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Biomedical subjects

P Legrand

Publications and source records attributed to P Legrand.

At least 37 records · Page 2Linked to original sources
PubMed2001

Leukocyturia as a predictor of tolerance and efficacy of intravesical BCG maintenance therapy for superficial bladder cancer.

OBJECTIVES: To examine leukocyturia as a predictor of tumor recurrence and occurrence of adverse events after bacille Calmette-Guérin (BCG) immunotherapy. The use of BCG immunotherapy, a very major advance in the management of superficial bladder cancer, is limited by the frequency of adverse events. As yet, we have no way of predicting the efficacy and tolerability of BCG instillation in clinical practice. This problem is even more acute during BCG maintenance therapy. METHODS: Adverse events in 72 patients who received 518 instillations were prospectively assessed using a four-class scale based on severity and duration. Urinary leukocytes were counted 3 days after each instillation, using the KOVA-Slide 10 method. RESULTS: High leukocyturia during BCG treatment (cutoff value 1.65 x 10(5)/mL urine) correlated with recurrence-free status (P = 0.009). The degree of leukocyturia correlated with the severity/duration of adverse events (P <0.0001); the median leukocyturia values associated with class I, II, and III adverse events were 4 x 10(4)/mL, 1.5 x 10(5)/mL, and 3.5 x 10(5)/mL, respectively. No class IV events occurred. The cutoff point indicating treatment cessation for adverse events was leukocyturia of 8.6 x 10(4)/mL. CONCLUSIONS: These results suggest a link between adverse events and efficacy during BCG maintenance therapy. Leukocyturia appears to correlate with both efficacy and tolerability in this setting. Prospective randomized studies are required to evaluate leukocyturia as a basis on which to adapt the BCG instillation schedule to individual patient susceptibility.

Administration, Intravesical↗
PubMed2001

Relationship between complement activation, cellular uptake and surface physicochemical aspects of novel PEG-modified nanocapsules.

The aim of our work was to examine the relationship between modifications of the surface of nanocapsules (NC) by adsorption or covalent grafting of poly(ethylene oxide) (PEG), and changes in their phospholipid (PL) content on complement activation (C3 cleavage) and on uptake by macrophages. The physicochemical characterization of the NC included an investigation of their properties, such as surface charge, size, hydrophilicity, morphology and homogeneity. This is the first time that such properties have been correlated with biological interactions for NC, a novel carrier system with a structure more complex than nanospheres. C3 crossed immunoelectrophoresis revealed the reduced activation for NC with longer PEG chain and higher density, although all formulations induced C3 cleavage to a lesser or greater extent. NC bearing PEG covalently bound to the surface were weaker activators of complement than plain PLA [poly(D,L-lactide)] NC or nanospheres (NS). Furthermore, the fluorescent/confocal microscopy of J774A1 cells in contact with NC reveal a dramatically reduced interaction with PEG-bearing NC. However, the way in which PEG was attached (covalent or adsorbed) seemed to affect the mechanism of uptake. Taken together, these results suggest that the low level of protein binding to NC covered with a high density of 20kDa PEG chains is likely to be due to the steric barriers surrounding these particles, which prevents protein adsorption and reduces their interaction with macrophages.

Adsorption↗
PubMed2001

Biodistribution of long-circulating PEG-grafted nanocapsules in mice: effects of PEG chain length and density.

PURPOSE: To study the pharmacokinetics and biodistribution of novel polyethyleneglycol (PEG) surface-modified poly(rac-lactide) (PLA) nanocapsules (NCs) and to investigate the influence of PEG chain length and content. METHODS: The biodistribution and plasma clearance in mice of different NC formulations were studied with [3H]-PLA. PLA-PEG copolymers were used in NC preparations at different chain lengths (5 kDa and 20 kDa) and PEG contents (10% and 30% w/w of total polymer). In vitro and in vivo stability were also checked. RESULTS: Limited [3H]-PLA degradation was observed after incubation in mouse plasma for 1 h, probably because of to the large surface area and thin polymer wall. After injection into mice. NCs prepared with PLA-PEG copolymers showed an altered distribution compared to poloxamer-coated PLA NCs. An increased concentration in plasma was also observed for PLA-PEG NCs. even after 24 h. A dramatic difference in the pharmacokinetic parameters of PLA-PEG 45-20 30% NCs compared to poloxamer-coated NCs indicates that covalent attachment, longer PEG chain lengths, and higher densities are necessary to produce an increased half-life of NCs in vivo. CONCLUSIONS: Covalently attached PEG on the surface of NCs substantially can reduce their clearance from the blood compartment and alter their biodistribution.

