Patterns of unintentional fatal drowning among children in North Tunisia : A 10-year study.

AIMS: This study aims to better understand patterns of unintentional fatal drowning among children in North Tunisia. METHODS: A cross-sectional retrospective study including all unintentional fatal drowning among children was conducted in the Legal and Forensic Medicine department in the Charles Nicolle Hospital, Tunis, between January 2010 and December 2019. Socio-demographic variables, as well as death circumstances, were documented for each victim and analyzed. RESULTS: A total of 200 casualties were included in this study. The highest rate of deaths was observed in the summer (N=44). Most of the victims were males and 55.5% were aged between 13 and 18 years. The drowning occurred in a canal or the sea in 33.5 % and 29.5 % of the cases respectively. The distribution of drowning sites varied significantly by season and place of living: drowning in the sea was more likely to occur in the summer and in urban areas (p < 0.05). In the first years of life, drowning occurred mostly in buckets and wells (N=9 and N=10, respectively) while between 7 and 18 years, it was more frequent in a canal or the sea. Swimming was the leading activity before death in 50% of the cases. CONCLUSIONS: Unintentional fatal drowning among children remains an underestimated major health problem in Tunisia especially in the population aged from 7 to 18 years. Effective prevention measures should be implemented nationwide, especially around seas and canals.

A cadaveric anatomical study of the adrenals: Relationship with the posterior abdominal wall muscles revisited.

AIM OF THE STUDY: The main difficulties during retroperitoneal laparoscopic adrenalectomies are due to its location. Our objective was to define the relationship of the adrenals with the diaphragm and the psoas muscle. METHODS: Our work is an anatomical dissection of 80 fresh cadavers' adrenals. To study the right adrenal, we performed a right nephrectomy and adrenal remained attached to the Inferior vena cava by its main vein. On the left, the edges of the adrenal have been identified by needles and the adrenal was reclined to study its projection on the posterior muscular wall. RESULTS: The right adrenal is located higher, 13mm [4-20mm] above the medial arcuate ligament (MAL) in 16 cases (40%). Its lower border was at the same level as the MAL in 18 cases (45%) and 11mm [10-17mm] below the MAL in 6 cases (15%). The posterior support of the right adrenal was the right crus of the diaphragm (Right-CD) in 34 cases (85%) and straddling the Right-CD and the psoas in 6 cases (15%). The study of the relationships of the left adrenal with the MAL showed that the lower edge of the gland was at its same level in 16 cases (40%) and below in 24 cases (60%) by 14mm [8-24mm]. The posterior support of the left adrenal was the left crus of the diaphragm (Left-CD) in 16 cases (40%) and straddling the Left-CD and the psoas in 24 cases (60%). CONCLUSIONS: Our results showed that the right adrenal is higher. The MAL is an important posterior element to the adrenal gland that could serve as an anatomical landmark to identify the adrenal during laparoscopic adrenalectomy.

[Circadian and septadian variation in sudden cardiac death : Autopsy registry of the Tunisian North]. / Variation circadienne et hebdomadaire de la mort subite d'origine cardiaque : registre autopsique du nord de la Tunisie.

INTRODUCTION: Several studies have suggested a circadian and septadian pattern of incidence of sudden cardiac death with a morning peak and a Monday peak. OBJECTIVE: To analyze the circadian and septadian pattern of occurrence of sudden cardiac death in the eight northern Tunisian governorates. METHODS: We prospectively collected epidemiological and autopsy data of sudden cardiac death victims occurring in the northern region of Tunisia between January 2013 and December 2019. RESULTS: The population included 1834 men (79.6%) and 468 women (20.4%) with a mean age of 56.5 ± 14 years. Smoking (53.9%) was the most prevalent cardiovascular risk factor. One-fifth (20.9%) of victims had known heart disease, and 3% had a family history of sudden death. ischemic heart disease was the leading cause of sudden death (46.8% of cases). One- fourth (25.7%) of autopsies were negative. Analysis of the circadian pattern of occurrence of sudden cardiac death identified a peak (36.1%, p < 0.001) between midnight and 6 am. This nocturnal excess mortality was significant (p < 0.001) and independent of sex (34.1 % in men and 43.8 % in women) and cause of death (39.3 % of cases of sudden ischemic death and 33.3 % of cases of nonischemic death). Moreover, there was a significant septadian variability in the occurrence of sudden death (p: 0.0015), with a peak on Friday (15.8 %, p: 0.042). CONCLUSION: This study showed a peak of sudden death between midnight and 6 am, and on Fridays, confirming the modification of the classic circadian and septadian pattern of sudden death occurrence. These results may help optimize the deployment of emergency mobile teams and structures during the most vulnerable periods.

