Effects of two sex steroids (17beta estradiol and testosterone) on proliferation and clonal growth of the human monoblastic leukemia cell line, U937.

We investigated the effects of two sex steroids (17beta estradiol and testosterone) on five human leukemia cell lines. We observed a statistically significant inhibition of proliferation, dose and time dependent, of the human monoblastic leukemia cell line U937. This inhibition was associated with a dose dependent decrease in the number of CFU-blasts in clonogenic cultures. Cytostatic effect was obtained with doses of 5 microM for estrogen and 10 microM for androgen and was not due to a non-specific cytotoxic effect, some cell viability remained high (> 90%) even after 6 days of incubation. More accurately, we demonstrated that growth inhibition was associated with a cell cycle arrest, U937 cells accumulating in G2/M phase. This blockade was dose related with a maximum number of cells accumulating at day 4. Sensitivity of these cells to an S-phase specific agent (hydroxyurea) was not increased, suggesting that these cells were blocked in G2/M and did not undergo mitosis. Expression in U937 cells of high affinity nuclear receptors for estrogen and androgen was negative which was in favour of a type II estrogen binding site, mediated mechanism. Moreover, a small fraction of these cells underwent apoptosis or differentiation with about 12% apoptotic cells and a significant increase (more than 30%) of two myelomonocytic markers (CD13 and CD64). These results demonstrate that the proliferation of some leukemic cells may be inhibited by micromolar concentrations of sex steroids, independently of nuclear receptor expression. The main mechanism seems to be a block in cell cycle associated with modulation of apoptosis and differentiation. It provided additional evidence for the potential value of sex steroids and their analogues in the treatment of leukemias.

Residual bodies stimulate rat Sertoli cell plasminogen activator activity.

Previous studies suggested indirectly that residual bodies (RB) may play a role in the regulation of the proteolytic system within the testis and in the coordination of the spermatogenetic process. In the present study, we examined the effects of RB recovered from adult rat testes by centrifugal elutriation on Sertoli cell plasminogen activator (PA) levels, by zymography and ELISA procedures. Addition of RB to Sertoli cell cultures prepared from 20-day-old rat testes resulted in a dramatic stimulation of PA. Effects were dose- and time-dependent. Phagocytosis of RB by Sertoli cells leads to a rapid stimulation of Sertoli cell interleukin-1alpha (IL-1alpha), a cytokine potentially involved in the regulation of spermatogenesis; the effects of IL-1alpha were investigated. We found that IL-1alpha augmented PA levels and that immunodepletion of Sertoli cell-RB cocultures with anti-IL-1alpha antibodies abrogated the stimulatory effects of RB on PA. Together, the present findings indicate that RB enhance Sertoli cell PA and that IL-1alpha may be involved in that control.

Plasminogen activator inhibitor-1 is expressed in cultured rat Sertoli cells.

Sertoli cells secrete plasminogen activators (PAs) on both sides of the blood-testis barrier, i.e., in the basal and apical compartments of the seminiferous tubules, whereas peritubular cells secrete plasminogen activator inhibitor-1 (PAI-1), a fast-acting and specific PA inhibitor. While it is likely that PAI-1 produced by peritubular cells counteracts the basal secretion of PA, the nature of the PA inhibitor acting in the apical compartment remains to be demonstrated. In the present study, we showed that Sertoli cells recovered from 20-day-old rats and cultured contained a transcript of 3-3.2 kilobases, which hybridized specifically to a PAI-1 cDNA probe (Northern blot). We verified that the observed PAI-1 transcript could not result solely from the peritubular cells (weakly contaminating the Sertoli cell cultures), by comparing PAI-1 mRNA levels of Sertoli and peritubular cells recovered from 20-day-old rats and cultured. We also demonstrated that cultured Sertoli cells secreted a protein that complexed with tissue-type PA (zymography), indicating that it was biologically active. This protein comigrated with purified PAI-1 as a doublet of 46 and 49 kDa (Western blot). The trophic hormone FSH decreased PAI-1 messenger RNA as well as immunoreactive PAI-1 protein (probably via the cAMP protein kinase A pathway), whereas transforming growth factor ss1 and basic fibroblast growth factor (in a nanomolar concentration) increased both of these. These observations support the hypothesis that PAI-1 is expressed by Sertoli cells and is under a complex hormonal (FSH) and paracrine and/or autocrine control exerted at least by basic fibroblast growth factor and transforming growth factor ss1.

