Melatonin-induced augmentation of collagen deposition in cultures of fibroblasts and myofibroblasts is blocked by luzindole--a melatonin membrane receptors inhibitor.

BACKGROUND: Melatonin has been proven to have a regulatory influence on collagen accumulation in different types of wound. It was found to inhibit collagen accumulation in the superficial wound model but increase it in the myocardial infarction scar. The aim of the study is to determine the mechanism of melatonin action in the two wound types in rats. METHODS: Cells were isolated from both the superficial wound (subcutaneously inserted polypropylene net) and myocardial infarction scar (induced by ligation of the left coronary artery) and were identified by electron microscopy. RESULTS: Long-shaped cells forming whirl-like structures in culture (mainly identified as fibroblasts) were isolated from the superficial wound model, while myofibroblasts growing in a formless manner were acquired from the infarcted heart scar. Melatonin (10(-7) M) increased collagen accumulation in both fibroblast and myofibroblast cultures. Luzindole (10(-6) M), the blocker of both MT1 and MT2 melatonin membrane receptors, inhibited the effect of melatonin on the two types of cells. CONCLUSION: Regardless of various healing potentials demonstrated by the tested cells (different cell composition, growth and organization), their response to melatonin was similar. Moreover, in the two investigated cultures, augmentation of the collagen content by melatonin was reversed by luzindole, which indicates the possibility of melatonin membrane receptor involvement in that process. The present results suggest that the increased melatonin-stimulated deposition of collagen observed in the infarcted heart of rats could be dependent on activation of the melatonin membrane receptors on scar myofibroblasts.

Changes in thickness of each layer of developing chicken cornea after administration of caffeine.

The aim of the study was the presentation of changes in thickness of each layer of a developing cornea, that came into being under an influence of caffeine which was administered to chicken embryos. Research materials were 26 chicken embryos from breeding eggs that had been incubated. Breeding eggs were divided into two groups: control (n=30) in which Ringer liquid was given, and experimental (n=30) in which teratogenic dose of caffeine was administrated - 3.5 mg/egg. In 36th hour of incubation solutions were given with cannula through a hole in an egg shell directly onto amniotic membrane. After closing the hole with paraffin, eggs were put back into incubator. On 10th and 19th day of incubation corneas were taken for morphometric and morphological analysis. In experimental groups reduction of corneal thickness, thickening of corneal epithelium and corneal endothelium as well as Bowman's and Descemet's membranes, decrease of thickness of corneal stroma in comparison with the control group have been observed. Caffeine causes thickness changes of all layers and decreases the total thickness of a developing cornea.

Minichromosome maintenance (MCM) and AgNOR proteins expression in desmoid tumours: a tissue microarray analysis.

In the present study, nuclear proliferative proteins: MCM2, MCM5, MCM7, Ki-67 and AgNORs expression was assessed in paraffin sections from sporadic desmoid tumours using a tissue microarray (TMA)-based immuno- and histochemistry, respectively. Nuclear expression of MCM7, where the percentage of positive cells was 0.87% (± 1.64) (range 0-5%), was found in 4/20 (20.0%) cases. In 32/32 (100%) of the examined desmoid cases no expression of nuclear proteins MCM2 and MCM5 was detected. Nuclear expression of Ki-67 was observed in 4/21 (19%) cases. Paraffin sections from 30 cases of desmoid tumours were silver-stained to visualize AgNORs. The following AgNOR parameters were calculated: mean AgNOR number per nucleus (N), mean AgNOR area per nucleus, mean AgNOR dot area per nucleus (A), and mean AgNOR content (C = N/A). In the investigated group the mean values of AgNOR parameters were the following number: 4.34 (± 0.11); area: 0.74 µm2 (± 0.19); dot area: 0.18 m2 (± 0.01), and AgNOR content: 23.73 (± 1.85). The mean AgNOR number per nucleus and mean AgNOR content in desmoid tumours were statistically significantly higher as compared to the controls (tonsil tissue) (p<0.001). This study observed low level of MCM7 and Ki-67 and lack of MCM2, MCM5 proteins expression which may explain commonly known low mitotic activity of desmoid tumour cells. The morphology of dots related to AgNORs (number, area) and their morphometric parameters point to elevated transcriptional activity of desmoid cells.

Analysis of pRb, p16INK4A proteins and proliferating antigens: PCNA, Ki-67 and MCM5 expression in aggressive fibromatosis (desmoid tumor).

