M. Allgöwer

The present status of research in burn toxins

Modern intensive care combined with current improvements in the specific, systemic and local therapy of burns has delayed the mortal effects of severe burns. Nor has there been any significant improvement in this mortality during the last decade.The occurrence of uncontrollable infection and sepsis due to gram-negative bacteria or fungi as the basic cause of death was not a satisfactory explanation. So, progress should only be expected from a new concept in burn treatment. This new concept should be to view the burn disease as being caused by toxic factors induced by thermal injury to the skin. Electron-microscope studies in mice and rats have revealed similar mitochondrial alterations in hepatocytes after either a sublethal controlled burn injury or an intraperiotoneal application of an equivalent dose, of a cutaneous burn toxin. The intraperitoneal injection of different amounts of the burn toxin indicated, that the extent of the mitochondrial changes correlated directly with the dose of toxin. Investigations of liver metabolism suggested an inhibition of the oxygenation chain. The incubation of isolated liver cells together with the burn toxin demonstrated by scanning electron microscopy a direct cytotoxic effect of the burn toxin. In animal tests the pathogenic effect of the burn toxin could be prevented by treatment with an antitoxic IgG generated in sheep.The fatal sepsis of severely burned patients is the consequence of a decreased host defence against infections, which is caused by a primary and general toxic alteration of the whole organism. One important aspect of treatment should therefore be the elimination of burn toxins. To achieve this management should include primary excision of the burns, local application of nonabsorbable protein-complex-binding substances and specific passive immuno-therapy with an antitoxic IgG.

Survival in major burn injuries treated by one bathing in cerium nitrate

Sixty-four patients aged 16-74 years with total body surface area burns (TBSA) ranging from 30 to 90 per cent, were given one bathing in 0.04 M cerium nitrate within 4 h of admission to hospital. Of 21 patients aged 16-30 years, one died (aged 28 with 90 per cent TBSA), and of those aged 31-74 years, two died, one (aged 50 years with 55 per cent TBSA) had multiple internal injuries, the other (aged 51 years with 55 per cent TBSA) had a pulmonary embolism at day 19. Two risk scores, developed from data on 11,200 burn patients treated by standard methods (Roi et al. 1983), were applied to the analysis of risk for 59 patients for whom both total burn surface (TB) and full thickness (FT) areas had been recorded. About 20 patients bore risk of 0.8 or greater on the FT scale and 1.0 on the TB scale, yet instead of 80 per cent deaths among these, only two died. No FT assessment had been made on the multiple injury death whose TB risk score was 0.66. Such survival results in high-risk patients should encourage the use of cerium nitrate for treating serious burn injury.

Influence of burn-induced lipid-protein complex on IL2 secretion by PBMC in vitro

Normal peripheral blood mononuclear cells (PBMC) were incubated with the lectins PHA and ConA to stimulate IL2 release into the culture supernatants. In the added presence of the lipid-protein complex (LPC) derived from burned skin, PHA and ConA produced much less bioavailable IL2, the combination with PHA being more inhibitory of its production than that with ConA at concentrations of 1 microgram and 5 micrograms lectin/ml. As LPC alone also elicited IL2 production the inhibition of active IL2 production with these lectins was seen as a synergistic reaction with LPC. This was not altered by incubating cells with PHA alone, followed later by LPC, suggesting that LPC affects later molecular events which develop in T-cell activation. However, after incubating LPC first and washing it from the cells, both lectins were able to stimulate secretion of higher levels of bioavailable IL2, but again, less IL2 was produced with PHA than with ConA. Since PHA and ConA are reported to react with the T-cell receptor (TCR) and CD3 T-cell surface antigens, respectively, although both react additionally with CD2, it appears that LPC interfered more directly with TCR-related reactions than those involving CD3, although the two antigens have been considered to be interdependent. LPC is a trimer of a complex of six proteins from skin cell membranes, which had coalesced under the influence of thermal energy. The six proteins have relative molecular weights of 40, 50, 65, 110, 120 and 160 kDa. By coincidence 40 kDa and 51 kDa are the weights of the heterodimer subunits of TCR alpha/beta, and CD2 is 50 kDa.(ABSTRACT TRUNCATED AT 250 WORDS)

