Identification of chromosomal loci associated with non-P-glycoprotein-mediated multidrug resistance to topoisomerase II inhibitor in lung adenocarcinoma cell line by comparative genomic hybridization.

In order to identify genomic changes associated with an etoposide resistance acquisition, we used comparative genomic hybridization (CGH) to compare a human lung adenocarcinoma cell line, A549 wild type, and three sublines, A549-VP1-3, exposed to increasing concentrations of the topoisomerase II inhibitor, VP16. R-banding karyotype, fluorescence in situ hybridization (FISH), and Southern blot for the MLL gene were also performed. The CGH analysis showed that the A549-VP3 cell line shared chemoresistance-specific abnormalities (amplification of 11q23-qter, loss of chromosome 17, and deletions of 2p14-pter and 2q23-q24). FISH analysis confirmed the loss of one chromosome 17 in the three resistant sublines and revealed an increased fragmentation of chromosome 2 in more than two segments, depending on the etoposide concentration. FISH with an MLL gene probe showed additional signals of MLL (from three in the A549-WT to seven in the A549-VP3 cell line) translocated onto several other chromosomes. Southern blot indicated an amplification of the MLL gene, dependent on the etoposide concentration, without gene rearrangement. The CGH results are suggestive of loci that could be associated with the acquisition of an etoposide-chemoresistant phenotype. Deletion of the 2p region has already been reported, without any candidate gene being identified. The role of MLL in leukemogenesis has previously been demonstrated, but its role in the development of other tumors or its significance in the chemoresistance process remains to be elucidated.

Human monocyte-derived macrophages and dendritic cells as targets for biomaterial cytocompatibility studies using an improved in vitro culture system.

Since macrophage plays a key role in the biocompatibility process, neoplastic macrophage cell lines and human blood monocytes are commonly used as target cells for in vitro biomaterial tolerance evaluation. However, tumor cells profoundly differ from normal tissue cells and monocytes are only precursors of macrophages. It has become possible to generate recently, under adherent-free conditions, fully mature macrophages and dendritic cells from human blood monocytes in the presence of GM-CSF and GM-CSF + IL4 respectively. In the present work, we examined the effects of titanium-alloy on morphology, adhesion, cell phenotype and TNF-alpha release activity of such differentiated cells grown in hydrophobic teflon bags. Scanning electron microscopy showed that macrophages substantially adhered and spread on titanium-alloy surface throughout the culture period, whereas only a few dendritic cells were adherent. The phenotype of both cell types remained unchanged in the presence of the tested material. However, titanium-alloy stimulated the secretion of TNF-alpha by the macrophages of some donors. This model of culture may offer new insights into the biomaterial evaluation and may be useful for studying individual responses induced by biomaterials.

Separation of dendritic cells from highly purified human monocytes by counterflow centrifugation induces tissue factor expression.

BACKGROUND: In vitro generation of dendritic cells (DCs) from human monocytes represents a promising tool in immunotherapy. However, it is not known whether the separation of DCs from monocytes induces tissue factor expression and therefore may trigger coagulation in patients receiving these DC preparations. The aim of this study is thus to analyze tissue factor expression on monocyte-derived DCs and to compare their ability to trigger thrombin generation to that of macrophages obtained from the same monocytes. STUDY DESIGN AND METHODS: Human monocytes are separated by leukapheresis and washed by using counterflow centrifugation in sterile, endotoxin-free conditions. Macrophages are grown from human monocytes in the presence of GM-CSF alone and immature DCs are grown in the presence of GM-CSF plus IL-4 for 5 days with fetal calf serum (IDC-FCS). Immature DCs are also grown from human monocytes for 7 days in the presence of GM-CSF plus IL-4 with human group AB serum (IDC-HS). The addition of prostaglandin E(2) and TNFalpha in this culture medium at Day 5 leads to mature DCs (MDC-HS). Tissue factor mRNA expression is studied by RT-PCR analysis. Tissue factor antigen is measured by ELISA in cell lysates and by direct flow cytometry. The procoagulant activity of intact cells is assessed by using an amidolytic assay or a chronometric assay. RESULTS: IDC-FCS express tissue factor mRNA and antigen and trigger thrombin generation. Procoagulant activity of IDC-FCS is dependent on both tissue factor expression and exposure to anionic phospholipid. Monocyte-derived macrophages cultured for 5 days with GM-CSF alone express lower levels of tissue factor mRNA, tissue factor antigen, and procoagulant activity than IDC-FCS. IDC-HS and MDC-HS also express high levels of tissue factor mRNA and antigen and support procoagulant activity. CONCLUSION: Monocyte-derived DCs express a high level of functional tissue factor and support procoagulant activity. This finding should be taken into account in clinical trials.

