Biomedical subjects
Publications and source records attributed to C Vigneron.
A study by an agarose technique of the chemotactic power of human alpha-thrombin on polymorphonuclear leukocytes reveals that this enzyme has a chemotactic activity. Hirudin, which inhibits the coagulant properties of thrombin, suppresses these chemotactic properties. This effect could be explained by the binding of hirudin to the structural domain of alpha-thrombin involved in its chemotactic activity.
Adequate storage of polymorphonuclear leukocytes would allow an easier in vitro study of their structure and their functions, an easier study of polymorphonuclear leukocyte diseases (e.g. chronic granulomatous disease) and an easier use of polymorphonuclear leukocytes as a clinical tool (e.g. for localizing infections). Unfortunately, polymorphonuclear leukocytes are nearly impossible to preserve, even in short-term storage. This study proposes a model for the study of polymorphonuclear leukocyte storage in a synthetic medium: Plasmion. For storage over a period of 24 h, we found that supplementation with each of the following additives: bicarbonate buffer, glucose, adenosine triphosphate (ATP), ascorbic acid, nicotinamide adenine dinucleotide (NADH), nicotinamide adenine dinucleotide phosphate (NADPH), alpha-tocopherol acetate, amikacin and ampicillin, significantly improves (p less than 0.05) one or several functions of the polymorphonuclear leukocytes. When samples were stored for 48 h, we found that the addition of bicarbonate buffer after 24 h significantly improves the maintenance of several functions of polymorphonuclear leukocytes, in particular chemotaxis. Preservation for 96 h was achieved by making additions of supplements on each day of storage, with a chemotaxis maintenance of 83% at 24 h, 59% at 48 h, 46% at 24 h and 20% at 96 h. In conclusion, by using the Plasmion medium, and adding the above-mentioned compounds on each day of storage, chemotaxis can be satisfactorily maintained over 4 days.
Histocompatibility assessment of 70 g/l stroma-free hemoglobin solutions pyridoxylated or not, and purified or not, was carried out using vascular perfusion of the intestine of rats. Mechanical and ultrastructural changes in the organ and the arteriovenous difference of PO2 were compared to those obtained with albumin, gelatin and dextran. Overlapping epithelium conserves its structure in the presence of hemoglobin, whereas it is partially or totally destroyed with the plasma substitutes. Nevertheless, with non pyridoxylated hemoglobin there is a strong detachment of the epithelium from the lamina propria. The intestine, irrigated by the hemoglobin solutions shows efficient peristalsis, but this totally disappears with plasma substitutes. For similar arterial PO2, for all the solutions, the arteriovenous difference was of 100 mmHg for the hemoglobin solutions, whereas they never exceeded 60 mmHg for the plasma substitutes. Hemoglobin pyridoxylation led to an arteriovenous difference significantly superior (p less than 0.001) to those obtained using non modified hemoglobin. With their O2 supply hemoglobin solutions appear able to limit the development of hypoxia in the tissue. The continuation of peristalsis and the weak ultrastructural modifications confirm the slight histological improvement gained when using pyridoxylated hemoglobin. Nevertheless an extravasation appeared constantly, as well as flow reduction during perfusion with the hemoglobin solutions.
To make stroma-free hemoglobin (SFHb) capable of carrying oxygen satisfactorily in vivo it is necessary both to improve its intravascular persistence and to reduce its affinity for oxygen. The method used up to now has consisted of chemical modification of SFHb to lower its oxygen affinity (fixation of permanent effector inside the phosphate binding site or intramolecular cross-linking of the deoxy form of SFHb), then polymerization or substitution with polymers. We have designed new functionalized polymers (from dextran and polyoxyethylene), capable of mimicking the effect of the natural intraerythrocyte effector, 2,3-disphosphoglycerate, i.e. of decreasing its affinity for oxygen, and we have linked these polymers chemically to oxyHb. All the resulting conjugates have lower oxygen affinity than SFHb and, when injected into rats, do not lead to hemoglobinuria. Preliminary in vivo tests also showed that these conjugates possess no acute toxicity. Further experiments with rats are now under way (60% and 80% hemorrhagic shocks) with the aim of evaluating whether these new products can be regarded as potential candidates for blood substitution.
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.
To test whether Tris is required for covalent binding of pyridoxal phosphate (PLP) to hemoglobin, we carried out the reaction in solutions of Tris homologues, carrying a blocked amine function. With the exception of Mono-Tris, these compounds permitted the synthesis of modified hemoglobins with acceptable spectral properties, P50 values, cooperativity and methemoglobin content, refuting Tris HCI participation during hemoglobin pyridoxylation.
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.