Algorithms↗
PubMed2001

Crystallization and 2.2 A resolution structure of R-phycoerythrin from Gracilaria chilensis: a case of perfect hemihedral twinning.

R-phycoerythrin, a light-harvesting component from the red algae Gracilaria chilensis, was crystallized by vapour diffusion using ammonium sulfate as precipitant agent. Red crystals grew after one week at 293 K and diffracted to 2.70 A resolution. Three serial macroseeding assays were necessary to grow a second larger crystal to dimensions of 0.68 x 0.16 x 0.16 mm. This crystal diffracted to 2.24 A resolution using synchrotron radiation at beamline BM14 of the European Synchrotron Radiation Facility (ESRF) at Grenoble, France and was used for structure determination. Data were collected at 100 K to a completeness of 98.6%. The crystal was trigonal, space group R3, with unit-cell parameters a = b = 187.3, c = 59.1 A, alpha = beta = 90, gamma = 120 degrees. Data treatment using the CCP4 suite of programs indicated that the crystal was twinned ((I(2))/(I)(2) = 1.41). Molecular replacement was performed with AMoRe using the R-phycoerythrin from Polysiphonia urceolata [Chang et al. (1996), J. Mol. Biol. 249, 424-440] as a search model. In order to overcome the twinning problem, SHELX97 was used for the crystallographic refinement. The twin fraction was 0.48, indicating a nearly perfect hemihedrally twinned crystal. The final R(work) and R(free) factors are 0.16 and 0.25, respectively. All the residues and chromophores of the alpha- and beta-chains are well defined in the electron-density maps. Some residues belonging to the gamma-linker are also recognizable.

Amino Acid Sequence↗
PubMed2001

Reduction of no synthase expression and tumor necrosis factor alpha production in macrophages by amphotericin B lipid carriers.

The present study compared the abilities of different lipid carriers of amphotericin B (AMB) to activate murine peritoneal macrophages, as assessed by their capacities to produce nitric oxide (NO) and tumor necrosis factor alpha (TNF-alpha). Although AMB alone did not induce NO production, synergy was observed with gamma interferon but not with lipopolysaccharide. This synergy could not be explained by the mobilization of the nuclear activation factor NF-kappaB by AMB. On the other hand, AMB induced TNF-alpha production without a costimulator and no synergy was observed. Anti-TNF-alpha antibodies did not influence NO production, and an inhibitor of NO synthase did not affect TNF-alpha production, indicating that the production of one of these effector molecules was independent of that of the other. The incorporation of AMB into lipid carriers reduced NO and TNF-alpha production with all formulations but more so with liposomes than with lipid complexes. NO production was correlated with the induction of NO synthase II, revealed by Western blotting. The extent of association of AMB with macrophages depended on the formulation, especially on the AMB/lipids ratio: the higher the ratio was, the greater the AMB association with macrophages. However, there was no clear correlation between AMB association with macrophages, whether internalized or bound to the membrane, and immunostimulating effects. These results may explain the reduced toxicities of lipid-based formulations of AMB.

Adjuvants, Immunologic↗
PubMed2001

[Urinary leukocytes as a new prognostic marker of therapeutic response and of adverse effects associated with the maintenance treatment with endovesical BCG, for the prophylaxis of superficial bladder tumors].