Plasma levels of tumor necrosis factor-alpha (TNF-alpha) are essentially dependent on visceral fat amount in type 2 diabetic patients.

TNF-alpha is considered as one of the potential determinants of insulin resistance. However several data suggest that TNF-alpha expression itself, could be modulated by the degree of adiposity and/or plasma insulin levels. To clarify the determinants of plasma TNF-alpha levels in type 2 diabetes mellitus, we studied the impact of intensive insulin treatment on plasma TNF-alpha levels in 16 type 2 diabetic subjects with failure to oral antidiabetic medication (HbA1c: 10.8 +/- 1.2 %). Furthermore, we analyzed the relationship between plasma TNF-alpha levels and total or regional body fat measurements using anthropometry, bienergetic absorptiometry and computed tomography in a cohort of 33 caucasian obese type 2 diabetic subjects (BMI: 32.2 +/- 4.4 kg/m(2) ). The plasma TNF-alpha level was neither affected by plasma glucose level variations nor intensive insulin treatment despite a 37 % decrease in daily insulin needs at the end of insulin therapy (total duration: 11.5 +/- 2.0 days). The plasma TNF-alpha level was similar in men and women and unrelated to age, fasting glycemia or HbA1c. A relationship was highlighted with BMI (r =0.39, p <0.02), but not with total fat mass. This relationship was only dependent on the intra-abdominal fat mass amount as assessed by the waist-to-hip circumference ratio (r =0.52, p <0.01) and the visceral adipose tissue area (r =0.56, p <0. 01). These results show that plasma TNF-alpha levels are essentially dependent on visceral fat amount, thus suggesting that TNF-alpha could be one of the factors mediating insulin resistance and cardiovascular risk in obese type 2 diabetic patients.

Human blood monocytes interact with type I collagen through alpha x beta 2 integrin (CD11c-CD18, gp150-95).

Human blood monocytes are attracted into connective tissues during early steps of inflammation and wound healing, and locally interact with resident cells and extracellular matrix proteins. We studied the effects of type I collagen on monocyte adhesion and superoxide anion production, using human monocytes elutriated from peripheral blood and type I collagen obtained from rat tail tendon. Both acid-soluble and pepsin-digested type I collagens promoted the adhesion of monocytes, whereas only acid-soluble collagen with intact telopeptides induced the production of superoxide. Adhesion and activation of monocytes on acid-soluble type I collagen depended on the presence of divalent cations. mAbs directed against integrin subunits CD11c and CD18 specifically inhibited adhesion and activation of monocytes on type I collagen. Protein membrane extracts obtained from monocytes were submitted to affinity chromatography on collagen I-Sepharose 4B, and analyzed by Western blotting using specific anti-integrin subunit Abs. In the case of both acid-soluble and pepsin-digested collagens, two bands were revealed with mAbs against CD11c and CD18 integrin subunits. Our results demonstrate that monocytes interact with type I collagen through CD11c-CD18 (alpha x beta 2) integrins, which promote their adhesion and activation. For monocyte activation, specific domains of the type I collagen telopeptides are necessary. Interactions between monocytes and collagen are most likely involved in the cascade of events that characterize the initial phases of inflammation.

Use of the PFA-100 apparatus to assess platelet function in patients undergoing PTCA during and after infusion of c7E3 Fab in the presence of other antiplatelet agents.

