[Use of human anti-Pseudomonas immunoglobulins in treatment of children with burns]. / Zastosowanie ludzkiej immunoglobuliny przeciwko Pseudomonas w leczeniu oparzonych dzieci.

This studies included 15 children with burns involving 10-55% of the whole body surface, treated at the two surgical departments in Poland. All patients have been given 0.5 mL of a 15% solution of anti-Pseudomonas immunoglobulin in a deep i.m. injections for 3 consecutive days. Immunoglobulin has generally been well tolerated, except short fever attacks. Human anti-Pseudomonas immunoglobulin prepared in the institute of Haematology and Transfusion in Warsaw prevented infections with P. aeruginosa in 12 burned children. There have been no cases of bacteremia produced by P. aeruginosa in 15 treated children with burns. The obtained results indicate efficacy of such therapy in burned children.

Cryoprecipitate of intermediate purity produced in a closed thaw-siphon system from DDAVP stimulated blood donor plasma.

The effects of intravenous administration of DDAVP to blood donors and the use of DDAVP plasma for the production of cryoprecipitate in the closed thaw-siphon system were evaluated. DDAVP treatment produced on the average a 3.2-fold rise in plasma levels of factor VIII. Von Willebrand factor antigen increased to a lesser extent. Cryoprecipitate prepared from 220-280 ml aliquots of DDAVP stimulated donor plasma contained 472 +/- 210 units of factor VIII and 276 +/- 130 units of von Willebrand factor antigen. The average yield of factor VIII was 57% of that in the prefrozen plasma. The specific activity of factor VIII in cryoprecipitate was 0.77 +/- 0.44 U/mg protein, comparable to that for intermediate purity concentrates. Thus, by the use of DDAVP and the thaw-siphon technique it is possible to produce cryoprecipitate 4-7 times as potent as conventionally manufactured preparations.

A new chromogenic assay for direct detection of staphylocoagulase.

Tube test for detection of staphylococcal coagulase, despite of many disadvantages, is commonly used in clinical microbiology for identification of Staphylococcus aureus. In this paper a new chromogenic method for detection of the coagulase directly in staphylococcal cultures is described and evaluated on the basis of a comparison with the standard tube assay. The chromogenic assay appeared to be as sensitive as the tube test but results of the former one can be read in a few hours without any apparatus.

Anticoagulant properties of extracellular slime substance produced by Staphylococcus epidermidis.

Slime produced by S. epidermidis strain KH 11 was extracted with phenol-saline. The saline phase was fractionated on a DEAE-Sepharose CL-6B column. The crude slime solution and its phenol-saline fraction were found to possess anticoagulant properties. They inhibited the coagulation of human plasma by thrombin, prolonged the activated partial thromboplastin time, but did not change the rate of plasma coagulation by reptilase. The anticoagulant effect of slime could be neutralized by rather high concentrations of protamine sulphate. In the presence of plasma, the staphylococcal slime also inhibited in a concentration dependent fashion the amidolytic activity of thrombin and factor Xa against synthetic chromogenic substrates. Both antithrombin III (AT III) and other plasma component(s), presumably heparin cofactor II, were required for the full expression of the slime effect. The anticoagulant action of slime was markedly less AT III dependent than that of heparin. The activity was resistant to heating (100 degrees C, 30 min). Slime and its fractions were stronger inhibitors of thrombin than of factor Xa. Fraction IV separated by DEAE-Sepharose chromatography and particularly rich in galactose and glucuronic acid had the highest inhibitory potency. It is conceivable that slime component(s) similar to glycosaminoglycans from other sources can carry the anticoagulant activity.

Effect of proteolysis on quantitation of plasma fibronectin concentration by two immunoassays (electroimmunoassay and immunoturbidimetric technique).