[Sudden cardiac death in women, data from the northern Tunisian sudden cardiac-death registry]. / La mort subite cardiaque chez la femme, registre du nord de la Tunisie.

BACKGROUND: Sudden cardiac death is a major public health problem. Epidemiological and clinical differences according to gender have been described in sudden cardiac death. The aim of this study was to examine the gender differences between autopsy findings and circumstance of occurrence associated with sudden cardiac death. METHODS: We prospectively collected epidemiological and autopsy data of victims of sudden cardiac death occurring in the northern governorates of Tunisia between January 2013 and December 2019. Symptoms preceding death, circadian, weekly and seasonal variations of sudden death were also analyzed. RESULTS: The study population included 1834 men and 468 women with a mean age of 56.5±14.2 years. All cardiovascular risk factors except smoking were significantly more frequent among women but ischemic heart disease was the most common cause of death in men (51.3 %, versus 28 %, P<0.001). Women were more likely to have a negative macroscopic autopsy than men (34 % versus 23.6 %, P<0.001). Chest pain preceding sudden death was more frequent in male (24 % versus 13.2 %, P<0.001). In contrast, women were more likely to have dyspnea (8.1 % versus 15.6 %, P<0.001). Sudden death in women occurred indoors more often than in men (63.9 % versus 54.5 %, P<0.001) and also more often during night (midnight to 6 am). We also recorded an excess cardiac mortality in winter in both sexes. CONCLUSIONS: Women had considerably more cardiovascular risk factors and more commonly negative macroscopic autopsy. Death occurred indoors and during night more often than in men.

A crosstalk between the Wnt and the adhesion-dependent signaling pathways governs the chemosensitivity of acute myeloid leukemia.

Relapses following chemotherapy are a major hindrance to patients' survival in acute myeloid leukemia (AML). To investigate the role of the hematopoietic niche in the chemoresistance of leukemic cells, we examined two pathways: one mediated by adhesion molecules/integrins, and the other by soluble factors of the morphogen Wnt pathway. In our study, both the adhesion of leukemic blasts to fibronectin and the addition of Wnt antagonists induced, independently, resistance of AML cells to daunorubicin in a cell survival assay. Using pharmacological inhibitors and siRNA, we showed that both resistance pathways required the activity of the glycogen synthase kinase 3beta (GSK3beta). Moreover, the AML cell protection downstream of GSK3beta was mediated by NF-kappaB. A link between the adhesion and the Wnt pathway was found, as adhesion of U937 on human osteoblasts, a component of the hematopoietic niche, triggered the secretion of the Wnt antagonist sFRP-1 and supported resistance to daunorubicin. The osteoblast-conditioned medium could also confer chemoresistance to U937 cells cultured in suspension, and this cell protective effect was abrogated after depletion of sFRP-1. In the context of this potential double in vivo resistance, modulators of the common signal GSK3beta and of its target NF-kappaB could represent important novel therapeutic tools.

[Anxiety and depressive disorders in 4,425 long term benzodiazepine users in general practice]. / Troubles anxieux et dépressifs chez 4 425 patients consommateurs de benzodiazépines au long cours en médecine générale.