The PFA-100 (Dade) is a new functional whole blood analyzer, the accuracy and reliability of which have been evaluated in von Willebrand disease and during acetyl salicylate acid therapy. This new test has the advantages of rapidity and simplicity. It may be useful to monitor new antiplatelet agents, such as GPIIb/IIIa receptor antagonists. The objective of this study was to assess the PFA-100 in comparison with aggregometry and with the percentage of blockaded receptors GPIIb/IIIa during and after c7E3 Fab infusion in fifteen patients undergoing PTCA. Our results showed a change of closure time values from normal to abnormal within a small margin of flow cytometric values (60-75% of blockaded receptors), and moreover a variable platelet response to long-term low dose aspirin treatment in agreement with aggregometry. No influence with heparin was observed. In conclusion, this study shows that PFA-100 may be helpful in the decision making for additional antiaggregant therapy before PTCA or in monitoring long-term GPIIb/IIIa receptor antagonist treatment.

[Secondary cutaneous effects of hydroxyurea: prospective study of 26 patients from a dermatologic consultation]. / Effets secondaires cutanés de l'hydroxyurée: étude prospective de 26 patients consultant dans un service de dermatologie.

PURPOSE: Hydroxyurea is a treatment of myeloproliferative syndromes. Its cutaneous side-effects are underestimated, because they are usually benign. We undertook a prospective study to evaluate their frequency. METHODS: During a 2-year period, all patients taking hydroxyurea for more than 6 months who had consultations at the dermatology department were systematically examined, regarding cutaneous side effects. RESULTS: Twenty-six patients were examined. All but one had cutaneous side-effects, including dryness (n = 16), moderate alopecia (n = 2), increased skin pigmentation (n = 5), melanonychia, single (n = 1) or multiple (n = 7), cutaneous atrophy (n = 4), leg ulcers (n = 8), plantar keratoderma (n = 3), pseudodermatomyositis (n = 1), lichen planus-like eruption on the dorsum of the hands (n = 2), actinic keratosis (n = 8), squamous cell carcinomas (n = 2), and mouth ulcerations (n = 1). CONCLUSION: This study shows that the frequency of hydroxyurea cutaneous side-effects diagnosed in 95% of studied patients is underestimated. They are usually benign, but some of them, in particular leg ulcers and squamous cell carcinomas, lead to modification of the treatment (39% of studied patients).

[Platelet-leukocyte interactions in coronary heart disease: pathophysiology, clinical relevance, pharmacological modulation]. / Interactions leucoplaquettaires au cours de l'insuffisance coronarienne. Physiopathologie, pertinence clinique, modulation pharmacologique.

The interactions between leukocytes and endothelial cells have been studied extensively under conditions of ischemia and reperfusion. In contrast, attraction of leukocytes by platelets at the site of damage is poorly understood. This recruitment facilitates inflammation and atherogenesis. Studies performed ex vivo in coronary artery disease show that neutrophil-platelet adhesion increases in unstable angina, coronary angioplasty and coronary artery bypass surgery, in comparison with stable angina. Experimental works have shown the major role of platelet P-selectin in platelet-leukocyte interactions, and of fibrinogen, which is the ligand of both platelets and leukocytes (B2 integrins). Studied performed in anti-GPIIb/IIIa-treated patients demonstrate a modulation, as inhibition, of platelet-leukocyte interactions. This new drug inhibits platelet function and coagulation, and moreover inflammation.

[Measurement of monocyte activation: perspectives in clinical application in the investigation of the diabetic patient]. / Mesure de l'activation monocytaire: perspectives d'application clinique dans l'exploration du patient diabétique.

Monocytes play a pivotal role in the complex processes of inflammation, immunologic responses and atherothrombosis. Clinical studies essentially reported an increased procoagulant activity in diabetes and coronary disease, suggesting an overexpression of tissue factor. This was further confirmed by the direct measurement of tissue factor on monocyte membrane by flow cytometry. Many receptors can be measured on monocytes by flow cytometry: beta 2 integrins (CD 11 a-b-c/CD 18) involved in adhesion, EPR-1 receptor, receptors for advanced glycation products, urokinase receptor U-PAR. Flow cytometry allows a cell analysis in whole blood. Modern methods allow a standardization of the procedures and a quantification of the number of sites expressed by the cell. However, the respect of preanalytical and analytical conditions is mandatory to obtain reliable data. Besides, clinical studies in diabetes should carefully define the subgroups of patients: type of diabetes, metabolic abnormalities, risk factors, infective complications.

