[Accident And Burns Of The Duke-King Stanislav, His Treatment And His Death. LunÉville, 5TH-23RD February 1766]. / L'Accident Et Les Brûlures Du Duc-Roi Stanislas, Le Traitment Médicamenteux Appliqué à Celles-Ci, Et La Mort Du Monarque. Lunéville, 5-23 Février 1766.

Early in the morning of Wednesday 5th February 1766, Stanislaw Leszcziynski (Leczinski), Duke of Lorraine and Bar, fell to the ground, very close to the fireplace in his room. He remained in this position for a long time, exposed to the flames, and suffered serious burns. During the first nine days, physicians used topical agents and dressings, associated with internal drugs, particularly quinine. But septicemia occurred after about two weeks. By February 20th, Stanislaw was in a very poor condition. Some stimulating drugs were administered, but he died three days later. He was 88 years old.

Review: behaviour of endothelial cells faced with hypoxia.

Hypoxia is a diminution of oxygen quantity delivered to tissue for cellular need to product energy. Hypoxia derives from two major conditions in health diseases: anemia and ischemia. Anemic hypoxia comes from damage to O(2) transport like red blood cells diminution or disease. Ischemic hypoxia is a diminution of blood flow following a diminution of blood volume after a hemorrhagic shock. After hypoxia, vessels dilate to increase blood flow allowing a better oxygenation of peripheral tissues. This vasodilation appears immediately after the beginning of hypoxia and can be maintained during several hours. Today, the molecular mechanisms of this vasodilation stay unclear. But it seems that potassic channels, ATP concentration and medium acidification in addition to vasodilator/vasoconstrictor balance play a great role to facilitate the oxygenation of the ischemic areas.As endothelial cells (EC) are lining the vasculature, they are always in contact with blood, which carries, amongst other compounds, oxygen. In this way, they are the first target for an oxygen partial pressure (PO(2)) diminution. EC, through different mechanosensors, can sense a variation in PO(2) and adapt their metabolism to maintain ATP production. Under hypoxia, EC switch into hypoxic metabolism, leading to the production of reactive oxygen species (ROS). Indeed, when PO(2) is low, the respiratory chain in the mitochondria runs slower. Furthermore, cytochrome C capacity to trap O(2) is reduced; this phenomenon alters the cellular redox potential and leads to the accumulation of electrons that induce the formation of ROS.This review presents an overview of the behaviour of endothelial cells face to hypoxia. We propose to focus on nitric oxide, hypoxia inducible factor (HIF), lactate and ROS productions. Then we present the different mode of culture of EC under hypoxia. Finally, we conclude on the difficulty to study hypoxia because of the various types of system developed to reproduce this phenomenon and the different signalling ways that can be activated.

[Hemoglobin-based oxygen carriers and trials to substitute red blood cells]. / Les transporteurs d'oxygène à base d'hémoglobine et les tentatives de substituer les globules rouges.

The idea to develop a blood substitute was stimulated by the need of military in the last two world wars and by transmission of pathogenic germs (Hepatitis B in 1960, HIV in 1980 and Hepatitis C in 1990) during blood transfusion that limited the donor blood transfusion. There are two main groups of blood substitutes: perfluorocarbon emulsions and hemoglobin-based oxygen carriers (HBOC). These latter are of natural origin: human, bovine or recombinant and undergo three modifications types: chemicals (intramolecular cross-linking, polymerisation, conjugation to macromolecules and combination of several chemical modifications), genetics or technological by microencapsulation. HBOCs are in different phases of clinical trials and some of them present side effects (hemodynamic and oxidative). The understanding of these effects and the possibility of correcting them, condition their use on a large scale and the economic consequences, which they can generate.

[Thoughts and proposals for quality assurance in orthopedics in the community pharmacy]. / Réflexions en vue de la mise en place de l'assurance qualité dans le domaine de l'orthopédie à l'officine.