Consumption rates of anxiolytic drugs, and especially of benzodiazepines, remain very high in France compared to other Western countries, whereas clinical guidelines limit their indications to short term treatments and only for some precise anxiety disorders. Recent epidemiologic surveys in the community indicated that more than 15% of people used once or more an anxiolytic drug in the past year. The issue of chronic treatments is particularly crucial because of their poor benefit/risk ratio in most anxiety disorders (limited efficacy, cognitive side effects, withdrawal and dependence problems). To address this important public health issue, and knowing that, in France, benzodiazepines are prescribed mainly by general physicians, our aims were to explore psychiatric diagnoses in GP's patients with chronic use of anxiolytic benzodiazepines. We included 4 425 patients consuming such drugs regularly for six months or more, and assessed their anxiety and depression symptoms through various clinical scales (Hospital Anxiety and Depressive scale - HAD, Clinical Global Impression scale - CGI, Sheehan Disability Scale - SDS, Cognitive Dependence to Benzodiazepines scale - CDB) and with the Mini International Neuropsychiatric Interview for DSM IV criteria. Only 2.2% of the subjects had neither anxious nor depressive symptoms as indicated by low scores on both subscores (less than 8) of the HAD scale, used as a screener. Nearly three quarters of the 4,257 subjects (73.2%), had CGI scores of at least 5 (markedly ill to extremely ill). Social and familial disability was also high in more than 40% of the sample (marked to extreme disruption according to SDS scores). About half of the sample had CDB scores suggesting a benzodiazepine dependence. According to the MINI, 85.1% of the patients had at least one current DSM IV diagnosis of affective disorder. The most frequent diagnoses were major depressive episode (60%), generalized anxiety disorder (61.2%), and panic disorder (22.5%). An anxiety and depressive comorbidity wad found in 41.9% of the subjects. Some methodological limitations must be taken into account in the discussion of our results, and especially the fact that the included patients were not supposed to be totally representative of all patients consuming anxiolytic benzodiazepines in general practice. However, the size of our sample is sufficiently large to limit possible biases in patient selection. The main result of this study is that a great majority of the patients had significant symptomatology, in particular major depressive episodes and generalized anxiety disorder, often with marked severity and disability. These data are in line with the knowledge of a lack of efficacy of benzodiazepines in depressive and most anxiety disorders, despite long term treatment. They also confirm the current guidelines which recommend prescribing serotoninergic antidepressants, and not benzodiazepines, when long term treatments are needed for severe and chronic affective disorders. This epidemiologic study leads to the conclusion that a specific and attentive diagnostic assessment should be done in all patients receiving benzodiazepines for more than three months, in order to purpose in many cases other long term therapeutic strategies.

Increased gadd153 messenger RNA level is associated with apoptosis in human leukemic cells treated with etoposide.

Treatment of leukemic cells with topoisomerase inhibitors can lead to growth arrest and subsequent apoptotic cell death. The relationships between cell cycle regulation and apoptosis triggering remain poorly understood. The gadd153 gene encodes the nuclear protein CHOP 10 that acts as a negative modulator of CCAAT/enhancer binding protein transcriptional factors and inhibits cell cycle progression. We have investigated the relationships between gadd153 gene expression and apoptosis induction in four human leukemic cell lines with different sensitivities to apoptosis induced by etoposide (VP-16), a topoisomerase II inhibitor. The gadd153 gene was constitutively expressed in the four studied cell lines. In U937 and HL-60 cells that were very sensitive to apoptosis induction by the drug, VP-16 induced a time- and dose-dependent increase of gadd153 gene mRNA expression. Using agarose gel electrophoresis and a quantitative filter elution assay, apoptotic DNA fragmentation was observed to begin when gadd153 gene expression increased. Equitoxic doses of VP-16 (as defined using a 96-h 3-4,5-dimethylthiazol-2,5-diphenyltetrazolium bromide assay) did not increase the gadd153 mRNA level in K562 and KCL22 cell lines that were more resistant to apoptosis induction by the drug. Nuclear run-on and mRNA stability experiments demonstrated that VP-16 treatment increased gadd153 gene transcription in the sensitive U937 cells. Cycloheximide did not prevent gadd153 expression increase. Both gadd153 mRNA level increase and internucleosomal DNA fragmentation were inhibited by N-tosyl-L-phenylalanine chloromethylketone, a serine threonine protease inhibitor, N-acetyl-leucyl-leucyl-norleucinal, an inhibitor of calpain, N-acetylcysteine, an inhibitor of oxidative metabolism, and overexpression of Bcl-2. Z-VAD and Z-DEVD peptides that inhibit interleukin 1beta-converting enzyme-like proteases suppressed DNA fragmentation without preventing gadd153 mRNA increase in VP-16-treated U937 cells. These results indicate that gadd153 gene expression increase occurs downstream of events sensitive to N-tosyl-L-phenylalanine chloromethylketone, calpain inhibitor I, and Bcl-2 and upstream of interleukin 1beta-converting enzyme-related proteases activation in leukemic cells in which treatment with VP-16 induces rapid apoptosis.

Influence of Bcl-2 overexpression on the ceramide pathway in daunorubicin-induced apoptosis of leukemic cells.

