Morphological parameters of the angiogenic response in pancreatic ductal adenocarcinoma--correlation with histological grading and clinical data.

The aim of this study was to evaluate angiogenesis in tissue of the pancreatic ductal adenocarcinoma and to correlate it with histopathological data such as tumour differentiation, tumour size, lymph node metastasis and patients survival. Tumour samples obtained during surgery from 36 patients were immunostained for the presence of blood vessels with monoclonal antibody against the CD31 molecule. Evaluation of microvasculature was perfomed by counting the microvessel density (MVD) in selected areas under light microscope as well as by computer assisted image analysis (CAIA). In the latter, the following parameters were used for assessment of microvessels: mean number/field, mean area of the vessels, total area/field and total perimeter/field. MVD values obtained under optical microscope and with CAIA were highly correlated. All parameters characterising microvasculature in CAIA also revealed a significant correlation with the histological grading of tumours; generally the less differentiated tumours manifested more extensive vascular network. No significant relationship was found between the tumour size and any of the CAIA parameters. The area of vessels (both total and mean values) revealed a significant, inverse correlation with the incidence of lymph node metastases. The same type of correlation was also found between the mean vessel area and the postoperative survival period. The results show that CAIA of microvessels offers new parameters with some predictive value for the outcome of patients with pancreatic cancer.

Helicobacter pylori-induced hyperproliferation: relevance for gastric cancer development in connection with mutagenic factors.

AIMS: Current data on Helicobacter pylori induced regenerative hyperproliferation of the antral gastric mucosa and significance in cancer development are still under discussion and investigation. An improved method for evaluation of the regeneratory process in antral mucosa is introduced and compared with the conventional method used for determination of proliferating cells in perpendicular sections of the gastric mucosa. METHODS: Using a combination of immunohistochemistry and PAS-staining the expression of Ki-67 (MiB1)-proliferation associated antigen was analyzed in 50 Helicobacter pylori (Hp) positive and 35 negative biopsies of the gastric antrum. PAS-staining was performed to identify the proliferative zone of the antral gastric glands. The degree of inflammation was evaluated by grades on routinely H & E-stained slides. RESULTS: Proliferative activity is significantly increased in Hp-positive cases (p = 0.00095) compared to negative ones. By using the conventionally applied proliferation index, every nucleus has to be counted and the proliferation zone is identified by at least one positive stained nucleus. The method presented here seems to be easier because the proliferative zone is clearly identified by PAS staining of neutral glycoproteins characteristic for the proliferation zone of antral glands. The density of labeled nuclei is determined and is expressed as a proliferation factor. This factor gives more distinct values, is easier to evaluate and shows a better correlation with the helicobacter status and the degree of inflammation. These results are discussed in relation to the data from the literature and with a view to other relevant factors in the course of carcinogenesis, such as growth factors, mainly EGF, p53 mutation and role of apoptosis, genetic instability and local production of oxidants. CONCLUSION: Helicobacter pylori induces an increase of regenerative proliferation activity. Under these conditions the chance of mutation is increased and time for DNA repair reduced. This could be at least a part of multiple step carcinogenesis. The newly introduced combination of staining procedures (PAS/MiBI) allows a more differentiated evaluation of the proliferation zone and its widening. This method can be more easily handled in follow-up studies than the method using perpendicular sections because in this method heavy irregularities of gland pattern induced by accompanying inflammatory processes considerably hinder evaluation.

Cystosarcoma phyllodes of the breast: prognostic significance of proliferation and apoptosis associated genes.

BACKGROUND: Cystosarcoma phyllodes (CP) is a rare fibroepithelial tumor of the breast. So far no valuable prognostic factors are available for predicting the biological behaviour and often unexpected clinical outcome of this tumor. PATIENTS AND METHODS: Using immuno-histochemistry, Mib-1 evaluated proliferative activity and expression of p53, Bcl-2, c-erbB1, c-erbB2, cytokeratin and vimentin were analysed in 17 benign and 12 malignant CP. RESULTS: Cell proliferation was significantly higher in malignant tumors compared to benign ones both in the epithelial and mesenchymal parts. In the epithelial part Bcl-2 positive cells were found more frequently in benign than in malignant lesions. Positive immunoreactivity for p53 was seen in only one malignant case. The reaction was positive in the both epithelial and mesenchymal compartments. Expression of c-erbB-proteins was not found in any case. The intermediate filament proteins indicate clearly the two components of the tumor. CONCLUSIONS: We concluded that increased proliferate activity and downregulation of Bcl-2 may be associated with dedifferentiation of cells and malignant progression. C-erbB-proteins are obviously not involved in carcinogenesis of CP.

