Effects of oral administration of Lonicera caerulea berries on UVB-induced damage in SKH-1 mice. A pilot study.

Solar ultraviolet radiation is a major environmental factor that has serious adverse effects on the structure and function of the skin. Although the UVB waveband (295-315 nm) represents only 5-10% of incoming UV light, it is very damaging to the skin. The aim of this study was to investigate the effect of Lonicera caerulea berries on UVB-induced damage to SKH-1 hairless mice. Mice were fed a L. caerulea berry-enriched diet (10%, w/w) for 14 days before a single UVB (1000 mJ cm(-2)) treatment. Effects on health status, antioxidant enzyme activity and expression, and DNA damage were evaluated. The bioavailability of L. caerulea phenolic components was also assessed. We found that feeding with L. caerulea berries prevented a decrease in catalase activity and stimulated NADPH quinone oxidoreductase-1, heme oxygenase-1, and gamma-glutamylcysteine synthetase catalytic and modulatory subunit expression in UVB exposed mice. Administration of the L. caerulea berry-enriched diet led to an increase in UVB-reduced interleukin-17 levels and a decrease in keratinocyte-derived chemokine protein expression that was enhanced after UVB treatment. Further, L. caerulea berries reduced UVB-induced DNA damage evaluated as number of single strand breaks, cyclobutane-pyrimidine dimer formation and H2AX phosphorylation, a marker of double strand breaks. Taken together, we provide evidence that oral administration of L. caerulea berries to mice affords at least partial protection from the adverse effects of a single UVB exposure via modulation of antioxidant enzyme activity/expression and reduction of DNA damage.

Hematopoietic stem cell separation for experimental purposes--methodic limitations.

BACKGROUND: Protooncogene CD117 is a cytokine receptor important for hematopoietic element development. Currently, we are not able to routinely separate a sufficient quantity of bone marrow CD117+ cells for experimental purposes. AIM: The aim of this study was to establish an immunomagnetic separation method for CD117+ hematopoietic stem cell isolation and to estimate the expression of chosen BCl-2 family protein members in these elements. MATERIAL AND METHODS: 120 samples of human and murine bone marrow were acquired using the magnetic separation system. The cells were stained for CD117, BCl-2, BAX, and CD33 by an indirect fluorescent immunocytochemistry. RESULTS: The flow cytometry analysis showed only 2.6% CD117+ cells from human as well as mouse bone marrow which is insufficient for further experiments. Cytospin was not good for morphologic characterization and immunophenotyping due to the fragility and destruction of the studied cells. Therefore, cell suspension staining was selected and by this method we found CD117 positivity in 70% of the mononuclerar (CD33 positive) elements in the case of chronic myeloid leukaemia. Labelling of the BCl-2 family in this case showed antiapoptotic BCl-2 expression in 80 %, proapoptotic BAX expression in approximately 5%. CONCLUSION: Our results show that CD117 immunomagnetic separation from bone marrow material is not acceptable for experimental purposes. They demonstrate that the only practical useful for the bone marrow cell examination (morphology and immunophenotype) is cell suspension staining which uncovers the distribution of both cytoplasmic proteins and surface antigens of immature blood elements.

Expression of p53, p63 and p73 in the orofacial region of human embryos.

We studied p53, p63, p73 protein expression in the orofacial region of five human embryos aged 7-18 weeks of intrauterine development using a three-step immunohistochemical method. Expression of proteins in various locations was evaluated semiquantitatively. A decrease in p53, p63 and p73 proteins occurred in the 13-week-old material with the exception of the tooth germ where a drop in p73 appeared in the ninth week.

The detection of myc proteins in the developing human kidney.