We have previously demonstrated that daunorubicin (DNR) induces apoptosis in some leukemic myeloid cell lines. We investigated a potential protective role for Bcl-2 in apoptosis induced by DNR in two leukemic cell lines, one myeloid and one lymphoid, overexpressing the anti-apoptotic gene Bcl-2. Parental cells treated with DNR exhibited classical features of apoptosis 6 h after drug exposure, all the cells being dead after 30-48 h. In contrast, overexpression of Bcl-2 significantly delayed, but did not prevent the occurrence of DNR-induced apoptosis, with no surviving cells 96 h after drug exposure. To elucidate the mechanism of the protection mediated by Bcl-2, we explored the signaling pathway which initiates DNR-induced apoptosis. In this report, we show that, in both the myeloid and lymphoid parental cell lines, DNR triggered a sphingomyelin (SM) hydrolysis after 10-15 min with a concomitant ceramide generation. Moreover, exogenous ceramide induced DNA fragmentation in these cells, with levels similar to those observed with DNR treatment. In contrast, Bcl-2 overexpression protected the cells against apoptosis induced by ceramide treatment, without preventing the early SM hydrolysis nor the ceramide generation in these cells. Our results strongly suggest that Bcl-2-mediated protection of DNR-induced apoptosis is effected downstream of the SM-ceramide signaling pathway.

Expression of the oncogenic NPM-ALK chimeric protein in human lymphoid T-cells inhibits drug-induced, but not Fas-induced apoptosis.

Anaplastic large cell lymphomas (ALCLs) are frequently associated with the t(2;5)(p23;q35) translocation, leading to the expression of NPM-ALK, a fusion protein linking nucleophosmin and anaplastic lymphoma kinase, a receptor tyrosine kinase. In ALCLs, dimerization of NPM-ALK leads to constitutive autophosphorylation and activation of the kinase, necessary for NPM-ALK oncogenicity. To investigate whether NPM-ALK, like other oncogenic tyrosine kinases, can inhibit drug-induced apoptosis, we permanently transfected NPM-ALK into Jurkat T-cells. As in ALCLs, NPM-ALK was expressed as a constitutively kinase-active 80 kDa protein, and could be detected by immunocytochemistry in nucleoli, nuclei and cytoplasm. Doxorubicin-induced apoptosis (assessed by cell morphology and annexin V-FITC binding) was significantly inhibited in two independent NPM-ALK-expressing clones (5.2+/-1.8 and 7.5+/-0.8% apoptosis), compared to control vector-transduced cells (36+/-6.7%). Similar results were observed with etoposide. In contrast, Fas-induced apoptosis was not inhibited. Cytochrome c release into the cytosol was delayed in doxorubicin-, but not anti-Fas-treated transfectant cells, indicating that apoptosis inhibition occurred upstream of mitochondrial events. Using NPM-ALK mutants, we demonstrated that inhibition of drug-induced apoptosis: (1) requires functional kinase activity, (2) does not involve phospholipase C-gamma, essential for NPM-ALK-mediated mitogenicity and (3) appears to be phosphoinositide 3-kinase independent, despite a strong Akt/PKB activation observed in wild type NPM-ALK-expressing cells. These results suggest that the NPM-ALK antiapoptotic and mitogenic pathways are distinct.

Basic fibroblast growth factor and hematopoiesis.

Basic fibroblast growth factor (bFGF or FGF-2) is an angiogenic and pleiotropic factor involved in the proliferation and differentiation of numerous cell types. It is expressed mostly in tissues of mesoderm and neuroectoderm origin, and plays an important role in the mesoderm induction, together with transforming growth factor-beta (TGF-beta). Although hematopoietic cells derive from the mesoderm, relatively few studies have addressed the role of bFGF in the hematopoietic system until recently. It appears that bFGF is expressed and produced by bone marrow stromal cells, as well as by cells from several mature peripheral blood lineages. It is released and stored in the bone marrow extra-cellular matrix. FGF-receptors (FGF-Rs) are expressed on nearly every cell of hematopoietic origin tested so far. Growing evidence shows that bFGF can positively regulate hematopoiesis, by acting on various cellular targets: stromal cells, early and committed hematopoietic progenitors, and possibly some mature blood cells. It synergizes with hematopoietic cytokines, or antagonizes the negative regulatory effects of another factor, TGF-beta, thus potentially playing a central role in hematopoiesis.

Expression of basic fibroblast growth factor (bFGF) and FGF-receptors in human leukemic cells.