UNLABELLED: The use of Bacillus Calmette-Guérin (BCG) vaccine as immunotherapy for superficial bladder cancer constitutes a major progress, although it remains limited by the development of adverse effects and problems related to safety. No individual tool is currently available in clinical practice to predict the efficacy or adverse effects of BCG therapy. These problems are accentuated in the case of maintenance therapy according to the protocol of D.L. Lamm. OBJECTIVES: To define whether the urinary leukocyte count observed after intravesical BCG instillation could be associated with adverse effects and efficacy of treatment. MATERIAL AND METHODS: A cohort of 72 consecutive patients (518 instillations) was studied prospectively. We defined four classes of adverse effects associated with BCG, according to their type, severity and duration. In combination with this classification, we performed systematic optical urinary leukocyte count on the third day after each instillation (KOVA-Slide 10). RESULTS: A high urinary leukocyte count (cut-off value: 165,000/ml) was correlated with absence of recurrence (p = 0.009). The adverse effect classification also demonstrated that urinary leukocyte count was related to the severity and duration of adverse effects (p < 0.0001). Median values observed for class I, II and III adverse effects were 40,000 leukocytes/ml, 150,000 leukocytes/ml and 350,000 leukocytes/ml, respectively. No class IV adverse effects were observed. The risk of developing class III adverse effects was increased when the urinary leukocyte count was greater than 86,000 leukocytes per ml. CONCLUSION: These results suggest a probable relationship between efficacy and safety of BCG, during maintenance therapy. Randomized prospective studies are necessary to evaluate urinary leukocyte count as a tool for adaptation and optimization of BCG therapy.

Adjuvants, Immunologic↗
PubMed2000

Self-assembly of the vascular endothelial cadherin ectodomain in a Ca2+-dependent hexameric structure.

Vascular endothelial cadherin (VE-cadherin) is a transmembrane protein essential for endothelial cell monolayer integrity (Gulino, D., Delachanal, E., Concord, E., Genoux, Y., Morand, B., Valiron, M. O., Sulpice, E., Scaife, R., Alemany, M., and Vernet, T. (1998) J. Biol. Chem. 273, 29786-29793). This molecule belongs to the cadherin family of cell-cell adhesion receptors, for which molecular details of homotypic interactions are still lacking. In this study, a recombinant fragment encompassing the four N-terminal modules of VE-cadherin (VE-EC1-4) was shown to associate, in solution, as a stable Ca(2+)-dependent oligomeric structure. Cross-linking experiments combined with mass spectrometry demonstrated that this oligomer is a hexamer. Gel filtration chromatography experiments and analytical ultracentrifugation analyses revealed the existence of an equilibrium between the hexameric and monomeric species of VE-EC1-4. The concentration at which 50% of VE-EC1-4 is in its hexameric form was estimated as 1 microm. The dimensions of the hexamer, measured by cryoelectron microscopy to be 233 +/- 10 x 77 +/- 7 A, are comparable to the thickness of adherens endothelial cell-cell junctions. Altogether, the results allow us to propose a novel homotypic interaction model for the class II VE-cadherin, in which six molecules of cadherin form a hexamer.

Animals↗
PubMed2000

Poly(D,L-lactide) nanocapsules prepared by a solvent displacement process: influence of the composition on physicochemical and structural properties.

Nanocapsules (NC) were prepared by interfacial deposition of preformed biodegradable polymer (PLA(50)) after a solvent displacement process. The influence of the composition used for the preparation of NC was evaluated in terms of particle size, polydispersity, zeta potential, homogeneity, and structural characteristics of the systems. The nature of the oil phase, polymer molecular weight, type and concentration of different surfactants were investigated to optimize the formulation to obtain NC suitable for intravenous administration. The influence of the physicochemical properties of the different oils used in NC preparation on the NC size was evaluated. The interfacial tension between the oil and water phases seems to have a greater effect on NC size than the oil viscosity. Miglyol 810 and ethyl oleate lead to the formation of smaller NC, probably because of the reduced interfacial tension. The polymer molecular weight plays only a small role in NC surface charge in the presence of lecithin, whereas NC surface charge, size, polydispersity, and short-term stability were highly influenced by lecithin purity. It appears that the absence of poloxamer 188 leads to smaller polydispersity, less contamination with nanospheres, and reduced formation of structures other than NC. Furthermore, electron microscopy and density gradient density techniques were used to examine the structure of the particles formed and their homogeneity. NC formation was evidenced by the bands with intermediate density between nanoemulsion and nanospheres; however, other bands of low intensity were observed. The presence of liposomes and multilayers in NC preparation was confirmed by electron microscopy. The percentage of carboxyfluorescein entrapped in different NC formulations allowed us to estimate the contamination by liposomes. It has been show that, under our experimental conditions, an excess of lecithin is an essential prerequisite for a stable preparation of PLA NC.