[Platelet activation in cardiology: methods, indications and therapeutic perspectives]. / Activation plaquettaire en cardiologie: méthodes, indications et perspectives thérapeutiques.

Platelet activation and/or platelet reactivity have been reported to be associated with coronary heart disease. Whole blood flow cytometry allows to analyze platelets in their physiological environment, while other assays need platelet separation, susceptible to induce platelet modifications. But flow cytometric assay also have limitations. We studied preanalytical conditions in healthy volunteers, using two monoclonal antibodies directed against CD62 and CD63 (two specific markers of platelet degranulation), and two markers which recognize GPIIb/IIIa activation (PAC1 and bound fibrinogen). Preanalytical requirements were as follow: 1) whole blood samples need antagonists of platelet activation i.e., a mixture of theophylline, adenosine and dipyridamole, since artefactual platelet activation rapidly occurred in citrated whole blood, 2) whole blood should be immediately immunolabeled when samples arrived to laboratory, because fixation did not prevent artefactual time dependent activation, 3) the stability of immunolabeling was determined for each monoclonal antibody: paraformaldehyde as fixative solution was mandatory for both CD62 and CD63, whereas it enhanced bound fibrinogen and PAC1 expression, 4) platelets can be easily identified and gating on a dual scatter (forward scatter x side scatter) dot plot with no specified labeling. The whole blood flow cytometric assay must be standardized in future clinical studies, especially regarding to preanalytical requirements.

Coexpression of tissue factor and tissue factor pathway inhibitor by human monocytes purified by leukapheresis and elutriation. Response of nonadherent cells to lipopolysaccharide.

BACKGROUND: Counterflow centrifugal elutriation is the method of choice for obtaining a large quantity of highly purified monocytes. In spite of the fact that this technique has been used for many years to isolate monocytes for cellular immunotherapy, it is not known whether the process of elutriation can stimulate tissue factor (TF) expression and therefore trigger coagulation in patients receiving these cell preparations. The aim of the present study is thus to identify TF and TF pathway inhibitor (TFPI) in elutriated monocytes and to evaluate their ability to trigger thrombin generation. STUDY DESIGN AND METHODS: Human monocytes are separated by leukapheresis and elutriation in sterile, endotoxin-free conditions. TF and TFPI mRNA is detected by reverse transcription-polymerase chain reaction. TF and TFPI are measured by enzyme-linked immunosorbent assay in cell lysates. TF antigen expression on cell surface is evidenced by direct-flow cytometry. Two functional tests (a chronometric test and an amidolytic assay) assess the capacity of monocytes to trigger thrombin generation. The response to lipopolysaccharide (LPS) is evaluated with each technique. Monocytic cell line THP-1 is used as a positive control. RESULTS: Elutriated monocytes coexpress TF mRNA and TFPI mRNA. The expression of TF mRNA is dramatically increased by LPS activation. This is correlated with a 100-fold increase in the amount of TF antigen in monocyte lysates. Flow immunocytometry confirms the expression of TF antigen on cell membrane in response to LPS stimulation, whereas TFPI mRNA is slightly increased after LPS stimulation. The amount of TFPI antigen in cell lysates is small when compared to that in plasma. Elutriated monocytes have a strong potential to trigger thrombin generation in response to LPS. CONCLUSION: In spite of the coexpression of TF mRNA and TFPI mRNA, elutriated monocytes are capable of supporting prothrombinase activity. This should be taken into account in the evaluation of the safety of adoptive cellular immunotherapy.

Inhibition of fMLP-triggered respiratory burst of human monocytes by adenosine: involvement of A3 adenosine receptor.