Differential expression of cerbB3 in naevi and malignant melanomas.

28 naevi, 43 malignant melanomas and 16 lymph nodes with melanoma metastases were stained immunohistochemically for the expression of cerbB3 oncoprotein. using antibody RTJ1. Positive reaction was found in 60% of naevi and in 25% of melanoma cases. The difference in frequency of the reaction occurrence between naevi and melanomas was statistically highly significant (p = 0.0017). In naevi, the percentage of cerbB3 positive cells (56.5%) was also significantly higher (p = 0.021) as compared with that of melanomas (37%). The cerbB3 positive cells were found only in cases without metastases. Positive reaction was also observed in 4 out of 16 investigated lymph nodes with melanoma metastases. The role of cerbB in the development of melanoma is discussed.

Antigen Ki-67 and c-myc oncogene as related to histoclinical parameters in pigmented skin lesions.

121 cases of cutaneous melanomas, 82 lymph nodes with melanoma metastases and 62 naevi were stained immunohistochemically for presence of Ki-67 antigen and c-myc oncogene. A significant correlation between expression of investigated markers, and survival time as well as presence of metastases, in cutaneous melanomas was found (p < 0.05). The role of Ki-67 antigen for differential diagnosis between naevi and melanomas is also demonstrated.

Immunohistochemical localization of CD44 variants 5 and 6 in human gastric mucosa and gastric cancer.

The aim of this study was to compare the distribution of CD44 variants 5 and 6 in normal mucosa and gastric cancer and to determine their relationship with histoclinical Parameters of the disease. Experimental material included 112 paraffin blocks of various human gastric carcinomas. Both variants of CD44 were detected immunohistochemically with primary antibodies deriving from Bender Med. Systems. In normal gastric mucosa positive reactions with both antibodies were observed in surface epithelium, parietal cells, myocytes and vascular endothelia. They were also found in intestinal enterocytes, esophageal epithelium and myoepithelial. Additional reactivity with antibodies against variant 6 was observed in Paneth cells. In gastric cancer, variants 5 and 6 were demonstrated in 91% and 64% of cases respectively, without significant correlation with the tumor type. The occurrence of variant 6 correlated positively with tumor size (p = 0.081) and negatively with histological grading (p = 0.093). The relationship with metastases was insignificant.

Comparative distribution of S-100 protein and antigen HMB-45 in various types of melanomas and naevi.

The content of S-100 protein and antigen HMB-45 in various types of melanomas and skin naevi was compared with respect to the frequency of two antigens, their distribution within lesions as well as diagnostic or prognostic relevance. The study material comprised 72 skin melanomas, 25 melanomas of the oral cavity and 63 non-malignant skin naevi. Formalin fixed, paraffin-embedded sections were used for immunohistochemical reactions with polyclonal rabbit anti-S-100 antibody (Dako) and monoclonal anti-HMB-45 antibody (Dianova) followed by PAP and APAAP Kits (Dianova), respectively. Detection of HMB-45 was preceded by a microwave treatment of sections. S-100 protein was found in all cases of melanomas irrespective of their location or histological type, but HMB-45 was missing in 8% of oral melanomas. Distribution of the antigens within tumors was heterogeneous and often mutually exclusive. S-100 protein was also present in all types of naevi, while HMB-45 was absent in intradermal naevi and strictly confined to epidermal component in the remaining ones. The intensity of staining for S-100 correlated inversely with the patients' survival. It was concluded that demonstration of HMB-45 might facilitate a differentiation of naevi from melanomas, whereas staining for S-100 protein might have some prognostic significance.

Immunohistochemical demonstration of S100 protein in malignant melanomas of the facial skin and oral cavity.

Immunohistochemical demonstration of S100 protein was performed in 56 cases of malignant melanoma of the facial skin and oral cavity. The depth of invasion was measured comparatively in HE sections and in sections stained for S100 protein. Comparison of measured melanoma invasion depth in S100- and HE-stained sections revealed a deeper invasion of the tumor in S100-stained slides than in slides stained routinely with HE according to Breslow's melanoma staging procedure. A reverse relationship between the intensity of immunohistochemical staining for S100 protein and survival rate was found in both melanomas of the facial skin and oral cavity. Although the presence of S100 protein has been demonstrated previously in skin melanomas, no similar investigations concerning the oral mucosa have been performed up to now.