Quantitation of fibronectin (FN) concentration is strongly influenced by FN fragmentation with trypsin, kallikrein and plasmin. Digestion by trypsin and kallikrein leads to a progressive decline in FN detectability by the immunoturbidimetric technique to zero values but is associated with an increase in the height of rockets in the Laurell's electroimmunoassay. Plasmin mediated FN fragmentation induces a strong overestimation of the FN content by the electroimmunoassay and, at very high enzyme concentrations, provokes an underestimation of FN by the immunoturbidimetric technique. The decline in FN reactivity in the immunoturbidimetric assay coincides with the disappearance of heavy fractions migrating only slightly faster than native FN in SDS-PAGE. The increase in the height of rockets in the electroimmunoassay is the highest when fractions of intermediate rate of migration prevail in the SDS-PAGE pattern. Concomitant use of these two immunoassays can distinguish native FN from its degraded form and may possibly provide a partial explanation for discrepancies in published studies on the concentration of circulating FN in various pathological states.

Plasma fibronectin concentrations in healthy and septic infants.

The concentration of plasma fibronectin was determined by Laurell's electroimmunoassay in 75 preterm or term newborns within the first 2 days of life, in 97 healthy infants aged from 3 days to 12 months, in 40 septic infants and in 38 healthy adult subjects. The mean fibronectin concentration in citrated plasma of normal adults was 318 +/- 84 ml/l. Healthy eutrophic term newborns 1-2 days old had approximately one-third of the fibronectin concentration of adults. There was no significant difference in the values between healthy term and eutrophic preterm newborns or between eutrophic and hypotrophic newborns. The plasma fibronectin increased strongly over the 1st month of life. No significant difference was observed between fibronectin levels in infant boys and girls. The values in septic newborns and septic older infants were significantly lower when compared with those of age-matched healthy controls. It is speculated that this deficiency, because of linkage to fibrin in disseminated intravascular coagulation or due to increased utilisation as a nonspecific opsonin and sequestration at sites of tissue injury, may contribute to organ failure in septicaemia.

Prothrombin activation by a metalloprotease from Staphylococcus aureus.

Formation of thrombin during incubation of purified bovine prothrombin with purified staphylococcal metalloprotease has been investigated. Thrombin activity was estimated by examination of clotting time and by digestion of a synthetic substrate, Chromozym TH. The metalloprotease caused direct activation of prothrombin which was inhibited by the addition of ethylenediaminetetraacetic acid. Metalloprotease produced by some strains of Staphylococcus aureus may simulate staphylocoagulase activity.

INfluence of Pseudomonas aeruginosa proteases on prothrombin, plasminogen and fibrinogen.

Out of 136 strains of Pseudomonas aeruginosa tested, 101 produced a procoagulant protease and 70 a protease with fibrinolytic activity. Strain No. 1800 producing both these proteases served as a source of the crude enzyme mixture. The crude enzyme was isolated from the supernatant and subjected to fractionation by isoelectric focusing. Five active components were isolated. Activities of individual proteases were compared. It was found that for prothrombin activation a protease characterized by isoelectric point of 7.0 is responsible; this protease is different from both collagenase and elastase. Protease available at isoelectric point of 8.8 is plasminlike and is also not identical with collagenase and elastase.

Separation of fibrinogen degradation products (FDP) by means of adsorption to insolubilized fibrinmonomer (FM-ag).

The phenomenon of complex formation between fibrinmonomer and fibrinogen degradation products was investigated by means of adsorption of FDP to insolubilized thrombin-modified fibrinogen (FM-ag). Since it could be demonstrated that there are different adsorption characteristics for early FDP and late FDP, the possibility of separation of FDP by means of affinity chromatography on FM-ag columns was evaluated using plasmic digests of 3H-Ac-labelled fibrinogen. The identification of FDP was performed by disc-electrophoresis. The results indicate that the adsorption of early FDP is comparable to the behaviour of fibrinogen, whereas late FDP show essential difference in the affinity towards FM-ag, evident by the result that fragment E adsorbs only to a minimal extents. Fragments D and E derived from fibrinogen as well as from non-crosslinked fibrin, revealed identical adsorption characteristics. Under specified conditions the procedure is suitable as a preparative method for the separation of fragments D and E.

