Proteinase-activated receptor-2 (PAR-2) is a ubiquitous surface molecule participating in many biological processes. It belongs to the family of G protein-coupled receptors activated by the site-specific proteolysis of trypsin and similar proteases. Altered function of PAR-2 has been described in different malignant tumors. In the present study, we investigated the expression of PAR-2 in breast cancer surgical specimens and the role of trypsin in breast cancer cell line MDA MB-231 proliferation and metabolism. A total of 40 surgical samples of infiltrative ductal breast cancer and breast cancer cell line were included in this study. We analyzed PAR-2 expression by immunohistochemistry, RT-PCR and western blot. Activation of PAR-2 on cell line MDA MB-231 was measured using calcium mobilization assay determined by flow cytometry. MTT cell metabolism assay and cell count analysis were used to assess the trypsin influence on breast cancer cell line MDA MB-231 proliferation. Immunohistochemical examination showed the expression of PAR-2 in all samples of breast cancer surgical specimens and high levels of cell lines which was confirmed by RT-PCR and western blot. Calcium mobilization assay corroborated the activation of PAR-2 on cell line MDA MB-231 either by trypsin or by an agonistic peptide. Cell metabolism assay and cell count analysis showed significant differences of proliferative activity of breast cancer cells dependent on the presence or absence of trypsin and serum in the culture medium. PAR-2 is expressed by high levels in infiltrative ductal breast cancer tissue specimens. PAR-2 is also strongly expressed in studied breast cancer cell lines. PAR-2 is activated by trypsin and also by agonistic peptide in the model of breast cancer cell line MDA MB-231. Activation of PAR-2 in vitro influences proliferative and metabolic activity of breast cancer cell line MDA MB-231. The action of trypsin is modified by the presence of serum which is a potential source of protease inhibitors.

Beranová M., P. Manìáková, P. ·íma, J. Slípka, F. VoÏeh, J. Koãová, M. âervinková, J. S˘kora: Morphology of Adrenal Gland and Lymph Organs is Impaired in Neurodeficient Lurcher Mutant Mice. Acta Vet. Brno 2002, 71: 23-28. There is a tight structural relation and functional co-operation between the nervous, endocrine and immune systems. A dense network of soluble neuro-endocrine and immune mediators exists to ensure close interactions. These hormones, cytokines and neurotransmitters all interact through positive and negative feed-forward and feedback loops. The mediators, once considered specific to the central nervous system (CNS), the endocrine system (ES) or the immune system (IS), do in fact act in all three systems, forming that way the united neuro-endocrine-immune system. The complex neuro-endocrine-immune networks operate under both physiological and pathological conditions.In the presented study microscopical analyses of selected immune organs (the thymus, spleen, inguinal and subscapular lymph nodes) and of the adrenal gland of the neurodeficient Lurcher mutant mice and control C3H mice were performed. In the neurodeficient mice the morphology of the immune organs was impaired. The changes followed in the spleen, especially the increased number of megakaryocytes, lead to the hypothesis of enhanced extramedullar hemopoiesis in the neurodeficient Lurcher mutant mice. Histopathological analysis of the adrenal gland showed the relative hypertrophy of the adrenal medulla. Regarding the adrenal cortex, the three cortical zones, zona glomerularis, fasciculata and reticularis, are difficult to be distinguished. It has been supposed that structural changes of adrenal medulla could document the increased secretion of catecholamines in the neurodeficient animals.Our observations confirm the idea of the tight cooperation of neuro-endocrine-immune structures and contribute to its better understanding, specifically in the conditions of postnatally progressing neurodeficiency. Lurcher mutant mice, homeostatic relatioships, neuro-endocrine-immune system, adrenal gland, megakaryocytesThe intrinsic condition for the individual survival is a balance of the internal enviromenthomeostasis. There are many proofs of the existence of a unified neuro-endocrine-immune regulatory system that is responsible for maintaining the homeostasis (Michael and Chapman 1990;Weigent and Blalock 1995;Besedovsky et al. 1983). The regulatory relations within that system are mutual and complex (Jankovic 1989;Csaba 1994; ·íma and Vûtviãka 1990;Provinciali and Fabris 1991;Pertseva 1991; Slípka 1961).To contribute to the explanation of these relations we used in our previous study "an experiment of the nature", gross-brain malformation -anencephaly -which eliminates the central nervous system and consequently the neuro-endocrine part of the homeostatic system (Slípka et al. 1997; B e ranová 1994). The spontaneously aborted human foetuses without any external malformation and human anencephalic foetuses were compared with

