpha1,3/4-fucosyltransferase (FT III), which is involved in synthesis of sialosyl Le(a) tetrasaccharide. It has been reported previously that, in contrast to the parental cells, the antisense-transfected CX-1.1AS5 cells do not express sialosyl Le(a) and do not adhere to E-selectin-expressing CHO cells. In the present work we have studied the formation of liver metastases by CX-1.1AS5 cells after their orthotopic or intrasplenic implantation into athymic nu/nu mice. After orthotopic implantation of sialosyl Le(a)-negative colon carcinoma CX-1.1AS5 cells, the number of mice with liver metastases was markedly lower (21% of mice) in comparison with their number after implantation of the parental CX-1.1 cells (86% of mice). However, no differences in ability to form colonies in liver were observed between parental CX-1.1 cells and antisense-transfected CX-1.1AS5 cells after intrasplenic inoculation. The liver metastases were formed in 89% and 84% of mice, respectively. Our data support the thesis on the importance of sialosyl Le(a) antigen expression in the development of liver metastases by colon cancer cells, and indicate the role of transplantation route and primary tumor localization in formation of metastases.

Mesoionic oxatriazoles (MOTA): NO-donating characteristics and pharmacology.

Biological role of nitric oxide (NO), functioning of isoforms of NO synthetases (NOS) and pharmacology of principle NO-donors were reviewed. NO donating characteristics and pharmacology of 23 mesoionic oxatriazoles (MOTA) were compared with those of 5-morpholinosydnonimine (SIN-1), S-nitroso-N-acetylpenicillamine (SNAP), sodium nitroprusside (NaNP) and glyceryl trinitrate (GTN). It is concluded that in vitro NO donating profile of MOTA hardly can be used as a predicting measure for their pharmacological activities either in vitro or in vivo. If anything, fast NO releasers seem to be stronger vasorelaxants than MOTA with slow NO releasing properties. Still, among representatives of this last category of MOTA one may find efficient antithrombotic and thrombolytic agents. For instance, MOTA 5-oxides were more potent thrombolytics than SIN-1, SNAP or NaNP. Also MOTA with potent anti-platelet action in vitro seem to be potent relaxants of tracheal strips. In summary, by manipulating the chemical structures of MOTA one may obtain relative selectivity towards vasorelaxant, anti-platelet, thrombolytic or tracheorelaxant properties. Thus different categories of MOTA might be designed with a hope of achieving hypotensive, antithrombotic, thrombolytic or anti-asthmatic drugs.