Pathophysiologische Untersuchungen an einem Verbrennungsmodell der Maus

Summary1.A new technique to apply high temperature burns and scalds under controlled conditions in living mice has been established.2.The difference between scalds and high temperature heat injuries with respect to the histological changes, the temperature profile within the skin and the mortality rate has been demonstrated. A mechanism for toxin formation shown to occurin vitro in mammalian skin under standardized energy application was reproducedin vivo.3.The toxic effect of the isolated product and the lethal burn injury(in vivo) upon kidney function measured by serum creatinine and urea has been shown.4.The relationship between the surface area injured and the animals body weight and total surface as a critical parameter with respect to the survival chance has been established.Zusammenfassung1.Es wurde ein neues Verbrennungsmodell der Maus entwickelt, das es erlaubt, Verbrennungen und Verbrühungen unter kontrollierten Bedingungen am lebenden Tier vorzunehmen.2.Der Unterschied zwischen Verbrennung und Verbrühung in bezug auf die Mortalität, den Temperaturverlauf in der Haut und die histologischen Unterschiede konnte gezeigt werden. Der mechanismus für eine Toxinbildung bei der Verbrennung wurde bewiesen.3.Der Einfluß des isolierten Toxins und der letalen Verbrennung auf die Nierenfunktion wurde an Veränderungen der Kreatinin- und Harnstoffwerte im Serum nachgewiesen.4.Das Größenverhältnis der durch thermische Energie geschädigten Haut zum Körpergewicht und der Oberfläche zeigte sich als kritischer Parameter der Mortalität.

Isolation and characterization of a toxic lipid-protein complex formed in mouse skin by controlled thermal energy: Comparison to an inactive precursor derived from thermally unexposed or native skin

Abstract 1. 1. A lipid-protein complex which has a lethal effect on recipient animals was isolated from mouse skin exposed to controlled thermal energy. 2. 2. A new isolation procedure was developed which takes advantage of the toxic activity present in thermally modified skin to trace the fate of the active compound. 3. 3. The physical and chemical properties of the toxic product were similar to those of the corresponding biologically inactive compound isolated from native skin by the same method. 4. 4. Equivalent results were obtained when germ-free, specific pathogen-free or randomly contaminated animals were used. 5. 5. The toxic lipid-protein complex and the corresponding derivative from native skin were shown to have an identical electrical charge and the same lipid (40%) and protein (60%) content. 6. 6. The lipid moieties of both derivatives were made up of six lipid classes and the apoprotein was composed of six different polypeptides. The qualitative “sub-structural” composition was also identical in both derivatives. 7. 7. The toxic lipid-protein complex differed from the non-toxic material by its larger size, higher density and its biological activity which resides in the apoprotein while the lipid moiety contributes to the toxic activity to a certain extent. 8. 8. The results suggest that the toxic compound is a polymer produced by thermal energy from a naturally occurring precursor.

Isolierung und physikalisch-chemische Charakterisierung eines Verbrennungstoxines aus der Mäusehaut