Localization of S-100 protein in pig ovarian structures.

Distribution of S-100 protein in porcine ovaries was revealed by an application of polyclonal antibodies against S-100 protein and PAP universal kit to formalin-fixed, paraffin-embedded tissues. The presence of S-100 protein was demonstrated in follicular cells of all ovarian follicles (including their atretic forms), with a decrease in intensity of the reaction parallel to an increase in follicle size. No reaction was found in oocytes, regardless of the developmental stage of the follicle. Weak expression of S-100 protein was observed in theca interna cells of mature follicles and in the youngest generation of theca-lutein cells of the corpus luteum. A distinctly positive reaction was also observed in groups of cells resembling hilus cells as well as in endothelia of arteries and some capillaries. Results are compared with data concerning the localization of S-100 protein in human and bovine ovaries. Possible relationship between the presence of S-100 protein and the level of cellular differentiation is discussed.

An application of MIB antibody to the retrospective study of melanomas of oral mucosa and facial skin.

A new monoclonal antibody prepared against a fragment of Ki-67 antigen MIB, from Dianova, was applied for investigation of malignant melanomas of facial skin (25 cases) and the oral cavity (25 cases), which were routinely embedded in paraffin. The values of the Ki-67 index (expressed as a percentage of positive nuclei) were correlated with TNM characteristics of tumors and patient survival. Significant correlation was found between the Ki-67 index and the level of lymph node involvement (N value), the presence of distant metastases and the time of patient survival. A positive relationship between the Ki-67 value and tumor size was also observed although it lacked statistical significance.

Influence of initial medium content (pH, serum and Ca++ concentration) on leukocyte migration test.

The aim of the study was to check to what extent inhibition of leukocyte migration in capillary tubes depends on alteration in pH, serum content and Ca++ concentration in medium in which the test is started. We found that relatively wide variations of pH (between 6.4 and 8.2) did not influence the final result. The best conditions for leukocyte migration were created by addition of 5% calf foetal serum, whereas a contamination of medium with autologous serum made a proper interpretation of the test difficult because of a resulting dispersion of cells. Neither removal nor addition of Ca++ ions had a significant effect on migration of leukocytes, unless combined with a simultaneous presence of calcium ionophore A 23187 in 0.5 microM concentration.

Correlation between the occurrence of Ki-67 antigen and clinical parameters in human breast carcinoma.

Cryostat sections of ductal breast carcinomas obtained during surgery from 48 patients were subjected to an indirect immunostaining with an antibody against Ki-67 antigen (Dako). The primary reaction was visualized by the APAAP technique with new fuchsin as chromogen. The percentage of positively stained nuclei was correlated with tumor size, number of metastatic lymph nodes, presence or absence of metastases in distant organs as well as with patient's age. The best correlations were achieved when the data were considered separately within the groups with low, medium and high Ki-67 values. A possible significance of Ki-67 evaluation for the prognosis and therapy is discussed.

Localization of intermediate filament proteins in various structures of the human placenta as revealed by immunoperoxidase technique.

Formalin-fixed, paraffin embedded fragments of normal human placenta were subjected to immunoperoxidase technique with the use of Dako Kits appropriate for detection of vimentin, desmin, and cytokeratins (K 660, 530, and 518 respectively). Distribution of the intermediate filament proteins in syncytiotrophoblast, connective tissue of villous stroma, walls of blood vessels as well as in cells of basal plate and placental islets was compared. General pattern of staining and its localization was characteristic for each of the 3 types of antibodies. In spite of that, most structures observed revealed the presence of more than one type of intermediate filament proteins, with the exception of syncytiotrophoblast containing cytokeratins only. Staining of blood serum and of connective tissue matrix with anti-desmin and anti-cytokeratin antibodies was also observed, whereas fibrinoid remained always negative.

Comparative distribution of four lysosomal enzymes within various structures of the human placenta.

