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.

Surfactant changes during experimental pneumocystosis are related to Pneumocystis development.

Pneumocystosis-related surfactant changes have been reported in both humans and corticosteroid-treated experimental hosts. As corticosteroids induce an increase in pulmonary surfactant, some findings could be considered as controversial. The aim of this study was to investigate whether the surfactant composition changes during experimental pneumocystosis were related to the Pneumocystis development. In this work two corticosteroid-untreated animal models were used: rabbits, which develop spontaneous pneumocystosis at weaning; and severe combined immunodeficiency mice, which were intranasally inoculated with Pneumocystis carinii. Surfactant phospholipid and protein content was explored by bronchoalveolar lavage. The in vitro effect of surfactant on P. carinii growth was also explored. In the two models, the surfactant phospholipid/protein ratio was significantly increased, whereas parasite rates were low. This ratio decreases with the slope increase of the parasite growth curve. These early surfactant changes suggested that Pneumocystis proliferation requires alveolar lining fluid changes, and that normal surfactant is not suitable for parasite development. In this way, in vitro experiments presented here have revealed an inhibitory effect of synthetic or seminatural surfactants on the P. carinii growth. Further studies are needed to determine how Pneumocystis induces the reported early modifications of the surfactant, and why the parasite development is inhibited by pulmonary surfactant.

Studies on ether-phospholipids of vascular smooth muscle cells. Identification of a rapid Ca(2+)-dependent hydrolysis of alkyl-phosphatidylethanolamine promoted by endothelin-l.

We have investigated the metabolism of 1-O-[3H]octadecyl-sn-glycero-3-phosphocholine ([3H]lyso PAF) and [3H] myristic acid in secondary cultures of aortic smooth muscle cells (SMC) to characterize the origin of second messengers generated upon stimulation with endothelin-l (ET-l). When cells were labelled with [3H]lyso PAF, we observed a transfer of the label from phosphatidylcholine (PC) to phosphatidylethanolamine (PE) In contrast, incubation with [3H]lyso PAF labelled mainly alkyl-subclasses while [3H]myristate was associated with diacyl-subclasses. Using these specific labelling procedures, we have shown that ET-l induced a strong hydrolysis of PE. This hydrolysis was specific for alkyl-PE with a maximum after 5 s of stimulation. We have also observed an extracellular Ca(2+)-dependent increase in diglyceride (DG), phosphatidic acid (PA) and mainly triglyceride (TG) concomitant to alkyl-PE hydrolysis. Thus, alkyl-DG generated from alkyl-PE appears to be a major product in ET-l stimulation of SMC. These results suggest a new level of complexity in the signal transduction cascade involving a specificity for phospholipid subclasses.

Expressed sequence tags identify human isologs of the ARF-dependent phospholipase D.

By searching into Expressed Sequence Tags databases (dbEST) using Blast X algorithm software and a plant phospholipase D as template, we have identified a cDNA from human brain (Z45777) which encodes for a protein similar to the amino acid region 743-929 of the human phospholipase D1 (PLD1), and a cDNA from human liver (R93485) which encodes for a protein similar to region 815-932 of PLD1. Sequence comparison between cloned phospholipases showed the presence of 3 conserved amino acid sequences: AFVGGIDLAYGRWD (box A), IIGSANINDRS (box B), and YIYIENQFFI (box C). Phylogenic analysis indicated that the cDNA from brain and liver encoded for human isologs of PLD1.

Distinct signal transduction pathways for activation of rabbit alveolar macrophages in vitro by cotton bract tannin.

These experiments were designed to study signal transduction pathways in alveolar macrophages stimulated by condensed tannin or zymosan. Condensed tannins, present in cotton mill dust, alter the host-defense function of alveolar macrophages and may contribute to the pathogenesis of byssinosis. We tried to determine the early steps in signal transduction mechanisms of cell activation by tannin. With the quantification of 51Cr release, we determined that tannin was cytotoxic for the cells after 30 min activation with 130 micrograms for 2 x 10(6) cells. 51Cr release was similar for control cells and zymosan- or 30 micrograms tannin-activated cells. Using the luciferine luciferase reaction, we showed that tannin markedly depleted ATP cell content. In inositol-labeled cells, tannin increased inositolphosphate release in a dose-dependent manner. In lysoPAF-labeled cells, tannin induced synthesis of phosphatidic acid and diglycerides. In the presence of ethanol, the level of tannin-induced phosphatidic acid was slightly reduced, and phosphatidylethanol was synthesized. No phosphatidylethanol was found in alveolar macrophages stimulated by zymosan in the presence of ethanol. GF 109203X, a specific inhibitor of protein kinase C decreased only tannin-induced phosphatidylethanol synthesis. In conclusion, tannin (at 30 or 130 micrograms/ml) activated an inositol phospholipase C in alveolar membranes. Phosphatidylcholine phospholipases C and D were found only at the higher concentration of tannin.

Differential labelings suggest two specific phospholipid subclass hydrolysis promoted by PDGF-BB in vascular smooth muscle cells.

We have used differential phospholipid subclass labelings performed with [3H]lyso PAF and [3H] myristic acid into vascular smooth muscle cells (vSMC) to characterize the subclasses of phospholipid substrates upon different stimulation times with platelet-derived growth factor (PDGF-BB). In cells labeled with [3H]lyso PAF, PDGF-BB induced a sustained hydrolysis of alkyl-PE. In contrast, in [3H]myristic acid-labeled cells, PDGF-BB promoted a rapid and transitory hydrolysis of diacyl-PC. This hydrolysis was concomitant with an synthesis of diglyceride (DG) and phosphatidic acid (PA). Thus, both diacyl-PC and alkyl-PE appear to be major targets in PDGF-BB stimulation of SMC. These results suggest that agonists could induce the hydrolysis of precise phospholipid subclasses leading to a new specificity into the signal transduction cascade.