SummaryA lipid-protein complex which has a lethal effect on recipient animals was isolated from mouse skin exposed to controlled thermal energy. The physical and chemical properties of the toxic product were similar to those of the corresponding biologically inactive compound isolated from native skin by the same method. Equivalent results were obtained when germ free, specific pathogen free (SPF) or randomly contaminated animals were used. From these results, bacteria were judged not to be involved either in the toxin formation or toxic activity. The toxic lipid-protein complex and the corresponding derivative from native skin were shown to have an identical electrical charge and the same lipid (40%) and protein (60%) content. The lipid moieties of both derivatives were made up of six lipid classes and the protein fraction was composed of six different polypeptides. The qualitative substructural composition was also identical in both derivatives. The toxic lipid-protein complex differed from the non-toxic material only by its larger size and higher density. The biological activity was shown to reside in the protein moiety, while the lipids contribute to the toxic activity to a certain extent. These results suggest that the toxic compound might be a polymer produced by thermal energy from a naturally occurring precursor.ZusammenfassungIsolierte Mäusehaut wird kontrollierter Einwirkung thermischer Energie unterworfen. Daraus wird ein im Tierversuch toxischer Protein-Lipid-Komplex isoliert. Ein analoges, nicht toxisches Kontrollpräparat wird mit demselben Isolierungsverfahren aus nativer Haut gewonnen. Nach Ultrazentrifugation im Dichtegradienten zeigt sich für beide Substanzen in der Gelelektrophorese eine Proteinbande. Bei gleicher elektrischer Ladungsdichte unterscheidet sich der toxische Lipid-Protein-Komplex von seinem nichttoxischen Analogen durch größere Dichte und ein um ein Mehrfaches höheres Molekulargewicht. Beide Produkte enthalten 40% Lipid und 60% Protein mit 1% Aminozucker. Beide Lipidanteile bestehen qualitativ und quantitativ identisch aus sechs verschiedenen Neutrallipiden, die Proteinanteile aus sechs verschiedenen Polypeptidketten. Das Molekulargewicht des nativen Komplexes liegt um 1×106, das des toxischen um 3–4×106. Die toxische Substanz wird als ein Trioder Tetramer eines in normaler Haut vorhandenen Membran-Lipid-Protein-Komplexes angesehen. Für die Toxicität ist die Toxin-Protein-Fraktion hauptverantwortlich. Der Lipidanteil trägt auf unspezifische Weise zur Toxicität bei. Bakterien als Ursprung dieser Komplexe oder als mitverantwortlich für die Toxicität konnten durch Verwendung keimfrei aufgezogener Tiere ausgeschlossen werden. Damit wurde erstmals am Tiermodell nachgewiesen, daß ein Verbrennungstoxin in thermisch geschädigter Haut auftreten kann.

Immuntherapeutische Untersuchungen mit Verbrennungstoxin am Modell der Maus

SummaryWith an experimental burn model in mice, we were able to demonstrate the identity of a specific burn toxin isolated from skin burntin vitro and producedin vivo under identical thermal injuries. Active and passive immunization showed a significant protective effect, and therefore, specific immunotherapy in burns may be possible.ZusammenfassungEs gelang am Verbrennungsmodell der Maus, die Identität eines aus in vitro verbrannter Haut isolierten spezifischen Verbrennungstoxins mit einem bei gleichartiger thermischer Schädigung in vivo gebildeten Toxin unter Beweis zu stellen und durch aktive und passive Immunisierung die Möglichkeit einer gezielten Immuntherapie von Verbrennungen aufzuzeigen.

Physical, chemical and biological properties of a specific toxic lipid-protein complex formed in thermally altered mouse skin

Abstract 1. 1. Analyses of a toxic isolation product from thermally injured mouse skin by gel filtration showed the presence of two compounds which are different in size. 2. 2. The effluent pattern and analytical disc electrophoresis demonstrated the purity and homogeneity of both products as well as the identity of the smaller inactive contaminant of the toxin with a non-toxic derivative from native skin. 3. 3. Disc electrophoresis in sodium dodecylsulfate, urea and 2-mercaptoethanol revealed that the apoprotein “cores” of the toxic and the non-toxic compounds were made up of six polypeptide chains with different size but the same pattern in both products. 4. 4. The minimum molecular weight of both apoproteins was shown to be 550 000. Virtually identical chemical analyses support the hypothesis of an energy-dependent polymerization of a structural membrane component as the mechanism of toxin formation. 5. 5. A nephrotoxic effect of the new compound has been demonstrated and the toxin was shown to possess a specific antigenic property protecting mice against a lethal burn injury after active immunization. Moreover, an even better protection was achieved with a therapy using a heterologous “anti-toxic” serum. 6. 6. A specific mechanism being responsible for toxin formation is suggested.

