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.

The toxicity and pharmacokinetics of dihydrosanguinarine in rat: a pilot study.

The quaternary benzo[c]phenanthridine alkaloid sanguinarine (SG) is the main component of Sangrovit, a natural livestock feed additive. Dihydrosanguinarine (DHSG) has recently been identified as a SG metabolite in rat. The conversion of SG to DHSG is a likely elimination pathway of SG in mammals. This study was conducted to evaluate the toxicity of DHSG in male Wistar rats at concentrations of 100 and 500 ppm DHSG in feed for 90 days (average doses of 14 and 58 mg DHSG/kg body weight/day). No significant alterations in body or organ weights, macroscopic details of organs, histopathology of liver, ileum, kidneys, tongue, heart or gingiva, clinical chemistry or hematology markers in blood in the DHSG-treated animals were found compared to controls. No lymphocyte DNA damage by Comet assay, formation of DNA adducts in liver by 32P-postlabeling, modulation of cytochrome P450 1A1/2 or changes in oxidative stress parameters were found. Thus, repeated dosing of DHSG for 90 days at up to 500 ppm in the diet (i.e. approximately 58 mg/kg/day) showed no evidence of toxicity in contrast to results published in the literature. In parallel, DHSG pharmacokinetics was studied in rat after oral doses 9.1 or 91 mg/kg body weight. The results showed that DHSG undergoes enterohepatic cycling with maximum concentration in plasma at the first or second hour following application. DHSG is cleared from the body relatively quickly (its plasma levels drop to zero after 12 or 18 h, respectively).

Apoptosis and expression of Bcl-2 in human endometrium in natural and artificial cycles.

Apoptosis plays a significant role in differentiation of many organs and helps to maintain homeostasis. The occurrence of apoptosis (using the apoptotic index) and expression of regulation protein Bcl-2 in the human endometrium was evaluated within the secretory phase of both the natural cycle, and an artificial one. Oral hormonal substitution used in this design induced similar, but more marked dynamic changes in Bcl-2 expression in the mid-secretory endometrium as were observed in the natural cycle, primarily in the surface and glandular epithelium of the endometrium. The apoptosis revealed similar a trend, but not significantly different.

Chemoprotective effect of plant phenolics against anthracycline-induced toxicity on rat cardiomyocytes. Part II. caffeic, chlorogenic and rosmarinic acids.

The chemoprotective effects of caffeic (CA), chlorogenic (CHA) and rosmarinic (RA) acids were tested against the toxicity of doxorubicin (DOX) in neonatal rat cardiomyocytes and the iron-dependent DOX induced lipid peroxidation of heart membranes, mitochondria and microsomes. The protectivity of these acids was compared with dexrazoxan, used as an adjuvant during DOX chemotherapy. The cytoprotective effects were assessed by enzyme (LDH and ASAT) and troponin I leakage, secondly by intracellular ATP content. All hydroxycinnamic acids proved non-cytotoxic, and they stabilized both membranes and the energetic status of cardiomyocytes. After preincubation of cardiomyocytes with the test compounds (100, 200 microm; 1 h) the cardiomyocytes were treated with the toxic agent, DOX (100 microm; 8 h). The test compounds protected cardiomyocytes against DOX induced oxidative stress (RA > CHA > or = CA) on all monitored parameters. Substantial preservation of monolayer integrity of the cardiomyocytes by test compounds was also found microscopically. All the acids were more effective in the assays used than dexrazoxan. RA showed the most effective cytoprotectivity. All the acids significantly reduced the iron-dependent DOX induced lipid peroxidation of heart membranes, although of the test compounds, CHA was found to be the most effective (IC(50) = 8.04 +/- 0.74/6.87 +/- 0.52 micro m for microsomes/mitochondria).

Sanguinarine and chelerythrine: assessment of safety on pigs in ninety days feeding experiment.

Sanguinaria canadesis, Chelidonium majus and Macleya cordata have been used for centuries as alternative medicines. Currently the extracts from these medicinal plants are components of veterinary and human phytopreparations, and of oral-hygiene agents. Sanguinarine and chelerythrine (SA/CHE) are biologically active components of these extracts. They display distinct antibacterial and anti-inflammatory properties, but, on the other hand, they have been reported as having adverse effects - genotoxicity and hepatotoxicity. This paper is aimed at evaluation of the effects of daily administration of the extract from Macleya cordata (2 mg and 100 mg in 1 kg feed, sanguinarine:chelerythrine 3:1) in the diet on the health status of swine. After 90-day administration, alkaloids were retained to a different extent in tissues. The highest SA/CHE retention was detected in the gingiva (0.55 microg/g) and liver (0.15 microg/g), no SA/CHE were detected in muscles. Plasma SA levels attained 0.11 microg/ml. Treated animals did not display any results of hematological, biochemical or histological assay different from controls. A (32)P-postlabeling assay proved that no DNA-adducts with SA/CHE were detected in pig livers. We did not observe any symptom linked to epidemic dropsy syndrome often attributed to sanguinarine. In conclusion, an average daily oral dose of alkaloids up to 5 mg per 1 kg animal body weight proved to be safe.

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.

Part of CD68+ macrophages in the clearence of apoptotic bodies in human metanephros.

According to recent research on mice, less on human material, cells responsible for clearing apoptotic cells away during development are, besides non-professional phagocytes, also tissue-fixed macrophages. The aim of our work was the determination of macrophage role in the phagocytosis of apoptotic bodies in neogenous zone of human metanephros. Histologicaly normal kidneys were collected from embryos and fetuses ranging from the 8th-28th week of IUD. These tissues were routinely processed. In the first step we detected CD68+ cells by means of standard indirect three-step immunohistochemical method having used MAb NCL-CD68-KP1 (macrophage marker) to find out whether such cells are actually present. In the second step tissue sections were labelled by double-staining principle (TUNEL technique for the detection of apoptosis and above mentioned macrophage marker) to judge co-localization of these two items. The slides were observed by using immersion objective and the amount of apoptotic cells was expressed in percents. CD68+ macrophages appeared dispersely as single cells or small groups in all the ages studied. According to our results, CD68+ macrophages phagocytose 37-75% of apoptotic cells present in neogenous zone and the number of engulfed apoptotic cells increases in the 12th week of the IUD, i.e. in the early fetal period and later it merely fluctuates.

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.