This review begins with an examination of the notion of quality in the industrial setting, its definition and the steps taken to achieve quality assurance. The review then considers what is meant by quality in the field of health, and in particular for orthopedic devices dispensed in a community pharmacy. Special attention is given to dispensing standard orthopedic devices; five aspects are considered: organization, personal competence, products, services, and relationship with the patient. The first aspect, organization, involves the selling area, stocks, different sizes and sale prices, and the introduction of new devices. Dispensing involves, reception and physical examination of the patient, information delivery, decision-making on the choice of an orthesis, measurement, trial and application, as well as complementary advice and completion of the patient's file. Quality is measured in terms of patient needs.

[Potential mechanism of dextran-conjugated hemoglobin penetration inside arterial wall]. / Mécanisme potentiel de la pénétration de l'hémoglobine conjuguée au dextrane dans la paroi artérielle.

Several reports indicated that cell-free hemoglobin induced vasoconstriction. This phenomenon was due to different pharmacological (NO trapping, vasoactive agents release and endothelial uptake...) and physical (viscosity and oxidative process of cell-free hemoglobin...) factors. We have previously showed that the blood pressure increase would be due to the presence of Dex-BTC-Hb inside arterial wall. However, we do not know how hemoglobin penetrate inside arterial wall. The objective of this study was to examine the new hypothesis of hemoglobin penetration inside arterial wall dependent of endocytosis. For this reason, an endocytosis inhibitor, cytochalasin D, was tested. We measured in anesthetized guinea pigs, the evolution of mean arterial pressure (MAP) and plasma hemoglobin concentration in presence or absence of cytochalasin D (1.6 x 10(-4) M). These measurements were carried out before and after 50% isovolemic exchange transfusion (IET) with two cell-free hemoglobins: Dex-BTC-Hb (300 kDa) and stroma-free hemoglobin (64.5 kDa). The administration of Dex-BTC-Hb or stroma-free hemoglobin induced an immediate increase in MAP that peaked within 17 min after IET and returned to baseline after 120 min. cytochalasin D attenuated the elevation of MAP when administrated before Dex-BTC-Hb but not when administrated before stroma-free hemoglobin. Furthermore, without cytochalasin D, plasma hemoglobin concentration after Dex-BTC-Hb or stroma-free hemoglobin administration decreased significantly 120 min after IET. In presence of cytochalasin D, plasma hemoglobin concentration stayed constant in Dex-BTC-Hb-treated animals but not in stroma-free hemoglobin-treated animals. cytochalasin D inhibits the endocytosis in case of Dex-BTC-Hb but not in case of stroma-free hemoglobin. This would be due to the molecular weight of cell-free hemoglobin. Based on these data, we suggest that endocytosis is one of the mechanisms by which cell-free hemoglobin with high molecular weight penetrated inside vascular endothelial cells. This endocytosis would have an impact on induced hypertension.

Modification of human hemoglobin by covalent association with soluble dextran.

Stroma-free Hb solutions present some drawbacks when used as erythrocyte substitutes, mainly because the protein has a short in vivo half-life, due to its small hydrodynamic volume. Covalent coupling of oxyHb with dialdehyde-dextran (Mw congruent to 40 000; Mn congruent to 25 000) leads to adducts whose properties depend upon the pH of the condensations. At pH less than 9.6, many labile imine linkages are formed and the conjugates have a high molecular weight at the end of the reaction. In contrast, the final products obtained as pH increases from 9.6 to 10 contain a low-molecular-weight adduct in an increasing ratio; in this case the bond between dextran and Hb is stable and this stability is assumed to result from the rearrangement of a specific imine linkage formed at an NH2 site of Hb, into a ketoamine group (Amadori rearrangement). Dextran-Hb conjugates have oxygen-binding properties characterized by increased oxygen affinity, and decreased subunit cooperativity and alkaline Bohr effect, relative to unconjugated Hb. These differences become less as the time of condensation reaction decreases and seem to be due to modification of amine groups involved in the salt bridges that stabilize the deoxy form of the protein. Taking into account their oxygen-binding characteristics, the low-molecular-weight conjugates can be regarded as potential erythrocyte substitutes.

