Hemorheological changes and characteristic parameters derived from whole blood viscometry in chronic heroin addicts.

A group of 15 chronic opioid addicts (DA) with mean age 26.5+/-7.3 years was studied by means of a rotational Contraves Low Shear 30 viscometer and the results have been compared with a control group of 19 healthy subjects. It was found that the mean whole blood viscosity values of the investigated group of heroin abusers (n=15) were elevated compared to that of healthy persons (n=19) over the whole shear rate range and fell by more than ten orders of magnitude (Savov et al., 2006). The present investigation uses the coefficients of the models of Ostwald-de-Walle (power law) and Herschel-Bulkley law, which describe whole blood flow curves (tau-gamma) within the shear rates range from 10(-2) to 10(2) s(-1) and itself incorporate whole blood viscosity data in the entire shear rate range. A significant difference in the mean yield shear stress tau(0) values of the drug abusers' group compared to the controls was found. A strong positive linear correlation was determined between the parameters of RBC aggregation in the group of heroin addicts confirming our previous results (Ivanov and Antonova, 2005; Savov, Zvetkova et al., 2007; Savov, Antonova et al., 2007) for intensive RBC and platelet aggregation and morphological changes in DA group.

Use of neopterin as a bone marrow hematopoietic and stromal cell growth factor in tissue-engineered devices.

The in vitro response of early haematopoietic progenitors or stem cells (CD34+)--common for myeloid (granulocyte, eosinophil, megakaryocyte) and marrow stromal cell lineages, to neopterin, exogenously added to the liquid mouse bone marrow cultures, at doses 12.5-25 microg/ml culture medium, has been studied. The results obtained show a significant stimulation of common--myeloid and stromal/ mesenchymal progenitor cell proliferation and differentiation, as early as 24h to the 96h after the in vitro treatment with neopterin. On day 4 of cultivation the granulocyte/macrophageal proliferation and differentiation has been attenuated giving place to the marrow stromal/mesenchymal cell growth and differentiation. A functional role of neopterin as hematopoietic growth factor--essential for the proliferation and differentiation of bone marrow common (hematopoietic and stromal) progenitors is not yet clear and remains to be elucidated. The in vitro and ex vivo applying of neopterin--alone or in specific combinations with other cytokines (e.g. FGF-2) for the induction of marrow stromal/mesenchymal cell proliferation and differentiation, merits further investigations with regards to its future use in regenerative medicine. The results provide a theoretical basis for the application of neopterin in tissue-engineered devices: incorporated into biodegradable polymer microparticles (with encapsulated early bone marrow progenitors and other special supplements), it could be experimentally applied for fast and easy induction of endothelial, osteoblastic/osteogenic, neuronal and other cell lineage differentiation as well as for improving tissue trophical processes and reparative microenvironment.

Whole blood viscosity and erythrocyte hematometric indices in chronic heroin addicts.

Whole blood viscosity (WBV) and hematometric indices of erythrocytes as red blood cell count (RBC), mean erythrocyte volume (MCV), hemoglobin (HGB), hematocrit (HCT), mean hemoglobin content of erythrocytes (MCH), HGB/HCT values (MCHC) and red blood cell distribution width (RDW) have been studied in a group of 15 chronic opioid addicts under methadone maintenance therapy with mean age 26.53 +/- 7.34 years. WBV elevation and changes in MCV, HGB, HCT, RDW were found in intravenous drug users compared to healthy individuals. As well, RBC was decreased leading to an increase in MCH and MCHC values. Correlation analysis suggested that the correlation among the RBC, HGB, HCT and WBV was the closest. Heroin macrocytosis (heroin macrocytic anemia) was established, related with the increased RDW in chronic heroin abusers. The results are in accordance with data revealing abnormal effects of alcohol and other drugs on whole blood rheology and hematometric/morphometric characteristics of erythrocytes.

In vitro effects of inhibin on apoptosis and apoptosis related proteins in human ovarian granulosa cells.

OBJECTIVE: To clarify the in vitro effects of inhibin A (I) on apoptotic cell death and its mechanisms in ovarian granulosa cells the immunoexpression patterns of the apoptosis markers caspase-3 and pro- and anti-apoptotic proteins (Bcl-2, Bcl-xl, Bak) were evaluated in ovarian granulosa cells collected from women with different hormonal status. MATERIALS AND METHODS: Granulosa cells were isolated from follicles of women participating in an in vitro fertilization (IVF) program, normally cyclic (NC) and premenopausal women (PrM). The obtained cells were cultured for 72 h with inhibin A (Sigma, USA)--10 ng/ml. The concentration of estradiol in the culture medium was determined by radioimmunoassay using the Coat-A-Count kit (Nippon, Japan), whose intra- and interassay coefficients of variations were 6,8% and 6,2% respectively. The expression of caspase-3, Bak, Bcl-2, Bcl-xl was investigated immunohistochemically. The percentages of immunopositive cells were calculated and Student's t-test was used for statistical analysis. RESULTS: Addition of inhibin A (10 ng/ml) to granulosa cells cultures resulted in increased estradiol production. Maximal stimulation was observed in granulosa cells collected from women participating in IVF whereas minimal effect of inhibin treatment on estradiol production was demonstrated in premenopausal women. Inhibin A exposition enhanced the immunoexpression of prooncogenes (Bcl-2, Bcl-xl) and reduced the expression of caspase-3 and pro-apoptotic protein Bak in ovarian granulosa cells from the three experimental groups. CONCLUSIONS: Our findings suggest that inhibin A in vitro stimulates the estradiol secretion by granulosa cells dependently of the woman hormonal status, while it inhibits apoptotic process in granulosa cells independently of the hormonal status.