Effects of retinoic acid on a new human erythromegakaryocytic cell line AP-217.

We have characterized a new human cell line AP-217, derived from the peripheral blood of a patient with chronic myeloid leukemia in blastic crisis. The analysis of cell surface antigens and ploidy showed that AP-217 was an erythro-megakaryocytic cell line. The effects of inducers of differentiation were studied and focused on retinoic acid (RA). Uninduced AP-217 cells produced a low level of hemoglobin (Hb) that showed a moderate but significant dose-dependent increase after 13 cis-RA induction (four times above the control at 10(-5) M). To outline this effect, AP-217 cells were cloned at limiting dilution. A subclone (clone 2) was isolated which expressed glycophorin A on 12% of cells, and showed a marked sensitivity to RA. After a 4 day induction with increasing concentrations of RA (1-10 x 10(-6) M) Hb production by clone 2 cells was enhanced 12 times over the control at the highest concentration (10(-5) M). No effect of RA on the Hb production of K-562 and HEL was observed. This increased Hb production occurred simultaneously with a growth inhibition in clonogenic cultures (20% reduction) associated with a drastic reduction of the colony size. Moreover, we demonstrated the expression of mRNA for the beta globin gene in clone 2 and AP-217-cells. This is the first report of a positive effect of RA on the erythroid differentiation of a human leukemic cell line.

Tumor necrosis factor-alpha regulates plasminogen activator inhibitor-1 in rat testicular peritubular cells.

We examined the regulation by tumor necrosis factor-alpha (TNF alpha) of plasminogen activator inhibitor-1 (PAI-1) in cultured peritubular cells recovered from 20-day-old rat testes. We demonstrated that TNF alpha in a nanomolar dose range stimulated PAI-1 messenger RNA (mRNA; Northern blots) as well as immunoreactive (Western blots) and bioactive (Stachrom) PAI-1 protein. Induction of PAI-1 mRNA started 4 h after the addition of TNF alpha (2.5-fold increase) and peaked (7-fold increase) after 24 h of treatment. Actinomycin D and cycloheximide inhibited the effects of TNF alpha on PAI-1 mRNA, suggesting that ongoing RNA and protein syntheses were required. The combined actions of transforming growth factor-alpha (TGF alpha), a potent inducer of PAI-1, and TNF alpha on PAI-1 were less than additive, suggesting the activation of some common pathway. TNF alpha action on PAI-1, like that of TGF alpha demonstrated previously, was masked by a preexposure to phorbol myristate acetate (a stimulator of protein kinase C) and strongly reduced by staurosporine (an inhibitor of the protein kinase C). Furthermore, using genistein to inhibit tyrosine kinase activity, we not only blocked the action of TGF alpha on PAI-1 [initiated upon binding to the tyrosine kinase epidermal growth factor/TGF alpha receptor (EGFR)], but also markedly reduced that of TNF alpha. Finally, TNF alpha, at a dose range that stimulated PAI-1, enhanced EGFR mRNA levels and EGF binding. Together, the present findings suggest that some of the biological effects of TNF alpha on PAI-1 might be secondary to de novo synthesis of EGFR. Because TNF alpha probably originates from testicular macrophages, such a regulation of PAI-1 by TNF alpha may occur in the context of physiological interactions between the testis and the immune system.

Plasminogen activator inhibitor-1 regulation in cultured rat peritubular cells by basic fibroblast growth factor and transforming growth factor-alpha.

