Garcia-Verdugo II) are not yet clear. For this reason, we studied short-and long-term effects of LPS on basal and agonist-stimulated secretory responses of rat AT II by using Ca 2ϩ microfluorimetry, a microtiter plate-based exocytosis assay, by quantitating PL and 3 H-labeled choline released into cell supernatants and by using quantitative PCR and Western blot analysis. Long term, but not short term, exposures to LPS led to prolonged ATP-induced Ca 2ϩ signals and an increased rate in vesicle fusions with an augmented release of surfactant PL. Most notably, the stimulatory effect of LPS was ATP-dependent and may be mediated by the upregulation of the purinergic receptor subtype P2Y 2. Western blot analysis confirmed higher levels of P2Y 2, and suramin, a P2Y receptor antagonist, was more effective in LPS-treated cells. From these observations, we conclude that LPS, probably via Toll-like receptor-4, induces a time-dependent increase in P2Y 2 receptors, which, by yet unknown mechanisms, leads to prolonged agonist-induced Ca 2ϩ responses that trigger a higher activity in vesicle fusion and secretion. We further conclude that chronic exposure to endotoxin sensitizes AT II to increase the extracellular surfactant pool, which aids in the pulmonary host defense mechanisms. endotoxin; lipopolysaccharide; lung; P2Y 2 PULMONARY SURFACTANT PREVENTS alveolar collapse and aids in the clearance of inhaled pollutants and pathogens. Its components (lipids and proteins) are synthesized and secreted by alveolar type II cells (AT II) via regulated fusion of lamellar bodies (LB) with the plasma membrane (PM). Many agonists have been described to activate LB-PM fusions and to release phospholipids (PL) in isolated AT II (1). Among these, nucleotides such as ATP are likely to be the most physiologically relevant ones (29). ATP exerts its effects through interactions with purinergic receptors, in particular with the G proteincoupled receptor P2Y 2 (37), which results in Ca 2ϩ mobilization and activation of PKC. On addition of ATP to cultured AT II, the first LB-PM fusion events initiate after the cytoplasmic calcium concentration ([Ca 2ϩ ] c ) increases above a low threshold level (23). Calcium is considered as the major second messenger to trigger LB-PM fusions and, importantly, to expand the exocytotic fusion pore to facilitate extrusion of the surfactant lipoprotein complex (15).Bacterial LPS, or endotoxin, is a potent proinflammatory molecule able to induce, in extreme cases, endotoxic shock (25). After inhalation of endotoxin, an increase of inflammatory mediators (e.g., TNF-␣, IL-1␤) in bronchoalveolar lavage (BAL) has been reported (40), which might reflect the inflammatory status of the alveolar epithelium after contact with it. In addition, exposure to endotoxin induces alterations of surfactant pool sizes and PL contents in BAL. Fehrenbach et al. (17) described the formation of giant LB and a reduction in PL content in BAL in isolated rat lungs perfused with LPS. Similarly, Viviano et al. (42) reported an increased po...

Background: Multiple myeloma (MM) is the second most common hematological cancer after lymphoma. It is characterized by the accumulation of clonal malignant plasma cells within the bone marrow. The development of drug resistance remains a major problem for effective treatment of MM. Understand the mechanisms underlying drug resistance in MM is a focal point to improve MM treatment. Methods: In the current study, we analyzed further the role of redox imbalance induction in melphalan-induced toxicity both in human myeloma cell lines (HMCLs) and primary myeloma cells from patients. Results: We developed an in-vitro model of short-term resistance to high-dose melphalan and identified that pretreatment with physiological concentration of GSH protects HMCLs from melphalan-induced cell cycle arrest and cytotoxicity. We validated these results using primary MM cells from patients co-cultured with their bone marrow microenvironment. GSH did not affect the ability of melphalan to induce DNA damages in MM cells. Interestingly, melphalan induced reactive oxygen species, a significant decrease in GSH concentration, protein and lipd oxydation together with NRF2 (NF-E2-related factor 2) pathway activation. Conclusions: Our data demonstrate that antioxidant defenses confers resistance to high dose melphalan in MM cells, supporting that redox status in MM cells could be determinant for patients’ response to melphalan.

Surfactant protein C (SP-C) consists of a hydrophobic alpha-helix inserted in pulmonary surfactant membranes, and a more polar N-terminal palmitoylated segment exposed to the aqueous phase. Previously, we showed that SP-C inserted in lipid vesicles interacts with bacterial lipopolysaccharide (LPS) and reduces LPS-elicited responses. As the N-terminal segment of SP-C was the most likely region responsible for these effects, a set of synthetic analogs of this stretch (SPC((1-13)) ) were studied. Binding studies showed that SPC((1-13)) binds LPS to the same extent as porcine SP-C under lipid-free conditions. In the absence of serum, both, palmitoylated and non-palmitoylated analogs enhanced the binding of tritiated LPS to macrophages as well as the LPS-induced production of TNF-alpha by these cells. These effects were reversed in the presence of serum; the analogs reduced the production of TNF-alpha in LPS-stimulated macrophages, probably by interfering with the formation of LPS/CD14/LBP complexes as suggested by analysis of the fluorescence emitted by a FITC derivative of Re-LPS. Our data indicate that water-soluble analogs of the N-terminal segment of SP-C can reduce LPS effects in the presence of serum, and thus might help in the design of new derivatives to fight endotoxic shock and pro-inflammatory events.