Pseudocoagulase activity of staphylococci.

A total of 245 strains of staphylococci isolated from various pathological specimens derived from cases of human infections was tested for staphylocoagulase activity. Test systems employing normal citrated rabbit plasma and the same substrate supplemented with inhibitors of thrombin and proteolytic enzymes (but not influencing the staphylocoagulase activity) were used for testing suspensions of bacteria and cell-free culture supernatants. A total of 237 strains clotted normal rabbit plasma; however, addition of Trasylol and heparin resulted in positive results in 222 strains, whereas plasma supplemented with Trasylol and hirudin was coagulated definitely by only 173 strains. It is postulated that proteolytic enzymes of staphylococci interfere with staphylocoagulase-induced clotting and may simulate coagulase-positive activity of staphylococci. To avoid such false results, a test system for detection of staphylocoagulase should include proteolytic enzyme inhibitors. Possible mechanisms of these findings are discussed.

The nature of clotting and fibrinolytic activities of Bacteroids melaninogenicus.

From the supernatant of B. melaninogenicus ss. asaccharolyticus culture, a protein fraction was isolated by ethanol precipitation. The fraction was tested for the presence of clotting and fibrinolytic activities by application of quantitative techniques and specific substrates for measurement of prothrombin and plasminogen activation, and collagenase and elastase activity. It is postulated that ability of Bacteroides melaninogenicus ss. asaccharolyticus extracellular factors to clot fibrinogen and activate plasminogen, are due to a limited proteolysis by the proteolytic enzymes produced by this microorganism and not to the existence of specific B. melaninogenicus coagulase of plasminogen activator.

Clotting and fibrinolytic activities of Bacteroides melaninogenicus.

106 strains of Bacteroides melaninogenicus ss. asaccharolyticus, intermedius and melaninogenicus were tested for production of extracellular blood clotting and fibrinolytic factors. 78.3% of tested strains caused clotting of rabbit plasma and 79.2% activated human plasminogen. Strains producing the clotting or fibrinolytic factor only were isolated. Both factors were quite active, as positive results for most strains were detected within one hour of incubation in controlled test system.

Studies on chemically modified fibrinogen.

Treatment of fibrinogen with maleic acid anhydride renders fibrinogen unclottable depending on the degree of modification of the molecule. According to radioactive studies the release of fibrinopeptides by thrombin or reptilase is undisturbed. The incoagulability is due to inhibition of the polymerization process of fibrinmonomers derived from modified fibronogen, mainly caused by the increase of electronegative charges upon the fibrogen molecule. According to discelectrophoretic analysis modified fibrinogen fails to produce fragments D and E following plasmic digestion, however, may be degraded to high molecular weight products. Modified fibrinogen reveals some similarities to abnormal fibrinogens in congenital dysfibrinogenemia with regard to its functional properties.

Bovine fibrinogen modified with 3H-acetic anhydride (3H-AcOAc).

The method is described for acetylation of bovine fibrinogen with 3H acetic anhydride (3H-AcOAc). Preparations acetylated at pH 7.8 with 10 to 40 molar excess of 3H-AcOAc were found to contain 8 to 13 moles of acetyl residues per mole of fibrinogen. The content of clottable protein and ultraviolet (UV) spectra were unchanged as compared with control unlabeled preparations. The rate of clotting with thrombin was only slightly affected. The investigations on distribution of 3H-acetyl groups in products of proteolysis of acetylated fibrinogen by thrombin demonstrated a preferential acetylation of fibrinopetide A and absence of radioactive tracer in fibrinopeptide B. Incorporation of the label into fibrinopeptide A opens the possibility for application of fibrinogen labeled with 3H-AcOAc as a convenient substrate for selective investigation on the enzymatic phase of clotting.