Recent reports have suggested that basic fibroblast growth factor (bFGF) could play a permissive role in hematopoiesis, in combination with specific colony-stimulating factors. We investigated the expression of bFGF and FGF-receptors (FGF-Rs) in leukemic cell lines of various hematopoietic lineages. Three protein isoforms of bFGF of approximately 18, 22 and 24 kDa were detected in the myeloid cell line K562, but not in myelomonocytic or lymphoid (T or B) cell lines. In vitro-induced differentiation of K562 cells did not change the pattern of expression of the different bFGF isoforms. Accordingly, the mRNA of bFGF was found expressed in K562, but not in other leukemic lines tested, as assayed by reverse transcript amplification (RT-PCR). Using the same technique, we searched for the presence of high affinity FGF-Rs on these cells: in eight out of ten cell lines tested, mRNA for at least one FGF-R among FGF-R1, FGF-R3 or FGF-R4 was expressed, whereas FGF-R2 was never detected. We found that two cell lines were responsive to bFGF in different biological assays: (i) in K562 myeloid cells induced to differentiate by hemin, preincubation with bFGF and heparin increased cell viability and decreased hemin-induced DNA fragmentation, without affecting erythroid differentiation; and (ii) in U937 monocytic cells, the production of plasminogen activator was increased by bFGF or aFGF in combination with heparin. Binding experiments with 125I-bFGF (up to 200 pM) in the presence of heparin revealed high affinity receptors on the K562 and U937 cell lines (1177 +/- 440 and 392 +/- 184 sites/cell, Kd = 61.7 +/- 8.6 and 43.1 +/- 13.5 pM, respectively). Thus our results strongly suggest that cells of hematopoietic origin could express functional FGF-receptors.

Phorbol myristate acetate-induced expression of high-affinity interleukin 2 receptors and production of interleukin 2 by human acute lymphoblastic leukemia T cells.

The effect of phorbol myristate acetate (PMA) on the expression of interleukin 2 receptors (IL-2R), production of IL-2 and IL-2-dependent proliferation of acute lymphoblastic leukemia T cells (T-ALL cells) from 10 patients was studied. First, the effect of PMA on the expression of cell surface markers was assessed: a decrease of CD3 and CD4, and an enhanced expression of CD8 molecule were observed on T-ALL cells. Moreover, PMA exhibited an heterogenous effect on various activation-associated molecules such as a decreased expression of transferrin receptor and T10 molecule and an induced expression of 4F2 and CD9 molecules. It is known that functional high-affinity IL-2R are composed of at least two IL2 binding molecules, the alpha (p55) and beta (p70) chains. We found that PMA induced the expression of both IL-2R alpha and IL-2R beta chains, as well as IL-2 production by T-ALL cells. These effects were time- and dose-dependent. Cross-linking experiments with 125I-labelled recombinant IL-2 (125I-rIL-2) revealed both p55 (IL-R alpha) and p70 (IL-2R beta) IL-2-binding polypeptides, whereas binding equilibrium assays on PMA-treated cells demonstrated the presence of a low number (31-413) of high-affinity binding sites/cell in five out of six cases analysed, as well as intermediate affinity IL-2R (1234-3919 sites/cell) in four out of six cases, according to the time of incubation with PMA. In two cases tested high-affinity IL-2R on PMA-treated T-ALL cells could internalize 125I-rIL-2 at 37 degrees C. PMA alone enhanced the spontaneous proliferation of T-ALL cells in three cases, whereas a clear synergy between IL-2 and PMA could be detected in three patients' cells. Moreover, exogenous rIL-2 enhanced cell proliferation of PMA-preincubated T-ALL cells in four cases studied. Taken together, these observations indicate that a short-term incubation of T-ALL cells with PMA can activate the IL-2/IL-2R system on these cells without inducing strong modifications of their differentiation status. These results thus suggest that this system may be involved in the proliferation process of some activated immature T cells.

Establishment of a novel anaplastic large-cell lymphoma-cell line (COST) from a 'small-cell variant' of ALCL.