Capsules↗
PubMed2000

Myristic acid, unlike palmitic acid, is rapidly metabolized in cultured rat hepatocytes.

This study was designed to examine and compare the metabolism of myristic and palmitic acids in cultured rat hepatocytes. [1-(14)C]-Labeled fatty acids were solubilized with albumin at 0.1 mmol/L in culture medium. Incubation with 24-hr cultured hepatocytes was carried out for 12 hr. Myristic acid was more rapidly (P < 0.05) taken up by the cells than was palmitic acid (86.9 +/- 0.9% and 68.3 +/- 5.7%, respectively, of the initial radioactivity was cleared from the medium after 4 hr incubation). Incorporation into cellular lipids, however, was similar after the same time (33.4 +/- 2.8% and 34.9 +/- 9.3%, respectively, of initial radioactivity). In the early phase of the incubation (30 min), myristic acid was more rapidly incorporated into cellular triglycerides than was palmitic acid (7.4 +/- 0.9% and 3.6 +/- 1.9%, respectively, of initial radioactivity). However, after 12 hr incubation, the radioactivity of cellular triglycerides, cellular phospholipids, and secreted triglycerides was significantly higher with palmitic acid as precursor. Myristic acid oxidation was significantly higher than that of palmitic acid (14.9 +/- 2.2% and 2.3 +/- 0.6%, respectively, of the initial radioactivity was incorporated into the beta-oxidation products after 4 hr). Myristic acid was also more strongly elongated to radiolabeled palmitic acid (12.2 +/- 0.8% of initial radioactivity after 12 hr) than palmitic acid was to stearic acid (5.1 +/- 1.3% of initial radioactivity after 12 hr). The combination of elongation and beta-oxidation results in the rapid disappearance of C14:0 in hepatocytes whereas C16:0 is esterified to form glycerolipids. This study provides evidence that myristic acid is more rapidly metabolized in cultured hepatocytes than is palmitic acid.

Journal Article↗
PubMed2000

In vitro reversion of amphotericin B resistance in Leishmania donovani by poloxamer 188.

A micellar formulation of amphotericin B (AmB) solubilized with poloxamer 188 was evaluated against an AmB Leishmania donovani-resistant line. A concave isobologram showed a synergistic effect of this association against promastigotes. This result was confirmed with amastigotes since the 50% effective concentration of the new formulation was 100 times less than that of the control AmB formulation.

Amphotericin B↗
PubMed1999

Hormonal regulation of stearoyl coenzyme-A desaturase 1 activity and gene expression in primary cultures of chicken hepatocytes.

Previous studies have provided evidence for the important role of liver stearoyl-CoA desaturase (SCD) in excessive adiposity in the chicken and suggest that the difference in SCD activity between fat and lean chickens could be explained by a difference in SCD1 gene expression. In the present study, the regulation of SCD1 gene expression was analyzed as the result of insulin and glucagon action, using primary cultures of 6-week-old chicken hepatocytes. Insulin increased SCD1 activity and mRNA levels, whereas glucagon decreased dramatically both the enzyme activity and the mRNA levels. Nuclear run-on transcription assays and mRNA stability investigations demonstrated that insulin and glucagon effects on SCD1 gene expression was primarily transcriptional. Furthermore, the results indicated that the glucagon-mediated inhibition of SCD1 gene transcription was more potent than just counteracting the insulin-mediated effect. These data represent the first demonstration that the glucagon effect on the SCD1 gene expression is primarily transcriptional. Moreover, among hepatic genes involved in lipid metabolism in chicken, SCD1 is the first gene shown to be regulated at the transcriptional level by insulin, in the absence of triiodothyronine. These data point out the potency of the growing chicken hepatocyte culture model in contrast with the embryonic cell culture model as regards the investigations of the insulin effect on gene expression.