[Relationship between arterial pressure evolution and free hemoglobin distribution into vascular wall in guinea pigs]. / Relation entre l'évolution de la pression artérielle et la distribution de l'hémoglobine libre dans la paroi vasculaire chez le cobaye.

Free hemoglobin (Hb) present at high concentration in plasma--in case of hemolysis, hemoglobin based oxygen carrier (HBOC) administration or in case of subarachnoid hemorrhage--induce hypertension as result of vasoconstriction. In this context, we studied on an exchange transfusion (ET) model at 50% of hematocrit with a HBOC, the distribution of this Hb inside abdominal aortic wall in guinea pigs in relation with mean arterial pressure (MAP) evolution. MAP was monitored during 180 min after ET and rings of abdominal aorta were taken at different times, when modifications of MAP were important, and analyzed by immunohistochemistry and confocal microscopy. Gelofusine 4%, used as control, did not modify MAP while free Hb increased MAP that reached its maximum (53% of hypertension) at t=17 min after the end of ET. MAP started to decrease (45% of hypertension) at t =60 min after ET, and recovered its baseline value at t=180 min. Confocal analysis of the vessel showed that: at 17 min (when hypertension was maximal), free Hb was present in endothelial cells (EC) and in vasa vasorum; at t=60 min (when hypertension decreased) and at t =10 min (when hypertension disappeared), free Hb was detected still in EC, but inside all abdominal aorta wall too. These results suggest in the first time that free Hb could induce a hypertension by direct interaction with EC but would also be unable to maintain this hypertension in spite of its massive tissue distribution.

[Prescription practices: an audit of prescription quality among Lorrain general practitioners]. / Qualité rédactionnelle des ordonnances. Résultats d'une enquête menée auprès des médecins généralistes lorrains.

Better medical practice begins with personal evaluation. A group of Lorraine medical practitioners too part in an evaluation of their prescription practices and their compliance with existing regulations. The prescription servers as a link between the physician, the pharmacists and the patient. Three broad topics were studies in this audit: patient identification, regulation per se, and general criteria such as legibility and computerization. Among the different mandatory inscriptions, the only item which was often missing was the patient's age. The results listed here present the average performance observed in a group of medical practitioners and the highest and lowest performances.

Synthetic carriers of oxygen.

During the last decade, construction of artificial carriers of oxygen for transfusion purposes has evolved in three main directions, which can be reviewed as follows. The first approach consists of modifying hemoglobin (Hb), the natural oxygen carrier, in order to lower its oxygen affinity and increase its intravascular persistence. To achieve this aim, two basic procedures have been used: molecular and environmental modification. In the first case, Hb is modified with chemical reagents; the second requires encapsulation of Hb to obtain artificial erythrocytes. The second approach is based on the use of synthetic oxygen-carrying chelates that mimic the oxygenation function of Hb. The main products in this class are metalloporphyrins, whose chemical environment is designed to render them efficient as reversible carriers of oxygen in vivo. Finally, the third approach deals with the perfluorochemicals used in emulsified form. Perfluorochemical liquids are excellent gas solvents, but some problems remain unsolved with regard to their development as oxygen carriers in vivo: low O2 dissolving capacity, toxicity, and excretion.

Involvement of neutrophilic granulocytes in the uptake of biodegradable non-stealth and stealth nanoparticles in guinea pig.