Adenosine (Ado) is a potent anti-inflammatory agent acting on a variety of cell functions. However, its effects on human monocytes have been less well characterized. We investigated the effect of Ado and its receptor-specific analogs on NADPH oxidase activity with the use of luminol-enhanced chemiluminescence (CL). Adenosine inhibited fMLP-triggered NADPH oxidase activity with a maximal inhibition of 55+/-5%. IB-MECA, a selective A3 Ado receptor agonist reduced fMLP triggered NADPH oxidase activity more potently than the A2 receptor agonist CGS 2180 HCl (CGS) and the A1 Ado receptor agonist N-2-phenylethyl-adenosine (R-PIA). The inhibitory effect of Ado was reversed by neither the A1 Ado receptor antagonist 1,3-dipropyl-8(2-amino-4chlorophenyl)-xanthine (PACPX) nor the A2 Ado receptor antagonist 3,7-dimethyl-1-(2-propynyl)xanthine (DMPX). It was significantly reversed by the A1/A3 Ado receptor antagonist xanthine amine congener (XAC). Pretreatment of monocytes by cytochalasin B reversed the effect of Ado but not of dibutyryl cAMP (dBcAMP) on fMLP-CL response. KT 5720, a specific cAMP-dependent protein kinase inhibitor completely counteracted the inhibition of NADPH oxidase activity by dBcAMP but not by Ado. Using flow cytometry, we observed that Ado did not inhibit intracellular oxidative metabolism, whereas dBcAMP did. Furthermore, the inhibition of NADPH oxidase activity by Ado was not mediated by changes in cytosolic calcium. These results demonstrated that Ado inhibited NADPH oxidase activity via A3 Ado receptor independently of cAMP elevation or changes in calcium mobilization.

Free and total platelet glycoprotein IIb/IIIa measurement in whole blood by quantitative flow cytometry during and after infusion of c7E3 Fab in patients undergoing PTCA.

A quantitative flow cytometry assay was used to evaluate the ex vivo kinetics of c7E3 Fab platelet effect in 16 patients undergoing PTCA treated with abciximab and compared with aggregometry assay. Immunolabeling of platelets was directly assessed on whole blood, using in parallel two monoclonal antibodies (Mabs) raised against GPIIIa, Mab1, the binding of which is inhibited by c7E3 Fab, and Mab2, the binding of which is not affected by c7E3 Fab. We found a severe and sustained inhibition of both GPIIb/IIIa receptors and platelet functions. The inter-individual variation in response to abciximab was low. A significant transient increase at H24 and H48 in the binding of Mab2 was found as an unexpected result, and confirmed in vitro. Results demonstrate that flow cytometry is a reliable method in agreement with aggregation. In addition, our results show that it is a standardized tool and a time-saving technique.

[Vascular disease and cellular activation: exploration of cell adhesion phenotype by quantitative flow cytometry]. / Pathologie vasculaire et activation cellulaire. Exploration du phénotype cellulaire adhérent par cytométrie en flux quantitative.

The pathogenesis of atheromatous and/or thrombotic vascular diseases involves rheological parameters, soluble mediators and cellular agents. The many studies that have tried to establish correlations between plasma factors, shear stress and the risk of ischemia have left some questions unanswered. Current exploration methods are now focusing on the determining role of cells. Activated cells express adhesion molecules on their membranes, which allow to communicate in a homo- or heterotypical manner. Quantifying adherence molecules on the surface of platelets, leukocytes and endothelial cells provides an assessment of the "adhesive phenotypical profile". Quantitative cytometry, using beads coated with a known amount of immunoglobulins as calibrators, is perfectly suited, through its multiple parameter analyses and the specificity provided by monoclonal antibodies, for the quantification of membrane antigens. Measuring the adhesive profile on the surface of cells that are implicated in vascular disease makes it possible to correlate that phenotype to the ischemic risk in such diversified pathologies or circumstances as intermittent angor, myocardial infarction, angioplasty, insulin-dependent diabetes or pre-eclampsia. In addition, that quantification permits monitoring the action of new therapeutical agents targeting adherence molecules.

Comparative inhibition of LPS-activated human monocyte-induced thrombin generation by unfractionated heparin and low molecular weight heparins.

Monocyte tissue factor may play a role in the physiological or pathological triggering of blood coagulation. It is well known that unfractionated heparin and low molecular weight heparins inhibit extrinsic thrombin generation. However, this notion has never been confirmed in a physiological model using tissue factor obtained from stimulated human monocytes. This is the purpose of this study. It was important to obtain a pure preparation of monocytes with no platelet contamination. This was possible by leukapheresis and elutriation. Under sterile and endotoxin-free conditions, the process does not activate tissue factor expression by monocytes. We adapted the technique of thrombin generation on an automatic analyzer and used human monocyte tissue factor to trigger thrombin generation. Our results show that unfractionated and low molecular weight heparins potently inhibit monocytic tissue factor induced thrombin generation. The comparison of low molecular weight heparins suggests that molecules with higher anti-IIa/anti-Xa ratios exert a stronger inhibitory effect. These data may be relevant to explain the therapeutic effects of unfractionated and low molecular weight heparins in cardiovascular disorders such as unstable angina.