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An efficient granulocytes preservation would allow an easier study of their structure and functions, of granulocytes functional diseases (e.g. chronic granulomatous disease) and an easier use of granulocytes as a clinical tool (e.g. for localizing infections or for transfusions). Unfortunately, granulocytes are nearly impossible to preserve, even in a short-term storage (less than 24 hours). This study proposes an experimental model which could improve in vitro granulocyte functions maintenance. Bicarbonate buffer, glucose, adenosine triphosphate, vitamins C and E, nicotinamide adenine dinucleotide, nicotinamide adenine dinucleotide phosphate, amikacin, and ampicillin supplementation significantly (p 0.05) improve maintenance of one or several granulocyte functions during storage.
Granulocytes transfusions set numerous problems and are less and less used. The very short maintenance of the granulocyte functions (chemotaxis) limits their use are a clinical tool (e.g. for localizing infections and for transfusions). Neither granulocyte collection by centrifugation methods (continuous or discontinuous flow) nor glucocorticoid premedication significantly alter granulocytes functions (in particular chemotaxis). Liquid preservation methods give better results than cryogenic methods. Optimal storage parameters are a temperature of 22 degrees C and a pH level close to 7.4. Chemotaxis is the most sensitive indicator of granulocyte damages in connection with storage. Bicarbonate buffer, glucose, proteins and reducing agents supplementation significantly improve chemotaxis maintenance. Cryogenic methods are less studied and less used. They allow storage of only little amounts of granulocytes. In conclusion, efficient granulocyte storage (over several days) would allow an easier use of these cells in many fields, but unfortunately, no solution is yet available.
The interactions between various dextran phosphates and Hb (hemoglobin) were studied by measuring the oxygen-binding parameters of the mixtures. The effector properties of polymers were found to depend on the concentration of monoalkylmonophosphate groups on the polymers and also on their molecular weights. The covalent fixation of dextran phosphates bearing aldehydic groups to oxyHb and deoxyHb was carried out. The oxygen-binding properties of the conjugates thus obtained depended upon the initial form of the protein. Thus, only the conjugates synthesized from deoxyHb exhibited a low oxygen affinity, which means that, in this case, the linkages between the dextran phosphate and the protein allow a permanent interaction of the phosphate groups with amines of the 2,3-diphosphoglycerate binding site. The Hill coefficient values of these conjugates were smaller than that of free Hb, corresponding to a loss of the cooperativity of the protein upon fixation of polymers. However, as these new conjugates are capable of unloading more O2 than blood when subjected to oxygen pressures corresponding to physiological conditions, they can be regarded as potential erythrocyte substitutes.
Human alpha thrombin at 1.1.10(-5) M is chemotactic for human polymorphonuclear leukocytes. This thrombin property disappears when the alpha thrombin (1.1.10(-5) M) hirudin (1.32.10(-5) M) mixture is realized. The same result is obtained when the thrombin at 1.1. 10(-5) M is inhibited by antithrombin III in a ratio of 1 mol of thrombin for 4.5 mol of antithrombin III. The hirudin and the antithrombin III appear therefore to mask, by their binding the structural domain responsible for the chemotactic properties of thrombin on polymorphonuclear leukocytes.
Chemotactic technique in agarose gel has exposed the attractive properties of human alpha thrombin with respect to human polymorphonuclear leukocytes. The observed chemotaxis is maximal between 1.4.10-5 M and 1.6.10-5 M but extends from 2.8.10-6 M to 2.2.10-5 M. Human prothrombin, in an identical concentration zone as that studied for thrombin shows no chemotactic activity on the polymorphonuclear leukocytes. During coagulation the formed alpha thrombin attracts the polymorphonuclear leukocytes to it's formation site.
Most workers studying haemoglobin solutions re-establish a P50 close to physiological values by covalent fixation of pyridoxal phosphate (PLP) using the method first described by Benesch. This is performed with deoxyhaemoglobin in a Tris-HCl buffer, considered to be necessary for transimination. To simplify the chemical procedure of the conjugation of a macromolecule to Hb-PLP we have performed the pyridoxalation in a phosphate buffer. Physico-chemical analysis of pyridoxalated haemoglobin solutions show a slight degradation of the protein, a amount of fixed PLP to the haemoglobin and a right-shift of the dissociation curve similar whether the coupling is performed in the phosphate or the Tris buffer. The pharmacological evaluation by total and isovolumic exchange transfusion in rats, of solutions prepared from both methods of pyridoxalation show identical survival times, vascular persistence and stability of the conjugates. Thus the pyridoxalation takes place with a comparable yield whatever reagent is used, proving that Tris is not essential to haemoglobin pyridoxalation.
Hemoglobin solutions will be of clinical interest only if they are easy to use, efficient and can be stored for long periods. While most studies are concerned with hemoprotein improvement (P50 and plasmatic half-life) few deal with long term stability of liquid state solutions. Physicochemical and physiological analyses were carried out on a 70 g/l ready-to-use hemoglobin solution after 1.5, 2.5 and 3 years storage at +4 degrees C, away from light and without any protective additives. The evaluation of hemoglobin stability by tests used in clinical biology shows few structural and functional alterations. However, after 1.5 year total transfusional exchanges carried out on rats cause rapid death, which seems to point to hemoprotein modifications undetected by biological techniques. It therefore appears that physicochemical tests do not provide adequate grounds for claiming that a hemoglobin solution kept for over a year and a half at +4 degrees C can still be used effectively in transfusions.