Anaplastic large-cell lymphoma (ALCL) is a distinct biological and cytogenetic entity with a broad spectrum of morphological features (common type, small-cell variant and lymphohistiocytic variant). Few cell lines of ALCL are available and they all originate from primary tumors demonstrating the common type morphology (ie large-sized lymphoma cells). We established a new ALCL cell line (COST) from the peripheral blood of a patient with a small-cell variant of ALCL, at diagnosis. Cells growing in vitro and in SCID mice consisted of two populations, that is, small- and large-sized cells as seen in the patient's tumor. Both large and small malignant cells were positive for CD43/MT1 T-cell associated antigen, perforin, granzyme B and TIA-1, but negative for CD2, CD3, CD5, CD7, CD4 and CD8 antigens. Standard cytogenetic studies as well as multiplex FISH confirmed the presence of the canonical t(2;5)(p23;q35) translocation, but also revealed additional numerical and structural abnormalities. The COST cell line is the first ALCL small-cell variant cell line, and thus provides a potentially useful tool for further functional and molecular studies that should improve our understanding of the small-cell variant of ALCL, which is more frequently complicated by a leukemic phase.

Interleukin 2 production and interleukin 2 receptor expression by human immature leukemic T cells.

In order to determine the role of interleukin 2 (IL2) on the proliferation of leukemic cells from patients with T-cell acute lymphoblastic leukemia (T-ALL) we studied the production of IL2, the function of IL2 receptors (IL2R) expressed on T-ALL cells and their IL-2-dependent in vitro proliferation. Leukemic cells from six out of 17 T-ALL/T-cell non-Hodgkin's lymphoma patients with a prothymocyte (stage I) or a mature thymocyte (stage III), but not with a common thymocyte (stage II) phenotype, could proliferate, in a dose-dependent manner, in response to recombinant IL2 (rIL2) and anti-Tac and TU27 moAbs as well as polyclonal anti-IL2 purified immunoglobulin G could inhibit this IL2-induced cell proliferation. Both crude or/and Amicon-concentrated media conditioned by T-ALL cells from 10 out of 13 tested patients contained IL2 activity as assessed by colorimetric biological and immunoenzymatic assays; this biologic activity was due to a 14.5 kDa molecule adsorbed by anti-IL2 antibodies in an immunoaffinity assay. Although less than 10% of fresh leukemic cells expressed IL2R alpha (Tac) chain, a 24 h cell incubation in the absence of any mitogenic stimulation induced IL2R alpha chain expression in five out of 13 patients (11-83% Tac+ cells). Morever, Tac mRNA transcripts could be detected in fresh cells from all 10 patients tested. Staining of fresh leukemic cells with an IL-2R beta-chain-specific monoclonal antibody and flow cytometry analysis revealed that 4-13% of leukemic cells were positive. Binding experiments with 125I-rIL2 showed a small number of high affinity IL2R on fresh cells from three T-ALL patients (114-200 sites/cell, dissociation constant = 101-181 pm). Finally, antibodies against IL2R alpha, IL2R beta and IL2 could inhibit both IL2 driven and spontaneous cell proliferation of most patients' T-ALL cells, although in some cases an heterogenous pattern of inhibition was observed. Taken together, these findings strongly suggest that an IL2/IL2R-dependent mechanism could be involved in the proliferation of some T-ALL cells.

Autocrine IL-2-dependent growth of a newly established CD3+, CD16-, CD56+, CD57+, J(H)-, TCRbeta-, TCRgamma- leukemia cell line (NOI-90).

Leukemic cells from a 45-year-old male patient with a CD3+, CD56+, CD57+, CD7+ acute lymphoblastic leukemia were cultured in vitro in the absence of any added growth factor for up to 6 years and a continuous lymphoblastoid cell line (NOI-90) was established. NOI-90 cells have the same phenotype and karyotype as initial leukemic cells. Southern blot of DNA from NOI-90 cells showed that TCRbeta, TCRgamma, and J(H) were in germ line. Two and 25% of NOI-90 cells were positive when stained with the IOT14 and 7G7/B6 moAbs, which recognize the CD25 molecule (IL-2R alpha chain); moreover, 4% and 13% of the cells were positive when stained with the TU-27 and mik beta3 moAbs which recognize the CD122 molecule (IL-2Rbeta chain). Equilibrium binding experiments with radiolabelled IL-2 revealed the presence of a small number of high affinity IL-2R on both fresh and continuously growing cells. Media conditioned by NOI-90 cells could induce proliferation of an IL-2-dependent cell line and this IL-2 activity could be detected by a sensitive immunoenzymatic assay using antibodies recognizing distinct epitopes of IL-2. Moreover, IL-2 activity could be adsorbed by immunoaffinity on anti-IL-2 polyclonal purified IgG and the retained molecule displayed a m.w. of 14.5 kDa in SDS-PAGE. In addition, IL-2 immunoreactive molecules could be revealed in the cytoplasm of the cells. Finally, IL-2 fixed on the cell membrane could be detected by indirect immunofluorescence. Although added IL-2 could not induce cell proliferation, monoclonal antibodies against CD25, CD122 and IL-2 could specifically inhibit spontaneous cell proliferation in a dose-dependent manner. NOI-90 cells failed to demonstrate any cytotoxic activity against the K-562, Raji or Daudi cells. These findings indicate that NOI-90 cells are of non-T, non-B, origin lacking NK activity but proliferate under an autocrine pathway which involves, at least partly, the IL-2/IL-2R system.