The aim of our work was the detection of c- and N- myc proteins in the developing kidney of human fetuses especially in the neogenous zone. These proteins being localized in nucleus and encoded by the cellular oncogene myc function as transcriptional factors. Myc gene represents the key control gene in the course of cellular proliferation and differentiation. It also participates in the regulation of apoptosis and the origin of some cancers. Both proteins have been proved to be inevitable for proper nephrogenesis in mice. Considering the dearth of studies dealing with human embryonic tissues, we attempted to map the localization and spatiotemporal relations of the expression of these proteins during human nephrogenesis. In addition, issuing from our previous observations, we tried to compare their ways of expression with those of Bcl-2 (antiapoptotic effect) and Bax (proapoptotic function) proteins. Histologically normal kidneys were collected from eight human fetuses ranging from the 10th-30th week of IUD. Tissue samples were fixed in methacarn and processed by routine paraffin technique. Standard indirect three-step immunohistochemical method was applied for the detection of N- and c-myc proteins. In the neogenous zone both proteins are markedly present in metanephrogenic blastema with declining intensity with the increasing age of fetus. Branches of ureteral bud are almost negative. Such localization is also typical for Bcl-2 protein whereas Bax positive cells are present mostly in branches of the ureteral bud. It is not clear if all these proteins collaborate in the course of regulation of apoptosis in human metanephros.

The occurrence of c-myc, p53 and Bcl-2 family proteins in the early phase of development of duodenal epithelium.

In last few years, numerous groups of proteins participating in the regulation of cell proliferation, differentiation and death during ontogenesis have been described. In this study we compared the occurrence of Bcl-2, p53 and myc protein families with the level of proliferative activity and apoptosis during development of duodenal epithelium. Paraffin embedded tissues of eight human embryos and foetuses aged from the 6th-18th week of IUD were used. For the detection of apoptotic cells the TUNEL method was performed, the proliferative marker PCNA and all the proteins studied were detected by means of indirect three-step immunohistochemical method. In the 6th and 8th week of intrauterine development we observed isolated TUNEL positive epithelial cells only and this was accompanied by the disperse presence of PCNA as well as by all the studied proteins: Bcl-2, Bax, Bcl-XL, c-myc, N-myc, p53, p63 and p73. In the early foetal period of duodenal development we registered changes in PCNA and TUNEL positivity in accordance with the constitution of the stem cell pool on base of villi, where more numerous Bcl-2 positive cells were also found. The separation of primitive crypts and villi was not accompanied by any differences in distribution of Bax, Bcl-XL, c-myc, N-myc, p63 and p73 proteins between those compartments: all the studied proteins showed dispersed character. P53 rapidly decreased in this period. In the 18th week of intrauterine development the balance between proliferation in crypts and apoptosis of villi epithelium was well established and no p53 positive cells were found. In the presence of Bcl-2, Bax, Bcl-XL, p63 and p73 we did not find any dramatic changes. The myc proteins were restricted within the epithelium of the Lieberkuhn crypts only.

Degradation phase of apoptosis during the early stages of human metanephros development.

Apoptosis as a vital process is necessary for human intrauterine development. Not only the induction and course of apoptosis, but engulfment of the apoptotic cells (bodies) were the centre of our interest. Macrophages were detected in the early stages of human intrauterine development and the role of macrophages in the clearance of apoptotic cells in the early stages of human metanephros development was confirmed.

Comparative study of apoptosis-detecting techniques: TUNEL, apostain, and lamin B.

The mechanism of the apoptotic process is generally recognized as being highly intricate. Unfortunately, so is its detection. The concept of detection sensitivity reflects not only the ability of a particular technique to label most of the cells displaying the appropriate marker but also the possibility of labeling cells in the earliest phase of the apoptotic process. For this study, we chose three techniques, visualized by means of immunohistochemistry: terminal dUTP-transferase-mediated nick end labeling (TUNEL), apostain, and lamin B. The number of apoptotic cells detected in the observed specimens with respect to the used technique differed for the majority of cases. The lowest apoptotic indices were usually obtained by the lamin B technique, the TUNEL technique followed, and the apostain technique gave the highest values. The comparison revealed that the TUNEL and apostain techniques were positively correlated in the majority of specimens observed. The linear correlation between TUNEL/apostain and lamin B was weaker. TUNEL, apostain, and lamin B techniques, variant in principle, give different yet correlating results, which is in accordance with the assumption that they describe the same phenomenon detected at different time periods of the apoptotic process.

Comparison of the TUNEL, lamin B and annexin V methods for the detection of apoptosis by flow cytometry.

The present study was performed in order to compare the sensitivity of 3 apoptosis-detection methods using a model cell culture after induction of apoptosis. Evaluation was performed by means of flow cytometry and light microscopy. It appeared that both TUNEL and annexin V methods are sensitive and specific and produced similar data in all measurements. The immunocytochemical detection of lamin B was less reliable than the other methods.