Vitamin D differentially regulates B7.1 and B7.2 expression on human peripheral blood monocytes.

The hormonal active form of vitamin D3, 1,25-dihydroxyvitamin D3 (1, 25(OH)2D3), inhibits (through an unknown mechanism) the ability of monocytes/macrophages to induce T-cell activation. For T cells to be optimally activated, recognition of antigen/major histocompatibility complexes (MHC) by the T-cell receptor (TCR) must be accompanied by a second costimulatory signal. Considerable experimental data now suggest that this costimulatory signal is predominantly generated by B7.1 and/or B7.2 molecules, expressed on antigen-presenting cells (APC), when engaged to their counter-receptor, CD28, present on T cells. To determine whether the inhibitory effect of 1,25(OH)2D3 on monocytes/macrophages might involve modulation of the expression of B7.1 and B7.2 molecules, we analysed (by flow cytometry) the influence of 1,25(OH)2D3 and an analogue, KH 1060, on the expression of these two molecules at the surface of resting human peripheral blood monocytes. In parallel, we tested the effect of these two agents on human monocyte expression of cell-surface markers (CD14 and CD4) and antigen-presenting molecules (MHC class I and MHC class II). Our results showed that both 1,25(OH)2D3 and KH 1060 inhibited the basal expression of B7.2 in a dose- and time-dependent manner, without affecting B7.1. Moreover, these two compounds increased CD14 and reduced MHC class II and CD4 expression. Furthermore, the effect of 1,25(OH)2D3 on B7 molecule expression in combination with lipopolysaccharide (LPS) or cytokines, including interleukin-10 (IL-10), interferon-gamma (IFN-gamma) and tumour necrosis factor-alpha (TNF-alpha), was studied. The 1,25(OH)2D3-induced B7.2 down-regulation was still detectable when monocytes were activated by IL-10, IFN-gamma and TNF-alpha but not with LPS. Moreover, the induction of B7.1 by TNF-alpha was inhibited by addition of 1, 25(OH)2D3. We conclude that the ability of 1,25(OH)2D3 to decrease B7.2 expression on human monocytes might contribute to its inhibitory effect on APC-dependent T-cell activation and to its immunosuppressive properties observed in autoimmune diseases and organ transplantation.

[Modulators of leukocytic functions]. / Les modificateurs des fonctions leucocytaires.

Polymorphonuclear neutrophils (PMN) and monocytes play a role in vascular diseases. Animal experimental models, using deleukocytation or injection of anti-CD11b-anti-CD18 monoclonal antibodies (inhibition of leukocytic adhesion and of interaction with the endothelial cell) have confirmed this role in the ischemia-reperfusion syndrome and in myocardial infarction. In man, increased production of oxygen radicals, PMN release of elastase, increased monocyte formation of tissue factor (TF) and integrins have been noted in coronary artery disease, in chronic arteriopathy of the lower limbs and in association with vascular risk factors such as diabetes. Pharmacological alteration of leukocyte hyperactivity therefore seems to be justified. Pentoxifylline, used with good effect in arteriopathy of the lower limbs, affects numerous leukocytic functions: diminution in adherence and in PMN production of free radicals, diminution in the formation of TF and cytokines (TNF). Gingkolides reduce leukocytic interactions and platelet activation through an anti-PAF (Platelet Activation Factor) action. Aspirin may interfere with free radicals and inhibit TF formation. alpha-tocopherol blocks the activation, by free radicals, of the transcription factor NF k B. By altering the TNF and IL-1 cytokines, leukocytic activation can be controlled. Other cytokines (IL-4, IL-10) have an immunosuppressive action and reduce the formation of TF. The pharmacological targets are therefore multiple. Their use in vascular diseases is only at a very preliminary stage.