Bronchoalveolar lavage phospholipid abnormalities in HIV-infected patients.

Our aim was to evaluate the quality of pulmonary surfactant, a nonspecific defence system, during the course of human immunodeficiency virus) infection. Protein and phospholipid composition were determined in 127 bronchoalveolar lavage (BAL) fluids from 89 HIV seropositive patients (54 acquired immune deficiency syndrome (AIDS), 35 non-AIDS) and 11 healthy controls. In all of the HIV BAL samples, biochemical abnormalities were found. In subjects with pulmonary infection or Kaposi's sarcoma, the phospholipid/protein ratio was decreased, mainly because of elevated protein levels (15.8 and 20, respectively, vs 7.2 mg.100 ml-1 for controls, p < 0.05). In subjects without obvious pulmonary involvement, phospholipid was decreased (1.3 +/- 0.2 vs 2.9 +/- 0.3 mg.100 ml-1 for controls, p < 0.001), whereas the protein was not altered. Phospholipid composition was also altered: the phosphatidylcholine percentage was decreased, whilst the other main phospholipids were increased. We conclude that the alveolar lining is altered, whatever the stage of HIV disease. In most patients, it results from an increase of vascular permeability, with an influx of serum proteins. However, changes in phospholipid composition suggest that, in some cases, surfactant is also altered.

Surfactant analysis during Pneumocystis carinii pneumonia in HIV-infected patients.

Pulmonary surfactant is altered in experimental Pneumocystis carinii pneumonia. Although P carinii is a major causative agent of pneumonia in immunocompromised patients, the pathophysiology of lung injury caused by this organism is poorly understood. Therefore, we studied bronchoalveolar lavage specimens obtained from 19 HIV-infected subjects with PCP compared with specimens from ten healthy control subjects. As iterative BAL was performed, 37 BAL specimens were analyzed for protein and phospholipid. The BAL samples were divided into two groups as follows: 22 BAL samples with the presence of P carinii and 15 BAL samples without P carinii. Compared to control subjects, HIV+ BAL presented a significant increase of PR and a decrease of total PL in both P carinii+ and P carinii- BAL, but in P carinii+ BAL, the fall of PL/PR ratio was significantly more pronounced compared to P carinii- (0.09 +/- 0.02 vs 0.19 +/- 0.04, p less than 0.02). The BAL performed during the recovery of PCP showed an improvement of initial biochemical abnormalities. Surfactant composition was also altered, with a phosphatidylcholine and phosphatidylglycerol drop and a sphingomyelin and lysophosphatidylcholine increase. The presence, even in P carinii- BAL, of less polar compounds of undetermined nature, was revealed. We concluded that in HIV+ patients, abnormalities of pulmonary surfactant were present before PCP, and that the development of PCP enhances these abnormalities. These surfactant alterations may contribute to the saprophyte-pathogen transformation of P carinii, but this hypothesis requires further investigation that is presently in progress.

Possible origins of PAF-acether and lyso-PAF-acether in rat lung alveoli secondary to hypoxia.

After 4 h hypoxia, platelet activating factor (PAF-acether or 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) and its deacetylated derivative, lyso-PAF-acether, accumulate in rat lung surfactant, the latter in a 1000-fold excess (Prévost, M.C., Cariven, C., Simon, M.F., Chap, H. and Douste-Blazy, L. (1984) Biochem. Biophys. Res. Commun. 119, 58-63). In order to determine the origin of these two phospholipids, rat lung alveolar lavages and rat lung macrophages were examined for phospholipid composition before and after 4 h of hypoxic treatment. Our data indicate an activation of phospholipase A2 in both compartments, as detected by the accumulation of lysophosphatidylcholine. The main effect was observed in lung surfactant, where phosphatidylcholine hydrolysis attained 13%. This change was concomitant with the activation of a calcium-independent phospholipase A2 present in lung alveolar lavages, which might be responsible for the accumulation of some lyso-PAF-acether, alkylacylcholine glycerophospholipids being present in low but significant amounts in lung surfactant. However, the main source of PAF and lyso-PAF-acether appears to be alveolar macrophages, which secreted significant amounts of the two phospholipids upon in vitro hypoxic treatment, although the participation of other cells, such as type II pneumocytes, cannot be excluded. The relative amounts of the two compounds might be regulated by both an intracellular and an extracellular acetylhydrolase, the two enzymes being distinct proteins on the basis of their different isoelectric points.

Platelet activating factor (PAF-acether) is released into rat pulmonary alveolar fluid as a consequence of hypoxia.

Hypoxia provokes pulmonary constriction and because PAF-acether is a very strong pulmonary constrictor, we looked for PAF-acether in lung alveolar lavage (LAL) with a biological method based on the measurement of rabbit platelet aggregation. We first demonstrated a PAF-acether secretion during bronchoalveolar lavage with sterile isotonic NaCl (pH 7.2). PAF-acether secretion was completely suppressed with isotonic NaCl containing 5 mM EDTA but lyso-PAF-acether was still present (1.9 +/- 0.55 nmoles). Upon hypobaric hypoxia, PAF-acether was detected in LAL (1.05 +/- 0.25 10(-2)nmoles). The amount of lyso-PAF-acether increased by 6 times (12.1 +/- 4.1 nmoles). These results are given for 10(4) nmoles phospholipids of LAL. They indicate that alveolar macrophages might be activated by hypobaric hypoxia, so they produce PAF-acether in the alveole. Such a process could be involved in the well-known bronchoconstriction accompanying hypoxia.