The in vivo behavior of monomethoxypoly(ethylene oxide)-poly(lactic acid) (MPEO20-PLA45/PLA (75/25)) nanoparticles in comparison with PLA ones was studied in guinea pig. Indeed, the aim of this study was to bring to the fore the in vivo stealth character of these copolymer nanoparticles and to identify the phagocytic circulating cells involved in their uptake. After the intravascular administration of fluorescent nanoparticles (rubrene), their phagocytosis by granulocytes and monocytes was assayed by flow cytometry. At the same time, the evolution of the number of these phagocytic cells was realized in order to identify their function in the nanoparticle uptake. Finally, a histological study of the spleen (30 h after the nanoparticle administration) was investigated to highlight the splenic trapping of these stealth nanoparticles. This study has shown that the phagocytic circulating cells involved in the nanoparticle uptake were mainly neutrophilic granulocytes and some of them were found in the spleen.

Cardiovascular and hemorheological effects of three modified human hemoglobin solutions in hemodiluted rabbits.

The cardiovascular effects of human albumin (Alb) and three human hemoglobin (Hb) solutions, dextran-benzene-tetracarboxylate Hb, alphaalpha-crosslinked Hb, and o-raffinose-polymerized Hb were compared in anesthetized rabbits undergoing acute isovolemic hemodilution with Hct reduction from 41.4 +/- 2.7 to 28.8 +/- 1.6%. The impact of the vasoconstricting properties of Hb was examined by measuring heart rate (HR), mean arterial pressure (MAP), abdominal aortic, and femoral arterial blood flow, vascular resistance (VR), and aortic distension during the first 3 h after hemodilution. The impact of the hemorheological parameters was assessed by measurements of hemodiluted blood viscosity. In contrast to Alb, the Hb solutions elicited an immediate increase in MAP (20-38%). The effects of Alb and Hb solutions on HR, as well as on aortic and femoral arterial blood flow, were similar. VR decreased with Alb (20-28%) and increased with all three Hb solutions (30-90%), but the MAP and VR rising trends were different with each Hb solution. Aortic distension decreased in Hb groups compared with the Alb group for the first 60 min. The viscosity of hemodiluted blood was similar for all groups at high shear rates but was dependent on the viscosity of the solutions at low shear rates. We conclude that the vasoconstriction elicited by the Hb solutions overrides the vasodilation associated with viscosity changes due to hemodilution and would be the major factor responsible to the cardiovascular changes.

Influence of experimental parameters on the characteristics of poly(lactic acid) nanoparticles prepared by a double emulsion method.

Nanoparticles were prepared by the double emulsion method (w/o/w), using methylene chloride as an organic solvent and polyvinyl alcohol (PVA) or human serum albumin (HSA) as a surfactant. Experimental parameters such as the preparation temperature, the solvent evaporation methods, the internal aqueous phase volume, the surfactant concentration and the polymer molecular weight were investigated for particle size, the zeta potential, the residual surfactant percentage and the polydispersity index. Preparation parameters leading to particles with well-defined characteristics such as an average size around 200 nm and a polydispersity index lower than 0.1 were identified. The conditions were optimized to ensure protein encapsulation: a cool temperature, a short processing time, a sufficient internal aqueous phase and careful washing. It appeared that the higher the surfactant concentration in the external aqueous phase was, the smaller the particles, the lower the polydispersity index and the higher the residual amount of surfactant were. For PVA or HSA, the agreement between the convenient surfactant concentration and its critical aggregation concentration could be emphasized. Otherwise, an increased polymer molecular weight led both to a slightly decreased particle size and to a lower polydispersity index. Moreover, multilayer absorption of PVA which does not depend on Poly(lactic-acid) molecular weight was exhibited. Finally, the zeta potential resulted from the polymer molecular weight and the residual PVA.

Freeze-drying of oxyhemoglobin: protection against oxidation in the presence of EDTA salts, sulfonic acid buffers, and pantothenic acid derivatives.