In the present study we examined the in vitro regulation of plasminogen activator inhibitor I (PAI-1) expression in peritubular cells recovered from 20-day-old rat testes. We tested two growth factors, basic fibroblast growth factor (bFGF) and transforming growth factor-alpha (TGF alpha). They are synthesized by Sertoli cells, and peritubular cells exhibit the corresponding high affinity receptors. After exposure to bFGF or TGF alpha (0.1-30 ng/ml), PAI-1 messenger RNA levels, as determined by Northern hybridization analysis, increased in a dose-dependent manner. The first significant effects were noted after 2-h exposure to bFGF or TGF alpha (10 ng/ml), and PAI-1 messenger RNA levels were maximally stimulated approximately 12-fold (bFGF) and 8-fold (TGF alpha) after 4 h. The two growth factors increased the amount of immunoreactive (Western blots) and biologically active (Stachrom) PAI-1 measured in the culture medium. Actinomycin D inhibited the effects of these factors, whereas cycloheximide augmented them. Phorbol myristate acetate, an activator of protein kinase C, mimicked the effects of bFGF and TGF alpha. Interestingly, long term (24-h) pretreatment with phorbol myristate acetate resulted in a severe loss of responsiveness to bFGF or TGF alpha. Staurosporine, an inhibitor of protein kinase C, also significantly reduced the effects of bFGF and TGF alpha. Given that PAI-1 inhibits Sertoli cell plasminogen activator activity and that bFGF and TGF alpha are synthesized by Sertoli cells, these factors are likely to interact to regulate protease activity in localized regions of the seminiferous tubule.

Retinoids induced t-PA synthesis by C6 glioma cells--role in tumoral haemorrhagic necrosis.

Treatment of rat C6 glioma with high doses of 13 cis-retinoic acid (cRA) was responsible for death related to haemorrhagic necrosis localized to the tumor. Our aim was to explore this adverse effect of retinoid treatment. We show that cRA-treated C6 glioma at 25 mg/kg/day for 18 days exhibits in vivo an increase T-PA activity, which is responsible for a localized tumor fibrinolytic activity. Production of t-PA is supported by specific enhancement of gene expression, as was shown by the increase in t-PA mRNA (x 2.3). This production is a direct effect of cRA when treating the tumor, since tumor cells themselves do not produce enough t-PA and treatment of control rats does not increase the t-PA level. T-PA production by rat C6 glioma is in vivo related to the specific synthesis of t-PA by the C6 cell-line. The stimulation of C6 cell-line by cRA in vitro is dose-dependent and reached a maximum for 3 and 30 microM at the 72nd h. So cRA-treated C6 glioma cells produce t-PA which appears to be the major species associated with the fibrinolytic activity-induced intra-tumoral haemorrhage after exposure to retinoid treatment.

Validation of electronic cell counts by quantitative buffy coat analysis (QBC).

A total of 458 eight blood cell counts, accompanied by blood film reviews of the same samples, were performed with an electronic cell counter and with the QBC. In the great majority of cases, the QBC gave fast and accurate control of the flags from the electronic counter, thus avoiding the necessity for manual validation with its associated risks of infection and contaminations. Furthermore, QBC non readability could be related to microcytosis and hypochromia and hence point to possible cases of congenital or acquired haemoglobinopathy.

A simultaneous evaluation of three multiparametric coagulation instruments: BFA, HEMOLAB and STA.

In the present paper, three multiparametric coagulation instruments were evaluated with regard to chronometric tests for aPTT (CK-Prest and automated APTT), PT (Recombiplastin and Thromborel), fibrinogen and factors of the prothrombin complex. Analysis of within-run precision and linearity and comparative studies showed the analytical performances of the instruments to differ according to the reagents used and emphasized the difficulty of finding the best compromise between instrument and reagent. On the basis of this study, the mechanical instrument appeared to be more versatile than the optical machines. This conclusion could however be modified after further evaluation of the recent new generation coagulation instruments.