Daunorubicin-induced internucleosomal DNA fragmentation in acute myeloid cell lines.

The study was designed to evaluate the implication of apoptosis in myeloid leukemic cell death induced by daunorubicin (DNR) and to identify the possible factors which may influence this process. DNR-induced apoptosis was characterized by morphology and DNA fragmentation in six leukemic myeloid cell lines which expressed different differentiation phenotypes. In phenotypically mature HL-60 and U937 cells, DNR induced typical apoptosis with characteristic morphological changes and intense internucleosomal DNA fragmentation within a narrow concentration range (0.5-2 microM). When these cells were treated with higher doses of DNR, large DNA fragments (100 kbp), but not internucleosomal fragments, were identified. DNR-induced DNA fragmentation in HL-60 and U937 was inhibited by antioxidants such as N-acetylcysteine (N-ac) or pyrrolidine-dithiocarbamate (PDTC). In the phenotypically immature KG1a, KG1, HEL and ML1 cell lines DNR induced no characteristic apoptotic morphological features as well as very low levels of internucleosomal DNA fragmentation, whereas large DNA fragments (200 kbp) were observed in KG1a treated with 7 microM DNR. Since the latter expressed P-glycoprotein (P-gp), the role of P-gp in the lack of apoptotic response to DNR was investigated. One P-gp inhibitor (verapamil) slightly improved DNR-induced DNA fragmentation in KG1a cells whereas the combination of verapamil and buthionine-sulfoximine (BSO), which depletes glutathion store, further increased internucleosomal DNA fragmentation. In conclusion, DNR induced internucleosomal DNA fragmentation in some but not all AML cells; the magnitude of this process being influenced by both intracellular drug concentration and oxidative balance.

Intracytoplasmic detection of the Tac (p55) chain of interleukin-2 receptor in pre-B leukemic cells associated with a constitutive expression of Tac mRNA.

The pre-B Reh-6 leukemic cells do not express membrane interleukin-2 (IL-2)-R alpha (Tac or p55) chain; however, their incubation with PMA induces the expression of both high and low affinity IL-2-R. Northern analysis of nonstimulated Reh-6 as well as leukemic cells from patients with acute B cell precursor lymphoblastic leukemia displayed a constitutive expression of p55 mRNA transcripts, which could be enhanced by PMA. Both actinomycin-D and cycloheximide could abrogate PMA-induced p55 membrane expression, suggesting the need for de novo mRNA and protein synthesis. The increased PMA-induced p55 mRNA accumulation was an early event (4 hr) and could be enhanced, specifically, by rIL-2 because anti-Tac moAb inhibited this rIL-2-mediated effect. Immunofluorescence and cross-linking studies using 125I-rIL-2 failed to reveal membrane-associated p55 protein on both Reh-6 and patients' leukemic cells. Conversely, immunogold staining and electron microscopy studies, revealed p55 immunoreactive molecules in the cytoplasm but not in the nucleus of all Reh-6 cells. Using a sensitive EIA, p55 molecules could be detected in cell lysates but not the culture supernatants of Reh-6 cells, suggesting that p55 was not released into the culture medium. These results indicate that constitutively expressed p55 mRNA on pre-B leukemic cells is translated into a relative immunoreactive protein that cannot be expressed on cell surface for unknown, yet, reasons.

Stability of a flow down an incline with respect to two-dimensional and three-dimensional disturbances for Newtonian and non-Newtonian fluids.