Isolation, Biological and Antigenic Properties of a Specific Toxin Formed in Thermally Altered Mouse Skin

Abstract. A lipid‐protein complex, which has a lethal effect on recipient animals, was isolated from mouse skin exposed to controlled thermal energy. A new isolation procedure was developed, which takes advantage of the toxic activity present in thermally modified skin, to trace the fate of the active compound. A bioassay was designed for this purpose by injecting Thorotrast into the recipient animals. The toxic lipid‐protein complex and the corresponding derivative from native skin were shown to have the same lipid (40%) and protein (60%) content. The lipid moieties of both derivatives were made up of six different lipid classes. The toxic compound differed from the non‐toxic material only by its larger size and higher density. Equivalent results were obtained on samples isolated, after the removal of surface lipids and soluble cell constituents, from the donor skins prior to the application of thermal energy i.e. processing “skin residues”. The biological activity was shown to reside in the apoprotein while the lipid moiety contributes to the toxic activity to a certain extent. The results suggest that the toxic compound is a polymeric form produced by thermal energy from a naturally occurring precursor. The toxin has a specific antigenic property protecting mice after active and passive immunotherapy against a lethal bum injury in vivo. This suggests strongly that the toxic compound is an etiological factor responsible for the high mortality after severe burns. It is significant that, if scalding was applied to the same extent as the dry heat burns in vivo, the injured animals survived. This was interpreted to support the hypothesis of a specific mechanism being responsible for toxin formation. The experimental results suggest that a specific therapy for the “late mortality” after severe human burns might be possible.

Chemical fractionation of immunosuppressive gamma-immunoglobulins from anti mouse-thymocyte serum (ATS)

SummaryIgG fractions with differing immunosuppressive activity were separated by repeated ion exchange chromatography on DEAE-Sephadex, using narrow salt gradients at constant pH. Charge differences, estimated by systematic disc electrophoresis of the column effluent, served as a parameter for pooling the fractions and characterizing the corresponding IgG species. 6 main fractions, each containing pure IgG in part and subsequently 4 highly purified subfractions were obtained. Differences of immunosuppressive activity were found in these fractions and a substantial enrichment of the specific immunosuppressive activity was achieved. Lymphocytotoxicity of the fractions having higher specific immunosuppressive activity was abolished, suggesting different factors to be responsible for these properties. Further, this suggested the possibility of eliminating undesirable side effects due to cytotoxic activity. Although the final yields were incommensurate with the 5% of the total IgG-population estimated to represent immunosuppressiverelevant antibodies, this approach may provide a promising technique for further purification.ZusammenfassungAus Antithymocytenserum (ATS) gegen Mausthymocyten wurden 6 IgG-Fraktionen mit verschiedener spezifischer immunosuppressiver Aktivität durch wiederholte Ionenaustauscherchromatographie an DEAE-Sephadex gewonnen. Dabei wurden extrem flache Salzgradienten bei konstantem pH verwendet. Als Kriterium zur Bildung einheitlicher Fraktionen des Säuleneluates dienten, an Stelle des üblicherweise bestimmten Proteingehaltes, Ladungsunterschiede der in der Polyacrylamidelektrophorese dargestellten Gamma-G-Banden. Aus einer so gewonnenen reinen IgG-Fraktion gelang die Auftrennung von 4 verschiedenen Unterfraktionen mit einer bis auf das 30fache gesteigerten spezifischen immunosuppressiven Aktivität. Gleichzeitig wurde jegliche lymphocytotoxische Wirkung aus diesen IgG-Fraktionen eliminiert. Damit scheint eine Möglichkeit gegeben, aus ATG die gewünschte immunosuppressive Aktivität erheblich anzureichern und gleichzeitig die cytotoxische Komponente abzutrennen.