The histochemical activity of 4 lysosomal enzymes, acid phosphatase (AP), nonspecific esterase (NE), aryl sulphatase (AS), and beta-glucuronidase (BG), was compared in following structures of the human placenta: syncytiotrophoblast, villous stromal cells, fetal capillaries and larger blood vessels, cells of basal plate, macrophages, and Hofbauer cells. In spite of a general similarity in distribution of the investigated enzymes, differences concerning particular structures were found. Thus positively stained granules in endothelia of capillary vessels were revealed only in the reaction for BG, although contours of capillaries were also outlined by the diffuse reaction product for AS. The muscular layer of larger vessels reacted strongly for AS and weakly for NE with the remaining reactions being negative. In syncytiotrophoblast, BG appeared much less active than the other 3 enzymes. The possible significance of the BG positive granules in endothelial of capillaries and of the occasional divergence in distribution of the classical lysosomal markers (AP and BG) is discussed.

Effect of the new theophylline derivative on leukocytes migration in vitro.

New theophylline derivative: 7-beta-hydroxy-benzylopiperazinopropyl-theophylline (R6) has been investigated for its influence on spontaneous migration of leukocytes, as well as for their response to calcium ionophore A23187 and phytohemagglutinin (PHA). Leukocytes from the peripheral blood of healthy donors were used and migration developed according to the routine capillary method. It has been found that R6 inhibited migration most effectively if the cells have not been treated with other agents. An addition of R6 to media containing A23187 has slightly modified an original action exerted by ionophore itself, the final effect being dependent on calcium concentration in the medium. When R6 was applied simultaneously with PHA its effect on leukocyte migration was negligible, if however R6 was added to leukocytes previously pretreated with PHA--an ultimate increase in migration inhibition was observed. It is suggested that R6 inhibits both spontaneous locomotion of leukocytes and their response to stimulating agents acting via an increase in intracellular cAMP level and/or by hindering the calcium transport across cell membrane.

Effects of seminal constituents in leucocyte migration tests on infertile couples.

Leucocyte migration tests were performed with cells from both partners of infertile couples in the presence of seminal constituents from the male partners. Leucocytes from fertile women were used as controls. Homogenized spermatozoa inhibited leucocyte migration in 36.1% of women and 20.7% of men from infertile couples and in 18.4% of control women. In all groups, occasional stimulation of leucocyte migration was observed (8.3%, 3.4% and 2.6% respectively). Although both types of changes in migration when considered together were statistically more frequent in the group of infertile women it was not possible to distinguish, on the basis of this test, between fertile and supposedly infertile individual women. It is suggested, however, that an inhibitory effect induced by spermatozoa on the men's own leucocytes may be of diagnostic significance. In contrast to spermatozoa, seminal plasma caused inhibition of leucocyte migration in the majority of cases -- 88% of women and 71.9% of men from infertile couples and 77.8% of fertile women. Those patients whose semen lacked this inhibitory property are deserving of further study.

Effect of the new theophylline derivatives on degranulation of mast cells and phosphodiesterase activity in smooth muscles.

Effect of the new theophylline derivative (beta-hydroxy-benzylopiperazinopropyl-theophylline), designed as R6 on degranulation of the mast cells from rat mesenteries, was tested in vitro and in vivo. In vitro it was found that R6 significantly inhibited mast cell degranulation induced both by the histamine liberator C 48/80 and by rabbit globulin directed against rat serum proteins. The effect was comparable to that revealed by mepyramine, but much stronger than the effects of DSCG and aminophylline. The influence of R6 on mast cells degranulation was less pronounced in the in vivo systems. It was demonstrated by histochemical methods that R6 did not change activity of lactic and succinic dehydrogenases, neither activity of lysosomal hydrolases. In separate system including small intestine from rat it was shown that R6 inhibited activity of the phosphodeisterase in smooth muscles. It was suggested that the effect of R6 on mast cells degranulation is mediated by an increase in cAMP as well as by indirect influence on Ca++ influx.

Acid and alkaline phosphatase activity in the morphotic elements of the amniotic fluid.

Acid and alkaline phosphatases were determined histochemically in the cells of the amniotic fluid obtained from the term pregnancies. Relatively high activity of the acid phosphatase was found within macrophages, few neutrophils and some polygonal cells from the intermediate layer of stratified epithelium. Moderate activity of this enzyme was demonstrated in sheets of superficial epitheliocytes and partly cornified cells of hair. Weak activity of the alkaline phosphatase was found in superficial epithelia, in neutrophils and clumps of meconium. It is suggested that demonstration of the acid phosphatase in the morphotic elements of the amniotic fluid might possess certain diagnostic significance.