Aggressive fibromatosis (desmoid tumor) is a mesenchymal lesion originating from fascial, aponeurotic and muscular connective tissue. It rarely becomes histologically malignant. In this study we analyzed the cell cycle regulation proteins: pRb, p16, and proliferating antigens: Ki-67, PCNA, MCM5 with immunohistochemical method in archival material derived from 27 extra-abdominal (E-AD), 18 abdominal (AD) and 5 intra-abdominal (I-AD) cases of desmoid tumor. None of the examined cases (n=50) of aggressive fibromatosis was pRb-immunonegative. Heterogeneous expression of pRb was observed in 51.85% (14/27) of Group AD cases and in 5.56% (1/18) of Group E-AD cases; positive expression in 48,15% (13/27) of Group AD cases, in 94.44% (17/18) of Group E-AD cases, and in 100% (5/5) of Group I-AD cases. There were no negative cases for p16 staining in any of the examined groups. The number of heterogeneous cases in individual groups was: 33.33% (9/27) in Group AD, 50% (9/18) in Group E-AD and 40% (2/5) in Group I-AD, and positive cases: 66.67% (18/27), 50% (9/18) and 60% (3/5), respectively. Overexpression of PCNA was noted in 98% (49/50) of cases. The positive staining for Ki-67 protein was noted in 25.93% (7/27) in Group AD, in 16.67% (3/18) in Group E-AD and in 60% (3/5) in Group I-AD. None of the examined cases was immunopositive for MCM5 protein. The noted levels of pRb and p16 expression in desmoid cells reflect their function in cell cycle regulation. Probably the unsettled cell cycle progression, especially in G1 phase, is not the cause of aggressive fibromatosis pathogenesis.

Analysis of TGF-beta protein expression in aggressive fibromatosis (desmoid tumor).

Aggressive fibromatosis, usually termed desmoid tumor, develops from muscle connective tissue, fasciae and aponeuroses. Aggressive fibromatosis located in various parts of the body demonstrates differentiated biological behavior. Abnormalities in TGF-beta expression are very common in many disease processes, including neoplasms. Immunohistochemical analysis employing a monoclonal antibody against TGF-beta was performed on archival material, consisting of 38 cases of aggressive fibromatosis, among which 23 represented abdominal, 11 extra-abdominal and 4 intra-abdominal localizations. The sections for immunohistochemical study were stained using the streptavidin-biotin (ABC) method. The average percentage of cells positively stained for TGF-beta protein was 40.2% in the group of extra-abdominal, 58.5% in the group of abdominal and 72.8% in the group of intra-abdominal localizations. There were significant differences observed between the analyzed groups of desmoid tumor (p<0.05). A positive cytoplasmic reaction for TGF-beta was noted in 65.8% (25/38) of the aggressive fibromatoses. Overexpression of TGF-beta protein was noted in 39.5% (15/38) of the aggressive fibromatoses. High expression noticed in desmoid fibroblasts might indicate that this protein plays a crucial role in the development of aggressive fibromatosis.

Quantitative analysis of cyclin E and protein p34 cdc2 expression in superficial bladder cancer.

In the present study, the expression of cyclin E and kinase p34 cdc2 was investigated in preinvasive bladder tumors. The study material consisted of bladder sections (grades: GI--16 cases, GII--10, and GIII--12) collected from 38 patients in the course of the tumor electroresection. Immunohistochemical examinations were carried out with immunoperoxidase method. Antigens were labeled with NCL-CYCLIN E or NCL-p34 cdc2 monoclonal antibodies (Novocastra, UK). Positive reaction was demonstrated using ABC-universal Kit (Novocastra, UK). Differences in the protein expression in relation to the tumor grade were determined with a non-parametric Mann-Whitney's test. Increasing grade of tumors was associated with down regulation of cyclin E visible as lower percentage of cyclin E-positive cells. These changes were statistically significant for GI group as compared to groups GII and GIII (p<0.001). There were no differences between the study groups in the p34 protein expression. Cyclin E expression was inversely correlated with tumor grade therefore may be helpful in establishing therapeutic procedure.

Analysis of p27KIP1 protein and Ki-67 expression in aggressive fibromatosis (desmoid tumor).

Aggressive fibromatosis (desmoid tumor) is an uncommon locally invasive non-metastasizing neoplasm lesion. Desmoid tumor consists of fibroblasts, miofibroblasts and a significant amount of extracellular matrix. p27KIP1 (p27) protein is a member of the universal cyclin-dependent kinase inhibitor (CDKI) family that regulates progression through the cell cycle. In various human neoplasms the decreased level of p27 was observed. There were analysed 42 specimens of aggressive fibromatosis, in which there were 24 abdominal and 18 extra-abdominal cases. There was performed immunohistochemical analysis employing a monoclonal antibody against p27 protein and Ki-67 (Novocastra, UK). The sections for immunohistochemical study were stained using the streptavidin - biotin method. The average percentage of cells stained positively for all cases for p27 and Ki-67 was 22.1% (SD=29.2) and 6.0% (SD=8.8) respectively. There was no statistically significant difference between Ki-67 or p27 expression in abdominal and extra-abdominal location. Analysis of p27 and Ki-67 expression levels might indicate that low proliferating activity of desmoid fibroblasts is connected with another mechanism than the one, in which p27 takes part.