Image analysis as a tool for quantitative enzyme determination at the cellular level: application for monocytic differentiation of the UM-384 cell line.

Image analysis has been used to determined enzyme activity at the cellular level in individual smeared cells. The counterstains used to visualize smeared cells were chosen to avoid overlap with the chromogene. The amount of the reaction product was quantified by computerised scanning cytophotometry when the conditions of incubation, time and temperature of the reaction, and substrate concentration varied. Under optimal conditions for time, temperature and substrate concentration, a linear relationship was found between enzyme activity determined on smeared cells and in cell lysate. Using these defined conditions, differentiation of UM-384 cells was studied by measuring enzyme activity. After a monocytic differentiation process, induced by sodium butyrate, non-specific esterase cell activity was compared either with differentiation markers (HLA-DR, plasminogen activator inhibitor type 2 and lysozyme) or with markers of proliferation (DNA content) or functional properties (nitroblue tetrazolium reduction and phagocytosis). The results show that, using image analysis, non-specific esterase seems to be a useful means for the assessment of monocytic differentiation whereas myeloperoxidase is not. More generally, quantification of enzyme activity at the cellular level using image analysis can be applied to the study of the differentiation process and may help in the classification of leukemic cells.

Absence of both subunits of cytochrome b558 in the UM384 cell line relative to the inability to generate superoxide anions.

Phagocytic cells are characterized by their ability to generate superoxide anions upon activation by appropriate stimuli. UM384, a myelomonocytic cell line, was shown to be defective in this oxidase activity as measured by nitroblue tetrazolium or cytochrome c reduction. Cytochrome b558, a unique pigment present in phagocytes and implicated in electron transfer from NADPH to O2, was absent in the differentiated UM384 cells. Both subunits of the cytochrome b558 appeared to be absent or present in strongly reduced amounts compared to the mother cell line U937, as indicated by immunocytochemistry or Western blot analysis using monoclonal antibodies (MABs). On the other hand, cytosolic factors also involved in NADPH oxidase activity were shown to be present, either immunologically or by using the capacity of the cytosol to activate the oxidase in a membrane fraction from bovine neutrophils. At the molecular level, the mRNA that encodes the gp91-phox was shown to be absent in the differentiated UM384 cells, whereas the mRNA that encodes the p22-phox was normally expressed. These results suggest that the defect in superoxide production by the UM384 cells is related to the absence of cytochrome b558, a situation mimicking that observed in phagocytes from patients with X-linked chronic granulomatous disease (X-CGD).

[Comparative and simultaneous evaluation of three automated counters in hematology: Coulter STKS, Sysmex NE 8000, Technicon H-2]. / Evaluation comparative et simultanée de trois automates d'hématologie: Coulter STKS, Sysmex NE 8000, Technicon H-2.

We evaluated and compared three automated blood cell counters, Coulter STKS, Sysmex NE 8000 and Technicon H-2. These perform both a complete blood cell count and a full white cell differential count. Carry-over was found to be acceptable, except for the leucocyte count by the Sysmex NE 8000 (1.5%; p < 0.001). Linearity over a wide concentration range for all of the measured parameters, haemoglobin, red blood cells, white blood cells and platelets, was excellent (r > 0.98). Within-run precision was verified with 22 samples covering a wide range of cell counts by repeated analysis (n = 20) of the same sample. Coefficients of variation (CV) were acceptable, < 4% for values within the normal range. The CVs for eosinophils and basophils were less good but without clinical impact. Analysis of 270 samples showed that the Coulter counter indicated the presence of atypical cell populations more frequently (19%; p = 0.02). Message of qualitative abnormalities displayed by the three analysers were often discordant. These three blood cell counters differed in their sensitivity and positive predictive value for detecting abnormal blood cells rather than in their specificity and negative predictive value. Other operating aspects of three instruments that are important in haematological practice are documented.

Monocyte tissue factor expression induced by Plasmodium falciparum-infected erythrocytes.