Animals↗
PubMed1999

Crystal structure of the oxidised and reduced acidic cytochrome c3from Desulfovibrio africanus.

Unique among sulphate-reducing bacteria, Desulfovibrio africanus has two periplasmic tetraheme cytochromes c3, one with an acidic isoelectric point which exhibits an unusually low reactivity towards hydrogenase, and another with a basic isoelectric point which shows the usual cytochrome c3reactivity. The crystal structure of the oxidised acidic cytochrome c3of Desulfovibrio africanus (Dva.a) was solved by the multiple anomalous diffraction (MAD) method and refined to 1.6 A resolution. Its structure clearly belongs to the same family as the other known cytochromes c3, but with weak parentage with those of the Desulfovibrio genus and slightly closer to the cytochromes c3of Desulfomicrobium norvegicum. In Dva.a, one edge of heme I is completely exposed to the solvent and surrounded by a negatively charged protein surface. Heme I thus seems to play an important role in electron exchange, in addition to heme III or heme IV which are the electron exchange ports in the other cytochromes c3. The function of Dva.a and the nature of its redox partners in the cell are thus very likely different. By alignment of the seven known 3D structures including Dva.a, it is shown that the structure which is most conserved in all cytochromes c3is the four-heme cluster itself. There is no conserved continuous protein structure which could explain the remarkable invariance of the four-heme cluster. On the contrary, the proximity of the heme edges is such that they interact directly by hydrophobic and van der Waals contacts. This direct interaction, which always involves a pyrrole CA-CB side-chain and its bound protein cysteine Sgammaatom, is probably the main origin of the four-heme cluster stability. The same kind of interaction is found in the chaining of the hemes in other multihemic redox proteins.The crystal structure of reduced Dva. a was solved at 1.9 A resolution. The comparison of the oxidised and reduced structures reveals changes in the positions of water molecules and polar residues which probably result from changes in the protonation state of amino acids and heme propionates. Water molecules are found closer to the hemes and to the iron atoms in the reduced than in the oxidised state. A global movement of a chain fragment in the vicinity of hemes III and IV is observed which result very likely from the electrostatic reorganization of the polypeptide chain induced by reduction.

Amino Acid Sequence↗
PubMed1999

Desulfovibrio desulfuricans iron hydrogenase: the structure shows unusual coordination to an active site Fe binuclear center.

BACKGROUND: Many microorganisms have the ability to either oxidize molecular hydrogen to generate reducing power or to produce hydrogen in order to remove low-potential electrons. These reactions are catalyzed by two unrelated enzymes: the Ni-Fe hydrogenases and the Fe-only hydrogenases. RESULTS: We report here the structure of the heterodimeric Fe-only hydrogenase from Desulfovibrio desulfuricans - the first for this class of enzymes. With the exception of a ferredoxin-like domain, the structure represents a novel protein fold. The so-called H cluster of the enzyme is composed of a typical [4Fe-4S] cubane bridged to a binuclear active site Fe center containing putative CO and CN ligands and one bridging 1, 3-propanedithiol molecule. The conformation of the subunits can be explained by the evolutionary changes that have transformed monomeric cytoplasmic enzymes into dimeric periplasmic enzymes. Plausible electron- and proton-transfer pathways and a putative channel for the access of hydrogen to the active site have been identified. CONCLUSIONS: The unrelated active sites of Ni-Fe and Fe-only hydrogenases have several common features: coordination of diatomic ligands to an Fe ion; a vacant coordination site on one of the metal ions representing a possible substrate-binding site; a thiolate-bridged binuclear center; and plausible proton- and electron-transfer pathways and substrate channels. The diatomic coordination to Fe ions makes them low spin and favors low redox states, which may be required for catalysis. Complex electron paramagnetic resonance signals typical of Fe-only hydrogenases arise from magnetic interactions between the [4Fe-4S] cluster and the active site binuclear center. The paucity of protein ligands to this center suggests that it was imported from the inorganic world as an already functional unit.