Hemoglobin was freeze-dried in the presence of salts of EDTA, sulfonic acids used as buffers, or derivatives of pantothenic acid. At 0.25 M most of the compounds effectively inhibited the formation of methemoglobin. The various model compounds used (sodium zinc EDTA, tris(hydroxymethyl) methylaminopropanesulfonic acid, and DL-pantothenol) produced similar decreases in methemoglobin formation as a function of the concentration of protective agent between 0.01 and 0.20 M. Experiments on the storage of the freeze-dried materials revealed substantial denaturation of oxyhemoglobin after 12 months at 4 degrees C. On the whole, these compounds were less effective than amino acid salts, during both desiccation and storage. The multiplicity of compounds that inhibit the denaturation of hemoglobin during freeze-drying indicates that their mode of action is nonspecific.

Protective effect of sucrose on spray drying of oxyhemoglobin.

As far as we know, spray drying has previously not been applied to oxyhemoglobin, undoubtedly because of the sensitivity of oxyhemoglobin to temperature and oxidation. Our experience with freeze drying encouraged us to perform spray-drying trials in order to compare the results of the two methods, in the absence and the presence of protective compounds. Spray drying of hemoglobin without a protective compound led, as in freeze drying, to formation of a percentage of methemoglobin (50%) that makes it unsuitable for transporting oxygen. In the presence of 0.25 M sucrose (optimum) and at 80-100 degrees C, the functional properties of the hemoglobin were well preserved (methemoglobin approximately 4%), and the residual humidity was limited to approximately 3%. Structural investigation by optical circular dichroism confirmed the results obtained by freeze drying: in the presence of an effective protector, the spectra were similar to those of control hemoglobin and the immediate environment of the heme did not undergo any major change. Electron spin resonance absorption bands in all samples were similar for each value of the spectral decomposition factor, g. This suggests that the structure of the heme is not altered by desiccation and that the protector does not penetrate into the heme pocket since it would have disturbed the symmetry of the crystalline field. Fundamentally, these results are equivalent or similar to those observed with freeze drying; since spray drying is a different process of dehydration, the results indicate a lack of specificity in the phenomena of oxidation or of protection affecting hemoglobin.

Stability and functional properties of haemoglobin freeze-dried in the presence of four protective substances after prolonged storage: dose-effect relationships.

Freeze-dried haemoglobin samples protected during the desiccation by sucrose, arginine aspartate, lysine aspartate, sodium-zinc EDTA and Ficoll 70 have been stored under air at 4 degrees C for 15 months. The analysis showed that sufficient concentrations of sucrose and of the amino-acid salts prevent the oxidation of haemoglobin and maintain its functional properties. Relationships between the concentrations of these compounds and the methaemoglobin levels before and after storage were calculated. They define theoretical concentration points where methaemoglobin would not be found after storage. Sucrose is slightly more effective than the amino-acid, but oppositely, EDTA and Ficoll 70 do not allow prolonged storage of freeze-dried haemoglobin.

Problems of haemoglobin freeze-drying: evidence that water removal is the key to iron oxidation.

Formation of methaemoglobin during freeze-drying of oxyhaemoglobin raises the question of the cause and mechanism of the oxidation. Haemoglobin with or without lyoprotector (250 mM glucose or amino acid salt) has been subjected to freeze drying changes in either or both of two constraints--vacuum and rise in temperature. A rise in temperature from -40 to +10 degrees C had no substantial denaturing effect on haemoglobin whether protected or not. Maintenance of a vacuum over frozen haemoglobin for 18 h often produced subtotal desiccation. Unprotected haemoglobin was partially oxidized (39% MetHb) whereas protected haemoglobin was not (less than 4% MetHb). Haemoglobin was also dried by rapid dehydration of thin films in a stream of air at room temperature (20 degrees C). The methaemoglobin content was then 43% whereas the amino acid salt or glucose limited it at 4 and 7%, respectively. Haemoglobin is oxidized, therefore, only because of the removal of water. Protectors, not specific in structure and action, probably work by holding or reinforcing the critical number of hydration layers around haemoglobin.

Total exchange transfusion in rats with hemoglobin solutions: influence of hemoglobin concentration.