Aims:The main feature of fibrosing idiopathic interstitial pneumonias (fIIPs) is the fibroproliferative potential of underlying pathogenetic process. We hypothesize that the concentration of potential markers of fibroproliferative healing, PAR-2, TGF-β1, TNF-α and IL-4Rα in bronchoalveolar lavage fluid (BALF) differ in patients with fIIPs compared to controls (C).Patients and Methods:10 patients with fIIPs and 9 controls (C) were included to the study.Concentrations of CD124 (IL4Rα), PAR-2, TGF-β1 and TNF-α in BALF were determined using the ELISA method.Results:We observed higher concentrations of IL4Rα (fIIPS 1499.4 pg/ml vs C 255.5 pg/ml; p < 0.05), PAR-2 (fIIPS 1807.9 pg/ml vs C 421.0 pg/ml; p < 0.05) and TGF-β1(fIIPS 283.0 pg/ml vs C 197.1 pg/ml; p < 0.01) in BALF in fIIPs versus C. The values of TNF-a in BALF did not differ significantly in fIIPs compared to controls. The ratios also showed differences in fIIPS and C: IL4Rα/TGF-β1 (fIIPS 6.19 vs C 0.68; p = 0.0143); TNF-α/IL4Rα (fIIPS 0.84 vs C 7.93; p = 0.043); TGF-β1/TNF-α (fIIPS 0.21 vs C 0.16; p = 0.0179) and protein/PAR-2 (fIIPS 0.06 vs C 0.28; p = 0.0033).Conclusions:We found that PAR-2, TGF-β1 and IL-4Rα are significantly up-regulated in the BALF of fIIPs compared to controls, therefore we suppose they could become biomarkers of fibroproliferative healing.

Background: Th-2 cytokine milieu including interleukin 4 (IL-4) was detected in fibrotic lung diseases. Chronic extrinsic allergic alveolitis (EAA) may be also accompanied by marked fibrogenesis. The aim of this study was to determine if IL-4 and its receptor (IL-4R-alpha) play any role in the clinical presentation and pathogenesis of chronic EAA.

Background: Th-2 cytokine milieu including interleukin 4 (IL-4) was detected in fibrotic lung diseases. Chronic extrinsic allergic alveolitis (EAA) may be also accompanied by marked fibrogenesis. The aim of this study was to determine if IL-4 and its receptor (IL-4R-alpha) play any role in the clinical presentation and pathogenesis of chronic EAA. Methods: Twenty patients originally investigated for interstitial lung disease and finally diagnosed affected with chronic EAA and sarcoidosis were prospectively enrolled into the study. Concentrations of IL-4, IL-4R-aplha and total protein were assessed in the bronchoalveolar lavage fluid (BALF) of all enrolled subjects as well as high resolution computed tomography (HRCT) scores and pulmonary function tests. Results: BALF IL-4R-alpha and total protein concentrations were significantly higher in chronic EAA patients (p < 0.05). Concentrations of BALF IL-4R-alpha were significantly higher in men than in women (p < 0.05) in EAA group. Total protein BALF levels were significantly elevated in ex-smokers with EAA compared to nonsmokers (p < 0.05). A positive correlation (p < 0.01) between IL-4R-alpha BALF concentrations and HRCT interstitial scores were observed in chronic EAA group; the IL-4R-alpha/total protein ratio showed the same significant positive correlation. A negative correlation between lung function results and IL-4R-alpha, and IL-4R-alpha/total protein as well, was also found (p < 0.05). Conclusions: We suggest a clinical relevance for the IL-4/IL-4R axis in the etiopathogenesis of chronic EAA. IL-4R-alpha could serve as a potential biomarker of lung fibrogenesis.

Objective: The nervous, endocrine and immune systems are functionally interconnected so that they may form a complex metasystem. Abnormalities within the neuroendocrine compartment can thus affect immune mechanisms and vice versa. The Lurcher-type mutation in mice has a profound impact on brain development in both homozygous and heterozygous individuals, which is followed by immune system changes. We investigated whether macroscopic changes in the thymus size were associated with an altered thymocyte development or with changes in peripheral T cell subset distribution. Methods: CD3, CD4 and CD8 expressions on thymocytes and peripheral T cells were compared with those in wild-type and Lurcher heterozygous C3H mice using surface immunophenotyping and flow cytometry. Galanthus nivalis agglutinin binding to thymocytes was measured at the same time. Results: While no differences between experimental groups were observed in 1-month-old mice, a critical reduction of numbers of thymocytes and namely double-positive cells occurred before the age of 3 months in Lurcher mice, which was accompanied by thymus involution. Interestingly, this was not accompanied by significant differences in major T subset proportions in the peripheral lymphatic tissue. Conclusions: We interpret our observations as hallmarks of premature thymus ageing in heterozygous Lurcher mice with only a marginal effect in the periphery in early adulthood.

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