Squire's theorem, which states that the two-dimensional instabilities are more dangerous than the three-dimensional instabilities, is revisited here for a flow down an incline, making use of numerical stability analysis and Squire relationships when available. For flows down inclined planes, one of these Squire relationships involves the slopes of the inclines. This means that the Reynolds number associated with a two-dimensional wave can be shown to be smaller than that for an oblique wave, but this oblique wave being obtained for a larger slope. Physically speaking, this prevents the possibility to directly compare the thresholds at a given slope. The goal of the paper is then to reach a conclusion about the predominance or not of two-dimensional instabilities at a given slope, which is of practical interest for industrial or environmental applications. For a Newtonian fluid, it is shown that, for a given slope, oblique wave instabilities are never the dominant instabilities. Both the Squire relationships and the particular variations of the two-dimensional wave critical curve with regard to the inclination angle are involved in the proof of this result. For a generalized Newtonian fluid, a similar result can only be obtained for a reduced stability problem where some term connected to the perturbation of viscosity is neglected. For the general stability problem, however, no Squire relationships can be derived and the numerical stability results show that the thresholds for oblique waves can be smaller than the thresholds for two-dimensional waves at a given slope, particularly for large obliquity angles and strong shear-thinning behaviors. The conclusion is then completely different in that case: the dominant instability for a generalized Newtonian fluid flowing down an inclined plane with a given slope can be three dimensional.

Peptides derived from the dependence receptor ALK are proapoptotic for ALK-positive tumors.

ALK is a receptor tyrosine kinase with an oncogenic role in various types of human malignancies. Despite constitutive activation of the kinase through gene alterations, such as chromosomal translocation, gene amplification or mutation, treatments with kinase inhibitors invariably lead to the development of resistance. Aiming to develop new tools for ALK targeting, we took advantage of our previous demonstration identifying ALK as a dependence receptor, implying that in the absence of ligand the kinase-inactive ALK triggers or enhances apoptosis. Here, we synthesized peptides mimicking the proapoptotic domain of ALK and investigated their biological effects on tumor cells. We found that an ALK-derived peptide of 36 amino acids (P36) was cytotoxic for ALK-positive anaplastic large-cell lymphoma and neuroblastoma cell lines. In contrast, ALK-negative tumor cells and normal peripheral blood mononuclear cells were insensitive to P36. The cytotoxic effect was due to caspase-dependent apoptosis and required N-myristoylation of the peptide. Two P36-derived shorter peptides as well as a cyclic peptide also induced apoptosis. Surface plasmon resonance and mass spectrometry analysis of P36-interacting proteins from two responsive cell lines, Cost lymphoma and SH-SY5Y neuroblastoma, uncovered partners that could involve p53-dependent signaling and pre-mRNA splicing. Furthermore, siRNA-mediated knockdown of p53 rescued these cells from P36-induced apoptosis. Finally, we observed that a treatment combining P36 with the ALK-specific inhibitor crizotinib resulted in additive cytotoxicity. Therefore, ALK-derived peptides could represent a novel targeted therapy for ALK-positive tumors.

T-cell colony formation in patients with T-cell malignancies: growth factor requirements for in-vitro proliferation of peripheral blood T-cell colony-forming cells.

Peripheral blood T colony-forming cells (T-CFC) from patients with T-cell malignancies are capable of proliferation in methylcellulose, in the absence of added growth factors or mitogenic stimulation. We show here that based on the spontaneous plating efficiencies of their T-CFC, two groups of patients can be established: Group A (10 patients) with a high colony number (more than 100 colonies/10(5) seeded cells), and group B (12 patients) with less than 100 colonies/10(5) cells. The addition of interleukin 2-(IL2) containing PHA-leukocyte conditioned medium enhanced colony growth from group B but not group A patients. Moreover, both biochemically purified and recombinant IL2 induced the colony growth from group B but not group A patients without any other stimulation. In addition, a monoclonal antibody (moAb) against the IL2 receptor (IL2-R; anti-Tac) inhibited the spontaneous colony formation from T-CFC of both groups of patients. These observations strongly suggest that IL2-R are involved in the spontaneous colony growth of T-CFC. To determine whether IL2 is also involved in the spontaneous colony formation, media conditioned (LCM) by unstimulated leukemic cells were tested for IL2 activity. A constitutive release of IL2 was detected in LCM from only 2 out of 10 patients tested, and some of them were capable to inhibit thymidine incorporation by IL2-dependent cells cultured in the presence of highly purified IL2. However, most of these LCM contained a T-cell colony-promoting activity (TCPA) inducing colony formation from both normal resting and PHA-stimulated E+ clonogenic cells without added IL2 or mitogenic stimulation. TCPA-containing LCM induced the expression of functional IL2-R and IL2 release by normal resting lymphocytes. TCPA was constitutively released by blast-enriched cell fractions suggesting that it is released by the leukemic cells.