In most published studies of the oxygen transport capacity of hemoglobin solutions for total or partial replacement transfusions, the hemoglobin concentrations have been in the order of 70 g/l. In this study we tried to identify the hemoglobin concentration that would give the best survival in rats at zero hematocrit. The longest survival time, of more than 4 h, was obtained with a hemoglobin concentration of 125 g/l, despite an oncotic pressure much higher than that of plasma. This observation suggests the use of more highly concentrated solutions of modified or unmodified hemoglobin than are presently recommended, in order to increase their oxygen transport capacity.

Importance of purification on transfusional efficacy of hemoglobin solutions.

It has only been realized quite recently how important is the purification of hemoglobin solution for use in transfusion and several techniques have been published. We used ion exchange chromatography with which the main "contaminants" (glycoproteins, enzymes, phospholipids) are absorbed by the gel, whereas hemoglobin is not retained. The solutions studied here are non-modified hemoglobin and its homologue pyridoxylated hemoglobin (PLP-Hb). Physico-chemical analyses, usually undertaken to characterize hemoglobin solutions, show no difference before and after purification, except that the enzymatic activity almost disappears. In order to appreciate the benefits of purification, total exchange transfusions were carried out on rats. Without reperfusion, purification of the hemoglobin solution allowed a significantly longer survival time which was even more significant with PLP-Hb solution. Urinary loss did not seem to be affected by purification. With reperfusion in order to compensate these renal losses, PLP-Hb solutions gave survival times up to three days. However, the inevitable death of the animals poses the problem of instability of these purified solutions following enzyme loss.

Physico-chemical and pharmacological comparison of pyridoxylated hemoglobin bound to polyoxyethylene or polymerized by glutaraldehyde.

Two modified hemoglobin solutions were assessed using the same physico-chemical and pharmacological techniques. The first was prepared by covalent binding of monomethoxypolyoxyethylene (MPOE) 1.9 kDa to pyridoxylated hemoglobin (PLP-Hb). The resulting conjugate had a molecular size of 100 kDa (MPOE-PLP-Hb). The solution was cleared of non-fixed MPOE through ion exchange chromatography on Spherodex, thus bringing viscosity and oncotic pressure back to physiological values. The second was prepared by limited polymerization of pyridoxylated hemoglobin with glutaraldehyde (POLY-PLP-Hb). Tangential flow ultrafiltration achieved a satisfactory polymer/oligomer return. Quality controls showed no difference between the solutions. Total isovolemic exsanguinotransfusions in the rat did not help differentiate the two solutions. Hemorrhagic shock (80% of blood volume, rat) gave definitive survival for 8 of the 14 animals tested with MPOE-PLP-Hb (57%) but only 3 of the 8 animals tested with POLY-PLP-Hb (38%). None of the chemical approaches to reduce hemoglobin loss proved any more efficient than another, with the evaluation techniques employed.

Hemoglobin solutions coupled with polyethylene glycol 1900: preparation, purification, quality control and pharmacological trials by hemorrhagic shock.

The usefulness of hemoglobin solutions as oxygen transporters is limited by their high affinity for oxygen and rapid elimination from the circulation. Various chemical modifications of hemoglobin aimed at overcoming these two handicaps have been suggested. We have developed a conjugate of pyridoxylated human hemoglobin with monomethoxypolyoxyethtylene 1900, whose preparation and properties are described. We present comparative results on short-term or definitive survival of Wistar rats which, during hemorrhagic shock due to the loss of 60 or 80% of their blood mass, were given a solution of native or modified hemoglobin, in some cases purified by ion-exchange chromatography to remove non-heme proteins, lipids, and some endotoxins. The more complex the treatment used to improve the properties and the purity of the hemoglobin solutions, the longer the animals survived. The loss of hemoglobin in the urine was greatly reduced after conjugation: after 20 h, less than 6% of the total infused.