Monocytes are active elements of the host response against Plasmodium falciparum. They are able to express tissue factor and trigger the extrinsic pathway of blood coagulation the activation of which remained unclear in malaria. Our aim was to assess the tissue factor expression of purified blood monocytes stimulated by cultured Plasmodium falciparum-infected erythrocytes. Malaria parasite induced an early generation of tissue factor with a peak between 8 and 12 h of stimulation. Maximum expression was observed for parasitemia ranging from 1 to 2%. Plasmodium falciparum culture supernatants had the same effect showing the existence of a soluble factor able to induce the tissue factor expression. These data, demonstrating an activation of the tissue factor pathway by the malaria parasite, emphasize thrombin generation. Therefore, thrombin could participate in malaria pathology either in the microcirculatory blockade via platelet and fibrinogen activation or as a mitotic.

Quantification by image analysis of immunocytochemical reactions: application for determination of lysozyme content in individual smeared cells.

The objective of the present study was to develop a cytophotometric technique to quantitate immunocytochemical reactions. Cell antigens were detected after immunophosphatase alkaline staining procedure. The amount of reaction product was quantitated by computerized scanning cytophotometry. The technical conditions (dilution of primary antibody; incubation time of the three antibodies; volume and pH of the enzyme substrate reaction; storage of the slides) required for optimal cytophotometric determination of the reaction product were determined. Under these optimally defined conditions, a linear relationship between cell protein content (lysozyme) and microdensitometric measure of the colored reaction product was found. This method could be used for other cells, antigens, and enzymatic indicators.

[Erythrocytes infected by Plasmodium falciparum activate human platelets]. / Les hématies parasitées par Plasmodium falciparum activent les plaquettes humaines.

Blood platelets are involved in Plasmodium falciparum malaria pathology as shown by thrombocytopenia and increased plasma level of two alpha granule proteins: beta thromboglobulin (beta TG) and platelet factor 4 (PF4). In this study we demonstrate that Plasmodium falciparum parasitized erythrocytes activate directly the secretion of beta TG and PF4 by human platelets. This secretion is related to parasitemia and occurs immediately after contact. Treatment of parasited erythrocytes by trypsin and diffusion chamber experiments suggest that platelet activation is triggered by parasitic substances shed on erythrocyte membrane and released in the culture medium.

Plasmodium falciparum growth inhibition by human platelets in vitro.

Platelets take an active part in immunological processes as well as in haemostasis, especially in the host-parasite relationship. Our aim is to assess the growth of Plasmodium falciparum, cultured in human erythrocytes in the presence of fresh washed human platelets, since thrombocytopaenia is frequently observed during malarial infections. Our results show that platelets induce a dose-related growth inhibition of P. falciparum. Both proliferation and maturation of intraerythrocytic stages of the parasite are inhibited. This growth inhibition is triggered by the parasite itself as neither specific antibodies nor any other components are needed to activate platelets. Activated platelets are directly toxic since complement is not involved. Furthermore, inhibition is not mediated by erythrocyte lysis or by toxic oxygen metabolites. Platelets induce an inhibition of P. falciparum growth, at least in vitro, although the importance of their role played in vivo in malarial immunity has yet to be evaluated.

Establishment and characterization of a granulocytic subclone (UM 384) from the monoblastic cell line U 937.

The human cell line U 937 spontaneously expresses monocytic maturation and can be induced into macrophage-like cells when treated with retinoic acid, sodium butyrate, or 2,3-O-tetra decanoylphorbol-13-acetate. We have selected a subclone, designated UM 384, that expresses granulocytic characteristics and can be induced to mature to granulocytes after exposure to retinoic acid, actinomycin D, and dimethylsulfoxide, and to monocyte-like cells when treated with sodium butyrate and phytohemagglutinin-stimulated leukocyte-conditioned medium. These cells retain the same constitutive markers as the parent line including histocompatibility leukocyte antigens and karyotype but share numerous chromosomal abnormalities, mainly t(X;8) (p21;q12).