Human blood is a very complex tissue. Therefore the idea of rediscovery its different cellular and plasmatic constituents would seem to be utopic. To be efficient the oxygen carrier, be it natural or by synthesis, must be stripped of antigenicity, be easily stockable and transportable. Thus these properties permit its use in urgent circumstances (accidents, natural disasters, war...), in those countries where there is a non existent or limited transfusional structure. This, under certain conditions, during very specific pathologies (localised ischemia for example). Among several hypotheses, they are two main lines of research that of "hemoglobin solutions" the oldest and the most physiological. This will be developed here in more lengthy terms due to our personal work on the subject. The second line of research concerns fluorocarbons, the most modern and artificial and without doubt better known to doctors and the public. 1. HEMOGLOBIN SOLUTIONS. Other than nephrotoxicity, which has proved affordable, research han revealed four large limitations with hemoglobin solutions (a high affinity for oxygen due to absence or loss of 2.3 DPG, a short half life due to vascular loss, rapid dimerisation and elimination of urine, insufficient concentration of prepared solutions (70 g/L) with as a result a weak oncotic pressure and oxygen supply, oxidation in methemoglobin). In order to overcome the two inconveniences, proposals were made to modify hemoglobin chemically, the idea coming from the putting into operation of potential analogues to or substitutes for 2.3 DPG which it is advisable to bring or to keep--by covalent bonding--near to the fixation site of the natural ligand. Thus our group has already deposed several patents and is now working on a complex hemoglobin-dextran benzine tetracarboxylate which appears promising. Today, due to the quality and reproduction of the results obtained on animals with chemically modified hemoglobin preparations clinical assays should be carried out soon. 2. FLUOROCARBONS. In this very different approach which uses totally synthetic compounds oxygen carrying can only be realised in dissolved form. Due to this fluorocarbons, even though they are remarkable solvents of gas, do not reach their full efficiency unless the patient breathes in a very rich oxygen atmosphere. This is therefore a considerable limiting factor. The other big problem is the insolubility of these compounds and therefore the need to emulsify them, but unfortunately these emulsions are difficult, if not impossible to stabilise.(ABSTRACT TRUNCATED AT 400 WORDS)
Study of the interface between blood and artificial surfaces reveals numerous undesirable reactions. For example, the formation of biologically active peptides, C3a and C5a (anaphylatoxins), is a result of complement activation via the alternative pathway. It cannot be denied that the reinjection of large quantities of these molecules could be at the origin of serious side effects. We report herein the anaphylatoxin levels found with 9 hemapheresis procedures (4 plasma filtration membranes, 3 systems for platelet collection, 2 plasma treatment techniques (dextran sulfate cellulose column and cascade filtration)). All the filtration procedures generated very high levels of anaphylatoxins, especially C3a. In contrast, the centrifugation systems produced lower levels. However, considerable amounts of C3a were found with the Cobe Spectra and V50 Haemonetics blood cell separators. The anaphylatoxins generated by the primary filter were partially retained by the dextran sulfate column. These high toxin levels enable an assessment of the degree of hemo-incompatibility of these systems. However, it remains difficult to evaluate whether these anaphylatoxins are dangerous or not.
We have studied the in vitro interactions versus some blood components of the hemoglobin niosomes whose preparation and physicochemical and oxyphoric properties have been published in a precedent paper (this journal, 1989, No. 7, p. 192). This work was devoted to the research of 1) Agglutination phenomena with ABO blood group substances, plasma, some of its components and three plasma expanders, finally main erythrocytic phenotypes. 2) Adsorption of plasma proteins by immunoelectrophoresis. 3) Effects of niosomes on blood coagulation by thromboelastography. 4) Interactions between niosomes and phagocytes by electron microscopy, chemotactic migration, oxygen consumption, superoxide generation and oxydases function. These assays allow to observe and conclude that: 1) The agglutination phenomena are almost constant except with red blood cells. The agglutinates are dissociable by shaking. The agglutination appears to be nonspecific of a niosome component but is not observed with "classical" DPPC-chol-DCP liposomes. 2) Albumin and eventually transferrin are adsorbed at the surface of niosomes but without destabilizing them. 3) The vesicules show no important effects on coagulation factors, the enhancement of clotting time appearing essentially the consequence of blood dilution. 4) Niosomes phagocytosis is important but all the measurements fail to show any cellular metabolism activation: cell oxygen consumption, oxygenated metabolites generation and oxydases activity are not enhanced whatever the "electric" charge or the niosomes/phagocytes ratio used.