Human bone marrow granulocyte-macrophageal colonies (GM-Cs) in vitro: myeloid cells in health and in cases of myeloid leukemia.

The morphological characteristics of granulocyte/macrophageal (GM-) colonies and clusters, obtained in vitro (in semi-solid agar cultures) from bone marrow hematopoietic myeloid progenitors pertain to leukocyte hemorheology of healthy persons and patients with myeloid leukemias. The morphological features of in vitro growing myeloid progenitors, granulocytes and macrophages of healthy persons differ in their cell size, shape and degree of differentiation from the cultivated marrow cells in cases of acute and chronic myeloid leukaemia. In this malignant disease, the rheological properties of leukocytes (granulocytes/macrophages) were found to provide diagnostic information. Further studies should be undertaken to examine whether the method could be useful in defining survival, prognosis and therapeutical approach in cases of myeloid leukemia.

Involvement of prostanoids in the inhibitory effect of endothelin-1 on progesterone production of ovarian granulosa cells.

The present study was undertaken to investigate the possible involvement of prostanoids in the inhibitory effect of endothelin-1 (ET-1) on progesterone production of ovarian granulosa cells. ET-1 (1 x 10(-7) M) decreased the basal and follicle-stimulating hormone- (FSH) stimulated progesterone production from both human and porcine granulosa cells. Indomethacin dose-dependently inhibited progesterone release, but did not alter the inhibitory effect of ET-1 (1 x 10(-7) M) on progesterone production of cultured ovarian granulosa cells. This study shows that ET-1 suppresses basal and FSH-stimulated progesterone production by ovarian granulosa cells, but this effect is not mediated by prostanoids.

Aqueous extracts of Crinum latifolium (L.) and Camellia sinensis show immunomodulatory properties in human peripheral blood mononuclear cells.

In Vietnamese and Chinese traditional medicine, hot aqueous extract of Crinum latifolium is used because of its antitumor activity. The genus Crinum is thought to possess antiviral and immunostimulative properties. Green and black tea derived from Camellia sinensis have similar qualities. A growing body of evidence suggests that moderate consumption of green and black tea may protect, e.g., against several forms of cancer, cardiovascular diseases, and bacterial infections. In this study, the immunomodulatory property of C. latifolium (L.) extracts should further be investigated and compared to those of black and green tea. Human peripheral mononuclear cells were cultured in the presence of tea extracts with or without mitogens or interferon-gamma. The effect of plant extracts on cultured cells was assayed by neopterin production, a sensitive marker reflecting the activation of cell-mediated immunity. Our experiments showed that extracts of C. latifolium (L.) slightly enhance neopterin production in unstimulated peripheral mononuclear cells, whereas an effective reduction of neopterin formation in cells stimulated with concanavalin A (Con A), phytohemagglutinin (PHA), or interferon-gamma (IFN-gamma) was observed. Green and black tea extracts displayed similar immunomodulatory properties in our in vitro system, whereas C. latifolium (L.) extracts seemed to be more effective in reducing neopterin formation in stimulated cells.

Aspects of granulocyte function in workers professionally exposed to pesticides.

Granulocyte function in 92 workers from a chemical plant for the production of pesticides was tested by means of the nitro-blue tetrazolium test (spontaneous and stimulated) and the phagocytosis test. Two opposite types of change were identified, namely increased and reduced functional activity. No reliable correlation between the studied parameters and length of service was found. In comparison with the routine hematological methods, the identified functional changes in polymorphonuclear neutrophil granulocytes serve as an early indicator of an impact on the leukocytes. The applied methods are accessible and may be used as an objective means of studying the dynamics of the unfavorable effect of zineb and of identifying groups at increased toxicological risk.

Methylene blue-fast green staining of hemopoietic colonies in agar cultures.

A simple technique is described for the routine in situ identification of the cellular composition of colonies and clusters in agar cultures of hemopoietic cells. The entire culture, dried and formalin vapor fixed within a Petri dish, is stained with a mixture of methylene blue and fast green. By this method cellular ribonucleoproteins (RNP), deoxyribonucleoproteins (DNP) and some cationic (arginine and lysine containing) proteins are detected. Different maturation stages of neutrophils, eosinophils, basophils, monocytes, and macrophages can be easily identified with colonies and clusters on the basis of the cytoplasmic and nuclear staining.