Amino Acid Sequence↗
PubMed1999

Knee cartilage topography, thickness, and contact areas from MRI: in-vitro calibration and in-vivo measurements.

OBJECTIVE: This study assessed the three-dimensional accuracy of magnetic resonance imaging (MRI) for measuring articular surface topographies and cartilage thicknesses of human cadaveric knee joints, by comparison with the calibrated stereophotogrammetric (SPG) method. METHODS: Six fresh frozen cadaveric knees and the knees of four volunteers were imaged with a three-dimensional spoiled gradient-recalled acquisition with fat suppression using a linear extremity coil in a 1.5 T superconducting magnet. The imaging voxel size was 0.47 x 0.47 x 1.0 mm. Both a manual and a semi-automated segmentation method were employed to extract topographic measurements from MRI. Following MRI, each of the six cadaveric knees was dissected and its articular surfaces quantified using stereophotogrammetry. The MRI surface measurements were compared numerically with the SPG measurements. RESULTS: For six cadaveric knees, the average accuracies of cartilage and subchondral bone surface measurements were found to be 0.22 mm and 0.14 mm respectively and the thickness measurements demonstrated an average accuracy of 0.31 mm. It was found that while most of the error may be attributed to random measurement error, the accuracy was somewhat affected by systematic errors. For each bone of the knee, accuracies were most favorable in the patella, followed by the femur and then the tibia. The more efficient semi-automated method provided equally good and sometimes better accuracies than manual segmentation. CONCLUSIONS: This study demonstrates that clinical MRI can provide accurate measurements of cartilage topography, thickness, contact areas and surface curvatures of the knee.

Adult↗
PubMed1999

Interactions between a macrophage cell line (J774A1) and surface-modified poly (D,L-lactide) nanocapsules bearing poly(ethylene glycol).

The interactions of naked and surface-modified poly(D,L-lactic acid) (PLA) nanocapsules (NC), where polyethyleneglycol (PEG) was adsorbed or covalently attached, have been studied with a macrophage-like cell line. The fluorescent oil marker, DiD, was successfully encapsulated in NCs in order to follow their interactions with cells. The cell-associated fluorescence obtained with PEG-PLA NC was about 3- to 13-fold lower than that obtained with naked-PLA NC. The effects of PEG chain length, its content as a percentage of total polymer and NC concentration in the culture medium were evaluated. PEG-PLA NC showed dramatically reduced fluorescence association with cells during an 18 h incubation compared with naked-PLA NC, showing that covalent attachment of PEG is important for the persistence of low uptake. The best results in reducing cell-associated fluorescence were obtained with a surface-modified PEG-PLA NC bearing a chain with 20000 MW. Increasing the percentage of PEG produced a reduction in marker association for a given PEG chain length. Moreover, when the PEG-containing poloxamer was simply adsorbed, marker association was dependent on the extent of dilution and the type of serum in the culture medium. Serum proteins, especially immunoglobulins, increased cell-associated fluorescence for PEG-adsorbed NC, but had very little effect on PEG-PLA NC. Marker association was only partially inhibited in the presence of cytochalasin B. The mechanisms of cell-NC interaction depended on the characteristics of the NC surface in each formulation. When the NC was physically separated from cells no diffusion of fluorescent marker in aqueous medium occurred. Nevertheless, collision-mediated transfer of DiD from NC to J774 cells was a non-negligible route of marker transfer, mainly for naked NC. However, this collision-mediated transfer was reduced for the PEG-PLA NC probably due to the restricted contact between NC and cells afforded by PEG steric hindrance at the surface.

Animals↗