Cytochemistry of nucleoproteids and some cathionic proteins in the peripheral blood leukocytes of patients with lung cancer.

In 40 patients with untreated lung cancer cytochemical studies of the peripheral blood leukocytes were conducted by means of a cytological method for the simultaneous staining of nucleoproteids (RNP and DNP) and some cathionic proteins (after Zvetkova and Zvetkov [60]). Changes were detected in the RNP cytoplasmic contents of lymphocytes, of which the most outstanding were the reduction and uneven distribution of RNP granules, their frequent extracellular expulsion by means of microclasmatoses, as well as changes in the staining of cathionic proteins of RNP accompanied by an increased nuclear chromatin condensation in the small and medium-sized lymphocytes. Parellel to reducing of the percentage of these cells in the peripheral blood of patients with advanced neoplastic disease an increased number of lymphoblastoid and monoblastoid cells is established with RNP diffusely stained, but reduced in quantity and localized in the cytoplasmic periphery and projections (compared to Downey type II atypical cells). By means of one of the variants of the method (modified type of Feulgen's reaction) a characteristic distribution and structuring of the nuclear chromatin is established in mono- and polymorphonuclear cells, most clearly expressed in the nuclei of monocytes and monoblastoid cells, as well as in nuclei of neutrophil granulocytes. In these cellular types a more specific nuclear modelling (microhypersegmentation) is observed resulting in multiple irregular nuclear projections on the nuclear surface, probably caused by subkaryolemal distribution of uneven chromatin thickenings. The changes are also recorded in the cathionic protein containing secondary cytoplasmic granules in granulocytes-neutrophils and eosinophils, probably associated with changes in the lysosomal and phagocytic functions of these cells in neoplastic diseases. The authors discuss the importance of the obtained results in connection with data on the participation of lymphocytes and neutrophils in the immune response to tumour antigenic stimuli during the course of the neoplastic process, as well as with data on the suppressive effect of antigenic (serum, viral) factors, possibly affecting the synthesis and the transport of cellular nucleoproteids (RNP and DNP) in leukocytes of cancer patients.

[2 new fluorochromation methods for blood smears and chromosomes using berberine sulfate following deoxyribonucleoprotein denaturation]. / Deux nouvelles méthodes de fluorochromisation des frottis sanguins et des chromosomes par la berbérine sulfate aprés la dénaturation des désoxyribonucléoprotides.

The authors reported a Berberine sulfate technique based on DNP-denaturation with modifications for the purposes of fluorescent cytochemistry in cytology of blood and vaginal smears. A similar fluorochromation technique for staining of metaphase chromosomes and chromosomes in meiotic division has been applied. The fluorescent specificities, probably due to the differences in the denaturation properties of the hetero- and euchromatin desoxyribonucleoprotein-complexes, are discussed in comparison with other fluorochrome techniques and in relation with differences in distribution of hetero- and euchromatin and amounts of proteins in DNP, as far as DNA denaturation and tinction properties are concerned. The weaker fluorescence of immature (or leucemic) nuclei in blood smears and certain chromosomal regions would be due to the greater amount of active euchromatin (DNA which is slow reassociating or unstable to denaturation), which obviously does not bind to a sufficient degree the fluorochrome applied. The differences established in the fluorescence of active euchromatin and inactive heterochromatin zones by post-denaturing fluorochromation with Berberine sulfate gave grounds to recommend the application of these techniques in haematological and cytological (normal and abnormal) practice and for the cytogenetical and microfluorimetrical analyses.

A cytological method for the simultaneous staining of nucleoproteids and some cathionic proteins.

A cytochemical method is suggested for the simultaneous and differential staining of cellular nucleoproteids [ribonucleoproteids (RNP) and desoxyribonucleoproteids (DNP)], as well as for the simultaneous contrast staining of some basic (arginine- and lysin-containing) proteins. The staining technique is based on DNA-denaturation procedures and the application of mixtures of basic dye--methylene blue and acid dyes--eosin or fast green at low concentrations. The combination of methylene blue with eosin is used for the staining of ribonucleoproteids (RNP) whereas methylene blue-fast green for the simultaneous detection of ribonucleoproteids and desoxyribonucleoproteids (RNP and DNP), as well as for the differential staining of nuclear DNP (after cold hydrolysis with 5 N HCl). The acid dyes eosin and fast green stain in pink resp. in green some cathionic proteins in the lysosomal (specific) granules of the neutrophilic and eosinophilic leucocytes in the cytoplasm of erythrocytes, and after cold hydrolysis in the cytoplasm of lymphocytes. A fluorescent variant of the method with sulfaflavin is also suggested for the fluorochromation of cytoplasmic cathionic granules in the luecocytes. Acid mucopolysaccharide components in the granules of basophilic leucocytes, tissue mastocytes and thrombocyres are stained intensively pink-violet (gamma-metachromatic). The possibilities for the application of the method in the quantitative analysis of blood and exfoliated cells, as well as for purpose of haematology, immunology and exfoliative cytology are discussed.